Fouad Pr Zouein

Ghali Rana

Rana Ghali

Thèse

Miss Nada Habeishi

Dr Cynthia Tannous

Dr Manal Fardon

Emna Abidi

Dr Ramzi Sabra

Dr Hana Itani

Miss Mr Ali Mrouheh

Maria Esmerian

Mrs Nahed Mougharbel

Mrs Ruwaydah Kabbani

Mr Georges Jabbour

Mrs Lina Kadi

Miss Mrs Rim Akika

Amira Bekdash

Mrs Rima

Rana Regards

Nada J Habeichi

Abdullah Kaplan

Rima Farhat

Abdo Jurjus

George W Booz

Ziad Mallat

Fouad A Zouein

Raffaele Altara

William E Louch

Alessandro Cataliotti

receptor CD Cluster of differentiation CLP Common lymphoid progenitor CHILP common helper innate lymphoid precursor CO cardiac output CRP C-reactive protein CSF Colony stimulating factor CTGF connective tissue growth factor CVD Cardiovascular disease CXC C-X-C motif Chemokine

Keywords: perspectives, limitations, and conclusion Interleukin-33, ST2, myocardial infarction, ventricular remodeling, ILC2, eosinophils

L'infarctus du myocarde (IM) est la principale cause de mortalité dans le monde. L'interleukine-33 (IL-33) est une cytokine présente dans la plupart des cellules cardiaques et sécrétée lors de la nécrose cellulaire, où elle agit comme un ligand fonctionnel pour le récepteur ST2. Bien que l'axe IL-33 / ST2 protège contre diverses formes de maladies cardiovasculaires, certaines études suggèrent des rôles potentiellement dommageables pour la signalisation cardiaque de l'IL-33. Le but de du présent travail de thèse était d'examiner l'effet de l'administration d'IL-33 sur la fonction cardiaque post-IM chez la souris. L'IM a été induit par la ligature de l'artère coronaire interventriculaire antérieure. Les souris ont été traitées avec IL-33 murin recombinant (1µg/jour) ou son véhicule (PBS) pendant 4 ou 7 jours. Les changements fonctionnels et moléculaires du ventricule gauche (VG) ont été évalués. Pour déterminer les cellules immunitaires impliquées, des suspensions unicellulaires ont été obtenues à partir de la moelle osseuse, du c r, de la rate et du sang périphérique, et évaluées par cytométrie en flux à 1, 3 et 7 jours après IM ou chirurgie sham, chez des animaux traités avec l'IL-33 ou son véhicule. Les résultats de la présente étude suggèrent que l IL-33 est associée à des réponses pro-inflammatoire et pro-fibrotique précoces réduites. Cependant, l'IL-33 est efficace pour établir un milieu riche en c okines de pe 2 dans le c r endommag , avec une infiltration myocardique prononcée d'éosinophiles et de cellules lymphoïdes innées de type 2, 7 jours après l'IM en comparaison avec les souris traitées avec le véhicule. Cette réponse immunitaire induite par l'administration d'IL-33 s accompagne d un remodelage cardiaque défavorable et d une détérioration de la fonction cardiaque après IM. L'administration d'IL-33 augmente la taille de l'infarctus, induit une mort accrue des cardiomyocytes, conduit à une hypertrophie du VG, et à une augmentation de la mortalité par rupture cardiaque. Cette étude révèle que, contrairement au rôle potentiellement bénéfique suggéré auparavant, l'administration d'IL-33 aggrave le remodelage ventriculaire et favorise l'insuffisance cardiaque après infarctus.

iii

Brief Overview and Main Aims and Results

Cardiovascular diseases, primarily acute myocardial infarction (MI) and stroke, represent the leading cause of mortality in the Western world and are on the rise in developing countries. Despite improved outcomes due to advances in medical and interventional treatments, a considerable number of MI patients still develop progressive left ventricular dysfunction [1]. MI, chiefly due to thrombus formation and obstruction of coronary arteries following atherosclerotic plaque disruption, is associated with decreased oxygen supply to the myocardium, resulting in myocyte loss and immediate activation of inflammation [2]. Because the mammalian heart has a reduced ability to regenerate [3], healing of the infarcted myocardium relies on a balanced activation of the sterile inflammatory response [4]. The healing process post-MI implicates a variety of cellular and molecular mechanisms that induce the replacement of necrotic myocardial tissue with a collagen-based scar [5,6]. Understanding the progression and resolution of the inflammatory processes occurring following an MI may reveal new strategies to preserve cardiac function and prevent ischemic cardiomyopathy, therefore complementing available neurohumoral blockade and interventional approaches.

Interleukin (IL)-33 is a constitutively expressed cytokine belonging to the IL-1 family, known to activate innate and adaptive immunities [7]. Following tissue injury, IL-33 is released as an alarmin from damaged cells and plays a role in the activation of immune cells and in promoting tissue repair [8,9]. The two known receptor isoforms for IL-33 are the long ST2 (ST2L) and the soluble ST2 (sST2) [10][11][12]. The long or transmembrane isoform (ST2L) is expressed on a variety of immune cells and is responsible for IL-33 signaling effects [13][14][15][16][17][18][19]. sST2, however, acts as a decoy receptor, thereby preventing IL-33 signaling [20]. sST2 has gained much attention as a cardiovascular biomarker, mainly predicting congestive heart failure and decreased survival in patients with acute MI [11,[21][22][23].

Studies performed over the past decade have shown that IL-33 is associated with the activation of various immune cells implicated in chronic inflammation, type-1 immunity, and infection, such as CD8+ T cells, NK cells, NKT cells, B cells, Th1 cells, neutrophils, and macrophages [24][25][26][27][28][29][30]. IL-33 participates in a wide range of diseases, including chronic obstructive pulmonary disease (COPD), infectious diseases (helminth, fungal, protozoa, viral, and bacterial infection), inflammatory bowel diseases, musculoskeletal diseases, fibrotic iv diseases, graft versus host disease (GVHD), diseases of the central nervous system (such as Alzheimer), cancers, obesity, diabetes, and cardiovascular diseases [31]. IL-33 is also responsible for the development of multiple allergic disorders and induces the activation of cells involved in type 2 immune responses [31]. In fact, IL-33 is the main activator of type 2 innate lymphoid cells (ILC2), which are part of the newly discovered innate lymphoid cell (ILC) family. The IL-33/ILC2 axis is implicated in allergic inflammation in multiple tissues (skin, nasopharynx, lung) and diseases (atopic dermatitis, asthma, chronic rhinosinusitis, allergic rhinitis) and is also associated with tissue remodeling and lipid metabolism in adipose tissues [17,[32][33][34]. Following their activation, ILC2 produce type 2 cytokines such as IL-5 and IL-13 [35][36][37], which participate in eosinophil homeostasis, recruitment, and proliferation [35,38]. ILC2 are present in the heart during inflammation settings such as myocardial ischemia and eosinophilic pericarditis [39,40].

In the heart, IL-33 is released from cardiomyocytes, coronary artery smooth muscle cells, and cardiac fibroblasts [7,23,39]. IL-33 is reported to have protective effects on cardiac function in many cardiovascular settings, including cardiac overload [41,42], abdominal aortic aneurysm [43] and atherosclerosis [44,45], as well as MI, and is portrayed as a potential novel therapeutic cytokine [46][47][48]. However, discrepant finding have also been published suggesting potential detrimental roles for IL-33 signaling. A study performed on atherosclerosis-prone apolipoprotein E (ApoE)-deficient mice reported that the IL-33/ST2 pathway is not implicated in the pathophysiology of atherosclerosis [49]. Another recent study involving patients with end-stage heart failure showed that the expression of the IL-33/ST2 pathway in the failing human heart is associated with increased profibrotic signaling proteins and overall cardiac fibrosis, suggesting a role of this pathway in profibrotic myocardial remodeling [50].

Moreover, exogenous administration of IL-33 has been shown to promote eosinophilic pericarditis, and adversely affected heart function even in healthy mice [51]. These studies in cardiovascular models underline the complexity of IL- Table 3-

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List of Tables

Epidemiology of cardiovascular disease

Cardiovascular disease (CVD) remains the main contributor to global mortality (31.5%), claiming more lives than all sorts of cancer with an estimated death rate of 17.9 million yearly [52]. On average, every 37 seconds, an American dies of CVD, and 2,353 deaths occur daily in the United States [53]. More than 4 million people die each year across the European continent due to CVD, including 1.4 million who die prematurely before reaching 75 [54].

Coronary artery disease (CAD), also known as ischemic heart disease, is the number one cause of morbidity and mortality attributable to CVD and causes more than 7.4 million deaths of total global mortality. CAD, including myocardial infarction (MI), remains a constant health issue and is considered as the top cause of disability and early death worldwide. Every year, more than 720,000 Americans are diagnosed with MI, out of which 46.5% are recurrent [55]. According o Fonda ion de France , 120,000 French patients are diagnosed with MI each year, out of which 10% die within the first hour of hospitalization and another 15% within the first year of diagnosis. Individuals with a first MI at the age of 45 and above are more prone (17-20%) to a recurrent MI, progression into heart failure, or even death from coronary heart disease within the first five years of diagnosis. Although over the last 15 years, advances in pharmacological treatments, critical care, and early reperfusion strategies have considerably decreased mortality post-MI, the risk of recurrent major cardiovascular events in surviving patients remains relatively high [56].

The lifetime risk for CAD varies depending on the incidence of several well known risk factors. Modifiable predisposing risk factors, including poor dietary habits, smoking, physical inactivity, hyperlipidemia, obesity, diabetes, hypertension, and stress, are on the rise within the global population and are tightly associated with an increased risk of MI development.

Identifying and controlling those risk factors through preventive measures has contributed to a considerable reduction in CAD and related mortality in both men and women [57]. Other non-modifiable risk factors include cardiovascular inheritance, age (above 55 years), and gender.

Men are four times more susceptible to develop MI than pre-menopausal women. Nevertheless, due to increased smoking habits and obesity, the prevalence of MI is currently rising among young women, and this risk becomes equivalent in both genders after menopause.

Pathophysiology of MI

The heart is the central organ of the cardiovascular system. This muscle, beating relentlessly, pumps oxygenated blood to the rest of the body and non-oxygenated blood to the lungs, whereas coronary arteries irrigate the heart with oxygen and nutrients required for its proper function. The heart muscle is supplied by two main coronary arteries, which branch off from the aorta: the left coronary artery and the right coronary artery. The left coronary artery branches into the left anterior descending artery (LAD) (which supplies the front of the septum and the front and bottom of the left ventricle), and the circumflex artery (which provides blood to the left atrium and the side and back of the left ventricle). The right coronary artery branches into the right marginal artery and posterior descending artery. The right coronary artery irrigates the right atrium and provides the main portion of blood to the right side of the heart, which irrigates the lungs. The right marginal coronary artery and the posterior descending artery, together with the branches of the circumflex artery irrigate the bottom of the ventricle and the back-side of the septum. The left and right coronary arteries further branch into arterioles, which in turn branch into countless capillaries that supply the heart with oxygenated blood.

Normally, blood flows unimpededly through coronary arteries. Over time and due to increased circulation of cholesterol and low-density lipoproteins (LDL), a process named atherosclerosis can arise. Endothelial dysfunction causes the deposition of these fatty substances within the arterial intima, which is a small area between the matrix and the endothelial cell layer of blood vessels. Oxidative modification of these LDLs triggers the activation of an inflammatory response and the attraction of monocytes and T lymphocytes into the subintimal region. This buildup of oxidized LDLs (ox-LDL) further damages the endothelial cells and results in the production of cytokines and oxygen-derived free radicals into the subintimal space [58]. Ox-LDLs are subsequently ingested by migrating smooth muscle cells and monocyte-derived macrophages, which transform into foam cells.

Accumulation of foam cells promotes the expansion of the subendothelial layer with accumulated lipids, apoptotic cells, and matrix materials. As these cells settle, they form a fatty plaque that hardens and causes reduced flexibility and narrowing of the arterial wall; a condition called atherosclerosis [59]. Since atherosclerosis develops in the lining of coronary arteries, this condition is known as CAD. Wall thinning associated with lesion instability of this fatty plaque makes it more susceptible to rupture [60] (Figure 1-1). Very often, a crack can occur at the site of plaque accumulation, causing the exposure of the highly thrombogenic content of the plaque to circulating blood, resulting in platelet aggregation and thrombus formation at the site of the crack. This thrombus can grow until it completely blocks the passage of blood in a coronary artery. Following blood flow obstruction into a coronary artery, the cardiac region once irrigated by this artery is affected and becomes deprived of oxygen and nutrients.

Figure 1-1:Plaque formation and rupture

Elevated levels of circulating cholesterol lead to the accumulation of fatty deposits, called plaques, within the arterial intima. Very often, a crack occurs at the site of plaque accumulation, causing the exposure of the fatty content to circulating blood and consequently leading to the formation of a blood clot (thrombus) at the site of the crack. This thrombus can grow until it completely obstructs the passage of blood in a coronary artery.

The heart is the organ with the highest oxygen consumption per tissue mass of all the body. To maintain cardiac viability, the coronary arteries must continuously supply oxygenated blood to the myocardium. The heart operates almost exclusively in an aerobic fashion with very little capacity to produce energy by anaerobic metabolism. While myocardial oxygen demand is defined as the quantity of oxygen required by the heart to maintain its proper function, myocardial oxygen supply is defined as the quantity of oxygen delivered to the heart through the blood circulating in coronary arteries. During resting conditions, the myocardium extracts up to 70-80% of the oxygen present within the blood flowing through the coronary arteries, compared with 25% of arterial oxygen extraction for the rest of the body.

Consequently, due to the limited capacity of the heart to further increase oxygen extraction in order to increase oxygen availability, any increase in myocardial oxygen demand (during stress or exercise) must be met by an equivalent increase in myocardial oxygen supply. Myocardial ischemia results when the coronary arteries fail to supply the metabolic needs of the heart muscle in terms of oxygen and metabolic substrates. Even a mild decrease in oxygen supply to the heart can impair cardiac electrical activity and result in anginal pain and regional interruption of myocardial contractile function. Continuous cessation of blood supply within a certain myocardial region will most likely result in an infarction [62].

MI can be classified into two main categories: type 1 MI and type 2 MI. While type 1 MI is defined as MI resulting from an acute coronary reduction or complete obstruction in coronary blood flow, type 2 MI is defined as MI caused by a mismatch in oxygen supply and demand and can occur without acute coronary obstruction. Type 1 MI can mainly result from a plaque rupture with thrombus formation, spontaneous coronary artery dissection, coronary embolism, vasospasm or microvascular dysfunction. Type 2 MI can be caused by several s ppl side abnormali ies s ch as h po emia, anemia, or h po ension, and demand side issues including severe hypertension and tachyarrhythmia [63].

Plaque rupture with thrombus formation is a type 1 MI that leads to the obstruction of a coronary artery supplying a region of the heart muscle with subsequent death of this region due to prolonged ischemia [64] (Figure 1-2).

Figure 1-2: How is the cardiac muscle affected by an infarct?

MI occurs when one of the heart's coronary arteries has an exceedingly low blood flow or is entirely blocked by the formation of a blood clot (thrombus). The blood clot usually forms inside a coronary artery that has already been narrowed by atherosclerosis. When blood flow irrigating a coronary artery becomes obstructed, the cardiac tissue surrounding this artery becomes deprived of oxygen and nutrients and can ultimately die (infarct).

The most vulnerable zones of the myocardial wall to ischemia are the endocardial and subendocardial regions because these two are the least perfused regions of the heart and are subjected to high mechanical stress. Depending on the anatomical and morphological thickness it affects, MI can exist in 2 different types: transmural and non-transmural. In transmural MI, the full thickness of the myocardial wall segment is affected and is characterized by necrosis extending from the endocardium through the myocardium to the epicardium (Figure 123). In a non-transmural MI, not all the thickness of the cardiac muscle is affected: Ischemic necrosis is therefore restricted to the endocardium and the inner myocardium.

Under anaerobic conditions, the mammalian heart is unable to generate enough energy to maintain cardiac function and survival. Ischemic injury causes decreased oxygen supply inside the cell, followed by an interruption of oxidative phosphorylation and subsequent decrease in high-energy phosphate generation. Within 10 seconds of occlusion, mitochondrial electron transport is interrupted, and anaerobic glycolysis becomes the only alternative source of adenosine triphosphate (ATP). This shift to an anaerobic metabolic pathway, associated with a coexisting lysosomal activation, promotes the depletion of intracellular ATP and

Normal artery with circulating blood

Artery with plaque buildup and thrombosis

Normal heart

Heart with AMI accumulation of lactic acid and calcium. These alterations increase cell osmolarity and initiate the development of intracellular acidosis and accumulation of free radicals. As a result, depletion of ATP promotes sodium pump failure and potassium efflux to the extracellular space, followed by an influx of water and sodium leading to cellular swelling. Ultrastructural cardiomyocyte alterations appear within minutes following the onset of ischemia, associated with glycogen depletion and mitochondrial and cellular swelling. Within 20 to 40 minutes of prolonged ischemia, sarcolemmal disruption accompanied by the presence of mitochondrial small amorphous densities are evidence of irreversible cardiomyocyte injury [65]. The ultimate result of this complex sequence of perturbations is a depression of cell function and, finally, myocardial cell death. Sections of the heart wall are composed of the inner lining (the endocardium), the heart muscle (the myocardium), and the outer surface (the epicardium). The heart is covered with an external sac (the pericardium).

Death of myocardial cells primarily takes place in the endocardium, which is the part of the myocardium that is farthest from the arterial blood supply (Figure 1-3). As the obstruction time increases, so does myocardial cell death, which leads to an enlargement of the infarcted area and its propagation from the endocardium to the myocardium and ultimately reaching the epicardium. This wavefront of myocardial cell death then extends to the collateral perfusion by lateral distribution. Within 6 to 7 hours of coronary obstruction, death of almost all the distal myocardium occurs [66]. Apoptosis is a programmed cell death that can equally affect cardiomyocytes, endothelial cells and interstitial cells and is characterized by cell shrinkage, chromatin condensation and DNA cleavage [67]. Two main pathways initiate apoptosis: the extrinsic pathway, which involves cell surface death receptors, and the intrinsic pathway, which is initiated through a wide range of intracellular non-receptor mediated stimuli and results in the change in the membrane permeability of the mitochondria. Both extrinsic and intrinsic pathways converge on caspases, which cleave several regulatory proteins that can lead to cell death. Several studies have demonstrated the essential role of apoptosis in MI. For instance, apoptosis occurs primarily in the peri-infarct area and largely contributes to cardiomyocyte cell death [68,69]. At the subacute phase of MI, apoptosis is also present, and is associated with the progression of LV remodeling [70,71]. Furthermore, increased apoptotic rates were associated with the deterioration of cardiac function and the induction of heart failure in patients with MI [70]. Apoptosis is therefore a major determinant of infarct size, LV remodeling and the development of heart failure post-MI. Necrosis is a passive process where dead cells swell and lose their membrane integrity. Upon necrosis, cardiomyocytes release alarmins, or danger signals, that trigger the activation of an extremely orchestrated immune response that clears the wound from apoptotic cells and necrotic debris, while preparing for the initiation of a reparative program [72]. Necroptosis is a type of necrotic cell death that has been depicted in multiple forms of heart failure [73] and was shown to partly mediate some associated cardiac damage [74]. Several heart failure mouse models, including LAD ligation [75,76] and chemical/drug-induced cardiomyopathy [77,78] have emphasized the role of necroptosis in worsening heart function and inducing adverse cardiac remodeling. Despite that, the exact mechanisms leading to the induction and execution of necroptosis during myocardial damage are not yet fully elucidated, both oxidative stress and inflammation have been largely associated with the development of an environment favoring necroptosis [76,79]. Recently, NLRP3-associated inflammasome has been reported to play a role in both the infarcted and noninfarcted areas post-MI, where necroptosis signaling was only depicted in the infarcted area [80]. Pathways executing necroptosis are likely to vary and depend on the triggering stimulus.

The current understanding of the mechanisms leading to necroptotic cell loss is largely based on experiments related to tumor necrosis factor receptor-1 (TNFR1) signaling, which follows the receptor interacting protein 1 and 3 and the mixed-lineage kinase domain-like (RIP1-RIP3-MLKL) pathway. This signaling cascade culminates with the disruption of the plasma membrane and cell lysis [81].

MI severity can be mainly determined by the level and length of coronary artery occlusion.

While proximal coronary occlusions could lead to increased myocardial damage and increased chance of pump failure or mechanical complications, irreversible myocardial damage is directly proportional to the length of coronary occlusion. The possibility of collateral circulation is another key determinant of MI severity, where availability of blood supply to the ischemic area from alternative blood vessels decreases the damage due to ischemia [66].

Reperfusion performed within the first few hours following MI could considerably enhance pa ien s outcome [82]. However, in most cases, this therapy is not performed early enough to prevent the negative consequences attributed to MI. Infarct size is the critical determinant of subsequent adverse remodeling and depends on the myocardial region that the infarct coronary artery irrigates. Large infarcts are generally associated with worsened remodeling that affects the infarcted as well as the residual non-infarcted myocardium [83]. Moreover, the severity of post-infarction remodeling is directly affected by the qualitative characteristics of wound healing (prolonged inflammation post-infarction can lead to enhanced protease activity associated with increased LV dilation. However, excessive matrix deposition can lead to fibrosis and ventricular stiffness, causing diastolic dysfunction) [72].

Ventricular remodeling and infarct expansion

Ventricular remodeling is defined as cellular, molecular, and interstitial cardiac alterations that manifest clinically as variations in mass, size, geometry, and function of the heart post-injury [84]. While studying myocardial ischemia, he ord remodeling as introduced for the first time in 1982 by Hochman and Bulkley to describe the replacement of a necrotic area with healed tissue [85]. Remodeling, was again used in 1985 by Janice Pfeffer to define LV dilation in rats post-MI [86]. In 1990, the expression en ric lar remodeling was created by Pfeffer and Braunwald to illustrate induced morphological alterations, more precisely ventricular chamber dilation leading to ventricular dysfunction following MI [87].

Ven ric lar remodeling has ever since been used in the literature to describe molecular and cellular changes occurring post-MI [2,65,66,88]. Ventricular remodeling is defined as alterations in the architectural structure of the heart associated with dilatation of the ventricular cavity, mainly causing wall thinning and expansion of the infarcted area (Figure 1234).

Histologically, this process includes hypertrophy and apoptosis of cardiomyocytes, transdifferentiation of myofibroblasts, and interstitial fibrosis [88].

At the organ level, remodeling results in increased LV mass and volume. Cardiac geometry is also affected where the heart becomes less elliptical and more spherical. These alterations have been shown to have adverse outcomes on cardiac function [66]. Cardiac remodeling is an adaptive response that enables the heart to preserve its function throughout the acute phase of cardiac injury. During the early phase following MI, the remaining viable cells forming the ventricular wall undergo rearrangements to help maintain CO stable.

However, this results in chamber dilation and alteration in the geometry of the left ventricle.

Chamber dilation is an adaptive response to MI that is associated with progression of systolic dysfunction, a higher incidence of arrhythmias, increased mortality, and adverse outcomes in patients with MI [89,90]. The early phase of MI is characterized by wall thinning and dilation of the infarcted area, followed by a complete transformation of this zone into a fibrous scar. Continuous ventricular dilation leads to a change in configuration (from an elliptical to a spherical isoform). This remodeling also affects the non-infarcted area leading to myocyte hypertrophy associated with increased apoptosis and interstitial collagen deposition.

Another process that accompanies post-infarction remodeling is infarct expansion, which is associated with increased mortality in patients with acute MI [91,92]. Expansion of the infarcted area is best described as elongation of the infarcted segment in the absence of additional loss and necrosis of cardiomyocytes [93]. Several processes are implicated in the pathogenesis of infarct expansion (Figure 1-5) and include:

1. Elonga ion of cardiom oc es accompanied b slippage or rearrangement of cardiomyocyte bundles leading to a decrease in the number of layers within the ventricular wall.

2. Chamber dilation induced by over-activation of matrix metalloproteinases (MMPs) and subsequent interstitial matrix degradation [94].

3. Perturbation in scar maturation resulting in increased chamber dilation due to contraction, spatial polarization, and alterations in the organization of infarct myofibroblasts. Infarct expansion is best described as elongation of the infarcted area without further loss of cardiomyocytes.

Cardiomyocyte slippage, as well as over-activation of MMPs leading to interstitial matrix degradation, are the leading causes of infarct expansion. Adapted from Frangoggiannis et al. 2015 [66].

Cellular death and the three phases of cardiac repair

Following infarction and despite their small number in the adult mammalian heart, immune cells are capable of expanding and promoting cardiac repair. As will be explained below, following MI, necrotic cells initiate an inflammatory response and activate reparative mechanisms that ultimately lead to infarct healing and scar formation. In most adult mammalian organs, the primary response to injury is tissue repair by fibrotic scar formation.

This mode of tissue repair can cause different levels of functional impairment and is, therefore, divergent from true regeneration. Although true regeneration of cardiac tissue is almost inexistent, it is essential to note the importance of non-regenerative repair mechanisms in maintaining heart functionality. In an oversimplified fashion, inflammation is often considered as harmful. However, in the heart, inflammation is an essential process that ensures the initiation of successful healing in response to injury. Without the inflammatory phase, ventricular dysfunction can occur as a consequence of MI.

Following cell death due to infarction, reparative mechanisms are initiated. These mechanisms are divided into three separate but overlapping phases and include the inflammatory phase, the granulation or proliferative phase, and the maturation or scar formation phase [65] (Figure 1 -6). In brief, the inflammatory phase is characterized by cytokine and chemokine activation and subsequent recruitment of neutrophils and macrophages into the infarct site. These leukocytes promote the clearance of the wound from necrotic cells and matrix debris. During the granulation phase, fibroblasts proliferate and start producing extracellular matrix (ECM) proteins. Inflammation subsides, and macrophages become activated and begin releasing cytokines and growth factors that help in the formation of neovascularized granulation tissue. With time, a collagen-based scar is formed and marks the onset of the maturation or scar formation phase [65]. signaling pathways [95]. Once released, these intracellular molecules stimulate the activation of a range of immune cells equipped with pattern recognition receptors (PRP) such as Toll-like receptors (TLR) or the receptors for advanced glycation end-products (RAGE) that consequently initiate the inflammatory response [66]. Examples of these alarmins include high mobility group box 1 (HMGB1), heat shock proteins (HSP), matrix protein fragments (like hyaluronan and fibronectin fragments) as well as ATP, IL-1 , DNA, and RNA. Following experimental MI, injured cells from ischemic tissues release IL-1 , which promotes the amplification of the inflammatory response. Mice deficient for IL-1 sho ed a marked decrease in inflammatory markers and neutrophil infiltration when compared to WT mice [96].

Dying cardiac cells release post-MI large quantities of DAMPS including self-DNA, which is sensed and taken up by macrophages. Phagocytosis of this self-DNA triggers maladaptive activation of interferon-regulatory factor 3 -type I interferon (IRF3-IFN) axis that amplifies the inflammatory response and leads to fatal complications post-MI [97].

The acute inflammatory phase

The release of alarmins from necrotic cardiomyocytes triggers a cascade of inflammatory events that initiate the clearance of the infarct site from dead cells and matrix 107]. Cardiac fibroblasts also produce the basic components needed to construct the ECM in order to maintain myocardial integrity [108].

Stimulated fibroblasts also enhance the expression of adhesion molecules and chemokines involved in neutrophil chemotaxis and are hence described as active participants in the progression of the inflammatory response post-MI [5,109]. Furthermore, fibroblasts can phenotypically transform into cells termed myofibroblasts following infarction [72]. In addition to cardiac fibroblasts, bone marrow-derived fibroblast progenitors, smooth muscle cells, pericytes and endothelial cells, all can transdifferentiate into mesenchymal cells, and participate in supplying the infarct with myofibroblasts [2].

Myofibroblasts are the most prominent cells implicated in myocardial fibrosis. These cells are not present in the healthy myocardium but are mainly produced during cardiac tissue injury and display characteristics of both smooth muscle cells and fibroblasts. The most important feature of myofibroblasts is their ability to migrate and contract. Their contracting phenotype is mediated by their de novo expression of contractile proteins such as non-muscle myosin and -smooth muscle actin ( -SMA) [66]. When activated, myofibrboblasts can produce several types of collagens, including collagen I and collagen III. While collagen I provides rigidity during conditions of fibrosis, collagen III provides elasticity, and promotes enhanced wall tension [110]. Collagen deposition is essential for increased wall stretching and therefore prevents ventricular rupture post infarction [99]. Myofibroblasts are also equipped with an extensive endoplasmic reticulum that allows them to produce large quantities of ECM proteins such as MMPs and their corresponding inhibitors, which are responsible for the structural tissue integrity of the myocardium. Cardiac fibroblasts and myofibroblasts are therefore regarded as key regulators of normal cardiac structure and tissue remodeling in response to injury. Furthermore, angiogenic signaling promotes the infiltration of endothelial cells and induces the formation of new capillary vessels in the infarct area. This neovascularization is crucial for oxygen and nutrient delivery to the myofibroblasts during the repair process [2].

Because the adult mammalian heart has a poor ability to regenerate following ischemic injury, dead cells are often replaced by a noncontractile collagen-based scar that preserves the structural integrity of the ventricle and prevents life-threatening events, such as cardiac rupture [66]. Remodeling of the neighboring myocardium follows, ultimately leading to impaired cardiac function. Remodeling includes hypertrophy (thickening) and fibrosis (stiffening) of the LV wall [99]. Cardiac fibroblasts are essential components for the normal structure of the heart.

However, during injury, pathological activation of cardiac fibroblasts induces excessive extracellular matrix deposition, leading to fibrosis, stiffness and decreased myocardial compliance and function [111]. In a mouse model of cardiac injury, targeting activated fibroblasts expressing the cell-surface fibroblast activation protein (FAP) by adoptive transfer of engineered T cells expressing a chimeric antigen receptor (CAR) specific for FAP, results in a substantial decrease in cardiac fibrosis and enhancement of cardiac function [112]. Two types of fibrosis, both mediated by fibroblasts and myofibroblasts, occur as a fibrotic response post MI: replacement fibrosis and reactive fibrosis. Replacement fibrosis or scar formation is an essential process that prevents LV wall rupture following an ischemic injury [72]. This beneficial scarring is achieved by the substitution of dead cardiomyocytes with fibrotic tissue. In contrast, reactive fibrosis is characterized by the hypertrophic growth of cardiomyocytes and occurs as an adaptive response to the increased ventricular wall tension induced by pressure or volume overload. By growing in size, cardiomyocytes strive to increase cardiac function and decrease wall tension. Nevertheless, cardiomyocyte hypertrophy associated with stiffness due to excessive collagen crosslinking; both gradually compromise cardiac function and may eventually lead to heart failure [99]. Reactive fibrosis is deleterious and is one of the target therapies during remodeling following MI. It is important not to target replacement fibrosis because inhibiting it could lead to a dramatic rupture in the ventricular wall.

Scar formation and maturation phase

The end of the proliferative phase and the transition to the maturation phase of cardiac repair are characterized by the formation of a stable network of structured matrix proteins. The ECM begins crosslinking through upregulation of enzymes like lysyl-oxidase [113] and the maturation of the scar follows. Replacement of the infarct with a collagen-based scar is followed by quiescence and death of all reparative cells including myofibroblasts, which become markedly decreased and may undergo apoptosis. The maturation phase is also marked by active suppression of inflammation, as macrophages ingesting apoptotic cells release large quantities of inhibitory mediators, such as 115]. Macrophages also participate in the scar maturation process by emitting signals that deactivate matrix repair, producing mediators responsible for microvascular coating and ensuring stability to the ECM [88].

Neurohormonal activation following myocardial injury

In response to myocardial injury, the heart cannot pump enough blood to meet the metabolic needs of the vital organs such as the brain, the lungs, the kidneys and the liver. The Over the past few decades, a number of novel immunomodulatory strategies have targeted the inflammatory response, with the main aim of alleviating detrimental outcomes associated with MI. Although many of these agents have given promising results in preclinical studies and early animal models, mainly showing reduced infarct size and decreased levels of inflammatory markers, application of these novel strategies on humans in large randomized clinical trials has been a challenge mainly due to the pathophysiological variation of the human condition. Bearing in mind the broad spectrum of reparative and detrimental actions induced by immune cells, inhibiting the inflammatory response post-MI may lead to significant health risks. This does not mean that the search for new immunomodulatory strategies targeting the improvement of cardiovascular outcomes must be abandoned, but should rather be reassessed in order to more deeply understand the fundamental contribution of inflammation in acute coronary syndrome. Hence, there is always a compelling need to develop new strategies aiming at controlling the inflammatory response with the main goal of counteracting the progression of MI-induced remodeling to overt heart failure.

Immune cells implicated in cardiac repair

The myocardium is a complex non-immunogenic organ consisting of a large array of cell-types. While cardiomyocytes occupy the largest proportion of the cardiac muscle space by volume (70 85%), they only constitute one third of the total number of resident cardiac cells .Microtubules (MTs) are an essential constituent of the cytoskeleton. MTs are connected to sarcomeres and together are subjected to a switch in mechanical behavior between high resistance buckling and low resistance sliding, a process partly mediated by desmin intermediate filaments. This switch between buckling and sliding is controlled by the detyrosination of microtubules, which induces the buckling behavior and provides a mechanical resistance that can hamper the contracting motion of cardiomyocytes [151,152].

Compared to other lymphoid organs, like the bone marrow, spleen, thymus, and tonsils, the heart contains a very limited amount of immune cells at steady state. For instance, the heart of an adult mouse contains less than 100,000 leukocytes at baseline. Among these cardiac leukocytes, 74% are myeloid cells, mainly macrophages, and 21% are lymphoid cells mainly B cells [153,154]. In this chapter, we will summarize the role of immune cells involved in myocardial injury and repair.

Chemokines

Chemokines are small cytokines expressed by several cell types in response to tissue

Cytokines

A number of pro and antiinflammatory cytokines are released in the heart following MI by resident cardiac or circulating inflammatory cells. These cytokines play a crucial role post-MI mainly by enhancing or dampening the recruitment of inflammatory mediators into the infarct zone. Among many others, type 2 cytokines are ideal biomarkers predicting MI severity and outcomes and are active participants in cardiac repair post-MI mainly by mediating tissue healing and regeneration. The main cytokines involved in MI pathophysiology are cited in this chapter and include: IL-1 , TNF-, IL-6, GM-CSF, TGF-, IL-10, IL-4, and IL-13.

IL-1

IL-1 is a prototypical proinflammatory cytokine that is markedly present in the

GM-CSF

Granulocyte/macrophage colony stimulating factor is a multifunctional growth factor that controls the production, proliferation, differentiation and survival of leukocytes. Although GMCSF is not required for hematopoiesis during normal physiological conditions, its levels rise significantly following injury. In the MI setting, while exogenous administration of GM-CSF exacerbates heart failure [179], elevated GM-CSF serum levels are observed in patients with MI and correlate with the severity of acute decompensated heart failure and subsequent ventricular remodeling [180]. However, in some patients, GM-CSF can have a beneficial role after MI mainly by improving neoangiogenesis [181]. In a mouse model of MI, GM-CSF acts on a specific bone marrow progenitor and controls monocyte and neutrophil production. After ischemic injury, GM-CSF stimulates the increased cardiac expression of CCL2 and CXCL2, which are chemokines attracting CCR2+ monocytes and neutrophils respectively. Increased expression of these chemokines leads to a massive recruitment of monocytes and neutrophils to the site of injury, leading to increased inflammation, tissue damage, and proteolysis, which ultimately result in heart failure and death [182].

TGF-

TGF-is a highly pleiotropic cytokine that plays an important role following infarction mainly orchestrating inflammation resolution and the transition to fibrous tissue deposition.

Experiments performed on mice with MI suggest that macrophages are the main source of

IL-13

IL-13 is another type 2 cytokine that induces the polarization of macrophages to the M2-like phenotype mainly by associating with the IL-4R and ini ia ing he ac i a ion of he signal transducer and activator of transcription (STAT) 6 signaling pathway [198]. In experimental MI, IL-13 increases in the mouse heart and peaks at 3 days post-MI [199]. In a mouse model of experimental MI, selective depletion of IL-13 was shown to decrease cardiac function mainly by inducing monocyte/macrophage recruitment to the infarct and border zone and reducing M2-like macrophage polarization. Interestingly, male MI mice with IL-13 deficiency showed increased mortality and LV dilation when compared with their female counterparts [199]. In contrast with its protective role in the experimental setting, IL-13 was implicated in the pathophysiology of chronic heart failure where increased levels of IL-13 were directly correlated with increased plasma levels of BNP and CRP and were associated with decreased LVEF [200].

Neutrophils

Monocytes

Monocytes arise from myeloid progenitors and are mainly produced by hematopoiesis in the bone marrow. Monocytes are also generated in the spleen by extramedullary hematopoiesis, which is a process not present during homeostasis but is only activated during embryogenesis and in certain disease settings like MI. As early as 30 minutes following infarction, circulating monocytes double in number and infiltrate the ischemic myocardium, mainly deriving from the splenic reservoir [210,211]. Different monocyte subsets are successively recruited to the site of infarction and regulate both the inflammatory and reparative processes occurring in the ischemic heart [98]. During the first four days of acute injury, increased expression of the monocyte chemoattractant CCL2 attracts inflammatory Ly6C(hi) monocytes expressing high levels of the CCR2 receptor [98]. Ly6C(hi) monocytes constitute the main monocyte subtype present during the early phase of infarction and participate in tissue inflammation, phagocytosis, and proteolysis. Starting day 5 post-MI, endothelial expression switches to CX3CL1 expression and the recruitment of Ly6C(low) monocytes predominates mainly through the CX3CR1 receptor [98]. In addition, Ly6C(hi) monocytes can also differentiate locally into Ly6C(low) monocytes, which participate in promoting tissue healing, ECM synthesis and angiogenesis [212].

Macrophages

CD4+ T-cells

Interleukin (IL)-33

IL33 is a 30 kDa non-chromosomal nuclear protein that belongs to the interleukin 1 family and is capable of regulating gene transcription and cell proliferation [257]. When released from cells, IL-33 is associated with its receptor, ST2, which mainly exists in two different isoforms [258]: a transmembrane receptor form (ST2L) and a short soluble receptor form (sST2) that can be found circulating in human serum [259]. IL-33 plays a key role in the pathophysiology of several diseases including asthma, allergies, arthritis, sepsis, and inflammatory bowel disease by mediating type-2 cytokine immunity and inducing inflammation [18,[260][261][262][263]. IL-33 signaling is mediated through a receptor complex, composed of IL-33, ST2L and the interleukin-1 receptor accessory protein (IL-1RAcP), that activates downstream tyrosine kinases and modulates inflammation through actions on nuclear factor kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) pathways [24,264,265]. In con ras o ST2L, sST2 ac s as a deco recep or for IL-33 that attenuates its beneficial effects when present in large amounts [7,42]. Full-length IL-33 is biologically active [266].

Upon necrosis or injury, nuclear IL-33 is passively released and acts as an alarmin or DAMP, warning the system of environmental stresses and pathogens that may lead to damage or necrosis of epithelial and endothelial cells [18,267,268]. However, during apoptosis, IL-33 is cleaved by caspase 3 or caspase 7, yielding biologically inactive isoforms [269,270]. Without cell death, bioactive IL-33 is released during mechanical stress by endothelial cells and fibroblasts [42,271,272].

Previous experiments have shown that the association of IL-33 with its transmembrane receptor (ST2L) could have many protective effects on the heart. For instance, in pressure overload, IL-33 treatment improves survival and decreases cardiac hypertrophy and fibrosis in WT but not in ST2-deficient mice. Administration of the decoy receptor sST2 reversed the antihypertrophic and anti-fibrotic effect of IL-33 [42]. In MI, IL-33 suppresses inflammatory cytokine expression; improves LV function, and decreases cardiomyocyte fibrosis, infarct size, and apoptosis [47,48]. In an ApoE-/-model of atherosclerosis, IL-33 decreases Th1 associated inflammatory response by promoting Th2 cytokine skewing [45] and decreasing foam cell formation [44]. protein was then named nuclear factor from high endothelial venules (NF-HEV) due to its abundant expression in the nucleus of HEVs, which are blood vessels specialized in the migration of naïve lymphocytes from the circulation into lymph nodes and secondary lymphoid organs. The location of the IL-33/ NF-HEV gene was also identified as a sequence present on the human short arm of chromosome 9 and precipitated a protein of 30kD [8]. Despite nonsignificant homology to any known protein at that time, NF-HEV had a strong similarity with the DVS-27 discovered earlier by Onda et al. [278]. During a computational structural database search for IL-1 family members, IL-33 was rediscovered as a cytokine ligand for the orphan receptor ST2. Upon signaling via the ST2 receptor, this cytokine was found to be a potent inducer of type 2 responses. IL-33 was then classified as the 11 th member of the IL-1 family, whose members are known for their three-dimensional structure in the carboxy-terminal end and their lack of a secretory N-terminal peptide sequence (BOX1) [7]. Among the different Several names have been attributed to IL-33 since its discovery. Important dates are indicated above and below the timeline. (adapted from Cayrol C. et al 2018 [31]).

IL-33 expression and release

IL-33 is constitutively present in almost all human tissues due to its prevalent expression in endothelial cells of small and large blood vessels [281,282]. In healthy mice and humans, IL-33 is constitutively expressed in the nuclei of non-hematopoietic cells [282] particularly in specialized epithelial and endothelial cells [281,283] [7,281,290]. In addition to endothelial and epithelial cells, IL-33 is also expressed in hematopoietic cells such as macrophages, dendritic cells and mast cells [291]. Moreover, keratinocytes, fibroblasts or smooth muscle cells also show IL-33 expression [7,42,281,292]. IL-33 induces the expression of cytokines and chemokines by many immune cell types, including Th2 lymphocytes, basophils, eosinophils, mast cells, NK cells and invariant natural killer (iNK) T cells [7,18,263,293,294]. IL-33 plays important roles in innate-type allergic airway inflammation as well as in type-2 innate immunity [279,[295][296][297]. Furthermore, IL-33 signaling plays a key role in the specification and development of a tissue-resident macrophage phenotype. For instance, downstream IL-33 signaling through activation of the adaptor protein MyD88 and ERK1/2 kinases is required for the optimal development of monocyte-derived iron recycling macrophages known as red pulp macrophages [298]. IL-33 is undeniably the major activator of ILC2 [35][36][37]299], which are cells that produce type-2 cytokines like IL-5 and IL-13 in response to IL-33 [35][36][37]. In blood and adipose tissue, ILC2 control eosinophil homeostasis [299,300]. Activation of ILC2 by IL-33 has been linked to intestinal helminth infection [35,36,295,296], atopic skin disease [301],

pulmonary influenza virus infection [302,303] and allergic airway inflammation [304,305].

The role of IL-33 is associated with major diseases, including allergies, particularly allergic asthma [18,306], rheumatoid arthritis (RA) [307], chronic ulcerative colitis [308], disorders of the central nervous system [309] and cardiovascular diseases [18,310]. In mucosal organs, IL- (Adapted from Baekkevold et al 2003, Lefrançais 2014and Cayrol 2018 [8, 31, 312]).

At the amino acid level, human and murine IL-33 share 55% homology, comprise 270 and 266 amino acids (Figure 1-8B) and are mapped on chromosomes 9 (9p24.1) and 19 (19qc1) respectively. By sequence analysis, human and mouse IL-33 were found to have almost identical molecular weights (30 kDa human and 29.9 kDa murine) [8]. IL-33 consists of eight coding exons: the first four (exons 1 to 4) encode for the N-terminal domain, whereas the last four (exons 5 to 8) encode for the typical IL-1-fold C terminal domain [24].

To be protected from cleavage, full-length IL-33 is stored within the nucleus [27], and is expressed intracellularly mainly in stromal cells including epithelial and endothelial cells, smooth muscle cells, and fibroblasts [281,313,314] (Figure 1-9A Therefore, by proteolysis at the N-terminal, the potency of the IL-33 protein can be rapidly amplified at the site of injury. Nevertheless, the bioactivity of the extracellular IL-33 is soon terminated after the proteolytic cleavage of the IL-1 family core structure [318]. Hence activated mast cells release proteases, such as chymases, that inactivate IL-33 and limit its role as an alarmin [319]. In conclusion, during apoptosis, full-length IL-33 is cleaved by caspase 3 and caspase 7 in the IL-1-like cytokine domain and becomes biologically inactive. However, during necrosis, IL-33 is processed by controlled proteolysis of the N-terminal domain into highly active mature forms, which are 10 to 30 times more bioactive. In addition, when the C-terminal core structure is truncated or oxidized, IL-33 loses its biological activity. (a soluble receptor), ST2L (a membrane receptor), ST2V and ST2LV (two variant forms of ST2). ST2L and sST2 are by far the most two abundant ST2 isoforms. ST2L is a membranebound receptor that includes an extracellular domain composed of three linked Ig-like domains, a single transmembrane domain and a cytoplasmic Toll-interleukin-1 receptor (TIR) domain [7,326,329] (Figure 1 -11). Most of the biological activity of IL-33 is mediated through the ST2L receptor, which forms a heterodimeric complex by its association with an accessory receptor protein named IL-1 receptor accessory protein (IL-1RAcP). The formation of the sST2 is composed of the extracellular cytokine-binding domain with an additional 9 amino acids at its C terminal sequence [332]. sST2 is also characterized by the absence of the transmembrane and cytoplasmic domains, which makes it a soluble secreted protein that moves freely in the peripheral circulation. sST2 is a decoy receptor that sequesters free IL-33 and prevents its binding to the ST2L receptor and consequent signaling on target cells (Figure 1 -11) sST2 is present in human serum and is used as a prognostic biomarker in patients with MI and heart failure [21,22]. Although expression of the ST2L is mostly constitutive, expression of sST2 is mainly inducible. Several inflammatory diseases have been linked with elevated serum sST2 levels including food allergy, asthma, lung fibrosis, chronic obstructive pulmonary disease (COPD), viral and bacterial infections, type 2 diabetes and systemic lupus erythematosus (SLE), as well as in some neurodegenerative diseases and certain cancers [95]. However, during adulthood, ST2LV expression decreases in all organs to remain pronounced in ocular tissue [333].

The ST2 receptor

ST2L is constitutively expressed on a large variety of immune cells, which are the main targets of IL-33 including: Th2 lymphocytes [335], mast cells [13], eosinophils [336], M2-like polarized macrophages [337], NK cells [30], iNKT cells [30], Treg cells [338], neutrophils

[261], basophils [339], cytotoxic CD8+ T cells [28] and dendritic cells [18,267,279]. ST2 is also expressed on ILC2 [24], which account for the type 2 detrimental allergic responses as well as the inflammatory responses in infection and tissue repair [294] (Figure 1 -12) . ST2 is associated with major diseases like pulmonary fibrosis, RA, asthma, collagen vascular diseases and septic shock [262,[340][341][342]. While ST2L was primarily found to be strictly expressed in hematopoietic cells, sST2 expression seemed initially exclusive to the retinal, mammary and osteogenic tissue (including that of fibroblasts) [10,343]. sST2 expression is increased during stimulation of mouse dermal tissue following exposure to ultraviolet radiation or the proinflammatory cytokines tumor necrosis factor (TNF), IL-1 and IL-1 . Unlike m rine iss es, sST2 e pression migh be more ab ndan in h mans o erall

[277].

IL-33 mechanism of action

As previously mentioned, IL-33 is released from the cell after tissue injury or necrosis and binds to the receptor complex composed of ST2L and IL-1RacP. Like many members of the IL-1 receptor family (IL-1R and IL-18R), the association of IL-33 with the receptor complex mediates recruitment of the adaptor protein MyD88 to the TIR domain present in the cytoplasmic region of the ST2 receptor [7]. This receptor complex, now associated with MyD88, activates interleukin-1 receptor-associated kinases (IRAK1 and IRAK4) by a mechanism that depends on TRAF6 recruitment in some cell types.

NF-B is an essential transcription factor that controls a wide range of genes implicated in inflammation, cell survival, adhesion, and growth control [66].

Degradation of the ST2L receptor by internalization

On the surface of unstimulated cells, the ST2 receptor has a duration of 5 hours. In parallel, both ST2L and sST2 are upregulated in rat cardiomyocytes and cardiac fibroblasts during biomechanical strain [42,346].

The role of the IL-33/ST2 axis in cardiovascular disease

Most previous data performed on various CVD models have shown improved outcomes following IL-33 induction or administration.

In a pressure overload model induced by transverse aortic constriction (TAC), administration of recombinant IL-33 to TAC induced WT mice, showed reduced cardiac hypertrophy and fibrosis, and improved contractility and survival in comparison with the ST2deficient mice. Furthermore, signaling of IL-33 through the ST2L receptor protected the myocardium during mechanical stretch by decreasing cardiomyocyte hypertrophy and cardiac fibrosis. Conversely, administration of sST2 reversed the anti-hypertrophic effect of IL-33 [42]. In vitro stimulation of cardiac fibroblasts with phorbol ester induced the production of IL-33 and sST2 and the pro-hypertrophic effects of angiotensin II or phenylephrine were abolished upon IL-33 administration to cultured rat and neonatal cardiomyocytes [42]. In IgE and IgG1 levels were markedly increased and IgG2a levels were decreased, consistent with the shift to a Th2 protective phenotype. Furthermore, the IL-33-treated group had increased levels of atheroprotective anti-ox-LDL antibodies suggesting an effect on B1 cells, which typically produce high amounts of IgM autoantibodies. Blocking IL-5 with a neutralizing antibody annulled the protective effect of IL-33 and dampened the production of ox-LDL antibodies suggesting that IL-5 might have a key role in the atheroprotective effect of IL-33 [45]. Another mechanism by which IL-33 decreased atherosclerosis is through the inhibition of macrophage foam cell formation in vitro [44]. Treating foam cell-derived macrophages with IL-33, reduced intracellular total and esterified cholesterol content by decreasing acetylated LDL and ox-LDL uptake and enhancing cholesterol efflux.

In mice subjected to MI, previous studies have shown that IL-33 reduces inflammatory responses and attenuates LV dysfunction and remodeling partly by suppressing p38MAPK and NF-kB pathways [47]. Others have shown that IL-33 improves survival, decreases infarct size, ventricular dilation and contractile dysfunction in WT but not in ST2 deficient mice [48].

In ischemia-reperfusion, IL-33 inhibits hypoxia-induced apoptosis in cultured rat cardiomyocytes in a dose dependent-manner by increasing the expression of a family of proteins kno n as inhibi ors of apoptosis (IAP) [48]. In a rat model of ischemia-reperfusion injury, IL-33 inhibits myocardial apoptosis by suppressing HMGB1 expression, associated with suppressed NF-kB signaling through undefined mechanisms [348,349].

IL-33 is also known to play a major role in preserving adipose tissue homeostasis.

Knocking out the IL-33 gene in mice fed with normal chow induces spontaneous obesity, whereas IL-33 -/-mice fed with high fat diet develop obesity associated with perturbed glucose homeostasis [34,350]. Furthermore, IL-33-/-mice exhibit reduced ILC2 presence and decreased IL-5 and IL-13 secretion in their white adipose tissue. In genetically obese diabetic mice, in vivo administration of recombinant IL-33 improves metabolic parameters, decreases adiposity and promotes polarization of macrophages to an M2-like phenotype [34,350,351],

whereas decreased levels of IL-33 in diabetic mice subjected to ischemia-reperfusion, were responsible for an exaggerated myocardial injury [352]. It is suggested that caloric expenditure is increased by IL-33 in an ILC2 dependent fashion where IL-33 regulates beige adipocytes recruitment and induces white adipose tissue beiging [34]. In humans, the presence of IL-33regulated ILC2 cells was detected in white adipose tissue and was suggested to control adipose tissue homeostasis [34].

In abdominal aortic aneurysm (AAA), daily intraperitoneal administration of recombinant IL-33, ameliorated AAA lesion collagen deposition, reduced inflammatory cytokine production, decreased T-cell and macrophage infiltration, skewed M2-like macrophage polarization, and reduced MMP activity, and cell apoptosis [43].

Soluble ST2 as a prognostic tool

Biomarkers are non-invasive tools used to detect early identification of certain diseases, reveal worsening signs of such clinical conditions and predict mortality in patients at risk [329].

High serum sST2 levels predict negative risk profiles in many diseases including heart failure [274,353], pulmonary arterial hypertension [275,354,355] and various respiratory diseases [276]. Elevated sST2 levels are associated with congestive heart failure and decreased survival in patients with acute MI [21,22]. Several studies have shown that increased sST2 expression can have a negative prognostic impact on the general cardiovascular risk profile. For instance, sST2 mRNA was markedly increased after biomechanical strain in cultured cardiomyocytes and fibroblasts and serum sST2 levels were transiently increased as early as four hours following coronary artery ligation in mice [346] and 12 hours in patients with acute MI [22].

In a study performed on 69 patients with acute MI, elevated serum levels of sST2 correlated positively with creatine kinase and negatively with LVEF [346]. In severe chronic heart failure, abnormally elevated levels of serum sST2 levels have been identified [259]. Variation in sST2 levels over a period of 2 weeks significantly predicted mortality in these patients, independent of pro-atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) [259]. Increased sST2 levels are also associated with post-infarct remodeling [356] and can predict heart failure and mortality within 30 days in acute MI patients [357]. Serum sST2 levels are also increased in patients with chronic and decompensated heart failure and can directly reflect disease severity and decreased survival [358,359]. The use of serum sST2 as a diagnostic tool is not only limited to ischemic diseases leading to heart failure but extends to all diseases that lead to heart failure. In patients with acute dyspnea, expression of sST2 is associated with cardiac structure and function on echocardiography. It is now well established that LV diastolic function is strongly associated with sST2 expression [360,361] and an increase in sST2 levels was found to be a strong predictor of mortality especially in those with preserved LVEF [360,362,363]. sST2 was even a better predictor of adverse outcomes than NT-proBNP, where baseline serum levels of sST2 independently predicted mortality at 1 year in these patients [362,363]. In hypertension, sST2 is considered as a cardiac biomarker of mechanical strain [361,364] and an increase in ST2 serum levels is associated with alterations of LV geometry

[364] and increased systolic blood pressure (SBP) [365,366]. In pulmonary arterial The results of this finding indicate that a genetic variation within ST2 could regulate its own expression and modify essential hypertension predisposition [409]. The third member of the IL33/ST2L receptor complex is IL-1RAcP. In a case-control study of the Han Chinese population, the rs4624606 AA genotype in the IL-1RAcP gene was found to be associated with increased prevalence of congestive heart disease (CHD). The AA genotype strongly correlated with increased risk of CHD particularly among hypertensive subjects [410].

In summary, these reports suggest that polymorphisms existing in or around the IL1RL1, IL-33, or the IL-1RAcP gene are directly associated with varied expression levels of ST2 (both sST2 and ST2L), IL-33, or IL-1RAcP. These genetic variants can have a major impact on the manifestation of a number of inflammatory and immune conditions. More studies are needed to establish a fundamental link between these genetic variants and CAD.

Innate lymphoid cells (ILCs) 1.4.1 ILC overview and discovery

Since 2010) [35][36][37], and their late discovery was mainly attributed to their scarcity in lymphoid organs (constituting less than 1% of total cells in the spleen and blood)

and to the lack of knowledge in ILC-specific surface markers. ILCs were renowned as a developmentally related but functionally diverse family of innate immune cells with phenotypic similarities to T helper cells [35][36][37][417][418][419][420]. In fact, the ILC family is reunited into three subsets of innate immune cells namely ILC1, ILC2 and ILC3 that mirror the cytokine expression profiles of CD4+ T helper cell subsets Th1, Th2 and Th17 respectively [421].

Compared to their adaptive counterparts, ILCs belong to the innate immune system but develop from the lymphoid lineage. Mature ILCs are known for their IL-7 R (CD127) and Thy1 In doing so, ILCs pro ide, bo h ph sicall and f nc ionall , he firs line of defense of iss eresident lymphoid cells against pathogens. Moreover, ILCs are fast responders to tissue damage and other non-immunological challenges, which are functions generally attributed to the myeloid lineage. With limited activation requirements, ILCs are capable of producing cytokines in response to any perturbation during homeostasis. ILCs are also known for their role in orchestrating lymphoid organogenesis [425,426], tissue repair after injury [17,[427][428][429], and regulation of host responses to inflammation or infection [35][36][37].

Similar to other myeloid cells in response to environmental stimuli, ILCs exhibit significant phenotypic plasticity. Ever since their recent discovery, ILCs have been the center of immunological research. While most of the information available about ILCs originates from mouse studies, many of their biological aspects have been explored and established in humans

How do ILCs differentiate?

The different groups that form the ILC family can be distinguished based on 1) their upstream cytokine activation of specific surface receptors, 2) the production of signature

Induction of ILC responses

As previously mentioned, unlike other lymphocytes, ILCs do not rely on patternrecognition receptors for pathogen detection, but rather react to the microenvironment by and ECM production as well as activation of M2-like macrophages [17,36,37,477]. ILC2 are present in several tissues including skin, gut, liver, lung, and adipose tissue [301,305,474,478]. The ILC family is reunited into three subsets of innate immune cells and include ILC1, ILC2 and ILC3 that reflect the cytokine expression profiles of CD4+ T helper cell subsets Th1, Th2 and Th17 respectively. ILCs are activated by tissue signals and produce cytokines that direct the immune response into one that is customized based on the original insult. adapted from Eberl G. et al., 2015 [493].

Implication of ILC subsets in cardiovascular disease

The basic understanding of ILC biology and their involvement in pathophysiology has grown substantially; nevertheless, their participation in CVD is still in its early discovery. Due

ILC2 and their potential implication in cardiac repair or injury post-MI

Several studies have demonstrated pivotal roles for ILC2 in adipose tissue function [519] and metabolic disorders such as atherosclerosis [506,520], obesity [34] and diabetes [521], which are all diseases pausing a direct risk for MI. At steady state, tissue-resident ILC2s

were depicted in the resting heart of naïve adult IL-5 reporter mice [38]. Although barely present in cardiac tissue during homeostasis, ILC2s are envisioned to have critical functions at low levels or to increase in numbers in response to MI. Furthermore, apart from ILC2 presence in the heart, their upstream activators IL-33 and IL-25 have been identified in the heart and were shown to positively contribute in diseases like atherosclerosis [45,522]. In MI, IL-13, which could be produced by ILC2, was also found to have protective effects in the murine heart [199]. Moreover, ILC2 activating cytokines, namely TSLP, IL-33 and IL-25 are recognized for their involvement in tissue fibrosis in both human disease and animal models. Contrary to their adaptive counterparts, ILC2 are rapidly responsive to tissue damage, thanks to their local production and secretion of cytokines, a virtue that best described cardiac ILC2 upon their identification [38].

Despite the presence of few substantial evidence involving ILC2 in the modulation of post-ischemic cardiac remodeling, several factors support this hypothesis: patients with heart failure were found to have elevated serum levels of IL-13 that inversely correlated with LVEF

Development, release and recruitment of eosinophils

The development of eosinophils occurs from GATA1 + granulocyte monocyte precursor (GMP) in the bone marrow [538] and is governed by transcription factors and cytokines such as GM-CSF, IL-3 and IL-5. The latter seems to be of particular importance in the persistence and survival of eosinophils in tissues, preventing their apoptosis and enhancing their cell activation [539,540]. CD34+ progenitor cells, Th2 lymphocytes, ILC2, mast cells, and NK T-cells are all source of IL-5 [38,541]. In tissues, ILC2-derived IL-5 promotes an eosinophilic-dependent metabolic homeostasis [495] and facilitates the homing of eosinophils into the small intestine during steady state conditions [38]. Furthermore, IL-5 can be released Normal levels of eosinophils are maintained by the activity of ILC2s, which in turn react to circardian cycling and to variations in energy intake [38]. Eosinophils enter primary and secondary lymphoid organs (including the thymus, the spleen, the lymph nodes, and the

Eosinophilia and tissue toxicity

During homeostatic conditions, eosinophils circulate in the blood in small numbers ( 350 eosinophils/mm 3 ) in both children and adults. The number of eosinophils increases under certain pathological conditions including allergic, infectious, or helminthic diseases [528].

While mild eosinophilia is defined as levels between 500-1500 eosinophils/mm 3 , moderate eosinophilia (or hypereosinophilia) ranges between 1500-5000 eosinophils/mm 3 , whereas levels above 5000 eosinophils/mm 3 are considered as severe eosinophilia [549]. Blood eosinophilia generally occurs due the enhanced production of eosinophils from the bone marrow, a phenomenon also known as eosinophilopoiesis. Peripheral blood eosinophilia can be further classified as primary, secondary, and idiopathic eosinophilia. Primary eosinophilia normally arises in the context of proliferative disorders and hematologic malignancies that lead to an increased number of progenitors and the consequent increase in eosinophil counts in the bone marrow. The most common form of eosinophilia is secondary eosinophilia, which usually occurs as a result of other primary diseases that involve an overproduction of IL-5 and the ensuing increased production of eosinophils. Idiopathic eosinophilia happens with no identifiable causes and is often associated with moderate to severe eosinophilia [542].

Even within the same organ, tissue-residing eosinophils vary dramatically in numbers.

For instance, in a healthy gastrointestinal tract, while no eosinophils are depicted in the normal esophagus, eosinophil counts gradually increase from the stomach to the colon [561]. Tissue eosinophilia normally occurs as a result of increased eosinophilic recruitment from the peripheral circulation. Once in the tissues, eosinophils are capable of causing tissue damage and organ dysfunction. Blood eosinophilia does not necessarily implicate tissue eosinophilia.

When activating signals are absent in the blood stream, blood eosinophilia can take place without any sign of increased tissue eosinophilia. In contrast, while blood eosinophil counts can be normal, enhanced migration of activated eosinophils from the circulation into tissues can still be observed. [542].

The amplitude of damage caused by the infiltration of eosinophils inside the tissues is associated with the type of stimulus that attracts eosinophils, the degree of activation of

IL-33 and eosinophils

Most allergic reactions, including eosinophilic rhinitis, asthma, chronic rhinosinusitis (ECRS), laryngitis, otitis media, and atopic dermatitis, are characterized by an expansion of As demonstrated in this chapter, eosinophils are key players in the pathogenesis and immunomodulation of several diseases and the understanding of the biology of IL-33 in relation with ILC2 and eosinophils is therefore a subject of significant controversy, showing a complex regulation that encompasses homeostasis and disease. and continued for 4 or 7 days thereafter. For flow cytometric analyses, mice were given a single (i.p.) daily injection of IL-33 or its vehicle right after sham-operation or LAD ligation (day0)

and continued for 1, 3 or 7 days thereafter (Figure 3-1).

Induction of MI

Mice were subjected to a permanent ligation (MI group) (n=167) or no ligation (sham group) (n=58) of the LAD coronary artery as previously described [594]. All surgical procedures were performed under a stereo-microscope using the Olympus SZX10. The mouse was first anesthetized with 3% isoflurane mixed with (95% O2; 5% CO2) in an induction chamber. After unconsciousness, the animal was then taped in a supine position to a temperature control pad to prevent hypothermia, and maintained on 2% isoflurane throughout the surgery. The neck and thorax were shaved using a chemical hair remover (Veet) and 70%

ethanol was applied three times to the surgical area to minimize infection. For pre-emptive and post-operative analgesia, buprenorphine (Reckitt Benckiser Pharmaceuticals, Richmond, VA)

was administered subcutaneously at a concentration of 0.1 mg/kg. The mouse was then For sham-operated mice, the same procedure was performed but without LAD ligation. Ribs and muscles were then closed in layers followed by skin closure with 6-0 prolene sutures. The mouse was then weaned from isoflurane anesthesia until full spontaneous breathing was restored. The mouse was finally extubated and allowed to warm up under an infrared lamp until full consciousness.

Echocardiography

To evaluate the progression of cardiac function or dysfunction, serial echocardiographic measurements were performed at baseline, day 1 post-surgery (LAD ligation or SHAM operation) and on the day of sacrifice (at 4 days or 7 days). All measurements and assessments were done on the Visual Sonics Echo System (Vevo 2100, VisualSonics, Inc., Toronto Canada) using the 22-55MHz linear transducer (MS550D), and performed by the same investigator. The mouse was first placed in an induction chamber and anesthetized using 3% isoflurane mixed with (95% O2; 5% CO2). After unconsciousness, the mouse was then placed in a supine position on a heating platform set at 40-42 o C and maintained unconscious with oxygenated 2% isoflurane. To monitor body temperature, a rectal thermometer was inserted, and the limbs of the animal were then taped to the electrocardiography metal leads primed with a conducting gel. To maximize visibility of the heart chambers, the chest was shaved using chemical hair remover, and ultrasound gel was applied to the thorax. The ultrasonic probe was then placed on the chest along the long-axis of the left ventricle and adjusted to obtain clear twodimensional brightness (B)-mode and motion (M)-mode parasternal long axis images. The animal was then allowed to rest until the heart rate stabilized to a range of 450-550 beats per minute (bpm) and recordings were then performed. B-mode tracings were used to assess the EF, CO, as well as LV volumes at end systole and end diastole (LVESV and LVEDV). Mmode tracings allowed the measurements of FS and the LV diameter at end systole and end diastole (LVESD and LVEDD).

Necropsy and tissue collection

Mice ere adminis ered 100 l heparin (Heparin Sodi m 1,000 I.U./ml) 30 min prior o sacrifice. Following deep anesthesia with 4% isoflurane, blood was collected by cardiac puncture, centrifuged at 2200 rpm for 10 min, and the plasma flash-frozen in liquid nitrogen and stored at -80 o C for future use. Mice were then subjected to cervical dislocation. Hearts were immediately injected with cardioplegic solution to arrest the LV chamber in diastole.

After excision, hearts were rinsed with physiological saline, weighed, and the left and right atria were resected along with the large vessels. The mid-section was then kept in 4% formalin, and the base and apex were snap-frozen in liquid nitrogen and stored at -80 o C for further experiments. Lungs were collected, weighed, and placed in a 37 o C oven overnight to assess pulmonary congestion ((Lungs Wet-Lungs Dry)x100/Lungs Wet). Spleens were collected, weighed, and stored in liquid nitrogen. Tibia length was also measured and used for normalization. For FACS tissue harvesting, single cell suspensions were obtained from mouse peripheral blood, bone marrow, spleen, and heart. Briefly, mice were given 100 µl of Heparin

(1,000 I.U./ml) 30 min prior to necropsy. Mice were then subjected to isoflurane anesthesia, blood was collected by cardiac puncture, and hearts perfused through the left ventricle with 5 ml of ice-cold PBS. Hearts and spleens were excised and placed in PBS (1% FBS) (FACS buffer). Femurs and tibias were also harvested, cleaned of all connective tissue and the extreme distal tip of each bone was cut off. Bone marrow was then collected by flushing through the bones with ice-cold FACS buffer. Heart specimens were cut in fragments of 2-3 mm 3 .

Fragments were suspended in 5 ml DMEM (10% FBS) containing an enzymatic cocktail (Collagenase D (450 U/ml), Roche/Sigma, cat# 11088858001; Dispase (1U/ml), STEMCELL Technologies, cat# 07923; and DNAse I grade II (60U/ml), Roche/Sigma, cat# 10104159001)

and dissociated using the Miltenyi GentleMACS for 15 sec. Dissociated tissue was digested for 30 min at 37 o C with gentle rotation. Whole blood was first subjected to immunofluorescence staining, then lysed using the red blood cell lysis buffer (Qiagen, cat# 158904). Spleens, bone marrow suspensions, and digested heart tissue were then triturated, and cells filtered through a 40 µm nylon mesh (BD Falcon). Cell suspensions were centrifuged (400g, 4 o C for 10 min) and subjected to red blood cell lysis for 3 min. The lysis reaction was stopped using 20ml FACS buffer and the mix was centrifuged (400g, 4 o C for 5 min). Total leukocyte numbers were determined using trypan blue. For each panel, 100 µl of cell suspension containing 1x10 6 cells was used from each organ.

Determination of infarct size

LV was separated from the right, and sliced transversally into three sections: Apex, midsection and base. Each section, including the right ventricle, was incubated with 2% 2,3,5 triphenyltetrazolium chloride (TTC) for 10 minutes at 37 o C. Sections were then photographed with a high-resolution digital camera (EOS rebelT3i DSLR, Canon, Tokyo, Japan). Infarct scar area, defined as unstained regions by TTC, was quantified using ImageJ 1.52 software and calculated as the ratio of infarct area to total LV area. All measurements were performed by an investigator who was blinded to the identity of the animals.

Survival

Starting with recovery from surgery, survival rate was assessed based on the animals that survived until the day of sacrifice. An autopsy was performed on each animal found dead.

Mortality was assessed using Kaplan-Meier survival curve analysis For dehydration, the inverse process takes place. Water bound to the tissue or found free in the tissue must be removed by immersing the sections in a series of increasing concentrations of ethanol [70% (2X 3 minutes); 95% (2X 3 minutes); 100% (2X 3 minutes)],

Histology

Rehydration and dehydration of fixed tissue

so that all the water present in the tissue is slowly replaced by alcohol. This incremental immersion in alcohol limits excessive tissue distortion. To achieve complete dehydration, sections are then immersed in xylene (2x3minutes), a process called clearing. Clearing refers to the use of solvents with high refractive index that allow tissue to become clear or transparent.

After xylene immersion, the tissue is dehydrated and ready for paraffin embedding.

Hematoxylin and Eosin staining

Hematoxylin and Eosin (H&E) staining was used to assess cardiomyocyte hypertrophy.

Hematoxylin is a basic dye, which stains nucleic acid in blue-black. However, Eosin is an acidic dye and stains proteins in pink. The process begins by rehydrating the tissue (see previous section), and staining it with Harris Hematoxylin for 2 minutes. Extra stain is washed off using 1% acid-alcohol and running water. Tissue is then immersed in Scotts Tap water substitute blueing reagen for 30 seconds, ashed in ap a er and counterstained using 1% eosin (Surgipath Medical Industries, Inc., UK) for 2 minutes. Sections are finally dehydrated (see previous section), mounted with DPX mountant and protected with a coverslip.

Masson trichrome staining

Western blot

Mouse cardiac tissues were ground with liquid nitrogen and proteins were extracted in

mRNA extraction and RT-qPCR

Total RNA was isolated from whole mouse heart using Trizol isolation reagent. Reverse transcription was performed using the QuantiTect Reverse Transcription Kit (Qiagen), and RT-qPCR was completed using the QuantiFast SYBR Green PCR Kit (Qiagen) according to the man fac rer s ins r c ions. Real-Time PCR was performed on a CFX-96 RT-PCR system (Biorad). Cycle quantification (Cq) values were assessed during the exponential amplification phase. The Cq value represen s he n mber of PCR c cles req ired for he sample s reac ion curve to cross the threshold line (or background signal) and detect a real signal. The quantified data were analyzed using the 2 C method and the relative expression levels of target genes, defined as fold change, were normalized to the corresponding fold change of the sham+vehicle group, which was defined as 1.0. The primers used for RT-qPCR are listed in Table 3-1. mRNA levels were standardized with the housekeeping gene hypoxanthine-phosphoribosyl transferase (HPRT), and all experiments were performed using n=3-4 for sham groups and n=5 or 6 for MI groups.

Assessment of cytokine levels by ELISA

The

TUNEL assay

Myocardial tissue sections obtained from sham-operated and LAD ligated mice (treated with IL-33 or its vehicle (PBS)) at two different timepoints (4 and 7 days) were analysed.

Single cell suspension and flow cytometry

Mice were sacrificed on day 1, 3, and 7 post-MI. A subset of 5 to 6 mice for the MI group and 3 to 4 mice for the sham-operated group per time point was used for flow cytometric analysis. Immunofluorescence staining was performed on single cell suspensions obtained from mouse peripheral blood, bone marrow, spleen, and heart as previously described [39,141]. Immune cell populations were defined based on previously published data [312,506,596,597]. For each panel mix, 100 µl of cell suspension were used from each organ and nonspecific binding was primarily blocked by incubating isolated cells with 0.5 μg Fc blocking antibody, anti-CD16/CD32 (clone 2.4G2, BD Pharmingen) at 4°C for 5 minutes. Cells were subsequently stained with 50 µl of the antibody mixes listed in Table 3-2 andTable 3-

Panel #1

In the first panel, DC, monocytes, and macrophages were identified. After the primary gating (Figure 3

Panel #2

In the second panel, neutrophils, B cells, NK cells, and eosinophils were identified.

After the primary gating (Figure 3

Panel #3

After the primary gating (Figure 3-26), a combination of lineage (Lin) FITC-conjugated antibody cocktail (Table 3-3) allowed the exclusion of T cells (including CD4+ and CD8+ Tcells), B cells, dendritic cells, neutrophils, NK cells, mast cells, basophils, and macrophages in a single dumping gate. ICOS positive cells but negative for Lin were subsequently subgated into CD25 vs SSC-A where CD25+ cells were defined as ILC2 (Lin-ICOS+CD25+) (Figure 3-29B).

Panel #4

After the primary gating (Figure 3-26), total leukocytes were gated as CD45+ cells (Figure 3-30A), total T cells as CD3+ cells (Figure 3-30B), then positive cells for CD4 cells but negative for CD8 were defined as CD4+T-cells (CD45+CD3+CD4+CD8-). In contrast, cells positive for CD8 and negative for CD4 were defined as CD8+ T-cells (CD45+CD3+CD4-CD8+) (Figure 3-30C).

Flow cytometric analysis

Samples were acquired on a BD FACSAria flow cytometer equipped with 4 lasers, and capable of detecting up to 17 fluorochromes. Data was analyzed using the FlowJo software 10.5.3 (Treestar,Ashland,OR). Voltages were set and the compensation matrix was computed based on unstained and single stained controls. Fluorescence minus one (FMO) controls were used to set gates. From each sample, a total of 0.5 x10 6 to 1x10 6 events were collected and doublets were excluded from the analysis using FSC (Area) versus SSC (Height). Dead cells were excluded using DAPI staining. All data percentages were calculated from total live cells or from CD45+ cells.

Statistical analysis

All analyses were performed in GraphPad Prism 8 (GraphPad Software, La Jolla, CA).

Data are presented as means ± standard error of the mean (SEM). Differences among experimental groups were analyzed for statistical significance using one-way analysis of variance (ANOVA) if there was one independent variable or two-way ANOVA if there were o independen ariables. To adj s for m l iple comparisons, e performed T ke s poshoc test, which main ains o erall pe I error ( ) at 5%. P-value <0.05 was considered statistically significant. Comparison of survival curves is analyzed using Log-rank (Mantel-Cox) test.

IL-33 worsens cardiac functions post-MI

IL-33 treatment worsens cardiac remodeling 7 days post-MI

All mice at baseline were the same age and ranged between 23 and 27g (25.1±2.3) g.

Induction of MI decreased total body weight over time but only significantly at 7 days in both MI groups to comparable levels (MI+vehicle=24.21±0.47; MI+IL-33=23.79±0.44) g (Table 3-

IL-33 treatment elicits eosinophilia

Eosinophils were identified as CD45+SiglecF+CD11c-. IL-33 is known to directly facilitate the maturation of eosinophils by enhancing their survival, activation, and adhesion

IL-33 treatment increases ILC2 levels at 7 days post-MI

ILC2 were identified as Lin-ICOS+CD25+. Although present in all organs in a very small percentage, ILC2 seemed to increase with IL-33 treatment. In the bone marrow, compared to

IL-33 administration modulates the activation of the adaptive immune system

DC were identified as CD11b+CD11c(hi). IL-33 increased DC production in the bone marrow to culminate at 7 days in both IL-33-treated groups (Figure 3 B cells were identified as CD45+B220(hi)IgM(hi)CD19+. In the bone marrow B cells constituted more than 7-14% of total leukocytes at day 1 in all experimental groups. At day 7, a decrease in B cell levels was observed between day 1 and day 7 in all studied groups, which was not significant in the vehicle-treated MI group (Figure 3

IL-33 treatment induces splenomegaly

In contrast to the normal spleen size and weight observed in the vehicle-treated sham and MI mice (ranging between 4.19±0.42 mg/mm and 4.81±0.19 mg/mm), administration of IL-33 for 4 and 7 days induced a significant increase in spleen weight in a time dependent manner regardless of the surgical intervention (Figure 3 -22). Increases of 1.73 folds and 2.6 folds in spleen weights were observed between the vehicle-treated and IL-33-treated sham-operated groups at 4 and 7 days respectively. The variation in spleen weights in the IL-33-treated MI groups was comparable to that observed in the sham-operated groups where increases of 1.7

folds and 3 folds in spleen weights were seen between the vehicle-treated and IL-33-treated MI groups at 4 and 7 days respectively. These findings are consistent with previous data showing increase in spleen size and weight with IL-33 treatment [7,273].

In this thesis, we explored the mechanism by which IL33 can cause splenomegaly and the implication of this on MI mice. Flow cytometry was performed in order to assess the impact of IL-33 administration on immune cell populations present in the spleen at 1, 3, or 7 days post sham-operation or LAD ligation. In summary, IL33 administration induced a decrease in T lymphocytes and monocytes while increases in the total number of splenic ILC2, eosinophils, and macrophages were observed, which might have participated in the increase in spleen weight and size with IL-33 treatment.

IL-33 treatment upregulates cardiac sST2 expression at 7 days in the presence and absence of MI

Patients with cardiac fibrosis, hypertrophy, LV dilatation, and decreased ventricular contractility have elevated sST2 levels, which is considered an independent predictor of Tables Table 3- The site of ligation of the LAD was determined 2mm below the left atrioventricular border. Using a tapered atraumatic needle, the LAD was isolated, ligated with a 7-0 prolene thread and tied with three knots. Successful ligation and ischemia were confirmed by visual discoloration and cyanosis of the ventricle distal to the occlusion. After the primary gating (Figure 3-26), (A) total leukocytes are gated as CD45+ cells, (B) total T cells as CD3+ cells, (C) then positive cells for CD4 cells but negative for CD8 are defined as CD4+T-cells (CD45+CD3+CD4+CD8-). In contrast, cells positive for CD8 and negative for CD4 are defined as CD8+Tcells (CD45+CD3+CD4-CD8+).

4 General Discussion and Conclusion

Discussion

Due to the limited capacity of the adult mammalian heart to regenerate, the healing process post-MI is phasic, relying predominantly on an early sterile inflammatory response to remove the debris of dead cells. Subsequently, the granulation and maturation phases follow with the main aim to replace lost cells with fibrotic tissue and promote the formation of a resilient scar. Depending on the clinical state, and the presence or absence of comorbidities, alteration in transition between phases such as inflammation prolongation or increased fibrosis deposition can worsen cardiac remodeling post-MI. Hence, providing a pharmacological platform to promote appropriate transition between early remodeling phases post-MI through immunomodulatory mechanisms is gaining significant attention and constitutes the basic aim of the experiments presented in this thesis project.

In the present study, we assessed the reparative actions of the alarmin IL-33 on the infarcted myocardium. IL-33 was administered for 4 and 7 consecutive days following LAD coronary artery ligation or sham-operation. We observed that IL-33 was indeed effective in activating a type 2 immune response in the damaged heart, consistent with the anticipated wound healing response, as evidenced by early (day 4) increased levels of type 2 cytokines Previous studies have shown that the expansion of the infarct is mainly due to muscle bundle slippage, which is more likely to occur in transmural infarctions [93], and is generally associated with increased prevalence in infarct wall rupture [87,605]. The exaggerated wall thinning and dilatation of the infarct area not only induces cardiac rupture but also triggers cardiac remodeling and ultimately leads to congestive heart failure [605,606]. Therefore, the frequency of myocardial rupture observed in the IL-33-treated MI mice is an indicator for the amplitude of infarct expansion and consequently for the extent of cardiac remodeling and progression to cardiac dysfunction in surviving mice. In this study, more than 50% of the IL-33-treated MI mice died of cardiac rupture. The latter might have been an extreme outcome of infarct expansion in which the dilated zone is insufficient to preserve the integrity of the ventricular wall before adequate deposition of collagen and the formation of a mature scar.

Despite decreased systolic function with IL-33 treatment, our FACS data did not show any major changes in monocyte and neutrophil cardiac infiltration pattern in the IL-33-treated MI group when compared with the vehicle-treated group at 7 days. These results are in agreement with previously published data showing no major increases in cardiac neutrophils and monocytes despite exacerbation in cardiac dysfunction and increased myocarditis with IL-33 treatment [51]. In the present study, increased cardiac hypertrophy and fibrosis were observed following IL-33 administration. This may also reflect a compensatory response to increased infarct expansion, although it is unsettled whether the fibrosis was productive or reflected decreased ECM turnover. Overall, IL-33 treatment was associated with a decrease in profibrotic markers in the infarcted heart (CTGF, TGF-) and in ac i a ed m ofibroblas s ( -SMA), as well as an increase in MMPs that degrade the ECM. Increase in MMPs is linked to the slippage of myocyte fascicles thereby promoting wall thinning in the infarcted area leading to cardiac rupture. Previous studies have shown that MMPs are not only involved in ECM degradation, but are also implicated in the progression to heart failure [607]. Targeted deletion of MMP2 or MMP9 decreases the incidence of cardiac rupture and attenuates LV enlargement following LAD ligation in mice [204,608,609].

We observed as well an increase in ILC2s with IL-33 treatment in the bone marrow, spleen, and blood, as well as the myocardium that was further enhanced at day 7 in the infarcted mice. ILC2 are main producers of type 2 immune cytokines and are known to play a central role in maintaining homeostasis in healthy lean adipose tissue, where they coordinate adipocyte, eosinophil, macrophage as well as innate like B-cell (IBC) function [32]. In adipose tissue, binding of IL-33 to the ST2 receptor present on the surface of ILC2 promotes the release of type 2 cytokines that play a key role in the improvement of obesity-induced insulin resistance. These cytokines enable the skewing of macrophages present in adipose tissue to an antiinflammatory M2-like phenotype [514,515]. Whether the cardiac ILC2s originated from a resident cardiac or pericardiac pool [39] or recruited from the mediastinal cavity [40] as suggested by others was not established. Along with Th2 cells, ILC2s are a major source of type 2 cytokines and mediators of the actions of IL-33 at barrier sites of the body [11], and in certain cases shown to amplify inflammation via activation of eosinophils [610,611]. Our results are in agreement with a previous study showing detrimental outcomes with IL-33 treatment. Daily administration of IL-33 induced systemic activation of ILC2 and increased Th2-biased immune responses, which were observed in the stomach and were associated with atypical gastric pathology. IL33-treated mice developed transmural inflammation in the stomach, associated with mucous metaplasia that was mediated by Th2/ STAT 3 signaling [603]. Although we did not see any increase in cardiac IL-4 and IL-13 protein expression in our study with IL-33 treatment in the sham-operated group at 4 days, MI mice showed a substantial increase in IL-4 and IL-13 protein levels with IL-33 treatment. In contrast, elevated levels of cardiac IL-5 mRNA were observed at 4 days and 7 days in the sham-operated and MI groups treated with IL-33, suggesting the involvement of this cytokine in the increased eosinophil levels observed in the heart.

As previously reported [7,273,600], IL-33 treatment induced splenomegaly, which we observed was associated with a delayed accumulation of eosinophils in the spleen. This finding is consistent with several reports stating that IL-33 administration is sufficient to induce eosinophilia in a number of tissues [7,600] In contrast with the lower levels of IL-1 obser ed in he hear a 7 da s in the IL-33-treated MI group when compared with the vehicle-treated group, IL-1 plasma le els were increased with IL-33 treatment at 7 days. Eosinophils are known to induce IL-1 pon ac i a ion [614,615]. Since eosinophils are abundant in the circulation of IL-33-treated mice at 7 days, they might have induced the increase in IL-1 le els in he plasma a 7 da s in this group. In addition, IL-33 was reported to induce degranulation and superoxide anion production of human eosinophils as effectively as . Nevertheless, differential effects of IL-33 on collagen deposition and MMP2/9 levels in sham versus MI hearts suggest that some other factor(s) might be involved in activating eosinophils. Enhanced secretion of GM-CSF from eosinophils increases the expression of the anti-apoptotic BCLXL in eosinophils and consequently inhibits their apoptosis [617,618]. GM-CSF can also promote the secretion of IL-4 and IL-13 from IL-33-activated eosinophils and lead to the differentiation of macrophages to an alternatively activated (M2-like) phenotype [572]. In this study, IL-33-induced increase by day 4 in IL-4 and IL-13 in the infarcted myocardium and the presence of GM-CSF may have contributed to this increase. Previous studies have shown a direct correlation between increased serum GM-CSF levels and impairment of the healing process post-MI by acting at a distance in the BM and locally in the heart to produce and enhance immune cells accumulation [182]. In this study, elevated serum GM-CSF levels were associated with strong eosinophil recruitment and cardiac dysfunction following IL-33 treatment post-MI. Our current data are in agreement with recently published data stating that the activation of ILC2 by IL-33 plays a pathogenic role in the heart by inducing cardiac eosinophilic infiltration and exacerbating cardiac dysfunction [40]. Earlier studies indicated that eosinophils are rapidly activated within 2-3 days following MI, infiltrate into the myocardium, and contribute to cardiac rupture in some patients [584,585]. Other studies have linked eosinopenia to an acute MI in patients presenting with acute coronary syndrome [590,592]. In addition, a lower eosinophil to neutrophil ratio in patients undergoing percutaneous coronary intervention (PCI) for ST-segment elevation MI (STEMI), 24 h after admission, was associated with a greater incidence of a major adverse cardiac event (MACE) up to 1 year later [586]. More recently, eosinopenia was associated with worse clinical outcome over a 3.5 year follow-up in STEMI patients with non-severe LV dysfunction undergoing PCI [619]. A decrease in blood eosinophils might be indicative of their accumulation in the damaged myocardium [620]. This scenario is further substantiated by evidence showing a positive correlation between higher baseline serum levels of eosinophil cationic protein (ECP) and follow-up occurrence of MACEs after PCI [587,588,621]. On the other hand, Shiyovich et al.

[591] observed a U-shaped association between blood eosinophils within 72 h of admittance and greater risk of death in patients with acute MI, with a greater count associated with lower 1 year mortality rates. This observation was interpreted to mean that eosinophils might help control the inflammatory response. These res l s s gges ha ph siological le els of eosinophils might be protective in the setting of MI, whereas increased eosinophil levels as observed in the present work could be detrimental.

Evidence suggests that eosinophils may contribute to thrombus formation that is associated with MI [589,590,622]. At least at the gross level, we saw no evidence for ventricular thrombi in IL-33-treated mice after MI. We did, however, observe increased expression of MMP2 and MMP9 at 7 days that may have contributed to wall thinning and rupture. These metalloproteases regulate ECM turnover and were shown to be produced by eosinophils and contribute to airway remodeling [623,624]. In the present study, the availability of cytotoxic CD8+T-cells in the bone marrow seemed to be more prominent during the early phase post-MI (day 1) and decreased in both vehicle and MI treated groups in a time-dependent manner. The reason of this decrease might be explained by an enhanced migration of these cells into the circulation or to secondary lymphoid organs. Although CD8+ T-cells were supposed to significantly increase in the heart at day 7 post-MI [161], neither MI groups (treated with vehicle or IL-33) showed significantly elevated levels at this timepoint. Both MI groups showed similar CD8+ T-cell levels in the heart suggesting no involvement of these lymphocytes in the differences observed between the IL-33 and vehicle treated MI groups.

As previously mentioned, infiltration of mature B-lymphocytes exerts detrimental outcomes in the myocardium post-MI [141]. B cells promote the secretion of CCL7, which is a chemokine orchestrating the mobilization of proinflammatory Ly6C(hi) monocytes from the bone marrow, consequently potentiating the proinflammatory response in the ischemic heart.

In this study, B cells seemed to be mobilized from the bone marrow at 7 days especially in the IL-33-treated groups. Although B-cells increased significantly in the heart at day 7 in both MI groups (treated with vehicle and IL-33), we did not see any increase in monocytes in these groups at this timepoint. In contrast, Ly6C(hi) monocytes were at their lowest levels in both MI groups at 7 days. Both IL-33 and vehicle-treated MI groups showed similar levels of B lymphocytes at 7 days indicating that these cells might not be involved in the detrimental effects observed with IL-33 treatment.

In this study, several factors might explain the different responses observed between the sham and MI mice treated with IL-33. Apart from being administered exogenously, IL-33 is a biomechanically induced protein [42] that is constitutively present in the nuclei of cardiomyocytes and cardiac fibroblasts [313] and may be released upon necrosis following MI [48]. In addition, several immune cells including dendritic cells, macrophages, B cells, eosinophils, and mast cells, are all known to induce the expression of IL-33 upon activation [632][633][634][635][636][637]. In contrast, IL-33 activity might be reduced by extracellular cysteine oxidation causing the formation of disulfilde bridges within the IL-33 molecule and the consequent disruption of its binding to the ST2 receptor [320]. Furthermore, the increased production of the decoy receptor sST2 in the MI setting can also influence the response [346]. All those complex variations in IL-33 availability, along with the cardiovascular events occurring post-MI might have participated in the differences observed at the molecular level between the sham and MI mice treated with IL-33.

Previous studies have shown that the association of IL-33 with its transmembrane receptor (ST2L) could have protective effects on the heart. In the MI setting, Yin et al showed that IL-33 significantly decreases macrophage infiltration and alleviates inflammation in the heart, making IL-33 a potential therapeutic cytokine for cardiac dysfunction post-MI [47,48]. Unlike Yin e al. ho sed -actin as housekeeping protein, total protein was used as loading control in this study for Western blot experiments. Previous studies have shown that the total protein normalization approach is typically better than the use of a single HKP [638][639][640][641].

Moreover, the use of Ponceau S as a total protein stain was recently proposed by the American Journal of Physiology and the Journal of Biological Chemistry to be the normalization method of choice for Western blot analysis [642,643].

Since the discovery of IL-33 over a decade ago, it became well established that this cytokine participates in tissue homeostasis, but can induce highly detrimental effects when activated inappropriately. Hence, several mechanisms have been developed to harness the activity and bioavailability of this versatile cytokine on multiple levels. Soluble receptor blockers of free IL-33 including the IL-33Trap and a form of the sST2 receptor have been developed to antagonize the unwanted biological functions mediated by . In allergic conditions involving hyperreactive immune responses, such as allergic asthma and atopic dermatitis, IL-33 is a disease sensitizer and mediates detrimental functions [18]. The use of recombinant sST2 or antibodies targeting IL-33 or ST2 to block the signaling of the IL-33/ST2 axis have proven beneficial outcomes in the management of allergic asthma and atopic dermatitis [647][648][649][650][651]. However, in a multitude of other processes, the IL-33/ST2 signaling pathway has pleiotropic immune functions and operates like a double-edged sword, presenting both tissue-reparative and pathological properties in many organs of the body. For instance, in the kidneys, lungs, skin, gastrointestinal system, central nervous system, and cardiovascular system, IL-33 mediates both inflammatory and reparative responses [18,652]. In addition, the IL-33/ST2 axis is implicated in several diseases, including colitis, fibrosis, lupus, rheumatoid arthritis, multiple sclerosis, central nervous system disorders, and age-related macular degeneration [95]. Given the diverse roles of IL-33 in tissue injury and repair, determining the appropriate time to manipulate the IL-33/ST2 axis can be a great challenge in alleviating inflammation. In myocardial infarction where the reparative process is associated with a great imbalance in immune responses, neither the physiological nor the pathological functions of IL-33 are fully elucidated and await further exploration.

Limitations and future perspectives

The present study has few limitations that should be acknowledged. Despite increased infiltration of ILC2 and eosinophils following IL-33 administration, our study did not explore the role of ILC2-deficient and eosinophil-deficient mice in the pathogenesis of MI. Moreover, the IL-33/ST2 axis is known to activate Tregs [510,613,653] as well as several signaling pathways including NF-kB, MAPKs and phosphoinositide 3-kinase (PI3K) [7,326,[654][655][656][657][658].

Previous studies have indicated that inhibition of the PI3K signaling pathway reduces inflammation and improves outcomes in several pathological events [659-662], including myocardial infarction [663]. The PI3K signaling pathway is also implicated in eosinophil chemotaxis and inhibition of the PI3K pathway was shown to impair release of eosinophils from bone marrow [664]. Future studies are warranted to explore the role of IL-33 daily administration in ILC2 and eosinophil KO mice with MI, and to examine whether Tregs are implicated in this pathophysiology. Furthermore, a thorough examination of the signaling pathways is required, particularly exploring whether inhibition of the PI3K signaling pathway improves outcomes following IL-33 administration in the presence and absence of MI. IL-33 is known to amplify M2-like macrophage polarization [337,665]. In this study, we saw an increased production of macrophages at day 7 with IL-33 treatment but we did not assess the type of macrophages implicated. Several marker genes such as iNOS (inducible NOsynthase), TNF-, and ca hepsin K are indica i e of he presence of classical M1-like macrophages whereas YM-1 (also known as chitinase-3-like protein-3), osteopontin, arginase-1, and CD206 are indicative of the presence of alternatively activated M2-like macrophages.

The addition of the CD206 antibody to our panel 1 would have determined whether those macrophages were classical (CD206 -cells) or alternatively activated (CD206 + cells).

In this study, IL-33 daily administration enhanced the production of type-2 cytokines.

Increased activation of eosinophils by IL-33 administration might have been another indirect

Conclusion

In conclusion, our findings indicate that IL-33 has opposing actions on the infarcted heart. On one hand, this alarmin creates a reparative phenotype in the infarcted heart through induction of a type 2 response, at the peak of inflammation-to-granulation transition, promoting a significant inflammatory resolution as early as day 4 post-MI, and inhibiting conventional prolonged inflammation by suppressing myocardial IL-1 and TNF-le els be een 4 and 7 days post-MI. On the other hand, IL-33 worsened cardiac remodeling and function and increased cardiac rupture and death. The adverse cardiac outcomes following IL-33 treatment were mostly related to an impaired transition to the granulation phase characterized by a significant decrease in appropriate fibrotic markers and myofibroblasts formation, both being essential for the establishment of a mature scar at an early phase post-MI. Granulation impairment seems to be concomitant with strong ILC2 and eosinophil infiltration. Of note, both neutrophil and Ly6C(hi) monocyte infiltration in the heart decreased significantly from day 3 to day 7 post-MI, and to comparable levels between the IL-33-treated and vehicle-treated MI groups, suggesting that these immune cells may not be implicated in the observed cardiac dysfunction. Whether ILC2 or eosinophils are ultimately and solely responsible for the adverse actions of IL-33 warrant further investigation. The potential use of IL-33 as an exogenous therapeutic strategy for the treatment of MI without activating unwanted immune responses remains a challenge. It awaits deeper understanding of the pharmacological dynamics to determine the ideal timing, route, and dosages in cardiac disease models. 395. Lu, X., et al., Genome-wide

Introduction

Cardiovascular diseases, primarily acute myocardial infarction (MI) and stroke, represent the leading cause of mortality in the Western world and are on the rise in developing countries. Despite improved outcomes due to advances in medical and interventional treatments, a considerable number of MI patients still develop progressive left ventricular dysfunction [1]. The healing process post-MI implicates a variety of cellular and molecular mechanisms that induce the replacement of necrotic myocardial tissue with a collagen-based scar [2,3]. Understanding the progression and resolution of the inflammatory processes occurring following an MI may reveal new strategies aimed at preserving cardiac function and preventing ischemic cardiomyopathy, therefore complementing available neurohumoral blockade and interventional approaches. Interleukin (IL)-33 (IL-33) is a constitutively expressed cytokine belonging to the IL-1 family, known to activate innate and adaptive immunities [4]. Following tissue injury, IL-33 is released as an alarmin from damaged cells, and plays a role in the activation of immune cells and in promoting tissue repair [5,6]. The two known receptor isoforms for IL-33 are the long ST2 (ST2L) and the soluble ST2 (sST2) [7][8][9]. The long or transmembrane isoform (ST2L) is expressed on a variety of immune cells and is responsible for IL-33 signaling effects [10][11][12][13][14][15][16]. sST2, however, acts as a decoy receptor thereby preventing IL-33 signaling [17]. sST2 has gained much attention as a cardiovascular biomarker mainly predicting congestive heart failure and decreased survival in patients with acute MI [8,[18][19][20].

IL-33 has been associated with a number of inflammatory, immune, and allergic disorders including arthritis, atherosclerosis, inflammatory bowel disease, obesity, and asthma [21]. IL-33 is also implicated in the activation of type 2 innate lymphoid cells (ILC2), which are a member of the newly discovered innate lymphoid cell (ILC) family and are involved in tissue remodeling and lipid metabolism in adipose tissues [14,[22][23][24]. Following their activation, ILC2 produce type 2 cytokines such as IL-5 and IL-13 [25][26][27], which participate in eosinophil homeostasis, recruitment, and proliferation [25,28]. ILC2 are found in the heart during inflammation settings such as myocardial ischemia and eosinophilic pericarditis [29,30].

In the heart, IL-33 is released from cardiomyocytes, coronary artery smooth muscle cells, and cardiac fibroblasts [4,20,29]. IL-33 is reported to have protective effects on cardiac function in many cardiovascular settings including cardiac overload [31,32], abdominal aortic aneurysm [33] and atherosclerosis [34,35], as well as MI, and is portrayed as a potential novel therapeutic cytokine [36][37][38]. However, a study performed on atherosclerotic-prone apolipoprotein E (ApoE)-deficient mice reported that the IL-33/ST2 pathway is not implicated in this pathophysiology [39]. Another, recent study involving patients with end-stage heart failure showed that the expression of the IL-33/ST2 pathway in the failing human heart is associated with increased pro-fibrotic signaling proteins and overall cardiac fibrosis, suggesting a role of this pathway in pro-fibrotic myocardial remodeling [40]. Moreover, an exogenous administration of IL-33 has been shown to promote eosinophilic pericarditis and adversely affect heart function even in healthy mice [41]. The aim of the present study was to assess the impact of IL-33 on the myocardial pathological changes, including ventricular remodeling, myocardial dysfunction, fibrosis, immune cell infiltration, and survival following MI.

Materials and methods

Experimental design

Animals were housed in sterilized cages and maintained on a 12-h (light:dark) cycle with access to autoclaved rodent chow and water ad libitum. Operating procedures were performed in the animal care facility of the American University of Beirut Medical Center (AUBMC). All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the AUB (IACUC # 18-2-RN560). Adult wild-type (WT) male C57/BL6J mice (12-14 weeks of age) were randomized into four different groups: sham+vehicle, sham+IL-33, MI+vehicle, and MI+IL33. In the sham and MI groups treated with IL-33, a single daily injection of mouse recombinant IL-33 (Bi-oLegend, San Diego, CA, U.S.A.) (1 µg suspended in 200 µl PBS) was administered intraperitoneally (i.p.) right after sham operation or left anterior descending (LAD) ligation (starting day 0) and continued for 4 or 7 days thereafter. IL-33 dose and route of administration were based on previous studies [36,37]. In the sham and MI groups treated with vehicle, a single daily injection of PBS (200 µl/day) was i.p. administered right after sham operation or LAD ligation (starting day 0) and continued for 4 or 7 days thereafter. For flow cytometric analyses, mice were given a single (i.p.) daily injection of IL-33 or its vehicle right after sham-operation or LAD ligation (day 0) and continued for 1, 3, or 7 days thereafter.

Induction of MI

Mice were subjected to a permanent ligation (MI group) (n=167) or no ligation (sham group) (n=58) of the LAD coronary artery as previously described [42]. In brief, mice were first administered a dose of 0.05 mg/kg buprenorphine (Reckitt Benckiser Pharmaceuticals, Richmond, VA) for pre-emptive and post-operative analgesia. Mice were then anesthetized with 2% isoflurane, intubated orotracheally, and ventilated with a MiniVent ventilator (type 845) (HSE Harvard, Germany). After shaving the chest wall, a small thoracotomy was performed between the left third and fourth intercostal space. The pericardial sac was cut open, the left ventricle (LV) visualized and the LAD isolated with a 7-0 prolene suture (Ethicon, Norderstdedt, Germany), 2 mm below the left atrioventricular border. Successful ligation was confirmed by visual discoloration of the ventricle distal to the occlusion site and confirmed by ST elevation on ECG using Indus MouseMonitor ® system. The same procedure was performed for sham-operated mice, but without LAD ligation. Ribs, muscles, and skin were closed in layers using 6-0 prolene sutures (Ethicon, Norderstdedt, Germany). The mouse was then weaned from isoflurane anesthesia, flushed with oxygen, extubated, and allowed to warm under an infrared lamp until full consciousness.

Echocardiography

To evaluate the progression of cardiac function or dysfunction, serial echocardiographic measurements were performed at baseline, day 1 post-surgery (LAD ligation or sham operation), and on the day of killing (4 or 7 days). All measurements and assessments were performed using the Visual Sonics Echo system (Vevo 2100, VisualSonics, Inc., Toronto, Canada) and the 22-55 MHz (MS550D) linear transducer (VisualSonics). Mortality rate was recorded for the entire study period. Mice were placed in the echocardiography room for at least 30 min before the examination. Mice were maintained unconscious using 2% isoflurane and placed in a supine position on a heating platform. The chest was shaved and ultrasonic gel was applied to the thoracic area to allow maximal visibility of the heart chambers. The ultrasonic probe was placed on the chest along the long-axis of the LV and adjusted to obtain clear two-dimensional B-mode and M-mode parasternal long axis images. Five minutes were allowed for each animal to stabilize in that position and before acquiring any measurement. Heart rate was maintained constant throughout the procedure (450-550 beats/min). For each parameter, three consecutive cardiac cycles were analyzed and averaged.

Necropsy and tissue harvest

Mice were administered 100 µl Heparin (Heparin Sodium 1000 I.U./ml) 30 min prior to killing. Following deep anesthesia with 4% isoflurane, blood was collected by cardiac puncture, centrifuged at 2200 rpm for 10 min, and the plasma flash-frozen in liquid nitrogen and stored at -80 • C for future use. Mice were then subjected to cervical dislocation. Hearts were immediately injected with cardioplegic solution to arrest the LV chamber in diastole. After excision, hearts were rinsed with physiological saline, weighed, and the left and right atria were resected along with the large vessels. The mid-section was then kept in 4% formalin, and the base and apex were snap-frozen in liquid nitrogen and stored at -80 • C for further experiments. Lungs were collected, weighed, and placed in a 37 • C oven overnight to assess pulmonary congestion (Lungs Wet -Lungs Dry) × 100/Lungs Wet). Spleens were collected, weighed, and stored in liquid nitrogen. Tibia length was also measured and used for normalization. For FACS tissue harvesting, single cell suspensions were obtained from mouse peripheral blood, bone marrow, spleen, and heart. Briefly, mice were given 100 µl of Heparin (1000 I.U./ml) 30 min prior to necropsy. Mice were then subjected to isoflurane anesthesia, blood was collected by cardiac puncture, and hearts perfused through the LV with 5 ml of ice-cold PBS. Hearts and spleens were excised and placed in ice cold PBS (1% FBS) (FACS buffer). Femurs and tibias were also harvested, cleaned of all connective tissue, and bone marrow was then collected by flushing through the bones with ice-cold FACS buffer. Heart specimens were cut in fragments of 2-3 mm 3 . Fragments were suspended in 5-ml DMEM (10% FBS) containing an enzymatic cocktail (Collagenase D (450 U/ml), Roche/Sigma, cat# 11088858001; Dispase (1 U/ml), STEMCELL Technologies, cat# 07923; and DNAse I grade II (60 U/ml), Roche/Sigma, cat# 10104159001) and mechanically dissociated using the Miltenyi GentleMACS for 15 s. Dissociated tissue was digested for 30 min at 37 • C with gentle rotation. Whole blood was first subjected to immunofluorescence staining, then lysed using the red blood cell lysis buffer (Qiagen, cat# 158904). Spleens, bone marrow suspensions, and digested heart tissue were then triturated, and cells filtered through a 40-µm nylon mesh (BD Falcon). Cell suspensions were centrifuged (400×g, 4 • C for 10 min) and subjected to red blood cell lysis for 3 min. The lysis reaction was stopped using 20 ml FACS buffer and the mix was centrifuged (400×g, 4 • C for 5 min). Total leukocyte numbers were determined using Trypan Blue. For each panel, 100 µl of cell suspension containing 1 × 10 6 cells was used from each organ.

Determination of infarct size

The LV was separated from the right, and sliced transversally into three sections (1-2 mm in thickness): apex, mid-section, and base. Sections, including the right ventricle, were incubated with 2% of 2,3,5 triphenyltetrazolium chloride (TTC) for 10 min at 37 • C in the dark. Sections were photographed with a high-resolution digital camera (EOS rebelT3i DSLR, Canon, Tokyo, Japan). Infarct scar area, defined as regions unstained by TTC, were quantified using ImageJ 1.52 software and calculated as the ratio of infarct area to total LV area. All measurements were performed by an investigator who was blinded to the identity of the animals.

Clinical Science (2020) 134 1191-1218 https://doi.org/10.1042/CS20200402

Survival

Starting with recovery from surgery, survival rate was assessed based on the animals that survived until the day of killing. An autopsy was performed on each animal found dead. Mortality was assessed using Kaplan-Meier survival curve analysis.

Histology

Formalin-fixed heart mid-sections were embedded in paraffin blocks, cut into transverse 5-µm sections and placed on microscope slides. After dewaxing and hydration steps using xylene and ethanol (100, 95, 70%), sections were stained with Hematoxylin and Eosin (H&E) and Masson's Trichrome (MTC) to assess cardiomyocyte hypertrophy and areas of myocardial fibrosis, respectively. Imaging was performed on an Olympus CX41 microscope, and quantification was assessed in a blinded fashion using ImageJ 1.52 software. A minimum of ten fields per section and three to four animals were analyzed per experimental group. The area of myocardial fibrosis was quantitatively measured from the photomicrographs of MTC stained slides by ImageJ 1.52 software. The fibrosis was identified using the differences in color (blue fibrotic area opposed to red myocardium) of MTC-stained slides. The results are presented as the ratio of fibrotic area to the whole area of the myocardium using 10× magnification. For cardiomyocyte hypertrophy, a 20× objective was used, and digital images taken from the subendocardial and subepicardial regions of the LV wall were analyzed. The cross-sectional area (CSA) of individual cardiomyocytes was measured by tracing boundaries of each cardiomyocyte on each image. CSAs were defined from an average of 100 cardiomyocytes per mouse.

Western blot analysis

Mouse tissues were ground with liquid nitrogen and proteins were extracted in RIPA buffer containing protease inhibitors (Roche, Basel, Switzerland), and quantified using Thermo Scientific NanoDrop 1000 UV-Vis spectrophotometer (Thermo Scientific, Waltham, MA) according to the manufacturer's recommended protocol. Concentrations were estimated based on an average obtained from triplicate measurements of the same sample. The Lowry colorimetric assay and Nanodrop 1000 methods were previously compared in our lab and both gave the same range of protein quantification. An equal amount of protein (100 µg) from each sample was heated at 95 • C for 10 min and electrophoresed under reducing conditions on a 15% SDS polyacrylamide gel and transferred on to a nitrocellulose membrane. Membranes were blocked with 5% nonfat milk 1 h at room temperature, and incubated with the primary antibody against transforming growth factor β (TGF-β) (1/500, cat# ab92486, Abcam); α smooth muscle actin (α-SMA) (1/200, Abcam, cat# ab5694); IL-1β (1/500, Abcam, cat# ab9722); tumor necrosis factor-α (TNF-α) (1:1000, Cell Signaling Technology, cat# 11948); and IL-4 (1/1000, Abcam, cat# ab9728); IL-10 (1/1000; Abcam, cat#ab9969) and IL-13 (1/500; Abcam; cat#ab106732) overnight on a rocker at 4 • C. After four washes with Tris-buffered saline-Tween (TBST) (0.02%), membranes were incubated for 1 h with species-appropriate peroxidase-conjugated secondary antibody (1/40000) at room temperature. Finally, membranes were washed twice with TBST (0.02%) and TBS, and immunoreactive bands were then visualized with enhanced chemiluminescence (Clarity Western ECL substrate, Bio-Rad) to detect protein bands using the ChemiDoc MP imaging system. Due to the instability of housekeeping proteins such as β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and β-tubulin in animals models of myocardial ischemic infarction [43], total protein levels were used as loading controls. The band densities of each protein of interest were normalized to the density of the total protein bands appearing in the same lane and on the same membrane. All data were then calibrated as fold change to the average of sham-operated vehicle-treated group.

Western blot bands and total protein representative figures are shown in (Supplementary Figure S4).

Ribonucleic acid extraction and reverse transcription quantitative polymerase chain reaction

Total ribonucleic acid (RNA) was isolated from whole mouse heart using TRIzol isolation reagent. Reverse transcription was performed using the QuantiTect Reverse Transcription Kit (Qiagen), and reverse transcription quantitative polymerase chain reaction (RT-qPCR) was completed using the QuantiFast SYBR Green PCR Kit (Qiagen) according to the manufacturer's instructions. Real-Time PCR was performed on a CFX-96 RT-PCR system (Bio-Rad). Cycle quantification (C q ) values were assessed during the exponential amplification phase. The quantified data were analyzed using the 2 -C t method and the relative expression levels of target genes, defined as fold change, and were normalized to the corresponding fold change of the sham+vehicle group, which was defined as 1.0. The primers used for RT-qPCR are listed in Table 1. mRNA levels were standardized with the housekeeping gene hypoxanthine-phosphoribosyl transferase (HPRT), and all experiments were performed using n=3 for sham+vehicle and n=5 or 6 for all other groups.

Assessment of cytokine levels by enzyme-linked immunosorbent assay

The concentrations of IL-5 (Stem Cell Technologies, cat# 02024); granulocyte macrophage-colony stimulating factor (GM-CSF, Invitrogen, cat# BMS612); and IL-33 (Abcam, cat# ab213475) were determined in mouse plasma using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions. All samples were analyzed in duplicates.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay

Myocardial tissue sections obtained from sham-operated and LAD-ligated mice (treated with IL-33 or its vehicle (PBS)) at two different time points (4 and 7 days) were analyzed. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was performed on 5-µm-thick paraffin-embedded heart sections. Tissue sections were deparaffinized and rehydrated, then cardiomyocyte DNA fragmentation was evaluated using the Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection (Thermo Fisher). The operation was performed according to the protocol provided by the manufacturer. Images were captured by the Zeiss Axio light microscope. Apoptotic nuclei were stained red with Alexa Fluor 647 and counterstained with DAPI (4,6-diamino-2phenylindole (blue)) to count the total number of normal nuclei per field. The total number of both TUNEL-positive myocytes and normal cardiomyocyte nuclei was counted using ImageJ (version 1.52) software.

Single cell suspension and flow cytometry

Mice were killed on days 1, 3, and 7 post-MI. A subset of five to six mice for the MI group and three to four mice for the sham-operated group per time point was used for flow cytometric analysis. Immunofluorescence staining was performed on single cell suspensions obtained from mouse peripheral blood, bone marrow, spleen, and heart as previously described [29,44]. Immune cell populations were defined based on previously published data [45,46]. For each panel, 100 µl of cell suspension was used from each organ and stained with 50 µl of the antibody mix. Each panel was used in the presence of 0.5 µg blocking antibody, anti-CD16/CD32 (clone 2.4G2, BD Pharmingen). Dead cells were excluded by the use of a viability dye (DAPI, Thermo Fisher). Samples were acquired on a BD FACS ARIA SORP cell sorter flow cytometer (BD Bioscience) and the raw data analyzed using FlowJo software (v10. 5.3,Tree Star). All antibodies were purchased from Biolegend, eBioscience, or BD Biosciences. Data were analyzed on cells within the live gate and reported as percentage of cell number in CD45 + cells (neutrophils and eosinophils) and as percentage of cell number in live cells (classical monocytes, macrophages, and ILC2). Flow cytometry panels showing antibodies and their corresponding fluorochromes are summarized in Table 2. Representative pseudocolor flow cytometric gatings of the population subsets are shown in (Supplementary Figures S5-S8).

Flow cytometric analysis

Samples were acquired on a BD FACSAria flow cytometer equipped with four lasers, and capable of detecting up to 17 fluorochromes. Data were analyzed using the FlowJo software 10.5.3 (Treestar,Ashland,OR). Voltages were set and the compensation matrix was computed based on unstained and single stained controls. Fluorescence minus one (FMO) controls were used to set gates. From each sample, a total of 0.5-1 × 10 6 events were collected and doublets were excluded from the analysis using FSC (Area) versus SSC (Height). Dead cells were excluded using DAPI staining. All data percentages were calculated from total live cells or from CD45 + cells.

Statistical analysis

All analyses were performed using GraphPad Prism 8 (GraphPad Software, La Jolla, CA). Data are presented as means or fold changes + -standard error of the mean (SEM). Differences among experimental groups were analyzed for statistical significance using one-way analysis of variance (ANOVA) if there was one independent variable or two-way ANOVA if there were two independent variables. To adjust for multiple comparisons, we performed Tukey's post-hoc test, which maintains overall type I error (α) at 5%. P-value <0.05 was considered statistically significant. Mortality rates were evaluated by the Kaplan-Meier method and survival curves were compared using the log rank test.

Results

IL-33 treatment worsens cardiac dysfunction 7 days post-MI

Induction of MI decreased ejection fraction (EF), fractional shortening (FS), and cardiac output (CO) significantly at D1 in all MI groups (****P<0.0001 vs. Baseline; Figure 1A-C) indicating successful ligation of the LAD coronary artery. There was a progressive decline in all hemodynamic parameters (EF, FS, and CO) between days 1 and 4 post-MI, but not to a significant extent (Figure 1A-C not observed in the vehicle-treated MI group. No significant changes in hemodynamic parameters were observed in any sham-operated groups (data not shown).

IL-33 treatment worsens cardiac remodeling 7 days post-MI

LV weight/tibia length, an index of hypertrophy, was measured post-mortem (4 and 7 days post-MI). A significant increase in LV hypertrophy was observed in the IL-33 treated MI group when compared with the vehicle-treated MI group at 7 days post-MI (6.30 + -0.24 vs 5.56 + -0.16 mg/mm; *P<0.05) (Figure 3A). Moreover, MI mice treated with IL-33 showed a significant increase in pulmonary congestion in comparison with the vehicle-treated MI group at 7 days post-MI as evidenced by wet-to-dry water percentage (78.66 + -0.57 vs 76.98 + -0.21%; *P<0.05) (Figure 3B). Infarct size and expansion was comparable between the MI groups treated with IL-33 or vehicle at 4 days post-MI (30.35 + -1.76 vs 28.17 + -3.78%). However, infarct expansion was more pronounced at 7 days in both MI groups, but to a greater extent in the IL-33 treated group (59.03 + -1.69 vs 40.58 + -3.51%; *P<0.05), indicating altered LV remodeling with IL-33 treatment following MI (Figure 3C,D).

IL-33 treatment increases mortality rate post-MI

Kaplan-Meier survival curve analysis showed a tendency toward decreased survival between days 1 and 4 in MI mice treated with IL-33 compared with vehicle-treated group (Figure 3E) (n=22 with 81.82% survival vs n=17 with 94.12% survival). However, a significant increase in mortality rate was observed by 7 days post-MI in the IL-33 treated group in comparison with vehicle-treated MI group (Figure 3F) (n=46 with 50.0% survival vs n=38 with 73.68% survival); most of the mice died between days 3 and 5 post-MI and upon autopsy, cardiac rupture was observed. None of the mice died from sham operation in both groups (IL-33 treated: n=29; vehicle-treated: n=29).

IL-33 treatment increases hypertrophy and fibrosis 7 days post-MI

Histological assessment revealed enlarged cardiomyocyte CSA and increased fibrosis in both MI groups (MI+vehicle and MI+IL-33) compared with the sham-operated groups at 7 days post-MI. Treatment with IL-33, however, further increased the degree of hypertrophy (399.5 + -13.2 vs 284.5 + -21.08 µm 2 ; *P<0.05) (Figure 4A) and fibrosis (30.

IL-33 treatment affects cardiac and plasma inflammatory cytokines post-MI

Protein expression of representative pro-inflammatory cytokines was quantified by Western blot and GM-CSF plasma levels were quantified by ELISA. Four days after MI, cardiac active IL-1β protein levels increased significantly in the vehicle-treated MI group when compared with the vehicle-treated sham-operated group (4.64 + -1.28 vs 1.00 + -0.18 fold change; *P<0.05) (Figure 6A). Cardiac active IL-1β tended to increase in the sham and MI groups treated with IL-33 without reaching significance (Figure 6A). At 7 days post-MI, cardiac active IL-1β protein levels returned to baseline in all groups except for the vehicle-treated MI group, which became significantly more elevated than the IL-33 treated MI group (3.38 + -1.03 vs 0.69 + -0.19 fold change; *P<0.05) (Figure 6B). Cardiac TNF-α expression increased significantly in the vehicle-treated MI group compared with the relative sham-operated group at 4 days (2.55 + -0.24 vs 1.00 + -0.05 fold change; *P<0.05) (Figure 6C). By itself, IL-33 increased cardiac TNF-α levels at 4 days vs 1.00 + -0.05 (sham+vehicle) fold change). However, TNF-α protein levels were attenuated in the IL-33 treated MI group 4 days post-MI when compared with the MI group treated with vehicle (0.59 + -0.14 vs 2.55 + -0.24 fold change; *P<0.05) (Figure 6C). At 7 days post-MI, TNF-α protein levels in all groups were at baseline with no significant differences among them (Figure 6D).

Plasma GM-CSF levels were significantly elevated in the IL-33 treated MI group at 4 days post-MI compared with all other groups (**P<0.01 to ****P<0.0001) (Figure 6E). Plasma GM-CSF decreased at 7 days post-MI 51.97 + -16.78 (pg/ml) (MI+IL-33 D7) vs (128.1 + -27.75 (pg/ml) (MI+IL-33 D4); **P<0.01), but remained insignificantly more elevated than all other groups at the same time point. The sham-operated group treated with IL-33 showed a slight but insignificant increase in GM-CSF plasma concentrations compared with all vehicle-treated groups at both 4 and 7 days post-MI (Figure 6E).

IL-33 treatment increases MMP2 and MMP9 mRNA expression 7 days post-MI

MMP9 mRNA levels significantly increased in the IL-33 treated MI group compared with all other groups at 4 and 7 days post-MI (*P<0.05, **P<0.01 and ****P<0.0001) (Figure 7A,B). MMP2, however, did not increase in any group at 4 days (Figure 7C), but significantly increased at 7 days (Figure 7D) in the IL-33 treated MI group compared with the vehicle-treated sham group (4.46 + -1.25 vs 1.00 + -0.07 fold change; *P<0.05). These results suggest that the administration of IL-33 in the presence of MI potentially enhances extracellular matrix (ECM) degradation. 95.10 + -0.55 (pg/ml) sham+IL-33 vs 0.51 + -0.29 (pg/ml) sham+vehicle) (****P<0.0001), respectively (Figure 8G). Seven days post-MI, IL-5 plasma concentrations decreased in the IL-33 treated groups compared with their counterparts at 4 days (97.56 + -4.94 (pg/ml) (MI+IL-33 D4) vs 23.92 + -6.73 (pg/ml) (MI+IL-33 D7) (****P<0.0001) and sham groups (95.10 + -0.55 (pg/ml) (sham+IL-33 D4) vs 56.19 + -6.74 (pg/ml) (sham+IL-33 D7) (***P<0.001)), but remained significantly more elevated compared with the vehicle-treated MI and sham groups at 7 days post-MI (23.92 + -6.73 (pg/ml) (MI+IL-33) vs 0.45 + -0.06 (pg/ml) (MI+vehicle); (*P<0.05)) and ( 56.19 + -6.74 (pg/ml) (sham+IL-33) vs 1.55 + -0.60 (pg/ml) (sham+vehicle); (***P<0.001)) (Figure 8G). No plasma IL-5 levels were detected in vehicle-treated MI and sham groups.

IL-33 treatment promotes type 2 cytokines release

IL-33 treatment modulates neutrophil and monocyte/macrophage production, mobilization, and recruitment

Neutrophils and monocytes were identified as CD45+CD11b+Ly6G(hi) and CD11b+Ly6C(hi)Ly6G-F480-cells, respectively. Compared with day 1, neutrophil and Ly6C(hi) monocyte production in the bone marrow peaked at 3 days in all experimental groups including the vehicle-treated sham-operated group (Figures 9A and10A). At 7 days, bone marrow neutrophil and Ly6C (hi) levels decreased significantly in the IL-33 treated MI group compared with the levels observed at day 3 (neutrophils: 46.37 + -1.58% (MI+IL33 D3) vs 29.85 + -1.94% (MI+IL33 D7) *P<0.05) (Ly6C (hi): 16.80 + -1.75% (MI+IL-33 D3) vs 5.59 + -0.99% (MI+IL-33 D7); ****P<0.0001) (Figures 9A and10A). Moreover, bone marrow neutrophils at 7 days were significantly lower in the IL-33 treated MI group in comparison with the vehicle-treated MI group at the same time point (29.85 + -1.94 vs 46.13 + -2.28%; *P<0.05) (Figure 9A). In the spleen, compared with day 1, neutrophils peaked at 3 days post-MI in the IL-33 treated group (10.72 + -1.47 vs 4.73 + -0.83% D1; *P<0.05) where levels were significantly higher than in the vehicle-treated MI group at the same time point (10.72 + -1.47 vs 5.01 + -0.93% (MI+vehicle D3); *P<0.05, Figure 9B). Neutrophil circulation in the blood culminated at day 1 in all experimental groups and reached its lowest point at 7 days where levels in the IL-33 treated MI group were significantly lower than in the vehicle-treated group at the same time point (7.07 + -2.95 vs 26.62 + -6.54%; *P<0.05) (Figure 9C). At 7 days, Ly6C (hi) levels in the spleen and in the blood were significantly lower in the IL-33 treated MI group when compared with the levels observed in the vehicle-treated MI group (Figure 10B,C; *P<0.05). Of note, neutrophils and monocytes increased in the hearts of both experimental groups (MI+vehicle and MI+IL-33) on day 1 post-MI, and decreased significantly and concomitantly on day 7 post-MI (Figures 9D and10D).

Macrophages were identified as CD11b+F480+Ly6C-. Compared with day 1, macrophages significantly peaked with IL-33 treatment in the bone marrow as early as 3 days post-MI in the IL-33 treated sham and MI groups, and remained elevated when compared with the vehicle-treated groups at 7 days post-MI (Figure 11A). In the spleen and blood, macrophages increased significantly with IL-33 treatment and culminated at 7 days post-MI (Figure 11B,C). In the heart, MI induced a significant increase in macrophage levels in the vehicle-treated group at 3 days post-MI when compared with day 1 (Figure 11D). However, a significant increase in macrophage levels was observed in the heart of the IL-33 treated MI group at 7 days when compared with the same group at day 1 and day 3 post-MI (Figure 11D). Furthermore, macrophage levels in the IL-33 treated MI group at 7 days were significantly more elevated than the vehicle-treated MI group at the same time point (Figure 11D).

IL-33 treatment elicits eosinophilia

Eosinophils were identified as CD45+SiglecF+CD11c-. IL-33 is known to directly facilitate the maturation of eosinophils by enhancing their survival, activation, and adhesion [47]. IL-33 treatment for 7 days induced a drastic increase in eosinophils in all organs (bone marrow, spleen, blood, and heart). Percentage of eosinophil out of CD45+ cells ranged between (8.81 + -1.23 and 42.37 + -5.89%) in the IL-33-treated sham-operated group and (10.02 + -1.74 and 62.26 + -5.56%) in the IL-33-treated MI group in all organs at 7 days (Figure 12A-D).

IL-33 treatment increases ILC2 levels at 7 days post-MI

ILC2 were identified as Lin-ICOS+CD25+. Although present in all organs in a very small percentage, ILC2 seemed to increase with IL-33 treatment. In the bone marrow, compared with day 1, a significant increase in ILC2s was observed at 3 days in the IL-33 treated sham-operated group (0.14 + -0.01% (D1) vs 0.46 + -0.11% (D7); ***P<0.001) (Figure 13A). Moreover, treatment of the sham-operated group with IL-33 increased ILC2 production in the blood at 7 days when compared with the vehicle-treated sham-operated group at the same time point (0.014 + -0.014 vs 0.30 + -0.06%; *P<0.05) (Figure 13C). In the spleen and in the heart, a tendency toward an increase in ILC2 percentage was observed in the IL-33-treated sham-operated group but without reaching significance. ILC2 significantly increased in the IL-33-treated MI group and ranged between (0.20 and 1.26)% of the total number of cells in all organs at 7 days (Figure 13A-D).

IL-33 treatment induces splenomegaly

In contrast with the normal spleen size and weight observed in the vehicle-treated sham and MI mice (ranging between 4.19 + -0.42 and 4.81 + -0.19 mg/mm), administration of IL-33 for 4 and 7 days induced a significant increase in spleen weight in a time-dependent manner regardless of the surgical intervention (Supplementary Figure S1). Increases in 1.73-folds and 2.6-folds in spleen weights were observed between the vehicle-treated and IL-33-treated sham-operated groups at 4 and 7 days, respectively. The variation in spleen weights in the IL-33-treated MI groups was comparable with that observed in the sham-operated groups where increases in 1.7-folds and 3-folds in spleen weights were seen between the vehicle-treated and IL-33-treated MI groups at 4 and 7 days respectively. These findings are consistent with previous data showing increase in spleen size and weight with IL-33 treatment [4,48]. IL-33 treatment up-regulated cardiac sST2 expression at 7 days in the presence or absence of MI mRNA levels of cardiac sST2 were measured at 4 and 7 days in all groups treated with IL-33 or vehicle. sST2 levels were not changed at 4 days in all sham and MI groups (Supplementary Figure S2A). However, at 7 days, sST2 mRNA levels increased significantly in the sham and MI groups treated with IL33 compared with the sham group treated with vehicle (P<0.01 and P<0.001, respectively). MI increased sST2 levels in the vehicle-treated group 7 days post-MI with no significance when compared with the relative sham group. Administration of IL-33 significantly up-regulated sST2 cardiac expression in the MI group in comparison with vehicle-treated MI group (P<0.01) (Supplementary Figure S2B). The receptor for IL-33 (ST2) is found primarily on many 'allergy associated' target cells, including, helper T cells (Th2), eosinophils, basophils, mast cells and ILC2 [49]. IL-33 participates in the synthesis and release of IL-5 from these target cells thereby possibly promoting eosinophilia [4].

IL-33 plasma availability peaked at 4 days and decreased at 7 days while remaining significantly more elevated than in the MI group treated with vehicle (Supplementary Figure S2C). It is possible that sST2 levels are increased in the plasma at 7 days, therefore acting as a decoy receptor to IL-33 and decreasing its availability in the plasma at 7 days. The dynamic expression of plasma IL-33 was measured by ELISA at 4 and 7 days post-MI (Supplementary Figure S2B). Plasma IL-33 concentrations peaked at 4 days in the sham and MI groups treated with IL-33 (Supplementary Figure S2C), and decreased significantly at 7 days in both groups but remained significantly higher than the vehicle-treated sham and MI groups (Supplementary Figure S2C).

Decreased protein and gene expression of profibrotic markers with IL-33 post-MI

Cardiac protein expression of the fibrogenic cytokine TGF-β was not altered by IL-33 or MI at day 4. However, at 7 days, levels of TGF-β increased in the sham+IL-33 and MI+vehicle groups without reaching significance. Moreover, TGF-β protein expression decreased significantly in the MI group treated with IL-33 when compared with the MI group treated with vehicle at 7 days (0.84 + -0.20 vs 2.29 + -0.50 fold change; *P<0.05) (Supplementary Figure S3A,B). The transdifferentiation of fibroblasts into myofibroblasts is characterized by the up-regulation of α-SMA [50]. α-SMA cardiac expression also decreased at 7 days post-MI in the IL-33 treated MI group compared with the MI group treated with vehicle (0.68 + -0.11 vs 1.63 + -0.31 fold change; *P<0.05) (Supplementary Figure S3C,D). Gene expression of connective tissue growth factor (CTGF), a matrix-associated protein, significantly increased in the MI group treated with vehicle compared with the sham vehicle-treated group at 4 and 7 days post-MI (16.39 + -2.69 vs 1.00 + -0.03 fold change; ***P<0.001). In contrast, IL-33 treatment significantly decreased CTGF gene expression at 4 days post-MI compared with the vehicle-treated MI group (6.07 + -1.22 vs 16.39 + -2.69 fold change; **P<0.01) (Supplementary Figure S3E). of note, CTGF gene expression remained more elevated in the vehicle-treated MI group at 7 days when compared with the sham-operated group (4.87 + -0.72 vs 1.00 + -0.21 fold change; *P<0.05) (Supplementary Figure S3F). These results indicate that administration of IL-33 in MI mice attenuates cardiac profibrotic markers in MI mice.

Discussion

Due to the limited capacity of the adult mammalian heart to regenerate, the healing process post-MI is phasic, relying mainly on an early sterile inflammatory response to remove the debris of dead cells followed by granulation and maturation phases to replace lost cells with fibrotic tissue and strengthen scar formation. Depending on the clinical state, and the presence or absence of comorbidities, alteration in transition between phases such as inflammation prolongation or increased fibrosis deposition can worsen cardiac remodeling post-MI. Providing a pharmacological platform to promote appropriate transition between early remodeling phases post-MI through immunomodulatory mechanisms is gaining significant attention and constitutes the basis of the experiments presented in this manuscript.

In the present study, we assessed the reparative actions of the alarmin IL-33 on the infarcted myocardium. IL-33 was administered for 4 and 7 consecutive days following LAD coronary artery ligation or sham-operation. We observed that IL-33 was indeed effective in activating a type 2 immune response in the damaged heart, consistent with the anticipated wound healing response, as evidenced by early (day 4) increased levels of type 2 cytokines and anti-inflammatory IL-10, and reduced levels of inflammatory TNF-α (at 4 and 7 days) and IL-1β (7 days). Nonetheless, IL-33 was associated with a pronounced decrease in systolic function at day 7 with ventricular dilation and infarct expansion, along with increased apoptosis (increased BAX/BCL2 and TUNEL+ nuclei). The overall results of the present study reveal that IL-33 is associated with worsened cardiac function and increased adverse cardiac remodeling in the MI mouse model. Administration of IL-33 increased mortality due to cardiac rupture, induced pulmonary congestion and cardiomyocyte apoptosis, and promoted LV hypertrophy. Despite decreased systolic function, our FACS data did not show any major changes in monocyte and neutrophil cardiac infiltration pattern in the IL-33-treated MI group when compared with the vehicle-treated group at 7 days. These results are in agreement with previously published data showing no major increases in cardiac neutrophils and monocytes despite exacerbation in cardiac dysfunction and increased myocarditis with IL-33 treatment [41]. With IL-33 treatment, increased cardiac hypertrophy and fibrosis were observed. This may reflect a compensatory response to increased infarct expansion, although it is unsettled whether the fibrosis was productive or reflected decreased ECM turnover. Overall, IL-33 treatment was associated with a decrease in profibrotic markers in the infarcted heart (CTGF, TGF-β) and in activated myofibroblasts (α-SMA), as well as an increase in MMPs that degrade the ECM. Increase in MMPs is linked to the slippage of myocyte fascicles thereby promoting wall thinning in the infarcted area leading to cardiac rupture. Previous studies have shown that MMPs are not only involved in ECM degradation, but are also implicated with the progression to heart failure [51]. Targeted deletion of MMP2 or MMP9 decreases the incidence of cardiac rupture and attenuates LV enlargement following LAD ligation in mice [52][53][54]. In this study, we observe increased expression of MMP2 and MMP9 at 7 days that may have contributed to wall thinning and rupture. These metalloproteases regulate ECM turnover and were shown to be produced by eosinophils and contribute to airway remodeling [55,56].

We observed as well an increase in ILC2s with IL-33 treatment in the bone marrow, spleen, and blood, as well as the myocardium that was further enhanced at day 7 in the infarcted mice. Our current data are in agreement with recently published data stating that the activation of ILC2 by IL-33 plays a pathogenic role in the heart by inducing cardiac eosinophilic infiltration and exacerbating cardiac dysfunction [30]. Whether the cardiac ILC2s originated from a resident cardiac or pericardiac pool [29] or were recruited from the mediastinal cavity [30] as suggested by others was not established. Along with type 2 Th2, ILC2s are a major source of type 2 cytokines and mediators of the actions of IL-33 at barrier sites of the body [8], and in certain cases shown to amplify inflammation via activation of eosinophils [57,58].

As previously reported [4], IL-33 treatment induced splenomegaly, which we observed was associated with a delayed accumulation of eosinophils in the spleen. This finding is consistent with a recent report stating that IL-33 positively affects eosinophil development [47]. IL-33 has also been implicated in activating mature eosinophils. For instance, IL-33 was reported to play a role in either the survival or death of human eosinophils depending upon context [47] and to stimulate nuclear factor-κB (NF-κB)-mediated inflammatory gene expression in murine eosinophils by both IL-4-independent and IL-4-dependent/autocrine mechanisms [59]. In addition, IL-33 was reported to induce degranulation and superoxide anion production of human eosinophils as effectively as IL-5 [60]. Nevertheless, differential effects of IL-33 on collagen deposition and MMP2/9 levels in sham versus MI hearts suggest that some other factor(s) might be involved in activating eosinophils.

IL-33 induced a delayed increase in eosinophils in the heart in both the absence and presence of MI. Increased plasma concentrations of IL-5 and GM-CSF (which are potent cytokines associated with eosinophil development and increased viability [61]) at 4 days may have facilitated this recruitment. The IL-33-induced increase by day 4 in IL-4 and IL-13 in the infarcted myocardium may have contributed as well [62]. Previous studies have shown a direct correlation between increased serum GM-CSF levels and impairment of the healing process post-MI by acting at a distance in the BM and locally in the heart to produce and enhance immune cells accumulation [63]. In this study, elevated serum GM-CSF levels correlated with strong eosinophil recruitment and cardiac dysfunction following IL-33 treatment post-MI. Earlier studies indicated that eosinophils are rapidly activated within 2-3 days following MI, infiltrate into the myocardium, and contribute to cardiac rupture in some patients [64,65]. Other studies have linked eosinopenia to an acute MI in patients presenting with acute coronary syndrome [66,67]. A decrease in blood eosinophils might be indicative of their accumulation in the damaged myocardium [68]. This scenario is further substantiated by evidence showing a positive correlation between higher baseline serum levels of eosinophil cationic protein (ECP) and follow-up occurrence of MACEs after PCI [69][70][71]. On the other hand, Shiyovich et al. [72] observed a U-shaped association between blood eosinophils within 72 h of admittance and greater risk of death in patients with acute MI, with a greater count associated with lower 1 year mortality rates. This observation was interpreted to mean that eosinophils might help control the inflammatory response.

Several studies have shown that increased sST2 expression can have a negative prognostic impact on the general cardiovascular risk profile [8,20]. For instance, sST2 mRNA was markedly increased after biomechanical strain in cultured cardiomyocytes and fibroblasts and serum sST2 levels were transiently increased as early as four hours following coronary artery ligation in mice [73] and 12 h in patients with acute MI [19]. Patients with cardiac fibrosis, hypertrophy, LV dilatation, and decreased ventricular contractility have elevated sST2 levels, which is considered an independent predictor of adverse outcomes in acute decompensated heart failure [74]. Moreover, in stable, well-treated congestive heart failure patients, elevated sST2 levels were associated with disease-specific end-points such as heart failure hospitalization and cardiovascular death [75]. Although in the present study we did not examine plasma sST2 levels, we did see an increase in cardiac sST2 mRNA levels at 7 days in our IL-33-treated MI mice, which corresponded with decreased IL-33 levels in the plasma at the same time point. Plasma IL-33 concentrations peaked at 4 days in the sham and MI groups treated with IL-33, and decreased significantly at 7 days in both groups but remained significantly higher than the vehicle-treated sham and MI groups. We therefore speculated whether sST2 levels are also increased in the plasma at 7 days, and are acting as a decoy receptor to IL-33 and decreasing consequently its availability in the plasma at 7 days.

Although the human and mouse IL-33 are only 55% identical, they both comprise 270 and 266 amino acids, respectively and have almost identical molecular weights (30 kDa human and 29.9 kDa murine) [5]. The IL-33 present in humans and mice have similar expression patterns and both orthologs are present in the nucleus and released extracellularly upon injury or necrosis [76]. Although previous investigators have used rat and human-derived IL-33 [4,32] and have shown variations in immune responses with IL-33 treatment, we used mouse recombinant IL-33 in our MI model. The administration of mouse IL-33 in the acute MI model in mice, can highly mimic the mechanisms occurring in human heart disease, and can therefore be a useful tool to elucidate the cardiovascular events arising following myocardial ischemia.

Previous studies have shown that the association of IL-33 with its transmembrane receptor (ST2L) could have protective effects on the heart. In the MI setting, Yin et al showed that IL-33 significantly decreases macrophage infiltration and alleviates inflammation in the heart, making IL-33 a potential therapeutic cytokine for cardiac dysfunction post-MI [37,38]. Despite similarities with IL-33 administration modality of this study, Yin et al. study differs in several aspects: (1) in the current study, hemodynamic parameters were collected at baseline, day 1, day 4, and day 7 post-MI. The results at day 1 clearly show that the hemodynamic parameters (EF, FS, CO, LV end-systolic volume (LVESV), LV end-diastolic volume (LVEDV), LV end-systolic diameter (LVESD), LVEDD) were in the same range in all MI groups, reflecting similar MI induction in all groups at day 1. Although no significant hemodynamic changes were observed at day 4, animals in the IL-33-treated MI group exhibited significantly decreased EF and increased LVESV, LVEDV, LVESD, LVEDD at day 7 post-MI in comparison with the vehicle-treated MI group. IL-33 administration and follow up was ethically limited to 7 days post-MI due to the very high mortality rate in the IL-33 treated MI animals (∼50%). In the Yin et al. study, LV function was assessed at day 14 and day 28 post-MI and no data were shown at day 1 post-MI to prove that both MI groups (treated with vehicle and IL-33) underwent the same degree of MI. Additionally, IL-33 treated MI groups showed extraordinarily improved cardiac function in comparison with the vehicle-treated MI group at 14 and 28 days post-MI. (2) In the present study, DNA fragmentation was assessed at 4 and 7 days post-MI and was shown to be significantly increased with IL-33 treatment post-MI. Yin et al. observed DNA fragmentation at an earlier phase (3 days post-MI only) and was shown to be decreased with IL-33 treatment.

3) The flow cytometry data in this study showed that macrophages increased in the heart 3 days post-MI in the vehicle-treated group, without being affected in any other organ such as the bone marrow, spleen and blood. These results suggest a possible local monocyte to macrophage differentiation, a process that can take place as early as day 1 post-MI [77]. No significant difference in macrophage levels in the heart were observed between the IL-33 and vehicle-treated MI groups at 3 days post-MI. However at 7 days post-MI, a significant increase in macrophage infiltration in the heart was observed in the IL-33 treated MI group in comparison with the vehicle-treated group at the same time point. In contrast, in the Yin et al. study, immunofluorescent-staining showed a significantly decreased F4/80+ macrophage infiltration in the heart in the IL-33 treated MI animals at 3 days post-MI. Macrophage levels might have changed in the heart post-MI with IL-33 treatment if they were assessed at later stages.

The present study has few limitations that should be acknowledged. Despite increased infiltration of ILC2 and eosinophils following IL-33 administration, our study did not explore the role of ILC2-deficient and eosinophil-deficient mice in the pathogenesis of MI. Moreover, the IL-33/ST2 axis is known to activate several signaling pathways including NF-κB, mitogen activated protein kinases (MAPK) and phosphoinositide 3-kinase (PI3K) [4,[78][79][80][81][82][83]. Inhibition of the PI3K signaling pathway is shown to reduce inflammation and improve outcomes in several pathological events [84][85][86][87], including MI [88]. The PI3K signaling pathway is also implicated in eosinophil chemotaxis and inhibition of the PI3K pathway was shown to impair release of eosinophils from bone marrow [89]. Future studies are warranted to explore the role of ILC2 and eosinophil KO mice and to see whether inhibition of the PI3K signaling pathway improves outcomes following IL-33 administration in the presence and absence of MI.

In conclusion, our findings indicate that IL-33 has opposing actions on the infarcted heart. On one hand, this alarmin creates a reparative phenotype in the infarcted heart through induction of a type 2 response, at the peak of inflammation-to-granulation transition, promoting a significant inflammatory resolution as early as day 4 post-MI, and inhibiting conventional prolonged inflammation by suppressing myocardial IL-1β and TNF-α levels between 4 and 7 days post-MI. On the other hand, IL-33 worsened cardiac remodeling and function and increased cardiac rupture and death. The adverse cardiac outcomes following IL-33 treatment were mostly related to an impaired transition to the granulation phase characterized by a significant decrease in appropriate fibrotic markers and myofibroblasts formation, both being essential for the establishment of a mature scar at an early phase post-MI (Supplementary Figure S9). Granulation impairment seems to be concomitant with strong eosinophil infiltration. Of note, both neutrophil and Ly6C (hi) monocyte infiltration decreased significantly from day 3 to day 7 post-MI, and to comparable levels between the IL-33-treated and vehicle-treated MI groups, suggesting that these immune cells may not be implicated in the observed cardiac dysfunction. Whether eosinophils are ultimately and solely responsible for the adverse actions of IL-33 warrants further investigation.

Clinical perspectives

• The IL-33/ST2 axis is associated with a number of in ammatory, immune, and allergic disorders but is reported to have protective effects on cardiac function in many cardiovascular settings including cardiac overload, abdominal aortic aneurysm, and atherosclerosis. In the present study we examined the effect of IL-33 administration on myocardial pathological changes, including ventricular remodeling, myocardial dysfunction, brosis, immune cell in ltration, and survival following MI.

• Our results suggest that IL-33 is effective in activating a type 2 cytokine milieu in the damaged heart, consistent with reduced early in ammatory and pro-brotic response. However, IL-33 administration is associated with worsened cardiac function and adverse cardiac remodeling in the MI mouse model. IL-33 administration increased infarct size, LV hypertrophy, cardiomyocyte death, and overall mortality rate due to cardiac rupture. Moreover, IL-33-treated MI mice displayed a signi cant myocardial eosinophil in ltration at 7 days post-MI when compared with vehicle-treated MI mice.

• The present study reveals that although IL-33 administration is associated with a reparative phenotype following MI, it worsens cardiac remodeling and promotes heart failure.

• Designing monoclonal antibodies targeting extracellularly released cytokines and their corresponding receptors has been a great tool in revolutionizing drug therapy in a wide range of disease areas [90].

Since the discovery of IL-33 over a decade ago, it became well established that this cytokine participates in tissue homeostasis but can induce highly detrimental effects when activated inappropriately. Hence, several mechanisms have been developed to harness the activity and bioavailability of this versatile cytokine on multiple levels. Soluble receptor blockers of free IL-33 including the IL-33Trap and a form of the sST2 receptor have been developed to antagonize the unwanted biological functions mediated by IL-33 both in vitro and in vivo [91][92][93]. In allergic conditions involving hyperreactive immune responses, such as allergic asthma and atopic dermatitis, IL-33 is a disease sensitizer and mediates detrimental functions [15]. The use of recombinant sST2 or antibodies targeting IL-33 or ST2 to block the signaling of the IL-33/ST2 axis have proven bene cial outcomes in the management of allergic asthma and atopic dermatitis [94][95][96][97][98]. However, in a multitude of other processes, the IL-33/ST2 signaling pathway has pleiotropic immune functions and operates like a double-edged sword, presenting both tissue-reparative and pathological properties in many organs of the body. For instance, in the kidneys, lungs, skin, gastrointestinal system, central nervous system, and cardiovascular system, IL-33 mediates both in ammatory and reparative responses [15,99]. In addition, the IL-33/ST2 axis is implicated in several diseases, including colitis, brosis, lupus, rheumatoid arthritis, multiple sclerosis, central nervous system disorders, and age-related macular degeneration [100].

Given the diverse roles of IL-33 in tissue injury and repair, determining the appropriate time to manipulate the IL-33/ST2 axis can be a great challenge in alleviating in ammation. In MI, where the reparative process is associated with a great imbalance in immune responses, neither the physiological nor the pathological functions of IL-33 are fully elucidated and await further exploration. Hence, the potential use of an anti-IL-33 antibody or the exogenous use of IL-33 as therapeutic strategies for the treatment of MI without activating unwanted immune responses remains a challenge. It awaits deeper understanding of the pharmacological dynamics to determine the ideal timing, route, and dosages in cardiac disease models.

I L-33 (interleukin 33) is type of cytokine designated an alar- min, which is released under stress conditions or by cell death to induce protective measures in neighboring cells. 1 It signals via the ST2 receptor, which is also expressed as a truncated and soluble form that acts as a decoy receptor soluble ST2 (sST2). 2 Substantial evidence supports the use of sST2 measurement as an indicator of cardiac stress and remodeling in hypertension, heart failure, cardiomyopathies, and aortic stenosis (AS). However, as discussed here, there are gaps in our understanding of the IL-33/ sST2 axis, including the source of the components, stimuli for their expression and release, what it actually assesses, and the role of immune cells. Moreover, as discussed, the fundamental assumption that Il-33 has beneficial actions that are thwarted by sST2 requires further scrutiny.

Background, Signaling, and Expression

IL-33 is constitutively expressed and mainly present in nuclei of epithelial and endothelial cells. 3,4 IL-33 was first discovered in 1999 as a nuclear protein of unknown function encoded by a gene upregulated in canine vasospastic cerebral vasculature after subarachnoid hemorrhage and termed DVS27. 5 In 2003, the same nuclear protein was found to be abundantly expressed in high endothelial venules that allow lymphocytes access into lymphoid organs and lymph nodes 6,7 and accordingly named nuclear factor from high endothelial venules. 8 Schmitz et al 9 determined 2 years later that DVS27 and nuclear factor from high endothelial venules were the same protein and noticed that its C-terminal domain contains a β-sheet trefoil fold structure similar to IL-1 family members. Indeed, the protein was subsequently observed to function as a cytokine that induces T helper (Th) type 2 responses through ST2, an orphan receptor of the IL-1 receptor superfamily. This novel IL-1 family member was then named IL-33. 9 The ST2 receptor exists as 2 isoforms, sST2 and transmembrane (ST2L), encoded by the same gene (IL1RL1) under a proximal and distal promoter, respectively. Oxidation and extracellular proteases terminate IL-33 biological actions. 10 Because of its expression at barrier tissues, IL-33 plays a key role in maintaining barrier tissue defense. 11 As an alarmin, IL-33 activates various cell types of the innate and adaptive immune system. Certain proteases, such as neutrophil and mast cell serine proteases, cathepsin G, chymase, and elastase, cleave IL-33 extracellularly and enhance its activity. 12-14 Resident immune cells constitutively expressing ST2L are a main target for IL-33 and include group 2 innate lymphoid cells (ILC2s), 15,16 mast cells, 17-19 and tissue-resident regulatory T cells. 20,21 Variations in ST2L expression affect the type of immune response that IL-33 produces. For instance, while expression of ST2L was decreased in ILC2 in a murine model of chronic obstructive pulmonary disease, it was induced in macrophages (with M2 polarization) and natural killer (NK) cells. 22 Other targets of IL-33 include Th2 cells, B cells, basophils, eosinophils, dendritic cells, and natural killer T (NKT) cells. 1 Th1 and CD8 + T cells also show induced expression of ST2L in certain cases. 23,24 Depending on context, IL-33 is therefore able to activate and perpetuate inflammation (eg, via Th2 or mast cells) 25 or reduce and resolve inflammatory responses (eg, via tissue-resident regulatory T cells or ILC2). 26,27 By activating Th2 cells, IL-33 elicits a type 2 immune response, which if exuberant may lead to tissue damage likely through activation of mast cells or eosinophils and development of pathological fibrosis. 28 In this way, IL-33 plays a role in the pathophysiology of several proinflammatory and autoimmune diseases. 29 In certain cells, IL-33 may induce expression of ST2L (eg, ILC2, eosinophils, and tissue-resident regulatory T cells) or sST2 (eg, mast cells). 1 Figure 1 summarizes some notable actions of IL-33 on immune cells that are potentially relevant to the heart.

Expression of IL-33 in nonimmune cells occurs normally throughout the human body. Endothelial cells constitute the main cell type that exhibits elevated levels of IL-33 at barrier surfaces. 3 IL-33 is present also in adipose tissue 30 and atherosclerotic plaques 31 and is constitutively present in secondary lymphoid organs. 3 Il-33 is constitutively expressed in endothelial cells of the vasculature tree of normal tissues, but not the microcirculation of glomeruli and brain. 3 Keratinocytes, epithelial cells, fibroblasts, and smooth muscle cells also express IL-33. 3,9,32,33 Under pathological conditions, increased IL-33 levels are detected in various adenocarcinomas, 3 and elevated levels are found in the circulation and tissues during several inflammatory diseases including rheumatoid arthritis, inflammatory bowel syndrome, asthma, and skin diseases. 34-37 Although IL-33 is primarily expressed by nonhematopoietic cells, activated dendritic cells and M2 macrophages may also express and secrete IL-33. 9,38 Expression of IL-33 at epithelial surfaces occurs in both humans and mice. 3,9,35 Unlike in humans, IL-33 is not normally present along the mouse vascular tree, although expression has been observed in a few vascular beds (ovaries, adipose tissue, and liver). 4,39-41 However, IL-33 expression was observed to be increased in endothelial (and interstitial) cells of the mouse heart after transverse aortic constriction. 42 In the human heart, IL-33 is mainly present in endothelial cells of the vasculature, although cardiomyocytes and fibroblasts of adults also constitutively express the protein.

Proinflammatory cytokines like IL-1β, TNF (tumor necrosis factor)-α, and IFN (interferon)-γ increase IL-33 in these cells, as well as in human coronary artery smooth muscle cells. 43 Very low levels of sST2 and ST2L mRNA are detected in human cardiomyocytes, vascular smooth muscle cells, and cardiac fibroblasts, whereas ST2 is widely expressed in endothelial cells of the cardiac vasculature, and both cardiac macro-and microvascular endothelial cells secrete sST2. 43 Expression of both IL-33 and sST2 is induced in rat neonatal cardiomyocytes and (more so) in cardiac fibroblasts on mechanical stress. 33,44 Hypertension/Pulmonary Hypertension Circulating sST2 levels may have diagnostic and prognostic value in hypertension. 45,46 Increased sST2 serum levels are associated with alterations of left ventricular (LV) geometry 46 and increased systolic blood pressure. 47,48 Among Framingham Heart Study participants, elevated blood levels of sST2 were associated with hypertension and diabetes mellitus 48 ; however, in healthy individuals from the general population, sST2 had little predictive value for cardiovascular events. 49 In pulmonary arterial hypertension (PAH), the role of the IL-33/sST2 axis would seem straightforward. Because sST2 release is potentiated during myocardial stretch, it is hypothesized that increased pulmonary arterial blood pressure and right ventricular (RV) afterload increase transmural wall stress, hence increasing myocardial stretch and sST2 release. Therefore, sST2 could be considered a cardiac biomarker of mechanical strain and act as a useful biomarker gauging RV dysfunction in PAH. 50 Indeed, sST2 expression reflects the size and function of the RV and independently predicts 1-year mortality in PAH patients. 51,52 Carlomagno et al 53 found that elevated sST2 expression in patients with PAH was associated with systolic dysfunction and RV dilatation. Elevated sST2 levels were also associated with poor prognosis in childhood PAH with sST2/NT-proBNP (N-terminal pro-B-type natriuretic peptide) biomarker combination predicting clinical condition and outcome. 54 Moreover, increased sST2 levels predicted disease severity in idiopathic PAH patients and correlated with hemodynamic parameters and other biomarkers. 50 In dyspneic patients, increased sST2 levels directly correlated with RV dysfunction and predicted worsened prognosis. 55 In patients with secondary pulmonary hypertension because of chronic obstructive pulmonary disease, increases in both sST2 and NT-proBNP correlated with RV dysfunction and possessed prognostic and diagnostic value. 51 However, the assumption that RV myocytes release sST2 when stretched is based on work with LV myocytes (see below), which have a different embryological origin. Alternatively, loss of nuclear IL-33 in pulmonary endothelial cells might explain elevated sST2 serum levels in idiopathic PAH patients. Nuclear IL-33 was found to be markedly reduced in endothelial cells of pulmonary arterioles of patients with idiopathic PAH, which has no cure and is associated with RV failure and death. 56 Circulating levels of IL-33 were unchanged, although accurate assessment of circulating IL-33 is fraught with challenges. 10 Further investigation revealed that nuclear IL-33 in human endothelial cells formed a complex with a transcriptional repressor and bound multiple putative homeodomain protein binding motifs in the proximal and distal promoters of ST2 genes to repress their expression. Nuclear IL-33 was also shown to function as a transcriptional repressor of other inflammatory genes, such as IL-6, in human endothelial cells. In addition, mast cells contribute to different types of PAH, 57 and active mast cells were found to accumulate in the RV of a mouse model of PAH in association with increased RV hypertrophy and fibrosis. 58 Mast cells constitutively express ST2L, and sST2 is strongly induced by IL-33. 1

Cardiac Hypertrophy

With hypertension, the LV undergoes hypertrophy, a risk factor for cardiovascular mortality. 59 Pressure overload of the mouse heart by transverse aortic constriction was found to induce IL-33 expression, and greater LV hypertrophy and adverse cardiac remodeling were observed in ST2 -/-mice, lacking both sST2 and ST2L. 33 Using cultured neonatal rat cardiomyocytes, IL-33 was demonstrated to antagonize agonistinduced cardiac hypertrophy, an action blocked by sST2 or an antibody to ST2L. 33 Moreover, treatment with recombinant IL-33 attenuated hypertrophy and cardiac remodeling in wildtype, but not ST2-knockout mice. Although IL-33 transiently activated NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in cardiac myocytes, its antihypertrophic actions were partially attributed to attenuation of NF-κB activation by hypertrophic agonists, secondary to suppression of ROS (reactive oxygen species) generation.

Systemic ablation of IL-33 exacerbated LV hypertrophy in mice in response to transverse aortic constriction and aggravated cardiac fibrosis and inflammation, including increased myocardial expression of Th1 cytokines, such as TNF-α. 60 However, subsequent findings on mice showed that pressure overload increases IL-33 expression in cardiac endothelial cells, and IL-33 released from endothelial cells of the heart (but not cardiac myocytes) was responsible not only for attenuating cardiac hypertrophy and fibrotic gene expression via ST2L but also paradoxically for inducing systemic inflammation. 42 Cardiac myocyte-derived sST2 was proposed to act locally to block the antihypertrophic actions of IL-33, whereas circulating levels of sST2 were postulated to arise from endothelial cells of the heart.

These preclinical findings support a model in which IL-33 is a mechanically induced protein in the heart that acts as a brake on cardiac hypertrophy and fibrosis. Thus, a correlation may occur between levels of IL-33 (negative) or sST2 (positive) and the degree of hypertension or extent of cardiac hypertrophy and fibrosis, and indeed, in vivo and in vitro evidence indicates that IL-33 has direct antihypertrophic actions on adult and neonatal rodent cardiomyocytes. 33,42 Whether the same is true in the adult human heart remains uncertain (Figure 2). Human coronary artery and heart microvascular endothelial cells from cardiomyopathy patients were found to express sST2, as well as IL-33 and ST2L. However, human cardiac myocytes, fibroblasts, and vascular smooth muscle cells were observed to express IL-33, but only minor amounts of ST2L or sST2 mRNA and not to secrete sST2 or be responsive to IL-33. 43 Explanted human cardiomyopathic hearts exhibited weak or no ST2L staining by cardiac myocytes or fibroblasts, respectively. In addition, no myocardial sST2 protein gradient was detected in heart failure patients. 61 Still, the biomechanical or inflammatory stress associated with myocardial infarction or pressure/ volume overload of the heart might induce sST2 release from cardiac myocytes. Mechanical strain, phorbol ester, IL-1β, and endothelin 1 were reported to increase production of sST2 by neonatal cardiac myocytes or fibroblasts. 33,44,62 Notably, in human LV hypertrophy because of AS and congestive heart failure of undefined cause, serum sST2 levels were found to correlate with diastolic load. However, in contrast to BNP, no correlation was observed with systolic wall stress, which may explain why sST2 in some cases may offer additional predictive value. 61 In heart failure, sST2 levels also correlate with serum levels of norepinephrine, natriuretic peptides, and CRP (C-reactive protein), and release of sST2 from human coronary artery endothelial cells is induced by certain inflammatory cytokines (IL-1β and TNF-α). 61 Thus, circulating sST2 levels may function as a measure of hemodynamic and inflammatory stress on the heart as primarily gauged by vascular endothelial cells. The importance of immune cells in contributing to the IL-33/ sST2 axis in the stressed heart is not defined. Although adult cardiac myocytes and fibroblasts express IL-33, its role in those cells may be largely intracellular. Extracellular IL-33/sST2, on the other hand, may enable crosstalk between endothelial and various immune cells that indirectly leads to cardiac remodeling. Treatment of human coronary artery endothelial cells with IL-33 was found to induce expression of adhesion molecules, produce the chemokine MCP1 (monocyte chemoattractant protein 1), and promote adhesion of human leukocytes. 31 Hypertensive patients with LV concentric hypertrophy have higher circulating sST2 levels than patients with normal heart geometry. 46 Another study by the same group reported that overall sST2 levels were higher in hypertensive patients with LV hypertrophy and correlated with both systolic and diastolic function, as well as RV remodeling. 45,46 However, although increased sST2 was associated with increased likelihood of LV hypertrophy in patients with metabolic syndrome, no association was noted with diastolic dysfunction. 63 These disparate conclusions could reflect disease stage, relative contributions of systemic versus cardiac inflammation, as well as extent and type of mechanical stress on the heart. 64 Such considerations may help explain why both correlations between circulating sST2 and cardiac hypertrophy and between sST2 and cardiac fibrosis (or diastolic dysfunction) need not be observed concurrently. For instance, in patients with hypertrophic cardiomyopathy, a correlation of sST2 with LV hypertrophy, but not LV fibrosis, was recently reported. 65

Heart Failure

Circulating sST2 levels are elevated in chronic heart failure and strongly associated with heart failure severity and adverse outcomes, such as cardiovascular events, cardiac hospitalization, increased risk of death or cardiac transplantation, and sudden cardiac death. 66 In contrast to levels of natriuretic peptides, sST2 levels are reported to be unaffected by body mass index, renal function, or age (although there are some reports of an association with age or body mass index). 48,67-72 Substantial evidence has shown that sST2 is a powerful prognostic biomarker in chronic heart failure with reduced ejection fraction (HFrEF). Blood sST2 levels have use to improve risk stratification in heart failure when added to established clinical variables or biomarkers, such as NT-proBNP. A review in mid-2014 of clinical trials on mostly HFrEF reported that sST2 levels, where assessed separately, offered incremental value as a predictor variable for primary outcome in 18 studies. 66 In 10 of these studies, sST2 offered independent prognostic information, and in 8 provided additive value to clinical variables and biomarkers for risk stratification. A recent meta-analysis assessing 6 of these studies, plus 1 from 2016, showed that sST2 is a predictor of both all-cause and cardiovascular death in heart failure patients. 73 The use of sST2 to predict clinical course of heart failure, all-cause and cardiovascular mortality, was observed in patients with diabetes mellitus as well. 74 Although shortcomings of these trials, such as limited multivariate analysis and narrow patient selection have been noted, 67 sST2 was included in the 2013 American College of Cardiology/American Heart Association Guidelines for management of patients with heart failure by additive risk stratification.

Other recent studies have supported the use of serial sST2 assessment 75-78 or a multi-biomarker profiling approach 68,79 in risk stratification of heart failure patients. In both cases, the lower biological variability for sST2 may be advantageous, 70,80 if properly combined with other biomarkers that are not redundant for the same pathophysiological process. 81 In the large (n=1449) CORONA study (Controlled Rosuvastatin Multinational Trial in Heart Failure), sST2 was independently associated with worsening heart failure. 82 In addition, clinical improvement in New York Heart Association (NYHA) III and IV heart failure was associated with a significant reduction in sST2 levels. 69 Thus, sST2 may provide insight into disease progression in heart failure comparable to NT-proBNP levels, 68 with added value over natriuretic peptides for accurately assessing disease management for monitoring or adjusting drug therapy. 83 sST2 was also demonstrated to have value for predicting clinical improvement in systolic heart failure patients. The ST2-R2 score, a composite of clinical variables plus serum concentrations of sST2 and complementary biomarkers, was demonstrated to predict reverse LV remodeling and functional improvement in systolic heart failure patients and mortality up to 4 years. 84,85 Interestingly, sST2 was the only studied biomarker independently associated with Whether IL-33 is beneficial or harmful is also not known. In the (human) heart, endothelial cells are likely the major source for IL-33 and sST2 release, with a lesser contribution from fibroblasts and myocytes. Although undoubtedly important, the contribution of immune cells is unexplored. The primary target of IL-33 in the heart would seem to be endothelial cells. A direct effect via ST2L on fibroblasts and myocytes in the adult human heart is less certain. However, in these cells, loss or increase in nuclear IL-33 levels may impact on inflammatory gene expression (inset). Although likely targeted, the role of immune cells in IL-33 actions on the heart awaits investigation. Dendritic cells and possibly other immune cells may internalize IL-33, but the cytoplasmic or nuclear consequences of this are not known. Finally, the contribution of extracardiac sources of sST2 and IL-33, such as adipose tissue, liver, large vessels, and lungs, remains a possibility. CBM indicates chromatin binding motif; H2A and H2B, core histones; IL1AcP, IL-1 receptor accessory protein; ILC2, group 2 innate lymphoid cells; Treg, tissue-resident regulatory T cells; and VSMC, vascular smooth muscle cells. October 2018 reverse remodeling. In the PARADIGM-HF trial (Prospective Comparison of ARNI With ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure), baseline sST2 level was an independent predictor of clinical outcomes even after adjusting for other predictors, including high-sensitivity troponin T (hs-TnT) and NT-proBNP. The association between baseline sST2 and outcomes was linear. 86 Notably, increases in sST2 at 1 month were associated with subsequent worse outcomes and decreased with better outcomes. Compared with enalapril, sacubitril/valsartan led to more reductions and fewer increases in sST2 levels over time.

More than half the patients with heart failure experience impaired cardiac relaxation, that is, diastolic dysfunction, in the face of a normal ejection fraction, a condition termed heart failure with preserved ejection fraction (HFpEF). 87,88 HFpEF, which is more prevalent among the elderly, is characterized by the presence of several comorbidities, such as obesity, type 2 diabetes mellitus, or hypertension. As there are no effective treatments for HFpEF based on the outcomes of clinical trials, HFpEF represents one of the largest unmet needs in medicine. Systemic vascular inflammation and coronary microvascular endothelial dysfunction are thought to be the basis for HFpEF. 89 In 2 studies of patients with stable or compensated HFpEF, sST2 blood levels were elevated compared with controls and roughly comparable to those of patients with HFrEF. 90,91 In 1 study, slightly higher sST2 levels for HFpEF (n=112; HFrEF, n=458) combined with a different biomarker profile, improved discrimination between the 2 HF subtypes in multivariable analysis. 91 However, no difference in sST2 levels was found between HFpEF (n=50) or HFrEF (n=51) patients and a control group (n=50) of randomly sampled free-living adults after adjusting for sex, age, clinical risk factors, and medications. 90 In contrast, multivariate analysis showed that elevated sST2 was independently associated with HFpEF in hypertensive patients (n=107), although levels provided no additional insight into the extent of diastolic dysfunction or heart failure functional class. 92 Nonetheless, evidence was reported that sST2 levels may have prognostic use in HFpEF, as well as HFrEF, for overall survival and heart failure hospitalization-free survival based on patient populations of predominantly NYHA class ≥III. 91 A correlation between sST2 and severity of disease, as assessed by blood NT-proBNP levels, E/E′ (a measure of diastolic dysfunction), left atrial (LA) volume, and galectin 3 (a measure of tissue fibrosis) was reported in HFpEF patients, although in a multivariable model after adjusting for confounders, only female sex, NYHA class, and LA volume were associated with higher sST2. 93 Increased LV diastolic stiffness in HFpEF might cause increased LA volume. Among HFpEF patients (n=174) enrolled in the RELAX trial (Phosphdiesterase-5 Inhibition to Improve Clinical Status and Exercise Capacity in Diastolic Heart Failure), sST2 was not associated with LV structure or LV systolic or diastolic function; however, higher sST2 levels were associated with the presence of diabetes mellitus, biomarkers of systemic inflammation and fibrosis, neurohumoral activation, myocardial necrosis, atrial fibrillation, systemic congestion, renal dysfunction, RV pressure overload (because of LV diastolic dysfunction [LVDD] and pulmonary congestion) and dysfunction, and exercise intolerance. 94 Interestingly, although type 2 diabetes mellitus was associated with elevated levels of sST2 (n=140 versus 18 patients without diabetes mellitus) and a correlation between glycemic control and sST2 was observed, patients with diabetes mellitus with LVDD (n=50) exhibited even higher sST2 levels. 95 The rather poor association of sST2 levels with ventricular function in the context of HFpEF may reflect its lack of cardiac specificity. The presence of multiple comorbidities, which may become more prominent with age, extracardiac sources of sST2, and its elevation in many other medical conditions likely limit its cardiac-related prognostic use in HFpEF.

A persuasive case can be made that clinical HFpEF is more reflective of adverse LA remodeling than LV concentric hypertrophy and fibrosis. HFpEF is characterized by greater LA stiffness compared with greater eccentric remodeling seen in HFrEF. 96,97 Preclinical and clinical evidence indicates that maladaptive LA remodeling occurs early during HFpEF development. 96,98,99 At early stages of HFpEF, LA dysfunction is associated with a higher risk of heart failure hospitalization independent of potential clinical confounders, although not independent of LV strain and filling pressure, suggesting a common link between LA and LV remodeling. 96 With more advanced-stage heart failure, LA dysfunction is a predictor of mortality in HFpEF. 97,100 Limited evidence implicates fibrosis in LA stiffness, 98,101 and increased fibrosis and wall stress could contribute to atrial arrhythmias, which is a common comorbidity of HFpEF. 97,101 Impaired LA function may explain the pulmonary congestion, shortness of breath, and exercise intolerance associated with HFpEF and could lead to right heart failure, 97,102 a leading cause of death in patients with HFpEF accounting for >50% of deaths. 103 Consistent with a primary role of LA stiffness in HFpEF are recent findings reported on 86 HFpEF patients from the prospective, multicenter KaRen substudy (Karolinska-Rennes). 104 Plasma sST2 levels were (inversely) associated with LA strain, but not LA size, nor LV geometry or systolic/diastolic function. Moreover, heart failure symptoms and outcome were independently predicted by reduced LA strain and not LV functional parameters. As discussed, 104 increased LA fibrosis (and sST2) in these HFpEF patients would seem to be reflective of inflammation, rather than attributable to increased hemodynamic load.

Although the prognostic use of sST2 is established in chronic heart failure, HFrEF or HFpEF, its predictive value in asymptomatic patients is uncertain. In a longitudinal observational study of 3915 community-dwelling elderly persons without established heart failure, the predictive value of sST2 for heart failure of any subtype or cardiovascular death was found to be modest compared with the existing risk models, despite strong cross-sectional associations with risk factors and underlying cardiac pathology. 105 Nor was sST2 associated with the development of either HFpEF or HFrEF.

With acute heart failure, plasma sST2 levels are higher than in chronic heart failure and also have prognostic use. 66,106 Multiple studies have reported that in acute heart failure, plasma sST2 levels at the time of hospital admission are strongly associated with measures of heart failure severity and with poor outcome. 66 Plasma sST2 levels in acute heart failure have been shown to provide independent and additive prognostic insight together with clinical variables and gold standard cardiovascular biomarkers, such as cardiac troponins and natriuretic peptides. The use of sST2 for predicting mortality in acute heart failure is observed regardless of whether the subtype is HFrEF or HFpEF, although sST2 concentrations may be lower in patients with HFpEF. 107 Others report that the prognostic value of sST2 in acute failure with HFpEF is less robust than with HFrEF, although in both subtypes prognostic accuracy is improved when combined with BNP. 108 Follow-up (out to 30 days) may strengthen the independent incremental prognostic value of sST2 in acute heart failure. 106 Moreover, persistently elevated sST2 at follow-up may identify a subset of patients with increased risk of death. A recent meta-analysis of 10 studies encompassing a worldwide population of 4835 patients with acute heart failure revealed that both admission and discharge plasma sST2 have prognostic use for cardiovascular and all-cause death, as well as the composite outcome of all-cause death or heart failure hospitalization. 109 Discharge sST2 was also predictive of heart failure hospitalization during follow-up. Unlike biomarkers of systemic inflammation, sST2 levels decrease rapidly with clinical improvement of acute heart failure. 110 Among the biomarkers assessed in this study, only postdischarge levels of BNP and sST2 had predictive value at 1 year by univariate Cox analysis. Serial measurements may improve the prognostic value of sST2 in acute heart failure to be additive or superior to natriuretic peptides. 66,78,111 A multi-biomarker approach that entails serial measurements of complementary biomarkers including sST2, along with standard clinical and laboratory parameters, might be used to guide personalized treatment strategies for acute heart failure. A strategy involving serial assessment of NT-proBNP, hs-cTnT (high-sensitivity cardiac troponin T), GDF15 (growth/differentiation factor 15), hs-CRP (high-sensitivity C-reactive protein), and sST2 was used to devise a model with very high prognostic value for 180-day cardiovascular mortality of patients with acute heart failure and mild-to-moderate renal impairment. 112

Cardiomyopathies

Other forms of cardiomyopathies have been associated with elevated blood sST2 levels (Table ). Diabetic cardiomyopathy is characterized by LVDD in the absence of other etiologic risk factors. Compared with healthy controls, type 2 diabetes mellitus (with or without LVDD) were reported to have higher serum sST2 levels that positively correlated with hs-CRP levels, a marker of systemic inflammation, and the extent of glycemic control (HbA1c). 95 Type 2 diabetics with LVDD had the highest levels of sST2. However, sST2 levels were found not to predict the occurrence of diastolic dysfunction 2 years later in patients with type 2 diabetes mellitus (n=48), when in fact sST2 was elevated further, although hs-CRP and triglycerides did. 120 These findings may suggest that increased levels of sST2 in diabetes mellitus are not causal for LVDD, but rather are reflective of a yet-to-be fully understood counter-regulatory phenomenon. 95

Aortic Stenosis

AS, which commonly develops during aging and often progresses without symptoms, is one of the most common and serious valve disease problems. 121,122 In the Western world, AS is the third most common cardiovascular disease (CVD) after coronary artery disease and systemic hypertension. 122 Surgical valve replacement is the standard of care for severe symptomatic AS. However, because of age, comorbidities, or frailty, upwards of a third of AS patients are not eligible for this surgery, and transcatheter aortic valve implantation can be an alternative treatment. 122,123 Severe AS is associated with increased morbidity and mortality, due in part to pressure overload-induced LV concentric hypertrophy and diastolic dysfunction that may progress to LV dilation and systolic dysfunction. 122 Inflammatory processes underlie the progression of AS, but the contribution of IL-33 is not well established. Analysis of aortic valve samples obtained from patients undergoing valve replacement surgery because of stenosis or regurgitation showed that sST2 protein expression was markedly upregulated in AS valves, whereas IL-33 was similar between the 2. 124 In addition, immunohistochemistry revealed the coexpression of both ST2 and IL-33 in CD68 + macrophages in AS valves.

An elevation in sST2 along with the biomarkers GDF15 and NT-proBNP was shown to predict 2-year mortality in patients (n=345) subsequently undergoing either transcatheter or surgical aortic valve replacement. 125 A more recent study reported that markedly increased preinterventional sST2 levels accurately predicted 1-year (but not 30 days) mortality in patients (n=274) who underwent transcatheter aortic valve implantation, even after correction for confounders in a multivariate analysis. 123 A similar smaller study (n=74 AS patients) reported that sST2 predicts major adverse cardiovascular events and 2-year survival after transcatheter aortic valve implantation. 126 In AS, sST2 may provide additional prognostic value as well. In a small cohort of patients with AS and preserved LV ejection fraction (n=86), both BNP and sST2 were associated with NYHA class, but sST2 more accurately identified asymptomatic patients or those who subsequently developed heart failure symptoms during follow-up. 127 Levels of sST2 were also associated with AS severity and degree of diastolic burden as assessed by left atrial area. However, the results of a larger study (n=461) showed that although sST2 independently associated with adverse outcome after transcatheter aortic valve implantation, it was not superior to NT-proBNP in predicting mortality. 128

Implications for Therapies and Future Directions

Accumulating evidence supports the diagnostic and prognostic use of sST2, particularly as a component of a multi-biomarker profiling approach, in chronic and acute heart failure, and for AS. In heart failure, measuring sST2 may offer added value for accurately assessing disease management for monitoring or adjusting drug therapy. Additional properly powered and rigorously designed clinical trials are needed to confirm its use in HFpEF, as well as for diabetes mellitus and other cardiomyopathies.

There is evidence that circulating sST2 levels function as a measure of hemodynamic and inflammatory stress on the heart as primarily gauged by vascular endothelial cells, although information on the contribution of immune cells is sorely needed. Preclinical studies involving cell-specific transgenic and knockout models are also required. A better understanding of the contribution of cardiac versus systemic sources of IL-33 and sST2, particularly as they impact on heart function and remodeling, is needed, not only for hypertension and diabetes mellitus but also for heart failure as well. October 2018 Genetic variants in and around the IL1RL1 gene are associated with differences in expression levels of sST2, ST2L, and IL-33, and polymorphisms on the surrounding gene cluster are associated with immune and inflammatory conditions. 10 However, more extensive genome-wide association studies are needed to establish a causal link with CVD. Other unresolved issues are the intracellular actions of IL-33 and how these are coordinated with its extracellular actions, and whether sST2 itself has biological actions.

A few pharmaceutical companies are developing anti-IL-33 and anti-ST2 biologics, as well as sST2-like drugs in the context of treating asthma and chronic obstructive pulmonary disease. Their impact on CVD warrants scrutiny, particularly on levels of natural IgM anti-oxLDL (oxidized low-density lipoprotein) antibodies that are linked to IL-33 and inversely associated with CVD. A better understanding of the differences in pharmacology between anti-ST2 and sST2 is desired. In this regard, the assumption that Il-33 has beneficial cardiovascular actions that are opposed by elevated sST2 in humans is unproven. For one thing, IL-33 can induce the expression of sST2 (as well as ST2L), indicating that chronically elevated sST2 may reflect unresolved actions of IL-33. IL-33 may increase its own No difference in sST2 on hospital admission between SCM and non-SCM patients; on follow-up (day 3), significant increase in sST2 in SCM patients. 119 Positive correlation of sST2 with hs-CRP at admission. 119 More elevated cTnI and BNP levels in SCM vs non-SCM patients. 119 sST2 and cTnI good combination to detect SCM in noncardiac MICU patients. 119 AMI indicates acute myocardial infarction; BMI, body mass index; CAD, coronary artery disease; cTnI, serum cardiac troponin I; DCM, dilated cardiomyopathy; DD, diastolic dysfunction; FC, febrile control; HC, healthy control; hs-CRP, high-sensitivity C-reactive protein; ICM, idiopathic cardiomyopathy; IL, interleukin; LV, left ventricular; LVEF, left ventricular ejection fraction; MICU, medical intensive care unit; NT-proBNP, N-terminal pro-B-type natriuretic peptide; RAP, right atrial pressure; RV, right ventricular; SCM, stress cardiomyopathy; and TTC, Takotsubo cardiomyopathy. Downloaded from http://ahajournals.org by on September 4, 2020 expression as well. 10 Evidence indicates that, although IL-33 has beneficial effects in atherosclerosis and adipocyte lipid homeostasis via ILC2 and Treg cells, IL-33 may activate an inflammatory response directly in endothelial cells, as well as by activating mast cells, eosinophils, and neutrophils. 10 Both concentration and spatiotemporal context are considerations in determining whether IL-33 favors an anti-or a proinflammatory outcome. A more rigorous assessment of circulating IL-33 levels using serum-certified ELISA assays that accurately assess active (nonoxidized and non-sST2 bound) levels is needed. 10 Depending on disease context, other treatments in combination with targeting sST2, ST2L, or IL33 will likely have additive effects. Synergistic interactions between IL-1β and IL-33 involving ST2L-mediated IL-1β and IL-33 expression, IL-1β-induced ST2L expression, and IL-33-induced/IL-1βinduced/ST2L-induced sST2 expression have been observed. 71 At the level of signal transduction, activation of ST2L in such cells as macrophages and mast cells may not only induce a Th2 response, but attenuate IL-1R1 (type I IL-1 receptor)-induced or TLR4 (Toll-like receptor 4)-induced Th1 responses by sequestrating the adaptor proteins Mal (MyD88-adapter-like) and MyD88 (myeloid differentiation primary response 88). 129 Thus, the recently reported cardiovascular benefit of targeting IL-1β shown in the CANTOS (Canakinumab Anti-Inflammatory Thrombosis Outcomes Study) 130,131 needs to be considered in exploiting the potential of IL-33-based/sST2-based therapies.

In conclusion, the clinical use of sST2 as a biomarker in CVD has recently gained recognition. However, further work is needed to better understand the accompanying physiological role of IL-33 at the cellular and molecular level that impacts on disease progression and outcome.

Introduction

Interleukin 33 (IL-33) is a member of the IL-1 family of cytokines, which strongly induces production of T helper-2 (Th2)-associated cytokines. Although regulation of transcription has been recently reported as an additional mechanism of IL-33 activity (see Section 3), classically active IL-33 functions as an 'alarmin' or stress-response cytokine that engages and regulates an immune response particularly at barrier sites in the body, where IL-33 is highly expressed by endothelial or epithelial cells. 1 Once released, IL-33 acts in an autocrine/paracrine manner to activate the ST2L (ST2 gene-like) membrane receptor on nearby cells, aka IL33R, and interleukin 1 receptor like 1 (IL1RL1). A soluble truncated form of ST2L without the transmembrane and intracellular domains, sST2, is secreted by endothelial and various immune cells either constitutively or upon stimulation (in some cases by IL-33). 2 sST2 is thought to function as a decoy receptor, thereby attenuating the actions of IL-33. 2 Evidence over the last decade has supported the conclusion that the sST2/ST2L/IL-33 triad plays an important role in cardiovascular disease (CVD). IL-33 is postulated to exert for the most part beneficial actions via ST2L that are related to cardiac repair or attenuation of adverse cardiovascular remodelling or atherosclerotic plaque progression. In the canonical model, sST2 attenuates the cellular and beneficial actions of IL- 33 in the cardiovascular system. Accumulating evidence has shown that elevated circulating levels of sST2 have evident prognostic utility for worse outcome in acute myocardial infarction (MI), 3 systemic and pulmonary hypertension, 4-6 coronary artery disease (CAD), 7 heart failure (HF), 8 and type 2 diabetes. 9,10 Most often, sST2, and not IL-33, was assessed due to its greater levels and stability.

New findings reveal that this view of IL-33 as strictly a protective or benign agent in CVD is over-simplistic. Neither is it established that sST2 is harmful because of its role as decoy receptor. As we assess in this review article, notwithstanding the evidence supporting the utility of sST2 as a CVD biomarker, there are gaps in our understanding of the functional significance of the IL-33/sST2 axis in cardiovascular and metabolic stress. Specifically, the focus of this review is on the vascular and metabolic aspects of the sST2/ST2L/IL-33 triad as a diagnostic and prognostic biomarker of stable CAD, MI, atherosclerosis, stroke, obesity, and type 2 diabetes. Also, we address the complicated question of whether IL-33/ST2 signalling functions simply as an acute 'alarmin' system or contributes to CVD progression under chronic or dysregulated conditions. In that context, the involvement of various immune cells and novel intracellular and extracellular signalling mechanisms in the actions of IL-33 are discussed.

Cellular expression

The membrane receptor for IL-33, ST2L is highly expressed by a wide variety of immune cells, including Th2 cells, regulatory T cells (Tregs), M2 polarized macrophages, mast cells, eosinophils, basophils, natural killer (NK) cells, invariant natural killer T (iNKT) cells, and type 2 innate lymphoid cells (ILC2s). 2 ST2L is constitutively expressed on cells of the cardiovascular system, in particular endothelial cells, 11 and can also be transiently induced in certain cases in other immune cell types, such as Th1 and cytotoxic T cells. 12 The notable actions of IL-33 on various immune cells are summarized in Table 1. In general, IL-33 is an important player in both innate and adaptive immunity as ST2L is expressed on most immune cells. By activating Th2 cells, IL-33 elicits a type 2 immune response, particularly at barrier sites. IL-33 also exerts protective and anti-inflammatory effects involving Treg and ILC2 (see Sections 8 and 10). However, if exuberant or dysregulated, type 2 inflammation may lead to tissue damage likely through activation of mast cells or eosinophils, and the development of pathological fibrosis. 13 In this way, IL-33 plays an indirect role in the pathophysiology of several pro-inflammatory and auto-immune diseases including asthma, allergies, arthritis, sepsis, and inflammatory bowel disease. 14 Whether a similar scenario also occurs in CVD is not known, and in fact the immune cellspecific role of IL-33 in CVD is not yet defined.

In healthy human tissues, IL-33 is mainly expressed by stromal cells, including endothelial and epithelial cells, and specialized fibroblasts. 53 IL-33 is constitutively present in the nuclei of cardiac fibroblasts, cardiac endothelial cells, cardiomyocytes, and coronary artery smooth muscle cells of human adults and is released during stress or with necrosis. 11 It is expressed only to a limited extent in mouse endothelial cells. 54 IL-33 can also be released from cells as a consequence of the cleavage of membrane phospholipids by secreted phospholipid A2 (sPLA2) enzymes, which is relevant to how venoms and inhaled allergens elicit a type 2 immune response 55 and likely relevant to atherosclerosis as well. In addition, IL-33 is a mechanically responsive cytokine secreted by living cells in response to stretch (Figure 1). 56 Pro-inflammatory cytokines such as tumor necrosis factor (TNF)-a, interferon (IFN)-c, and IL-1b increase IL-33 expression. 11 In humans, ST2L and sST2 mRNA on the other hand were reported to be expressed at low levels in cardiomyocytes, cardiac fibroblasts, and vascular smooth muscle cells, but widely present in endothelial cells of the cardiac vasculature. 11 ST2L is prominently expressed by ILC2s, mast cells, and Tregs expressing the GATA3 transcription factor, as well as by activated Th2 lymphocytes. 2 Levels of ST2L are enhanced by IL-33 in ILC2s and Tregs, but neither expresses sST2. ST2L is expressed weakly as well by dendritic cells, neutrophils, and uncommitted macrophages (and enhanced by IL-4/IL-13). 2 Taken together these findings would suggest that the primary direction for communication of the IL-33 alarmin system is from parenchyma or endothelium to the endothelium and immune cells, with production of sST2 by endothelial cells and certain pro-inflammatory immune cells serving a protective or damping role. Uncertain, however, is how ST2L expression in cardiovascular cells is affected by disease state.

Novel signalling

Two modes of action have been identified for IL-33, an extracellular one as a cytokine or alarmin, and a nuclear one as a regulator of transcription. Pro-and anti-inflammatory actions have been attributed to both modes of action, which are cell-and context-dependent. IL-33 localizes to the nucleus due to the presence of two bipartite nuclear localization sequences in the predicted helix-turn-helix structure of the homeodomain-like N-terminus. 57 Deubiquitination of IL-33 has been implicated in its nuclear stability, yet ubiquitination of IL-33 has also been implicated in its activation of transcription. 58,59 A better understanding of the different ubiquitination profiles of IL-33 and their significance is needed.

IL-33 associates with chromatin to ostensibly repress gene expression via protein-protein interactions, involving a short chromatin-binding motif that binds the acidic pocket made by the histone heterodimer H2A-H2B at the nucleosome surface. 60 However, the nuclear actions of IL-33 are diverse and incompletely understood. Binding of IL-33 to promoterbound homeodomain proteins, such as histone methyltransferase SUV39/HI, was implicated in IL-33-mediated suppression of IL-6 and sST2 expression in human atrial endothelial cells. 61 IL-33 was reported to induce transcription of the type 2 inflammatory cytokine IL-13 in HEK293T cells by binding a conserved non-coding sequence before the transcription initiation site. 58 In addition, IL-33 was reported to function as a transcriptional regulator of NF-jB p65 expression in endothelial cells and participate in the inflammatory response by binding the p65 promoter. 62 In contrast, IL-33 was reported to act as a transcriptional repressor of NF-jB in synoviocytes of patients with rheumatoid arthritis. 63 In some cases, IL-33/NF-jB p65 protein-protein interactions may impair NF-jB DNA binding and thus interfere with NF-jB-dependent transcription. 64 Thus, both pro-and anti-inflammatory actions have been ascribed to nuclear IL-33. 65,66 However, in many cell types, the role of nuclear IL33 is still unknown. 67 IL-33 is constitutively expressed in many non-haematopoietic tissues, but its expression can be induced in both non-haematopoietic and some haematopoietic cells. 2,60 Th1 and Th2 cytokines were reported to regulate intracellular levels of the precursor or full-length IL-33 in fibroblasts of healthy human lungs by activating or inhibiting, respectively, its proteasomal degradation. 68 Notably, full-length IL-33 was found to promote inflammation in the lung, but not a Th2 response, in an ST2-independent fashion. 69 Importin-5 (IPO5) was identified as an intracellular binding partner of full-length IL-33 that protects it from proteasomal degradation, but IPO5 is not required for nuclear localization of IL-33 and does not control its secretion. 70 Full-length IL-33 is released into the extracellular space on cell damage or necrosis, whereas caspases 3 and 7 cleave and inactivate intracellular IL-33 during apoptosis (Figure 1). 71 Alternative transcript splicing with deletion of exons 3 and 4 may confer cytoplasmic localization and facilitate secretion. 72 The release of IL-33 from cells in the absence of damage or necrosis is not well understood, but in bronchial epithelial cells was shown to be under the regulation of ATP-induced P2 purinergic receptor stimulation and calcium influx. 73 Extracellular IL-33 activates the membrane receptor ST2L, which together with the co-receptor IL-1R accessory protein (IL-1RAcP) recruits MYD88, IRAK1, IRAK4, and TRAF6, followed by activation of multiple signalling pathways, including MAPK1/ERK2 and/or MAPK3/ERK1, p38a MAPK, JNK1, and NF-jB (Figure 2). 60 An extensive quantitative phosphoproteomic analysis of IL-33-mediated signalling was recently reported. 74 There is evidence as well that extracellular IL-33 may suppress activation of the p38 MAPK and NF-jB pathways in the heart 3 days post-MI, but this is likely indirect. 75 A number of mechanisms act to localize and limit both temporally and spatially the actions of extracellular IL-33 so as to make less likely an uncontrolled Th2 inflammatory response. Unlike most IL-1 family members, IL-33 has a comparatively long pro-peptide sequence of 110 amino acid residues at the N-terminus. Contrary to original thinking, IL-33 bioactivation does not seem to be dependent upon caspase1/inflammasome-mediated processing within the cell, nor is cleavage necessary for secretion. 76,77 Rather, a number of extracellular proteases are involved in its activation, with the cleaved sequence targeted within the N-terminal domain or central domain being protease-specific. 71 These include proteases that are released by neutrophils and mast cells, such as neutrophil proteinase 3 (PR3), elastase, and cathepsin G. Moreover, it was recently proposed that full-length IL-33 functions as a biochemical sensor of the proteolytic activities of a large variety of environmental aeroallergens. 78 While short-term exposure enhances the activity of IL-33, longer exposure to some proteases promotes further degradation and loss of activity by targeting the C-terminus IL-1-like cytokine domain. • Increases surface levels of maturation markers MHC-II, CD40, CD80, CD86, OX40L, and CCR7 18-20 • Increases production of pro-allergic cytokines and chemokines IL-4, IL-5, IL-13, CCL17, TNF-a, and IL-1b • Promotes type 2 cytokines production 27,28 • Expands in vivo 27,29 Invariant natural killer T (iNKT)

• Causes expansion and activation 30 • Enhances production of several cytokines, including both IL-4 and IFN-c and induces IFN-c instead of IL-4 upon TCR engagement in cooperation with IL-12 30,31 M2 polarized macrophages

• Amplifies the expression of M2 markers 32,33 • Enhances activation by upregulating lipopolysaccharide (LPS) receptor components TLR4 and MD2, soluble CD14, and MyD88, thus increasing LPS-induced cytokine production 33 Mast cells

• Induces production of inflammatory cytokines MCP-1, TNF-a, IL-1, and IL-6 34 • Enhances IgE-mediated activation 34 • Promotes survival 35,36 • Promotes mast cell activation and maturation, and induces GM-CSF, IL-5, IL-13, CXCL8, CCL17, CCL22, and CCL2 secretions 36,37 • Induces production of various type 2 cytokines 38-40 • Promotes Th17 response during airway inflammation 41 Natural killer (NK) cells

• Increases IFN-c synergistically with IL-12 30,31 Regulatory T cells (Treg)

• Enhances protective ability/increases immunomodulatory function 42,43 • Expands/increases directly or via IL-33-induced DC production of IL-2 21,44-50 Th2 cells

• Increases production of type 2 cytokines IL-5 and IL-13 51 • Chemoattractant 52

. . . . . . . . . . . . Furthermore, IL-33 is also rapidly oxidized within the extracellular milieu resulting in the formation of two intramolecular disulfide bonds that disrupt the ST2L binding site. 79 Besides impairing function, IL-33 oxidation might alter its immunoreactivity and confound assays that rely on antibody detection. Thus, oxidation should be taken into consideration in measuring IL-33 especially under conditions of heightened inflammation and oxidative stress, as seen for instance with cigarette smoke, a major CVD risk factor. In the canonical model, sST2 functions as a decoy receptor for IL-33, thereby preventing the cellular actions of IL-33 mediated by interaction with the membrane receptor ST2L (Figure 2). However, there are a few intriguing reports that sST2 may have actions of its own on certain cells.

Evidence was reported that sST2 has direct anti-inflammatory actions on macrophages by downregulating Toll-like receptors. Treatment with an ST2-human IgG fusion protein induced cellular signalling and downregulated expression of TLR4 and TLR1 in bone marrow-derived macrophages. 82 In addition, administration of the fusion protein to mice attenuated LPS-mediated mortality and serum levels of IL-6, IL-12, and TNF-a, while an anti-ST2 antibody worsened the toxic effects of LPS, which are known to be mediated by TLR4. Others reported that sST2 suppresses LPS-induced IL-6 production in a human monocytic leukaemia cell line. 83 Evidence (based on an ST2 Fc chimaera protein) was also reported to support the conclusion that sST2 may contribute to adverse aortic remodelling seen in obesity by stimulating VSMCs to produce collagen type I, fibronectin, and profibrotic factors, as well as increase activities of MMPs. 84 Note, however, that because of the IgG portion of the molecule, sST2-fusion proteins (unlike sST2) could theoretically undergo dimerization, which might impact on their actions.

IL-33 and sST2 as biomarkers in coronary artery disease and myocardial infarction

The results of several studies summarized in Table 2 support the conclusion that serum levels of IL-33 decrease with increasing CVD severity. 85-87 The opposite pattern was reported for either the pro-inflammatory cytokine IL-6 or the extracellular protease matrix metalloproteinase (MMP)-28, 85-87 supporting the proposal that combining their assessment with that of IL-33 might be useful in gauging the severity of CAD. However, the number of CAD/acute coronary syndrome (ACS) cases were small in these three studies (n = 83/40, 103/27, and 70/20). Others did not find a difference in IL-33 between patients with ACS (n = 195) and stable CAD (n = 178), but in this study the highest quintile of IL-33 predicted mortality (mean follow-up 3.6 years) in patients with STEMI. 88 Although the number of participants was larger, it was still relatively small and the number of patients with adverse clinical events at follow-up was very small (37 deaths). Moreover, serum IL-33 was undetectable in more than 50% of study participants. Accurate assessment of IL-33 in human serum is difficult for a number of reasons, including lack of sensitivity and specificity of available ELISA assays, interference by the presence of sST2, and the use of non-serum certified kits. 116 There is also a necessity to differentiate between oxidized (inactive) and reduced (active) forms of IL-33, which is now possible through the development of specific ELISAs. 79 In contrast, a clear pattern of increasing serum sST2 levels with greater severity of CAD events has been consistently observed [healthy < stable angina < unstable angina < non-ST elevation MI (NSTEMI) < STEMI < sudden cardiac death]. Several studies have reported the prognostic value of sST2 in patients with stable CAD. In the Ludwigshafen risk and cardiovascular health study, sST2 did not correlate with the angiographic severity of CAD; however, on long-term follow-up (median time of 9.8 years), higher levels of sST2 were an independent predictor on multivariate analysis for all-cause mortality and cardiovascular death after adjusting for clinical variables (including age, sex, body mass index (BMI), hypertension, smoking status, and diabetes) and biomarkers. 7 Soluble ST2 within the normal range had prognostic value additive to NT-proBNP and hs-cTnT, supporting its utility in a multimarker approach. Results of a 2-year follow-up from the ARTEMIS (international Ambulatory blood pressure Registry: TEleMonitoring of hypertension and cardiovascular rISk project) study, involving a study population of 1243 patients and 649 controls, revealed that in multivariate analysis only sST2 and hs-C-reactive protein (CRP) predicted the primary endpoint of cardiac death or HF hospitalization in both diabetic and non-diabetic patients with CAD. 89 Results of the KAROLA study showed that after multivariable adjustments sST2 levels in a cohort of 1081 stable CAD patients independently predicted both short-term (4.5 years) and long-term (12.3 years) risk for total mortality, and short-term risk for fatal cardiovascular disease-related events, but not non-fatal cardiovascular events. 90 Circulating sST2 levels have diagnostic and prognostic value after STEMI. sST2 levels measured within 1 day post-MI correlated positively with peak creatinine kinase, an estimate of the extent of necrosis, and negatively with pre-discharge left ventricular ejection fraction (LVEF). 92,93 Early sST2 positively correlated with infarct size and expansion, as well as greater infarct transmurality and endocardial extent, microvascular obstruction, and plasma aldosterone levels. 94 Early values were a significant predictor of cardiovascular death and HF over the following 30 days after STEMI, independent of baseline characteristics or NT-proBNP levels and, in combination with NT-proBNP, improved risk stratification. 93,95 Interestingly, unlike NT-proBNP, sST2 levels on presentation were not associated with clinical conditions linked to increased LV wall stress, such as age, hypertension, previous MI, or prior MI; however, higher levels were associated with diabetes mellitus. 93 In a recent report on multimarker risk stratification for STEMI involving upwards of 1258 patients enrolled in the Clopidogrel as Adjunctive Reperfusion Therapy-Thrombolysis in Myocardial Infarction 28 (CLARITY-TIMI 28) trial, sST2 was a significant predictor of HF or shortterm cardiovascular death (out to 30 days) along with two other biomarkers, troponin T, and myeloperoxidase (MPO). 96 Soluble ST2 had greater prognostic value than hs-cTnI for 30 day cardiac mortality in both STEMI and NSTEMI patients. 97 Another study showed that elevated sST2 levels with STEMI were associated with increased all-cause mortality up to 1 year and improved risk stratification using a multimarker approach. 98 sST2 levels were reported to be elevated in patients with STEMI and NSTEMI, with levels markedly higher in those with STEMI. 88 In addition, the highest quintile of sST2 predicted mortality in patients with STEMI, but not those with NSTEMI. Others reported that elevated sST2 predicated long-term major adverse event in NSTEMI patients but did not improve risk stratification for established markers. 99 In a recent study of 1401 first-ever MI patients involving mostly (79%) NSTEMI, higher sST2 values were associated with increased risk of death and HF over a 5 year follow-up, independent of other prognostic indicators. In this study, higher values of sST2 were associated with age, female sex, and hypertension, in addition to diabetes mellitus. 100 Findings of a crosssectional, population-based study revealed that sST2 also positively correlates with markers of type 2 diabetes and endothelial dysfunction, but not established cardiovascular risk factors. 10 This suggests that activated/ stressed vascular endothelial cells are the source of sST2 in diabetes. While pathology-related increases in circulating sST2 have clinical value, others reported that sST2 levels in healthy men and women added little long-term predictive information for cardiovascular events or all-cause mortality. 117 Overall, there is strong evidence for the diagnostic and/or prognostic utility of sST2 in CAD and MI (both STEMI and NSTEMI), particularly in combination with established biomarkers. Key studies supporting this conclusion are listed in Table 2. The observation that circulating levels of IL-33 and sST2 exhibit an opposite pattern of change with increasing severity of CAD event, together with MI preclinical studies (see below), underpins the conclusion that enhancing cardiovascular activity of IL-33 may be beneficial for limiting cardiovascular events.

Pathophysiological role of IL-33/ ST2 signalling in preclinical studies of myocardial infarction

The strong association between increased circulating levels of sST2 and poor prognosis in patients with MI provides circumstantial evidence for a protective role of IL-33 in the heart under stress that is borne out by preclinical studies. Biomechanical strain induces expression of sST2 and IL-33 in both cardiac myocytes and fibroblasts, with cardiac fibroblasts being more responsive. 118 Similarly, IL-33 was mostly expressed by interstitial cells (likely myofibroblasts) in pressure overloaded mouse hearts. 118 Levels of IL-33 in human adult cardiac myocytes and fibroblasts are also increased by inflammatory cytokines. 11 IL-33 was found to protect neonatal rat cardiomyocytes from cell hypoxia-induced caspase-3 cleavage and apoptosis, and this was associated with increased expression of anti-apoptotic proteins (XIAP,cIAP1,survivin,. 119 The addition of sST2 blocked these protective actions of IL-33. Others reported evidence for the attenuation of reactive oxygen species (ROS) generation by IL-33, and the subsequent sequential activation of PKCbII and JNK, in the protection of neonatal mouse cardiomyocytes from apoptosis after anoxia/reoxygenation. 120 In vivo preclinical evidence also indicates that IL-33 protects the heart from infarction. IL-33 treatment was found to decrease fibrosis, infarct size, and apoptosis after ischaemia-reperfusion (I/R) in the rat and improve cardiac function. 119 In addition, IL-33 reduced ventricular dilation, improved contractile function, and increased survival following coronary artery ligation in wild type, but not in ST2 -/-mice. 119 IL-33 treatment was associated with a decrease in mast cell density in the infarct area, as well as an increase in Th2 and decrease in Th1 genes in the infarct. Many of these beneficial actions of IL-33 are probably secondary effects on the myocardium, since as discussed IL-33 by itself may have profibrotic actions. In addition, IL33 activates mast cells and a reduction in cardiac mast cells was reported to attenuate myocardial contractility after MI. 121 Thus, the reduction of mast cells in the study of Seki et al. 119 is probably secondary to general reduction of inflammation and unrelated to improved cardiac contractility.

Another study on MI (permanent LAD occlusion) in mice also reported similar beneficial effects of post-treatment with IL-33 on cardiac function and structure, as well as reduced myocardial macrophage infiltration and inflammatory cytokine production, and suppression of p38 MAPK and NF-jB activation. 75 However, the exact involvement of p38 MAPK signalling in the cardioprotective actions of IL-33 is likely a matter of timing and model of injury. Others recently implicated activation of p38 MAPK in the anti-apoptosis and anti-inflammatory actions of pre-treatment with IL-33 in protecting the heart, including decreased expression of the cytokine/alarmin high mobility group box 1 protein (HMGB1), in a rat model of I/R-induced cardiac injury. 122 Diabetes mellitus increases the vulnerability of the heart to I/Rinduced injury. This has been attributed in part to increased PKCbII activity, which is enhanced by diacylglycerol (DAG). 123 Cellular levels of DAG are in turn regulated by DAG kinase (DGK), which catalyses its conversion to phosphatidic acid. Diabetes-related exaggerated apoptosis and dysfunction of the myocardium that is observed with I/R was attributed to increased PKCbII activity due to reduced expression of DGKzeta. 123 The later was linked to reduced levels of IL-33, which was shown to induce DGK-zeta expression in the heart and isolated cardiomyocytes. Thus, IL-33 may negatively regulate PKCbII activity in cardiac myocytes both by attenuating oxidative stress and by enhancing expression of DGK-zeta. Evidence was reported that the reduced IL-33 levels in the diabetic heart result from high glucose-induced secretion of HMGB1 from cardiac myocytes. 124 HMGB1 in turn stimulates TLR4 receptors on fibroblasts to reduce their IL-33 production, thereby leading to enhanced collagen production and cardiac fibrosis. However, the means by which IL-33 suppresses fibrosis in the heart is not known and likely indirect. Surprisingly, IL-33 was found not to directly inhibit collagen I/III or periostin production by adult rat cardiac fibroblasts, or their proliferation; rather, IL-33 stimulated expression of cytokines and chemokines (IL-6 and CCL-2) associated with cardiac inflammation and fibrosis, although the migratory ability of the cardiac fibroblasts was attenuated. 125 Interestingly, in a mouse infarction model, myocyte-targeted ablation of TGFb signalling markedly augmented IL-33 expression in what appeared to be perivascular interstitial cells, but no impact on collagen deposition in the infarct was seen. 126 In summary, despite the fact that stressed and injured cardiac myocytes may secrete IL-33, they produce factors (e.g. HMGB1) that reduce IL-33 production by cardiac fibroblasts, which may favour ROS-induced PKCbII/JNK activation, inflammatory cytokine, and apoptosis gene expression. Several rodent studies reported a protective effect of IL-33 supplementation on the heart, delivered either before or after MI, which is attributable to reduced ROS production. The cell type(s) mediating the cardioprotective effects of IL-33 is (are) not yet defined. Paradoxically, Abston et al. 127 have reported that IL-33 treatment in healthy mice induces inflammatory cytokines in the heart, and independently induces eosinophilic pericarditis and impairs heart function. Strain differences or dosing regimen cannot explain the discrepant findings between this study and the ones involving MI, so other factors such as diet, surgical procedure, pre-existing injury, need to be considered. In any event, the findings of Abston et al. caution against taking a broad approach in IL-33 delivery for protecting the infarcted heart.

IL-33/ST2 signalling in the pathophysiology and clinical outcome of stroke

In patients (n = 206) who suffered acute ischaemic stroke, serum IL-33 levels were elevated; however, lower levels were associated with greater stroke severity and large infarction volume. Levels were higher in patients with a favourable outcome, and IL-33 levels were an independent predictor for functional outcome. 103 On the other hand, higher sST2 at the time of admission was reported to be associated with allcause mortality 90 days after acute ischaemic stroke (n = 721), but did not offer prognostic value in multivariate analysis. 104 Larger, adequately powered and well-designed studies across multiple centres with proper controls are needed to assess the prognostic value of IL-33/sST2 in ischaemic stroke.

In preclinical models, treatment with IL-33 was shown to be protective in ischaemic stroke 128,129 and spinal cord injury 130 by causing a shift towards the M2 microglial/macrophage cell phenotype and attenuating inflammation. Expression of IL-33 in oligodendrocytes and astrocytes increases with ischaemic injury in the mouse, along with ST2L expression in microglia and astrocytes. Yang et al. 131 provided evidence that the neuroprotective actions of IL-33 in ischaemic stroke are due in part to its stimulation of anti-inflammatory cytokine IL-10 production by microglia cells.

In summary, despite serum IL-33 being increased in ischaemic stroke, an association of lower IL-33 and higher sST2 with worse outcome was observed. Although based on a single study, this is consistent with the idea that in ischaemic stroke IL-33 has protective actions that are dampened by sST2 (Table 2), as supported by animal studies. However, by themselves early serum IL-33 levels may reflect mostly the extent of injury, rather than serving as a measure of the extent of protection mounted. Paradoxically, its induced target sST2 is likely a gauge of both extent of injury and blockade of protection. For that reason and technical issues previously discussed, greater confidence ought to be placed in reported sST2 values in MI and stroke studies.

Genetic variants in IL-33 and ST2 genes and relationship with CAD

A prospective study of 2991 Framingham Offspring Cohort participants revealed that much of the variation in sST2 production among individuals is due to genetic factors. 132 The IL1RL1 gene encodes for both the membrane-bound receptor isoform (ST2L) and the soluble protein (sST2) through alternative promoter activation and splicing. 133 Multiple single-nucleotide polymorphisms (SNPs) within IL1RL1 were found to correlate with sST2 levels in a genome-wide association study, and five missense variants mapping to the intracellular domain of ST2L, which is not present in sST2, correlated with higher sST2 levels. 132 Experiments on cultured cell lines expressing the intracellular variants attributed the increase in sST2 levels to an autocrine loop of increased IL-33 induction and enhanced ST2L responsiveness. Briefly, increased sST2 was ascribed to (i) increased induction of IL-33 by ST2L because of enhanced NF-jB and AP1 signalling, which also selectively activated the proximal promoter of IL1RL1 linked to sST2 expression and (ii) a selective increase in ST2L expression due to an increase in endogenous IL-1b levels resulting from enhanced constitutive ST2L-mediated inhibition of a counter-regulatory PI3K/AKT/mTOR signalling axis that attenuates IL-1b levels. In light of the recently reported outcome of the Canakinumab Antiinflammatory Thrombosis Outcomes Study (CANTOS), 134,135 the potential synergistic interplay between IL-1b and IL-33 in vivo merits investigation.

An earlier study linked two polymorphisms in the distal promoter of IL1RL1 that drives ST2L expression to enhanced CAD severity, but no sST2 measurements were made. 136 Another SNP in IL1RL1 was linked to increased risk for CAD without defining its functional impact. 137 Yet another SNP of IL1RL1 was associated with lower circulating sST2 levels; however, in affected individuals with CAD or peripheral artery disease, increased sST2 levels were an independent predictor of all-cause mortality by multivariable Cox regression analysis, but not for secondary endpoints of CV death, MI, hospitalization for HF, stroke, and amputation. 138 Unfortunately, the impact of this SNP on IL-33 levels or ST2L expression was not determined. An SNP within the promoter region of the IL-33 gene was associated with increased circulating levels of IL-33 and increased risk for CAD. 137 Another IL-33 gene polymorphism that was linked to decreased IL-33 production was associated with a decreased risk for developing premature CAD or central obesity. 139 Others reported the opposite effect of this SNP genotype on serum IL-33 levels in patients with rheumatoid arthritis and thus no causal relationship can be drawn. 140 In addition, a direct causal relationship between IL-33 levels and CAD is not established as neither of the studies on IL-33 gene variants reported values of sST2. Nonetheless, an SNP in the IL-1RAcP gene was also linked to CAD risk. 141 In summary, limited reports suggest that genetic variants in or around the IL1RL1 gene are associated with differences in expression levels of both sST2 and ST2L, as well as IL-33. Polymorphisms in the gene cluster within which IL1RL1 resides have been associated with a number of immune and inflammatory conditions, 132 but more extensive GWAS are needed to establish a causal link between IL1RL1 variants and CAD. This is the case for the IL-33 gene as well.

Mechanistic insights into the role of IL-33/sST2 in atherosclerosis

Increased IL-33 expression has been detected in human atherosclerotic plaques, emphasizing the importance of IL-33 in vascular biology and remodelling (Figure 3). 105 Atherosclerosis is characterized by a chronic arterial wall inflammation that plays a major role in atheroma formation. 142 The presence of oxidized low-density lipoproteins (ox-LDL) in the vessel induces the production of pro-inflammatory mediators like cytokines and growth factors from surrounding tissues that further fuel the inflammatory response and atherosclerosis progression. 143 Miller et al. revealed that IL-33 administration to ApoE -/-model of atherosclerosis in mice, induced a shift from the Th1 pro-atherosclerotic immune response to a Th2 protective and pro-resolving immune response by significantly increasing Th2 cytokine production and decreasing IFNc levels, a typical Th1 cytokine. 144 Th1 to Th2 polarization resulted in a reduction of aortic atherosclerotic lesions when compared to vehicle-treated group. 144 Of note, atherosclerotic plaque formation and progression is multifactorial and T cell infiltration can either increase (Th1) inflammation in plaques or decrease (Th2/Treg) it depending on the dominant phenotype. 145 In addition to polarizing effects, IL-33 increased levels of atheroprotective natural IgM type anti-ox-LDL antibodies suggesting a potential effect on B1 cells. Neutralizing IL-33 effects via sST2 administration to ApoE -/-mice resulted in aortic plaque expansion when compared to control IgG-treated group. Additionally, blocking IL-5 with a neutralizing antibody negated the protective effect of IL-33 and dampened the production of ox-LDL antibodies suggesting that IL-5 might have a key role in the atheroprotective effect of IL-33. 144 In vitro studies on the other hand, showed that IL-33 atheroprotection might have occurred via inhibition of macrophage foam cell formation through decreased acetylated LDL and ox-LDL uptake and enhanced cholesterol efflux. 146 Recently, the ability of IL-33 to protect macrophage-derived foam cells from cholesterol overload was attributed to the induction of IL-10, which helped IL-33 in an autocrine manner to increase expression of ATP-binding cassette transporter (ABCA1), potentially promoting cholesterol efflux. 147 Multiple lines of evidence support the concept that IL-33 may also be atheroprotective by engaging ILC2s and activating downstream type 2 immunity, mainly IL-5 and IL-13. 148,149 IL-5 may stimulate B1 cell proliferation and production of atheroprotective natural IgM antibodies against the phosphorylcholine (PC) head group of oxidized phospholipids within LDL. 149-151 Besides inducing the expansion of ILC2s, recent evidence indicates that IL-33 promotes the egress of ILC2s from the bone marrow and possibly from secondary lymphoid organs, 152 which further lends weight to the idea that administration of IL-33 at pharmacological levels may be necessary to reveal its role in atherosclerosis. Consistent with this possibility, loss of either endogenous IL-33 or its receptor ST2 was found to have no impact on development of atherosclerosis in ApoEdeficient mice. 153 Activated ILC2s may also attenuate the progression of atherosclerosis by producing IL-13, which polarize macrophages towards the 'M2' like phenotype. 154 In addition, the actions of ILC2s in regulating adipose tissue homeostasis and limiting obesity (see Section 10) may be an additional means by which IL-33 exerts atheroprotective effects.

IL-33 may also contribute to an increase in Treg cells, which exert anti-atherogenic effects by limiting both adaptive and innate immune responses. 155-157 This function of IL-33 may be compromised in atherosclerosis due to both increased serum levels of sST2 and reduced levels of CD4 þ ST2 þ cells. 158 Recent evidence shows that expression of ST2 is also a feature of a sizable number of tissue-resident Treg cells that are important for tissue repair and promoting organ homeostasis. 159 Their expansion and activation is stimulated by IL-33. 159,160 These ST2 þ Tregs exert anti-inflammatory actions and suppress CD4 T cell proliferation through the release of IL-10 and TGF-b. 161 This pool of Treg cells is especially prominent in visceral adipose tissue, where Treg cells support metabolic functions and possibly adipocyte differentiation. 162,163 Little information is available concerning the expression pattern of the IL-33/ST2L axis within the atherosclerotic plaque. In an immunohistochemical study on endarterectomy samples, Marzullo et al. 107 observed that ST2L was expressed in atherosclerotic plaques to a similar extent in asymptomatic and symptomatic patients on both T cells and endothelial cells of neo-angiogenic vessels (much more so than the endothelial cells covering the residual lumen of the vessel). In contrast, expression of ST2L on macrophages was more remarkable in symptomatic patients. Based on these observations, the authors hypothesize that the IL-33/ ST2L axis drives plaque development and eventual rupture; however, the sample size in their study was small, and causality was not studied. Others have recently suggested that IL-33 may contribute to plaque progression in part by inducing expression of the chemokine CXCL1 (see Section 9). 106 On the other hand, in patients with primary hypertension, a major risk factor for atherosclerosis, circulating levels of sST2 were found to be high, whereas IL-33 levels were low. 108 Moreover, sST2 was identified as a risk factor for subclinical atherosclerosis and its levels were positively correlated with the standard atherosclerosis risk factors, LDL cholesterol, CRP, and carotid intima-media thickness. 108 The overall evidence supports the conclusion that IL-33 has atheroprotective effects. Several mechanisms may explain these actions. These include a shift in T cell polarization from Th1 to Th2 and an increase in Treg cells, increased levels of natural IgM anti-ox-LDL antibodies, inhibition of macrophage foam cell formation, stimulation of ILC2s, and polarization of macrophages towards the 'M2' like phenotype.

Vascular inflammation

Several studies have reported direct pro-inflammatory effects of IL-33 on endothelial cells. For instance, IL-33 induces the secretion of the inflammatory cytokines IL-6 and IL-18 from human umbilical vein endothelial cells (HUVECs), 164 as well as the expression of chemoattractants for leucocytes (CXCL1 and CCL2). 106 Also, IL-33 promotes the adhesion of human leucocytes to human endothelial cells and induces vascular cell adhesion molecule-1, intercellular adhesion molecule-1, endothelial selectin, and CCL2 mRNA and protein expression in human coronary artery and umbilical vein endothelial cells in vitro and human explanted atherosclerotic plaques ex vivo. 105 These effects of IL-33 on endothelial cells and immune cells may explain why increased IL-33 serum levels after coronary stent implantation are associated with coronary in-stent restenosis, 165 as leucocyte activation is a critical step in development of restenosis after PCI. 166 Interestingly, Pollheimer et al. 167 observed that the pro-inflammatory actions of IL-33 on cultured HUVECs was greater Other studies have demonstrated that IL-33 promotes angiogenesis and vascular permeability in vitro and in vivo, notably within the context of inflammation. 168-173 The pro-inflammatory actions of IL-33 on the vasculature, and endothelial cells in particular, may contribute to the pathogenesis of giant-cell arteritis (GCA), which is an inflammatory disease of blood vessels that occurs in the elderly. The exact basis for GSA is uncertain, but ageing-related alterations in the immune system in genetically predisposed individuals seem to be involved. 174 Recently, increased expression of both IL-33 and ST2, chiefly in endothelial cells of newly formed vessels, was found in GCA arteries. 175 IL-33 expression correlated with the degree of vessel wall inflammation and was reduced in arteries from steroid-treated GCA patients. In addition, a positive association was observed between IL-33 and the numbers of neovessels, suggesting that IL-33 participates in the pathogenesis of angiogenesisdependent inflammation in GCA. Although no Th2 cytokines were detectable, expression levels of IL-33 correlated with the presence of M2 macrophages, the latter being reported to promote angiogenesis in vivo. 176 Recently, the rs7025417 polymorphism in the IL-33 gene, which was noted to be associated with increased IL-33 plasma levels, was identified as a risk factor for GCA in a large meta-analysis involving a total of 1363 biopsy-proven GCA patients and 3908 healthy controls from four European cohorts. 177 GCA and other inflammatory or infectious conditions increase the risk for having an acute aortic dissection. Other risk factors include hypertension, smoking, atherosclerosis, and certain genetic diseases. In a recent large retrospective study with a prospective validation cohort, sST2 was found to have overall superior diagnostic utility for detecting acute aortic dissection among emergency room patients with suddenonset severe chest pain, which is easily misdiagnosed. 178 This finding and those related to GSA and diabetes (see Sections 4 and 10) highlight the utility of IL-33/sST2 as a biomarker of vascular health.

In summary, IL-33 has been implicated in vascular inflammation via upregulation of adhesion molecules and chemokines for leucocytes. The pro-inflammatory actions of IL-33 on endothelial cells contribute to the pathogenesis of GCA and are seemingly more prominent in angiogenesis. Further studies are needed to establish the role IL-33-induced endothelial inflammation in restenosis, as well as plague and post-ischaemic neoangiogenesis.

Obesity and type 2 diabetes

Obesity and its common consequence, type 2 diabetes are major risk factors for cardiovascular disease that are marked by chronic systemic and vascular inflammation. 179,180 Obese adipose tissue is characterized by an inflammatory immune environment consisting of classically activated M1 macrophages, cytotoxic T cells, and pro-inflammatory Th1-type cytokines (such as TNF-a and IFN-c). 181 In contrast, lean fat tissue is characterized by an anti-inflammatory environment of alternatively activated M2 macrophages, eosinophils, Th2 cells, Tregs, and ILC2s, along with antiinflammatory Th2-type cytokines (such as . IL-33 was recently shown to regulate white adipose tissue (WAT) homeostasis, a process that when dysregulated results progressively in the proinflammatory state, obesity, insulin resistance (IR), and the metabolic syndrome. 77 Production of IL-33 by WAT is stimulated by the sympathetic nervous system, with IL-33 exerting positive reinforcement by inducing the upregulation of tyrosine hydroxylase, a rate-limiting enzyme in catecholamine biosynthesis. 182 Compared to wild type mice fed a high fat diet, ST2 knockout mice fed a high fat diet have a higher body weight and greater fat mass, along with more impaired insulin secretion and glucose tolerance. 183 The major orchestrators in the actions of IL-33 on adipocyte function and metabolic homeostasis in both rodents and humans are ILC2s, which may actually be the major source of the type 2 cytokines in WAT, rather than Th2 cells. 184,185 IL-33 that is released most likely by adipose tissue endothelial cells, and perhaps adipocytes themselves, maintains ILC2 cells in WAT and stimulates them to initiate a number of actions that limit adiposity by increasing caloric expenditure. 77,185,186 The overall process is known as beiging or browning of WAT and involves upregulation of uncoupling protein 1 (Ucp-1) in adipocytes. 77 ILC2 cells were proposed to recruit eosinophils and M2 macrophages, which support optimal beiging of WAT through the release of type 2 cytokines. ILC2 cells also produce methionine-enkephalin peptides that directly act on adipocytes to promote beiging. 184 IL-33 may also exert positive regulatory actions on WAT mass and milieu via the development and maintenance of ST2 þ visceral adipose tissue-Treg cells, which are diminished in obese mice and implicated in preserving insulin sensitivity and glucose tolerance through dampening actions on pro-inflammatory M1 macrophages and CD8 þ T cells. 162 On the other hand, while M1 macrophage-driven inflammation subserves obesity-associated IR, fat-resident ST2 þ Treg cells have been implicated in promoting age-associated IR. 187 One possible explanation would be that some degree of inflammation is favourable for adipose tissue remodelling and metabolic function.

Serum IL-33 levels are lower in non-lean individuals compared to those who are lean, and negatively correlated with BMI and body weight in those who are lean and overweight, but not obese. 115 In addition, IL-33 was found to be negatively correlated with HbA1c in non-diabetic persons, but not diabetics, and to be associated with a protective lipid profile. On the other hand, severe obesity is associated with increased expression of both IL-33 and ST2 in endothelial cells of adipose tissue of both humans and mice, although the significance of this observation to endothelial function or inflammation is unclear. 109 Plasma sST2 levels are also reported to be elevated with obesity in humans, suggesting an attenuation of the beneficial actions of IL-33 in obesity. 109 Several studies report higher circulating sST2 levels in individuals with type 2 diabetes. 4,10,110-112 A recent study reported a positive association between sST2 levels and various risk factors for developing diabetes after adjusting for age and sex and implicated the highest increases in sST2 with increased risk for developing diabetes. 9 Among diabetic patients, only hs-TnT and sST2 were found to be independently associated with cardiovascular and all-cause mortality during a 5 year follow-up. 113 Levels of sST2 among diabetics are increased further by LV diastolic dysfunction. 111,114 In summary, IL-33 has been shown to limit adiposity by increasing caloric expenditure via ILC2s and prevents IR and impaired glucose tolerance by taming WAT inflammation via WAT Tregs. Plasma sST2 levels are increased with obesity and are a risk factor for development of type 2 diabetes. Increased circulating sST2 in type 2 diabetes may be reflective of microvascular endothelial inflammation.

Unresolved issues and future directions

Accumulating evidence supports the conclusion that sST2 is a biomarker of vascular health with diagnostic and/or prognostic value in various cardiovascular diseases, including CAD, MI, atherosclerosis, GCA, acute aortic dissection, and ischaemic stroke, as well as obesity and diabetes. However, the role of IL-33 is more complicated, as this alarmin may have both pro-and anti-inflammatory actions depending upon which cell type is engaged (Figure 4). Overall, the actions of IL-33 in vivo are pleiotropic and must be viewed in pathophysiological context.

In pursuing the pharmacological potential of IL-33/ST2, it is important to acknowledge the detrimental vs. protective effects of IL33/ST2 signalling. There is a need for additional experimental studies in various contexts to better comprehend the role of IL33/ST2 signalling. For example, the cell-specific effects of IL33 in vivo; the impact of the microbiota; the impact of acute injury (IL33 can be secreted after MI, and atherosclerosis can be accelerated after MI; does IL33/ST2 signalling play a distinct role in this context?), the interaction with other CV risk factors (does IL33/ ST2 signalling affect atherosclerosis differently in obese or diabetic conditions?), etc. Additionally, there is a need for GWAS studies to address causality between IL33/ST2 signalling and CVD. To exploit the translational potential of IL-33/ST2-based therapies, a better understanding of differences in pharmacology between sST2 and anti-ST2 is needed. 188 Also, caution must be exercised in assessing the translational relevance of studies with injection of recombinant IL33, which might not reflect endogenous levels. Several strategies that aim at blocking IL33 signalling are nowadays feasible in patients. A few pharmaceutical companies are developing anti-IL33 mAb, anti-ST2, or sST2, mainly for asthma and chronic obstructive pulmonary disease. Obviously, these approaches may lead to potential CV side effects; it might be wise to measure natural IgM antiox-LDL antibodies in these patients as the levels of those antibodies are inversely associated with CVD in humans.

It is increasingly appreciated that the pathophysiological importance of IL-33 is highly dependent on cellular and temporal expression. The actions of IL-33 are likely to be pleiotropic in a dose-dependent manner, depending as well on which immune cells are activated and for how long or whether endothelial cells are engaged. The final outcome would reflect the contribution of its protective and anti-inflammatory actions mediated by Treg cells, the inflammatory actions of various recruited immune cell types, and the injury-related response of stromal/parenchymal cells, all of which are modulated by the dampening actions exerted by sST2. In many cases, the levels of either ST2 or sST2 are positively affected by IL-33 in a dose-dependent manner. IL-33 may also increase levels of myeloid-derived suppressor cells (MDSC), which potently suppress T cell responses. 189 Additional in vivo studies involving immune cell type-specific knockouts and transgenics are desired to better define the role of IL-33/ST2 axis in various diseases.

The importance of spatiotemporal context in IL-33 signalling is illustrated by the actions of IL-33 on mast cells in asthma. On the one hand, IL-3 acts on mast cells via ST2 to increase bronchial hyper-responsiveness in part by boasting FcR-mediated degranulation. 190 The released proteases generate forms of IL-33 with increased biological activity, thus establishing a positive feedback loop. On the other hand, mast cell sST2, which dampens the actions of IL-33, 2 is strongly induced by IL-33, and long-term exposure to IL-33 also induces a mast cell phenotype with decreased degranulation. Moreover, recent evidence shows that in smaller peripheral airways IL-33 protects against bronchial hyper-responsiveness by inducing PGE2 formation by mast cells, which has relaxing effects on airway smooth muscle and anti-inflammatory actions on mast cells. 191 While ST2/IL-33 signalling in ILC2s, Tregs, and IL-10 producing B cells protects against inflammation, IL-33 clearly contributes to pathogenesis as a regulator of a type 2 immune response in certain settings (e.g. allergic diseases and asthma). Although initially beneficial in dealing with certain pathogens, chronic, excessive, or dysregulated type 2 immunity may contribute to tissue damage and fibrosis. 13 As an early component of tissue injury and inflammation, IL-33 plays an important role in tissue repair, but in certain cases, IL-33 may contribute to excessive acute sterile inflammation and tissue damage. For instance, IL-33 from liver sinusoidal endothelial cells was found to exacerbate I/R-induced hepatic sterile inflammation, a contributor to organ damage in liver surgeries, by stimulating neutrophil extracellular trap formation. 192 Moreover, ST2 expression by neutrophils was markedly increased by IL-33, thereby amplifying its inflammatory actions. Both the identity of the cell type engaged and the magnitude of its response will impact on the outcome seen with IL-33.

Unrecognized until recently are the different potencies of the various proteolytic forms of extracellular IL-33 that are generated in vivo. Which forms are actually elevated in various disease conditions is largely unknown. There are great gaps also in our understanding of the nuclear roles of IL-33 and how these are coordinated with its extracellular actions. The processes involved in the secretion of IL-33 are also poorly understood. Finally, the potential actions of sST2 on its own, independent of its role as an IL-33 decoy receptor, need to be better established.

In conclusion, IL-33 serves as an important local link between tissue injury or metabolic disturbances and a physiological response of limiting or repairing tissue damage. In CVD, IL-33 exerts beneficial actions that are attenuated by its sST2 decoy receptor, which in many cases is induced by IL-33 and can serve as a biomarker of tissue stress/damage. IL-33 supplementation is atheroprotective and may be beneficial in treating MI and ischaemic stroke. IL-33 may also prevent obesity and type 2 diabetes by regulating lipid metabolism. The mechanisms behind these beneficial actions are not fully defined, but are now known to involve Treg, ILC2 cells, and type 2 immune responses. On the other hand, IL-33 appears to drive endothelial inflammation and angiogenesis, which is relevant to metabolic syndrome, type 2 diabetes, and GSA. Moreover, as in several pro-inflammatory and auto-immune diseases, exuberant IL-33 signalling may cause tissue damage due to recruitment/activation of mast cells or eosinophils. Thus, a cellular or targeted approach is needed to exploit the beneficial therapeutic potential of IL-33 in CVD.

  33 and the necessity to establish how IL-33 administration can modulate the inflammatory and/or regulatory responses involved in the control of post-MI remodeling. The aims of this Ph.D. project were 1-To examine the effect of IL-33 administration on cardiac function and remodeling post-MI. 2-To focus on better understanding the underlying mechanisms behind the immune response post-MI generally, and the role of IL-33dependent immunomodulatory impact, specifically. For cardiac function, two different time viii interleukin-1 receptor accessory protein IL-1LRL interleukin-1-like receptor ligand ILC innate lymphoid cells iNKT Invariant natural killer T iNOS inducible Nitric oxide synthase ix inducer LTiP lymphoid tissue inducer precusor LV left ventricle LV mass left ventricular mass LVEDD left ventricular end diastolic diameter LVEDV left ventricular end diastolic volume LVESD left ventricular end systolic diameter LVESV left ventricular end systolic volume LW lung weight Ly6C lymphocyte antigen 6 complex M MACE major adverse cardiovascular events MAPK mitogen-activated protein kinase MCSF macrophage colony stimulating factor MCP-1 monocyte chemoattractant protein-1 MerTK myeloid-epithelial-reproductive tyrosine kinase MHC major histocompatibility complex MI myocardial infarction MMP matrix metalloproteinase mRNA messenger ribonucleic acid MTC Masson s richrome s aining N NADPH nicotinamide adenine dinucleotide phosphate NET neutrophil extracellular trap NF-HEV nuclear factor of high endothelial venules NF-kB nuclear factor kappa-light-chain-enhancer of activated B cells NFIL3 nuclear factor IL-3 regulated NK natural killer NO nitric oxide NLS nuclear localization sequence O oxLDL oxidized low density lipoprotein P PAMP Pathogen associated molecular pattern PAH pulmonary arterial hypertension x PAI plasminogen activator inhibitor PBS phosphate buffered saline PCI percutaneous coronary intervention PLZF promyelocytic leukemia zinc finger pNK natural killer cell precursors PVR pulmonary vascular resistance R RA rheumatoid arthritis RAG recombination-activating gene RAGE receptor for advanced glycation end-products RAR rearranged antigen receptor ROR retinoid acid-related orphan receptor ROS reactive oxygen species RNA ribonucleic acid RT-qPCR reverse transcription quantitative polymerase chain reaction S SBP systolic blood pressure SEM standard error of the mean SIGIRR single immunoglobulin IL-1R-related molecule SLE systemic lupus erythematosus SMC smooth muscle cell sST2 soluble ST2 STAT signal transducer and activator of transcription ST2L long transmembrane ST2 STEMI ST-elevation MI T TAC transverse aortic constriction TBST tris-buffered saline-Tween T-bet T-box transcription factor TCF1 T-cell factor 1 TCR T cell receptor TGF-transforming growth factor-Th T helper TIMP tissue-inhibitor of metalloproteinase TIR toll-interleukin-1 receptor TL tibia length TLR toll like receptor TNF tumor necrosis factor Tol-DC tolerogenic dendritic cell TOX thymocyte selection-associated high mobility group box protein Treg Regulatory T cells TSLP thymic stromal lymphopoietin TSP1 thrombospondin 1 xi TTC triphenyltetrazolium chloride TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling V VEGF vascular endothelial growth factor VLDL very low density lipoprotein VSMC vascular smooth muscle cell W WHO world health organization WT wild type xiv 3.4 IL-33 treatment increases hypertrophy and fibrosis 7 days post-MI ...................... 3.5 IL-33 treatment increases cardiac BAX/BCL2 mRNA levels and DNA fragmentation 101 3.6 IL-33 treatment increases MMP2 and MM9 mRNA expression 7 days post-MI ... 3.7 Decreased protein and gene expression of profibrotic markers with IL-33 treatment post-MI ............................................................................................................................... 3.8 IL-33 treatment affects cardiac and plasma inflammatory cytokines post-MI ....... 3.9 IL-33 treatment promotes type 2 cytokines release ................................................ 3.10 IL-33 treatment modulates neutrophil and monocyte/macrophage production, mobilization, and recruitment ............................................................................................ 3.11 IL-33 treatment elicits eosinophilia ..................................................................... 3.12 IL-33 treatment increases ILC2 levels at 7 days post-MI ................................... 3.13 NK levels are decreased in the heart and spleen at day 7 post-MI with IL-33 treatment ............................................................................................................................ 3.14 IL-33 administration modulates the activation of the adaptive immune system . 3.15 IL-33 treatment induces splenomegaly ................................................................ 3.16 IL-33 treatment upregulates cardiac sST2 expression at 7 days in the presence and absence of MI ..................................................................................................................... 4 General Discussion and Conclusion .............................................................................. 4.1 Discussion ............................................................................................................... 4.2 Limitations and future perspectives ........................................................................ 4.3 Conclusion ............................................................................................................... References .............................................................................................................................. xv

Figure 1 - 1 : 4 Figure 1 - 2 : 6 Figure 1 - 3 : 7 Figure 1 - 4 : 11 Figure 1 - 5 : 12 Figure 1 - 6 : 18 Figure 1 - 7 : 42 Figure 1 - 8 :Figure 3 -

 1141261371411151216181742183 Figure 1-1:Plaque formation and rupture ................................................................................... 4 Figure 1-2: How is the cardiac muscle affected by an infarct? .................................................. 6 Figure 1-3: Anatomy of the cardiac wall. .................................................................................. 7 Figure 1-4: Schematic representation of ventricular remodeling following MI. ..................... 11 Figure 1-5: Infarct expansion in the sham vs. infarcted heart. ................................................. 12 Figure 1-6: The three phases of cardiac repair. ........................................................................ 18 Figure 1-7: History of the discovery of IL-33. ........................................................................ 42 Figure 1-8: Human and murine structure of IL-33. ................................................................. 44 Figure 1-9: IL-33 during homeostasis, apoptosis, and necrosis............................................... 46 Figure 1-10: Inactivation of IL-33. .......................................................................................... 47 Figure 1-11: ST2: the receptor of IL-33. ................................................................................. 49 Figure 1-12: ST2+ immune cells activated by IL-33. .............................................................. 50 Figure 1-13: Mechanism of action of IL-33. ........................................................................... 52 Figure 1-14: Differentiation of innate lymphoid cells (ILCs). ................................................ 65 Figure 1-15: Activation of ILC subsets and their corresponding functions: ........................... 69 Figure 3-1 Study design. ........................................................................................................ 117 Figure 3-2 LAD ligation procedure. ...................................................................................... 118 Figure 3-3: Functional cardiac parameters 4 and 7 days post-MI in vehicle-treated and IL33treated mice. ................................................................................................................... 119 Figure 3-4: Increased left ventricular volume and diameter with IL-33 treatment 7 days post-MI. .................................................................................................................................. 120 Figure 3-5: Increased adverse left ventricular remodeling and death with IL-33 treatment post-MI. .................................................................................................................................. 121 Figure 3-6: Administration of IL-33 in mice significantly enhances cardiac remodeling (fibrosis and hypertrophy) in vivo at 7 days. ............................................................................... 122 Figure 3-7: IL-33 administration increases cardiomyocyte apoptosis at 4 and 7 days post-MI. ........................................................................................................................................ 123 Figure 3-8: Upregulation of matrix metalloproteinases with IL-33 treatment at 7 days. ...... 124 Figure 3-9 Decreased profibrotic protein and gene expression with IL-33 treatment. .......... 125 Figure 3-10: IL-33 treatment affects inflammatory cytokine protein expression. ................. 126 Figure 3-11: Increased type 2 cytokine production with IL-33 treatment. ............................ 127 Figure 3-12: Comparable neutrophil resolution in the heart between IL-33-treated and vehicletreated MI groups at 7 days post-MI. ............................................................................. 128 Figure 3-13: Dynamics of proinflammatory monocytes at different timepoints with and without IL-33 treatment. ............................................................................................................. 129 Figure 3-14: Increased macrophage levels with IL-33 treatment at 7 days post-MI. ............ 130 Figure 3-15: IL-33 induces eosinophilia at day 7 regardless of the surgical intervention. .... 131 Figure 3-16: Treatment with IL-33 increases ILC2 levels at 7 days post-MI. ...................... 132 Figure 3-17 NK cells are not increased in the heart at 7 days post-MI with IL-33 treatment. ........................................................................................................................................ 133 Figure 3-18 Dynamics of dendritic cells at different timepoints with and without IL-33 treatment. ....................................................................................................................... 134 Figure 3-19 Dynamics of CD4+ T-cells at different timepoints with and without IL-33 treatment. ....................................................................................................................... 135 Figure 3-20 Dynamics of CD8+ T-cells at different timepoints with and without IL-33 treatment. ....................................................................................................................... 136

Figure 3 -

 3 Figure 3-21 Dynamics of B cells at different timepoints with and without IL-33 treatment. Figure 3-22 Variation of spleen weight normalized to tibia length at 4 and 7 days post-LAD ligation or sham-operation. ............................................................................................ Figure 3-23 Effect of IL-33 administration on the size and complexity of immune cell populations observed in the spleen. ............................................................................... Figure 3-24 Cardiac sST2 and plasma IL-33 levels............................................................... Figure 3-25 Western total protein membranes.......................................................................Figure 3-26 Primary gating. ...................................................................................................Figure 3-27 Gating strategy for panel #1. ..............................................................................Figure 3-28 Gating strategy for panel #2. .............................................................................. Figure 3-29 Gating strategy for panel #3. .............................................................................. Figure 3-30 Gating strategy for panel #4. ..............................................................................

Figure 3 -

 3 Figure 3-21 Dynamics of B cells at different timepoints with and without IL-33 treatment. Figure 3-22 Variation of spleen weight normalized to tibia length at 4 and 7 days post-LAD ligation or sham-operation. ............................................................................................ Figure 3-23 Effect of IL-33 administration on the size and complexity of immune cell populations observed in the spleen. ............................................................................... Figure 3-24 Cardiac sST2 and plasma IL-33 levels............................................................... Figure 3-25 Western total protein membranes.......................................................................Figure 3-26 Primary gating. ...................................................................................................Figure 3-27 Gating strategy for panel #1. ..............................................................................Figure 3-28 Gating strategy for panel #2. .............................................................................. Figure 3-29 Gating strategy for panel #3. .............................................................................. Figure 3-30 Gating strategy for panel #4. ..............................................................................

Figure 3 -

 3 Figure 3-21 Dynamics of B cells at different timepoints with and without IL-33 treatment. Figure 3-22 Variation of spleen weight normalized to tibia length at 4 and 7 days post-LAD ligation or sham-operation. ............................................................................................ Figure 3-23 Effect of IL-33 administration on the size and complexity of immune cell populations observed in the spleen. ............................................................................... Figure 3-24 Cardiac sST2 and plasma IL-33 levels............................................................... Figure 3-25 Western total protein membranes.......................................................................Figure 3-26 Primary gating. ...................................................................................................Figure 3-27 Gating strategy for panel #1. ..............................................................................Figure 3-28 Gating strategy for panel #2. .............................................................................. Figure 3-29 Gating strategy for panel #3. .............................................................................. Figure 3-30 Gating strategy for panel #4. ..............................................................................

Figure 3 -

 3 Figure 3-21 Dynamics of B cells at different timepoints with and without IL-33 treatment. Figure 3-22 Variation of spleen weight normalized to tibia length at 4 and 7 days post-LAD ligation or sham-operation. ............................................................................................ Figure 3-23 Effect of IL-33 administration on the size and complexity of immune cell populations observed in the spleen. ............................................................................... Figure 3-24 Cardiac sST2 and plasma IL-33 levels............................................................... Figure 3-25 Western total protein membranes.......................................................................Figure 3-26 Primary gating. ...................................................................................................Figure 3-27 Gating strategy for panel #1. ..............................................................................Figure 3-28 Gating strategy for panel #2. .............................................................................. Figure 3-29 Gating strategy for panel #3. .............................................................................. Figure 3-30 Gating strategy for panel #4. ..............................................................................

1

 1 Primers used for RT-PCR analysis. ....................................................................... 112 Table 3-2 Flow cytometry panels showing antibodies and their conjugated fluorochromes. 113 Table 3-3 Lineage cocktail for Panel#3. ................................................................................ 114 Table 3-4 Characterization of immune cell populations and their corresponding conjugated fluorochromes. ............................................................................................................... 115 Table 3-5 Variation of weight over time with and without IL-33 treatment. .......................

Figure 1 - 3 :

 13 Figure 1-3: Anatomy of the cardiac wall.

  of the heart A cell surrenders to death either gradually (in a regulated fashion), or dies immediately following exposure to extremely unfavorable stimuli. In the infarcted heart, cardiac cell death is under the control of various receptors and signaling. Depending on extrinsic or intrinsic stimuli, cardiac cells are subjected to different cell death mechanisms. A myocardial cell can either have an intact or a disrupted membrane during cell death. While death by apoptosis preserves the integrity of the cell membrane, necrosis and necroptosis induce a disruption of the cellular membrane and the subsequent release of the cellular contents, including endogenous cytokines. Release of these cytokines during cell death induces the activation of stress signals that notify nearby cells and promotes the subsequent activation of various proinflammatory signaling pathways.

Figure 1 - 4 :

 14 Figure 1-4: Schematic representation of ventricular remodeling following MI.

Figure 1 - 5 :

 15 Figure 1-5: Infarct expansion in the sham vs. infarcted heart.

1 . 1 . 4 . 3

 1143 debris and promote dead tissue healing by the formation of a collagen-based scar. During the inflammatory phase, that extends from 1 to 4 days post-MI, neutrophils and inflammatory Lymphocyte antigen 6 complex (hi) (Ly6C(hi)) monocytes are the first two cellular waves recruited into the inflammation site to clear the cardiac tissue from cellular debris. These immune cells migrate to the injured site following a chemotactic cytokine gradient generated by chemokines. Chemokines are soluble proteins synthesized by leucocytes and necrotic tissue (endothelial cells and cardiomyocytes) that allow intercellular communication during the inflammatory phase. Ly6C(hi) monocytes/M1-like macrophages are recruited to the inflammatory site by CC chemokines like monocyte chemoattractant protein CCL2 via the CCR2 receptor[98]. Neutrophils are recruited by the CXC chemokines containing a glutamic acid-leucine-arginine (ELR) tripeptide sequence (ELR + -CXC chemokines). Lymphocytes subpopulations that exert antifibrotic and angiostatic actions are recruited by ELR --CXC chemokines[66, 99]. Following ischemia, the main functions of recruited inflammatory cells are to phagocytize dead cells and degrade the ECM. The release of granules containing MMPs like collagenase MMP-8 and gelatinase MMP-9 by neutrophils and macrophages contributes to the lysis of the preexisting ECM and promotes inflammatory cell infiltration inside the infarcted area. Neutrophils and macrophages also generate reactive oxygen species (ROS) and release proinflammatory cytokines (TNF-, IL-1 , IL-6) that activate and recruit other inflammatory cells.Inflammation plays a pivotal role in the repair process. Proper inflammation induces timely activation and recruitment of cytokines and chemokines that allow the clearance of the infarct from dead cells and tissue debris. Downstream reparative pathways are consequently initiated leading to a stable scar formation. Spatial containment of inflammation is also of extreme importance: successful healing is determined by the prevention of the inflammatory response to propagate into the viable myocardium. Insufficient inflammation can equally hinder normal cardiac repair by inducing infarct expansion; whereas prolonged and excessive inflammation can accentuate cardiac injury by promoting continual tissue destruction leading to increased adverse remodeling and cardiac rupture[2, 72]. The granulation or proliferation phase Following the phagocytic engulfment of dead cells, also known as efferocytosis,[100] and the clearance of the wound from matrix debris, the proliferative phase begins. Neutrophils start undergoing apoptosis, inflammation subsides and recruitment switches to antiinflammatory Ly6C(lo) monocytes, initiated 4-7 days post-MI [101]. Moreover, the presence of soluble antiinflammatory mediators like IL-10 promotes a shift from a proinflammatory (M1-like) to an alternatively activated (M2-like) macrophage phenotype from within the granulated tissue. This antiinflammatory Ly6C(lo) monocyte/(M2-like) macrophage phenotype promotes angiogenesis and ECM synthesis by releasing vascular endothelial growth factor (VEGF) as well as transforming growth factor beta (TGF-). Suppression of inflammation is of extreme importance during the proliferative phase because persisting inflammatory responses can induce serious myocardial damage. Inflammatory cells decrease in number as soon as proinflammatory signals are suppressed. Profibrotic signaling takes over, where fibroblasts become the main cell type in the injured area. Fibroblasts constitute an abundant population of resident cardiac cells, which are present in interstitial and perivascular areas of adult mammalian hearts. During the granulation phase, fibroblasts start migrating, proliferating and adopting a secretory myofibroblast phenotype [72]. In the homeostatic heart and during embryonic development, fibroblasts are generated from the epicardium and endocardium through epithelial-to-mesenchymal transition, which continues to adulthood [102, 103]. In an adult resting heart, fibroblasts are found within the myocardial insterstitium of the cardiac skeleton and are also abundant in the muscular compartment [104]. Fibroblasts constitute the most abundant non-cardiomyocyte component and are necessary for ECM construction and maintenance [105]. During tissue damage, fibroblasts are stimulated by DAMPS released by necrotic cells and matrix fragments and start releasing proinflammatory cytokines such as IL-1 , TNF

Figure 1 - 6 :

 16 Figure 1-6: The three phases of cardiac repair.Myocyte death triggers the release of DAMPS that activate inflammation. During the inflammatory phase, cytokine and chemokine activation takes place followed by the recruitment of neutrophils and macrophages into the infarcted site. These leukocytes promote the clearance of the wound from necrotic cells and matrix debris. During the granulation phase, fibroblasts proliferate into myofibroblasts and start producing ECM proteins. Inflammation subsides, and macrophages become activated and start releasing cytokines and growth factors that help in the formation of a neovascularized granulated tissue. With time, a collagen-based scar is formed marking the onset of the maturation or scar formation phase.

  heart tries to compensate to these hemodynamics changes by activating the sympathetic nervous system (SNS) and the renin angiotensin aldosterone system (RAAS), which together are referred o as he ne rohormonal ac i a ion . Angiotensinogen produced by the liver is converted to the decapeptide angiotensin I by the action of renin. Angiotensin-converting enzyme (ACE) then cleaves angiotensin I to form the potent vasoconstrictor angiotensin II (Ang II), an octapeptide. Simulation of the angiotensin II receptor type 1 (AT1) by angiotensin II, leads to the release of aldosterone, which plays an important role in the pathogenesis of heart failure. The main signaling molecules of the SNS and the RAAS are the cathecholamines (epinephrine and norepinephrine), Ang II and aldosterone. As the heart fails to compensate, circulating levels of these signaling molecules increase in an incremental fashion leading to vasoconstriction and water and salt retention. Maladaptive cardiac remodeling is also stimulated and is associated with chamber dilation and increased interstitial fibrosis. As the left ventricle changes in conformation and becomes more spherical in shape, a decline in mechanical competence becomes more pronounced. Positive feedback loops occurring between these compensatory systems further deteriorate cardiac function. The remaining net effect is a vicious cycle that causes a progressive decline in cardiac function and can lead to sudden cardiac death, repeated hospitalization and premature mortality[116].1.1.6 Treatment strategies in MIOver the last four decades, many treatment modalities have been introduced with the main aim of improving prognosis in patients with acute MI. Restoring coronary flow by reperfusion, either by percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG), is still the main strategy of treatment for patients with myocardial ischemia.If performed early following infarction, PCI or CABG can significantly improve survival and prevent further cardiomyocyte necrosis. The recurrence of new thrombotic events can be reduced by the administration of anticoagulation and antiplatelet agents, which also showed improved outcomes in MI patients. For many years, conventional pharmacological therapies have remained the most effective treatment modality for patients with MI. Most of these therapies include angiotensin-converting enzyme (ACE) inhibitors or receptor blockers (that decrease myocardial afterload through vasodilatation), beta-blockers (that decrease the force and rate of myocardial contraction and decrease myocardial oxygen demand), statins (that lower cholesterol level and prevent the buildup of atheroma) and aspirin (that breaks up blood clots and prevents additional platelet activation). The angiotensin receptor-neprilysin inhibitors (ARNI) is a drug combination used for the treatment of HF. Natriuretic peptides (NP) are small peptide hormones mainly produced by cardiomyocytes in response to stretching and play a key role in cardiorenal homeostasis[117]. In fact, NPs induce natriuresis, vasodilation, modulation of the renin-angiotensin-aldosterone systems, vascular remodeling and possess antiproliferative effects[118]. Due to their fast degradation by the membrane-bound endopeptidase neprilysin, NP cannot by administered exogenously[119]. Apart from inducing NP degradation, neprilysin also breaks down other vasoactive peptides including Ang II, a potent vasoconstrictor[118]. Inhibiting neprilysin can increase Ang II levels therefore countertbalancing the vasodilating benefit that could be provided by the increased availability of NPs. To overcome this unwanted effect, adding angiotensin receptor blocker to neprilysin inhibitors (ARNI) was found to give beneficial outcomes in the treatment of HF[118].Apart from the conventional cardioprotective effects, some of these pharmacological therapies induce direct modulation of inflammation. For instance, mineralocorticoid receptor (MR) inhibition [120], angiotensin receptor blockade [121], angiotensin converting enzyme inhibition [122], administration of statins [123], and -adrenoceptor blockade [124], all involve some immunomodulatory effects. For example, signaling via the leukocyte-specific 2adrenoceptor was demonstrated to play a key role in leukocyte infiltration following acute cardiac injury [125]. In a mouse model of ischemia-reperfusion, the infarct-limiting effects offered b -blockade with metoprolol were abolished by either neutrophil depletion or genetic knockdown of genes implicated in neutrophil: platelet association [124]. However, bearing in mind the effect of neurohormonal activation on all cardiac cell populations post-MI, the relative impact of these drugs on the immunomodulatory response remains unclear. A possible therapeutic approach to limit infarct expansion and prevent adverse cardiac remodeling is to dampen the inflammatory response and enhance the ensuing antiinflammatory tissue reparative process. It is well established that chronic or persistent inflammation occurring post-MI, may lead to detrimental cardiac remodeling, offering a possible therapeutic target to prevent heart failure post-MI [126]. Cyclosporine is a potent cyclophilin B inhibitor known to protect ischemic cardiomyocytes from dying by inhibiting the opening of the mitochondrial permeability transition pore (mptp). In pilot studies performed on STEMI patients, treatment with cyclosporine reduced infarct size [127] but in a large randomized double-blind controlled trial, administration of cyclosporine showed no beneficial outcomes in STEMI patients undergoing PCI [128]. Loss of the integrin receptor CD11/CD18, that allows the binding and adhesion of neutrophils to endothelial cells, was associated with decreased infarct size in MI animal models [129-131]. However, clinical trials have shown that blocking the CD11/CD18 integrin receptor with either Hu23F2G (LeukoArrest) or rhuMAb CD18 did not affect infarct size in STEMI patients who underwent primary angioplasty [132, 133]. Other antiinflammatory strategies targeting neutrophils with the main aim of reducing post-infarct injury were shown to hamper the overall healing response. In a mouse model of MI, neutrophil depletion resulted in decreased cardiac function associated with pronounced fibrosis and progressive increase in heart failure-related biomarkers [134]. Those detrimental effects were associated with decreased expression of myeloid-epithelial-reproductive tyrosine kinase (MerTK) in the heart. MerTK is a phagocytic receptor expressed on the surface of macrophages and promotes the clearance of dead cardiomyocytes during infarction [135]. Induction of MerTK expression was associated with increased capacity of macrophages to induce efferocytosis, which was mainly mediated by neutrophil-gelatinase associated lipocalin (NGAL), a type of gelatinase secreted by neutrophils [134]. Several other therapeutic strategies targeting the inflammatory response post-MI have been explored and a number of which have failed to show beneficial clinical outcomes. In the clinical setting, treatment with non-steroidal anti-inflammatory drugs (NSAIDS) was primarily associated with adverse remodeling, including myocardial thinning, impaired myocardial repair, and cardiac rupture [136]. Later experiments have shown that prolonged NSAIDs treatment promoted the formation of aneurysms, which were mainly caused by the inhibition of the reparative response along with the intended inhibition of the acute inflammatory response [137, 138]. A number of studies have also investigated the role of the inhibition of the complement cascade in cardiac remodeling post-MI. Pexelizumab is a monoclonal antibody that binds to the C5 component of the complement cascade and prevents its cleavage. The proteolytic cleavage of C5 yields C5a and C5b-9, the main two components of the membrane attack complex. Many small and large clinical trials exploring the effect of Pexelizumab have failed to gain any clinical benefit either in CABG or in reperfused STEMI patients [139, 140]. Targeting the adaptive immune response as part of an immunomodulatory strategy post-MI has also been investigated. Preclinical trials have shown that mature B lymphocytes produce CCL7 and selectively mobilize and recruit proinflammatory monocytes into the heart, leading to enhanced infarct expansion and worsening in cardiac function post-MI. In addition, elevated circulating levels of B cell activating factor (BAFF) and CCL7 were associated with increased mortality in patients with recurrent MI. Either antibody-mediated (BAFF or CD20-specific antibody) or genetic (BAFF receptor deficiency) depletion of mature B lymphocytes decreased monocyte recruitment and enhanced cardiac function [141]. The efficacy of Rituximab (an anti-CD20 antibody) is currently being investigated in the RITA-MI clinical trial (NCT03072199) to test its safety and clinical efficacy in MI. Targeting inflammation in chronic coronary artery disease by inhibiting interleukin-1 with the monoclonal antibody canakinumab has been widely investigated in the Canakinumab Antiinflammatory Thrombosis Outcomes Study (CANTOS). Treatment of patients with canakinumab was shown to decrease elevated levels of high sensitivity C-reactive proteins and to significantly lower the rate of recurrent cardiovascular events in comparison with patients treated with placebo. However, despite being performed on stable patients with previous myocardial infarction and not on patients at the acute phase of MI, the CANTOS study showed an increased incidence of fatal infections following canakinumab administration, arising concerns about the risk-benefit of the treatment [142]. An alternative approach for the inhibition of inflammation was evaluated by the administration of low doses of methotrexate, a non-specific anti-inflammatory treatment in the Cardiovascular Inflammation Reduction Trial (CIRT) [143]. The results of the CIRT study showed that methotrexate did not decrease high-sensitivity C-reactive protein levels nor lower cardiovascular events in treated patients when compared to placebo, leaving unanswered the question of whether interfering with inflammation can have a beneficiary effect on cardiovascular outcomes. Another clinical trial, the Colchicine Cardiovascular Outcomes Trial (COLCOT), tested the use of low-dose colchicine (an anti-inflammatory agent known to inhibit NLRP3 inflammasome-derived IL-1 and IL-18 production) as a means of secondary prevention post-MI. The results of the COLCOT study have shown that administration of colchicine decreased the soft cardiovascular events, like recurrent non-fatal MI or hospitalization, but had no impact on the hard events like fatal MI [144].

[

  145]. The remaining two-thirds are composed of non-cardiomyocyte cells mainly consisting of smooth muscle cells, fibroblasts, autonomic motor neurons, endothelial cells, and immune cells [146]. Cardiomyocytes possess innate conduction abilities that mediate the contractile force of the heart, whereas non-cardiomyocyte cells are responsible for tissue degradation and replacement, autonomic regulation and angiogenesis. During development, major cell types in the heart form a wide range of cell-cell interactions, which hold crucial hints to cellular functions during adulthood. For instance, both macrophages and fibroblasts couple to spontaneously beating cardiomyocytes via gap junctions and propagate inputs received from cardiomyocytes to other neighboring cells [147, 148]. Hence, macrophages and fibroblasts play an essential role in maintaining normal cardiac conduction and contribute to the synchronization of cardiac contraction. The myocardial ECM constitutes the scaffold that withholds cardiomyocytes and intramural coronary vasculature from undergoing myofibril slippage, and supports the mechanical function of the cardiac tissue [149, 150]. The ECM contains a vast array of signaling molecules and proteins that maintain the integrity of the cardiac structure [150]. In addition, the ECM provides a connection between intercellular and intracellular cytoskeletal proteins, allowing the heart to deliver biochemical signals through mechanosensation [149].

  injury or inflammation, and are actively involved in leukocyte trafficking post-MI. The expression of chemokines on the surface of endothelial cells plays a central role in providing key directional signals for the recruitment of leukocytes and their migration inside the tissue[66]. Depending on the chemokines being expressed, endothelial cells are capable of recruiting specific leukocytes in response to different inflammatory cues. Chemokines are divided into subfamilies based on the number of conserved cysteine (C) residues and amino acid (X) residues that separate them and include: CC, CXC, and CX3C. For instance, CC chemokines have the first two cysteine residues adjacent to each other, whereas ELR + -CXC chemokines have the tripeptide ELR sequence and the first two cysteine residues are separated by an amino acid. CC chemokines such as CCL2 and CCL7 serve as attractants for mononuclear cells[141, 155], whereas ELR + -CXC chemokines are mainly involved in neutrophil chemoattraction to the site of injury[156]. In contrast, ELR --CXC (such as interferon--inducible protein CXCL10) are lymphocyte-attracting chemokines that exert antifibrotic actions[157], and are rapidly but briefly upregulated following infarction. Apart from leukocyte trafficking, certain chemokines are also capable of activating several other responses that affect leukocyte activation, degranulation and apoptosis.

  infarcted myocardium during the early phase of inflammation. Proinflammatory monocytes and macrophages are the main source of IL-1 [158], which requires two signals for its production in the infarct area: First through pattern recognition receptor (PRR) such as TLR signaling that induces gene transcription of pro-IL-1 , and second through the assembly of inflammasomes. The main role of inflammasomes is to cleave biologically inactive pro-IL-1 to its active form [159]. In the setting of MI, IL-1 is ac i a ed and peaks 6-24 hours in the infarct-related area and decreases thereafter [160, 161]. In the non-infarcted area, increased IL-1 levels can still be observed as late as 12 weeks following coronary artery ligation [160]. IL-1 promo es he prod c ion of chemo ac ic media ors and initiates the recruitment of immune cells inside the infarct site [162]. In addition, IL-1 stimulates matrix degradation in cardiac fibroblasts and mitigates TGF-induced myofibroblast transdifferentiation.[158]. Elevated serum levels of IL-1 obser ed af er MI ha e been associa ed with systolic dysfunction and adverse cardiac remodeling [163]. Administration of a recombinant IL-1 receptor antagonist (IL-1R1a; Anakinra) in non-ST segment elevation acute coronary syndrome (NSTEMI-ACS) leads to decreased levels C-reactive protein, a marker of inflammation used to predict continued risk of MI and other cardiovascular events [164].

  TGF-[183] . Early inhibition of TGF-enhances c okine s n hesis, e acerba es en ric lar dilation and increases mortality[184], whereas late inhibition of TGF-red ces cardiac fibrosis and attenuates LV remodeling[184, 185]. TGF-decreases he s n hesis of c okines b macrophages, reduces the expression of chemokines and adhesion molecules by endothelial cells and promotes the differentiation of regulatory T (Treg) cells[186]. Furthermore, TGFinduces fibroblast activation, myofibroblast transdifferentiation, and increases the synthesis of ECM proteins (including fibronectin, collagen, and matricellular proteins)[5, 187]. TGF-also promotes the suppression of matrix degradation mainly by increasing the expression of protease inhibitors such as tissue-inhibitor of metalloproteinase 1 (TIMP-1) and plasminogen activator inhibitor (PAI)-1[188]. is an antiinflammatory cytokine that plays a role in the suppression of inflammation[189]. Activated T cells and monocyte/macrophage subsets are the main source of IL-10 in the infarcted myocardium[189, 190]. Elevated serum levels of IL-10 are observed within 24h following angioplasty in patients with acute MI and are associated with decreased incidence of heart failure progression[191]. In experimental MI, the expression levels of IL-10 gradually increase in the infarct and peak day 7 following injury[161]. Treatment of LADligated mice with IL-10 reduces inflammation and improves LV function[192], whereas IL-10 deficiency enhances neutrophil infiltration, promotes myocardial necrosis and increases infarct size[193]. is a type 2 cytokine associated with antiinflammatory and cardioprotective properties. The main role that IL-4 plays in cardiac repair is to induce the polarization of macrophages to the antiinflammatory M2-like phenotype. For instance, administration of the IL-4 plasmid DNA in the peri-infarct area of MI mice enhances cardiac function mainly by promoting a timely polarization of proinflammatory M1-like macrophages into the M2-like reparative phenotype[194]. Similarly, administration of an IL-4 complex at an early phase (1h) post-MI, improves M2-like macrophage availability in the infarct and peri-infarct zone, increases M2-like mediated tissue repair and improves cardiac function. Furthermore, IL-4 induces fibrotic tissue formation, a process mainly mediated by M2-like macrophages rather than cardiac fibroblasts. Those IL-4-mediated effects are not observed at a late stage (7 or 28 days) post-MI implying that the cardioprotective effects of IL-4 are only effective during the early phase post-MI[195]. In humans, previous studies have shown that elevated IL-4 serum levels were associated with reduced risk of CVD[196] whereas, decreased levels of IL-4 were depicted in MI patients and were associated with later development of LV dysfunction[197].

  CD4+ T-cells are bone marrow-derived lymphocytes maturing in the thymus and are known for mediating cellular immunity. Activation of T cells requires antigen presentation via MHC II complex present on the surface of APCs (such as macrophages, DCs, and B cells), a process normally taking place in draining lymph nodes of organs. Priming of CD4+ T cells requires the ligation of the peptide-MHC complex to the T-cell receptor (TCR), including the presence of CD28 and CD40 which are two membrane-bound costimulatory molecules necessary for T-cell costimulation. Meanwhile, locally released cytokines activate T cells toward a T helper profile (either Th1, Th2, or Th17), which then migrates to the injured tissue. Naïve CD4+ T-cells can differentiate into Th1, Th2, Th17, and Treg cells, which are adaptive immune cells having specialized functions. Th1 cells secrete IL-2 and IFN-, Th2 cells secrete IL-4, IL-5 and IL-13, whereas Th17 produce IL-17 and IL-22. Treg cells constitutively express CD25 and are regulated by the forkhead box P3 (FoxP3) transcription factor. Treg cells have immunomodulatory functions and are implicated in globally suppressing the activation of the immune response. Increased cardiac expression of TBX21 (a Th1-related transcription factor) and IFN-was observed in the myocardium 7 and 14 days post-MI. In contrast, no significant change in GATA 3 (a Th2related transcription factor) nor IL-4 cardiac expression was observed at any time point post-MI indicating an apparent predominance of Th1 response post-MI [161]. Th1 polarization in the MI setting is associated with detrimental outcomes including increased cardiomyocyte apoptosis, decreased myofibroblast differentiation and imbalanced ECM turnover, leading to cardiac rupture [236]. Th17 are involved in late stage ventricular remodeling post-MI.

  members of the IL-1 cytokine family, IL-33 shared similar amino acid sequence homology with IL-18 and exhibited similar biological functions[279]. Based on these characteristics, was finally the name proposed bySchmitz et al. to this novel member of the IL-1 family in 2005[7] (Figure1-7). One year later, IL-33 was identified as a chromatinassociated nuclear factor and was demonstrated to be identical to the NF-HEV previously characterized[280].

Figure 1 - 7 :

 17 Figure 1-7: History of the discovery of IL-33.

The IL- 33

 33 protein is composed of the N-terminal domain (amino acids 1 111) and the C-terminal domain (amino acids 112 270). The N-terminal domain contains a chromatin binding motif (CBM), a bipartite nuclear location sequence (NLS), and a central domain, which is the cleavage/activation site essential for IL-33 biological activity (Figure 1-8A). While the N-terminal domain ensures the localization of IL-33 into the nucleus [280], the C-terminal domain is an IL-1-like trefoil domain that ensures the binding of IL-33 to the ST2 receptor.

Figure 1 - 8 :

 18 Figure 1-8: Human and murine structure of IL-33. (A) IL-33 is composed of the N-terminal and C-terminal domain. Alignment of the human amino acid sequence of IL-33 (hIL-33) and its murine (mIL-33) orthologue. Conserved or identical residues are marked by a black do nder he amino acid. The 1-12 indicate the 12 strands of the C terminal region organized in an IL-1-like ß trefoil. The IL-33 chromatin binding motif (CBM) (orange line), consensus nuclear localization sequence (NLS) (pink line), and cleavage sites for inflammatory proteases (green arrows) and caspases (red arrow) are indicated. (B) The human and murine sequence of IL-33 comprises 270 and 266 amino acids respectively.(Adapted fromBaekkevold et al 2003, Lefrançais 2014 and Cayrol 2018 [8, 31, 312]).

Figure 1 - 9 :

 19 Figure 1-9: IL-33 during homeostasis, apoptosis, and necrosis. During homeostasis, full-length IL-33 is constitutively present in the nucleus of cells (A). During programmed cell death, IL-33 is kept intracellular and is cleaved in the IL-1 like cytokine domain by caspace 3 or 7 resulting in its inactivation (B). During necrosis, IL-33 is released extracellularly and is truncated in its central domain yielding highly active residues (C).

Figure 1 - 10 :

 110 Figure 1-10: Inactivation of IL-33. Oxidation and formation of disulfide bridges within the full-length and cleaved IL-33 molecule. Adapted from Cayrol C. et al 2018 [31].

  ST2L and IL-1RAcP complex is essential for IL-33 signaling via ST2L. Previous studies have demonstrated that the affinity of ST2L for IL-33 increases four times more in the presence of the IL-1RAcP [330]. Mast cells of IL-1RAcP-null mice resulted in the failure of IL-6 release upon IL-33 exposure [330]. Similar findings were depicted during IL-33 administration in IL-1RAcP-deficient mice, which failed to achieve the typical cellular hyperplasia and inflammatory-cell infiltration observed in their WT counterparts [331].

Figure 1 - 11 :

 111 Figure 1-11: ST2: the receptor of IL-33.The full-length transmembrane form ST2L, and the soluble short form sST2 are the two most abundant ST2 isoforms. ST2L is a membrane-bound receptor that comprises an extracellular domain composed of three linked Ig-like domains, a single transmembrane domain and a cytoplasmic Toll-interleukin-1 receptor (TIR) domain. sST2 is a soluble receptor for IL-33 and acts as a negative regulator for the IL-33 mode of action, by preventing the binding of IL-33 to the ST2L receptor and consequent signaling on target cells. The interleukin-1 receptor accessory protein (IL-1RAcP) is a co-receptor for ST2L. The formation of the ST2L and IL-1RAcP complex is essential for IL-33 signaling via ST2L.

Figure 1 - 12 :

 112 Figure 1-12: ST2+ immune cells activated by IL-33. Many immune cells constitutively express the ST2 receptors and include: dendritic cells (DC), type 2 T-helper (Th2) cells, cytotoxic CD8+T-cells, alternatively activated macrophages (M2-like Mø), Regulatory T-(Treg) cells, natural killer (NK) cells, invariant (iNK) cells, type 2 innate lymphoid cells (ILC2), basophils, mast cells, neutrophils, and eosinophils.

  During homeostasis, NF-B dimers are present in the cytoplasm and are inactive due to their association with inhibitory proteins, named inhibi ors of B (I B), of hich he mos known are I B , I B , and I B .Following their activation by the IL-33 complex, IRAKs mediate the phosphorylation, polyubiquitination, and degradation of the inhibitor IkB , which subsequently results in the liberation of NF-B and its translocation of to the nucleus. Alternatively, activation of the mitogen-activated protein kinase (MAPK) pathway (through the extracellular signal-regulated kinase (ERK), Jun-N terminal kinase (JNK), and p38) by IRAKs induces and activates the activator protein (AP)-1[7, 344]. The activated AP-1 then translocates to the nucleus and, like the nuclear NF-B, binds to promoter regions of target genes and initiates the transcription of a wide array of genes, including chemokines and proinflammatory cytokines, leading to the biological activity of (Figure1-13). IL-33 is essential in mediating innate and adaptive Th2 cytokine responses mainly through the production of IL-4, IL-5, and IL-13 from cells associated with innate immune signaling[7].

Figure 1 - 13 :

 113 Figure 1-13: Mechanism of action of IL-33.Association of IL-33 with its cell surface receptor (composed of ST2L and IL-1RAcP) mediates recruitment of the adaptor protein MyD88 to the TIR domain of ST2 present in the cytoplasm[7]. This receptor complex, now associated with MyD88, activates IRAK1 and IRAK4 by a mechanism that depends on TRAF6 recruitment in some cell types. IRAKs then mediate the phosphorylation and degradation of IkB-a. This results in the translocation of NF-kB to the nucleus. Alternatively, activation of the MAPK pathway through ERK, JNK and p38 induces and activates the activator protein (AP)-1[7, 344]. Then the transcription factors of NF-kB and AP-1 initiate nuclear gene expression, leading to the biological activity of IL-33 on cells.

  However, upon activation by its ligand IL-33, ST2 is soon degraded by internalization in a proteasome-dependent fashion. After stimulation, ST2 is phosphorylated by glycogen synthase kinase 3 (GSK-3 ) at Ser442 forming a docking domain for the E3 ubiquitin ligase F-box 19 (FBXL19). Ligation of FBXL19 to the C-terminal region of ST2 promotes polyubiquitination at Lys 326 and subsequent cytoplasmic degradation of the ST2L receptor [345].1.3.6 IL-33 and ST2 expression in the heart IL-33 and ST2 have different expression patterns in the human heart. IL-33 is constitutively present in the nuclei of cardiomyocytes and cardiac fibroblasts of human adults and is released upon necrosis during mechanical stress [272]. Proinflammatory cytokines such as IL-1 , TNF-, and IFN-significantly increase IL-33 expression in these cells as well as in human coronary artery smooth muscle cells [313]. Increased mRNA expression of both ST2 isoforms (ST2L and sST2) along with an increased production of the sST2 protein were detected in the cardiac vasculature mainly in endothelial cells of the aorta, coronary arteries, and cardiac capillaries. In contrast, cardiomyocytes, cardiac fibroblasts and vascular smooth muscle cells (VSMC) barely express ST2 mRNA and do not produce any ST2 protein [313].

  another study performed on mice with cardiac overload,Chen et al. investigated the relationship between cardiac hypertrophy and the specific deletion of IL-33 from either cardiomyocytes or endothelial cells. They found that a specific deletion of either IL-33 from endothelial cells or ST2 from cardiomyocytes worsened cardiac hypertrophy during pressure overload[271]. Another group has shown that complete ablation of IL-33 in mice subjected to mechanical overload exacerbated cardiac remodeling and impaired cardiac function. IL-33 -/mice exhibited amplified LV hypertrophy and fibrosis, increased inflammation and chamber dilation and impaired survival in comparison to WT mice[41].In atherosclerosis, chronic arterial wall inflammation plays a major role in plaque progression[347]. The presence of oxidized low-density lipoproteins (ox-LDL) in the vessel induces the production of proinflammatory mediators like cytokines and growth factors from surrounding tissues. T cells can also infiltrate the vessel wall during atherosclerosis and depending on their phenotype can either increase inflammation in plaques (Th1) or decrease it (Th2/Treg)[59]. Previous studies have shown that intraperitoneal administration of IL-33 in an ApoE / mouse model induced a shift from the Th1 pro-atherosclerotic immune response to a Th2 protective immune response. This Th1 to Th2 skewing resulted in reduced aortic atherosclerotic lesions when compared to the vehicle-treated group[45]. Conversely, neutralization of IL-33 signaling by administering the decoy receptor sST2 in ApoE / mice, resulted in aortic plaque expansion when compared with control IgG-treated group. In serum and lymph node cells, IL-33 treatment significantly increased Th2 cytokine production (IL-4, IL-5 and IL-13) and decreased IFN le els, a pical Th1 c okine. Moreover, total serum IgA,

1 . 3 . 9

 139 hypertension (PAH), increased pulmonary vascular resistance (PVR) levels characterize this devastating disease, which progresses into right-ventricular failure or premature death[367].Despite the main concentration on increased sST2 levels in left-heart failure, sST2 expression can also reflect the size and function of the right ventricle and can independently predict 1 yearmortality in PAH patients[368, 369]. Carlomagno et al found that elevated sST2 expression was observed in patients with PAH and was associated with systolic dysfunction and right ventricular dilatation[275]. In patients with childhood PAH, elevated sST2 levels were associated with poor prognosis and the combination of sST2 and pro-BNP could be a useful biomarker predicting clinical condition and outcome[370]. Moreover, increased sST2 levels predicted disease severity in idiopathic PAH patients and correlated with hemodynamic parameters and other biomarkers[355]. In dyspneic patients, increased sST2 levels directly correlated with right ventricular dysfunction and could predict long-term decreased survival[371]. In patients with secondary pulmonary hypertension, Agoston-Coldea el al also showed that sST2 concentrations were a strong predictor of right ventricular dysfunction due to chronic obstructive pulmonary disease[368]. Since the sST2 release is potentiated during myocardial stretch, it is hypothesized that decreased right ventricular function might increase ventricular filling and transmural wall stress, hence increasing myocardial stretch and sST2 release. sST2 might, therefore, be a useful biomarker predicting right ventricular function in diseases such as pulmonary arterial hypertension[355]. Epigenetics of the IL-33/ST2 axis Genetics control cytokine activity, and any alteration in certain genes encoding these cytokines could lead to increased or decreased cytokine production, which can affect the predisposition to certain diseases or their clinical outcomes[372]. Genetic dysregulation of the IL-33/ST2 axis seems to affect the susceptibility to many diseases including inflammatory bowel disease[290, 373], atopic dermatitis[374], asthma[375][376][377], allergic rhinitis [378], nasal polyposis [379], ischemic stroke [380] and Al heimer s disease (AD) [381]. Furthermore, IL-33 gene polymorphisms have been associated with many autoimmune diseases such as ankylosing spondylitis [382], Behce s disease (BD) [383], RA [384], and SLE [385]. With respect to gene polymorphisms of IL-33, Li et al. demonstrated that the CC genotype of SNP rs7044343 was directly associated with RA in a three-stage case-control study performed on Northern Han Chinese population [384]. In ankylosing spondylitis, Fan et al. identified statistically significant differences between cases and controls in the South Han Chinese population at the level of the IL-33 gene polymorphism rs1929992. The frequency of the A allele was more elevated in the affected group than in the controls [382]. In a cohort of Turkish patients with BD, IL-33 serum levels were found to be lower in active BD patients in comparison with the inactive BD patients and healthy controls. In terms of allelic and genotypic distributions of rs1929992 and rs1157505 polymorphisms, no significant differences were observed between BD patients and controls. However, rs7044343 and rs11792633 polymorphisms of the IL-33 gene were found to be protective for the susceptibility to BD in the examined population [383]. In a study performed in a Chinese Han population, polymorphism in the IL-33 gene rs1929992 was associated with increased susceptibility to SLE [385]. Sabashita et al demonstrated that a single nucleotide polymorphism in the rs1929992 gene of IL-33 was strongly associated with cedar pollinosis [386]. Moreover, J. Chapuis et al. indicated that in AD patients, the expression of IL-33 was decreased and that three SNPs in the IL-33 gene (rs1157505, rs11792633 and rs704343) were strongly linked to AD in Caucasian populations [381]. The impact of ST2 genetic polymorphism was also explored in several inflammatory disease settings: Ali et al associated asthma in children with a polymorphism in the distal promoter region of the ST2 gene (-26999 G/A allele) [387]. Others have shown that the same polymorphism could be associated with an increased risk of AD [374]. Another member of the IL-33/ST2 axis is the IL-1RacP, the signaling receptor subunit that is required for ST2 to induce downstream IL-33 signal transduction [331]. The IL-1RAcP gene is located on chromosome 3q28. Polymorphisms in the IL-1RAcP gene have been linked to many diseases like persistent hepatitis B virus [388], prostate cancer [389] and Kawasaki disease [390]. Other studies have confirmed the association between IL-33 gene variation and some cerebrovascular and cardiovascular diseases. In a northern Chinese population, Guo et al have demonstrated that the genetic variation of rs4742170 in the IL-33 gene was significantly associated with an increased risk of ischemic stroke [380]. CAD, the leading cause of morbidity and mortality worldwide, is an intricate trait caused by several environmental and genetic factors and their interactions [391]. In a longitudinal study performed on more than 20,000 Swedish twins, Zdravkovic et al. assessed that the total heritability for CAD was 0.43 in women and 0.39 in men for angina and 0.38 in women and 0.57 in men for death [392]. Nearly 50 risk loci for CAD have been identified by large-scale genome-wide association studies (GWASs) [393-398]. Nevertheless, these risk loci justify only 10.6% of the overall CAD heritability [398-400]. Many inflammatory factors have been shown to be involved in the progression to coronary heart disease (CHD) and atherosclerosis in several population-based studies [401-403]. In a case-control association study performed on a Chinese Han population, Tu et al. have demonstrated that two promoter SNPs (rs7025417 in IL33 and rs11685424 in IL1RL1 (which is the gene encoding ST2)) were significantly associated with CAD. This CAD association was not only limited to patients with severe coronary stenosis but also included the subgroup of revascularization and MI. These two SNP variants interact with each other in a biological fashion and together display a much greater effect associated with CAD than that of a single variant. Moreover, rs7025417 genotype influenced circulating IL-33 levels, which suggests that rs7025417 may affect the progression of CAD by regulating IL33 expression [404]. In the context of MI, Yang et al have suggested that the IL-33/ST2 pathway could affect the progression to MI by showing that an IL-33 gene polymorphism (rs11792633) could be associated with an increased prevalence of hypertension in patients with MI [405]. Other investigators have suggested that the IL-33/ST2 pathway might genetically be associated with CAD by demonstrating the correlation between the distal promoter variants in ST2 (-27307 T/A and -27614 C/A) and the angiographic severity of CAD [406]. In a genome-wide associated study, Ho et al have investigated the determinants of sST2 plasma concentrations and found that apart from environmental and clinical factors, genetic factors predominantly explained the variations in sST2 levels [407]. Genetic variations in IL1RL1 have been associated with a number of inflammatory and immune diseases [408]. Deciphering the genetic variants of ST2 may bring about new perceptions into ST2 regulation pathway and may helpin the discovery of new therapeutic approaches for the treatment of cardiovascular and immune diseases. Five missense mutations of IL1RL1 mapped to the membrane-bound ST2L were found to be associated with elevated sST2 concentrations. These genetic variations in the transmembrane ST2 not only affected sST2 expression but also increased the responsiveness of ST2 to IL-33 itself. This draws the conclusion that polymorphisms in the IL1RL1 gene, that alter intracellular ST2 transmembrane signaling, could affect ST2L responsiveness to IL-33 and increase sST2 expression therefore influencing inflammatory and immune regulation[407]. In a case-control study, Wu et al analyzed the genetic association between ST2 variants and the risk to essential hypertension. They discovered that rs6543116 and rs1168524 genetic variations within the promoter region of ST2 affected the development of essential hypertension by interfering with the expression or function of ST2. The C allele in the rs3821204 could enhance sST2 mRNA expression and increase circulating sST2 levels by disrupting the miR-202-3p binding site within the 3 0 -UTR of sST2. Therefore, patients with the G>C genetic variant in rs3821204 had significantly more elevated levels of plasma sST2.

  the groundbreaking discovery of leukocytes in 1843 [411], identification of distinct cell lineages has emerged including that of myeloid and lymphocyte subsets. In 1986, Th1 and Th2 lymphocyte subsets were identified by Tim Mosmann and Robert Coffman who demonstrated that CD4+ T cells were subdivided into two groups characterized by distinct cytokines and functions [412]. Almost two decades later, Th17 cells were discovered [413] and formed, with Th1 and Th2 subsets, the helper T cell paradigm which is a theory that separates polarized CD4+ T cells based on their production of specific cytokines. With the emergence of a new cell population belonging to a distinct arm of the innate immune system, named innate lymphoid cells (ILCs), a new paradigm was born. Ever since their discovery in 1975, Natural killer (NK) cells were the only known ILCs available [414]. NK cells are involved in cytotoxicity and promote primary immune responses against viruses and tumor cells [415]. Nearly 2 decades later (in 1997), lymphoid tissue inducers (LTi) were classified as a different cell type of ILCs and were found to be essential for the formation of lymph nodes and Pe er s pa ches d ring embr ogenesis [416]. Additional subsets of the ILC family were much later unraveled (

(

  CD90) expression; however, they lack lymphoid and myeloid lineage marker expression. ILCs are tissue resident cells, which are relatively rare in lymphoid tissues but are mainly present at mucosal and non-mucosal barrier surfaces such as the skin, the lungs, the intestine, and the adipose and mucosal-associated lymphoid tissues[299, 422, 423]. It is now well established that ILC subsets are important orchestrators of an early innate immune response to bacteria, viruses and fungi as well as to intracellular and extracellular parasitic invasions at barrier tissues. Recruitment of ILC subsets at barrier surfaces occurs during embryonic development and in the context of ongoing inflammation; additional migration of ILCs is likely to occur.Despite their similarities in effector programs, ILCs and helper T cells are distinctly regulated. Naïve CD4+ T cells are equipped with recombination activating gene (RAG)dependent rearranged antigen receptors (RAR). RAR ensure the priming of naïve CD4+ T cells by recognizing peptide-loaded MHC and costimulatory molecules, both present on the surface of an APC. In contrast, ILCs lack somatically rearranged antigen-specific receptors and hence do not require adaptive antigen receptors for costimulation or activation, but rather rely on their ability to recognize soluble mediators through their surface receptors. Examples of these soluble mediators are alarmins, proinflammatory cytokines, lipids and hormones, which are released by epithelial, stromal and myeloid populations. Moreover, different amounts of time are required for CD4+ T cells and ILCs to mount an immune response. For instance, to be primed, CD4+ T-cells need to undergo activation and clonal expansion in lymphoid tissues, then develop effector functions and generate surface molecules that ensure their migration to non-lymphoid tissues. This process takes place over several days. In contrast, at steady state, most ILCs are constitutively present in non-lymphoid tissues[424], and upon stimulation, are capable of producing large amounts of cytokines without the need for further differentiation.

[

  430]. The role of ILCs in human pathology has been well defined, extending from autoimmune diseases such as asthma, allergy and inflammatory bowel disease [431] to infectious diseases [432] and graft-versus-host disease development post-hematopoietic stem cell transplantation [433, 434]. Independently from hematopoietic supply, helper-like ILCs residing in nonlymphoid organs are self-maintained through local proliferation and are relatively long-lived [424].

cytokines and 3 )

 3 the expression of distinct transcription factors. Similar to their adaptive counterparts, ILCs express and rely upon transcription factors for their development and function. Coordinated expression of these transcription factors promotes activation or repression of target genes that are critical for the programming of ILCs into different subsets. Upon activation, these differentiated subsets generate distinct patterns of expression of signature cytokines that help in the execution of distinct effector functions. ILCs arise from common lymphoid progenitors (CLP) initially in the fetal liver and then develop in the adult bone marrow [435] (Figure 1-14). As their name implies, CLPs are capable of differentiating into cells of the adaptive (T cells and B cells) and of the innate immune system (ILCs). For CLP to develop into global innate lymphoid progenitors (GILP) rather than adaptive progenitors [435], several transcription factors are required and include the inhibitor of DNA binding 2 (Id2), the avian erythroblastosis virus E26 homolog-1 (ETS1), the nuclear factor IL-3 regulated (NFIL3) and the thymocyte selection associated high mobility group box protein (TOX). At first, GILP give rise to two precursors: natural killer cell precursors (pNK) and common helper innate lymphoid precursors (CHILPs). While the development of CHILP requires GATA-binding protein 3 (GATA3), interleukin 7 (IL-7) and T-cell factor 1 (TCF1), the development of pNK requires Id2, ETS1 and NFIL3. pNKP and CHILP give rise to NK cells and innate lymphoid cell precursors (ILCP), respectively. From CHILPs, lymphoid tissue inducer (LTi) cells develop from precursors (LTiP) expressing the retinoid-related orphan receptor gamma (ROR t), TOX and Id2. CHILPs also develop into ILC precursors (ILCP), a process that depends on the transcription factor promyelocytic leukemia zinc finger (PLZF). PLZF is a transcriptional repressor that plays a role in cell cycle control, differentiation and development of myeloid cells [435]. Killer ILCs, represented by conventional NK cells are IL-7R -and are considered the innate counterparts of CD8+T cells because they both express the transcription factors eomesodermins (Eomes), T-box transcription factor (T-bet) and Id2 and share related functions such as interferon (IFN-) production and cytotoxicity. All the remaining ILCs are effector cytokine-producing subsets and are known as helper-like ILCs that express the IL-7R (CD127) [436]. Helper-like ILCs share many properties with T helper cells including the use of common transcription factors, production of comparable cytokine profile and mediation of the same physiological functions. ILC1 are similar to Th1 cells of the adaptive subsets because they both express T-bet and fight intracellular pathogens by producing IFN-[437, 438]. Th2 and their innate counterparts ILC2 express retinoid acid-related orphan receptor alpha (ROR ) and (GATA-3) [439] and secrete IL-4, IL-5, and IL-13, as well as the epidermal growth factor EGF-like molecule amphiregulin (Areg). ILC3 comprise LTi cells and both groups express ROR t. ILC3s are similar to Th17 due to their expression of (ROR t) and production of IL-17 and IL-22.

Figure 1 - 14 :

 114 Figure 1-14: Differentiation of innate lymphoid cells (ILCs).ILCs arise from common lymphoid progenitors (CLP) which develop into global innate lymphoid precursors (GILP). GILP serve as a precursor to natural killer (NK) cells and the common helper innate lymphoid progenitors (CHILP), which themselves develop into lymphoid tissue inducer progenitors (LTiPs) and innate lymphoid cell precursors (ILCP). LTiPs give rise to LTis, whereas ILCPs differentiate into ILC1, ILC2 and ILC3. Figure adaptedfrom Vivier E. et al, 2018 [440].

  relying on inflammatory mediators, stress ligands, microbial compounds, and cytokines produced by local cells for activation. Recent data performed at the single-cell level from different tissues reveal that ILCs display a large array of unique surface receptors, which are encoded by specific transcriptomic signatures and are capable of recognizing chemokines, cytokines, growth factors, hormones, neuropeptides and metabolic signals[441, 442]. Each ILC subset is therefore conditioned to sense local endogenous signals that control their specification by regulating the expression of specific patterns of inhibitory or activating receptors. For instance ILC2 express Nmur1, which is a specific receptor for neuromedin U (NMU). NMU is a neuropeptide with pleiotropic roles and is mainly involved in modulating the sense of satiety and mediating intestinal peristalsis[443]. NMU is present in the gastrointestinal tract and is highly secreted by cholinergic neurons of the lamina propria[444].Activation of the mucosal neurons during a parasitic worm infection with Nipposgtrongylus Brasiliensis, induces the secretion of NMU, which is sensed by the receptor Nmur1 present on the surface of ILC2. Stimulation of the Nmur1 by NMU promotes the release of IL-5, IL-13 and amphiregulin by ILC2[444, 445] and the subsequent expulsion of the parasite, a process mainly mediated by the specific recruitment of mast cells, basophils, eosinophils, and the induction of goblet cell hyperplasia[37]. Deficiency in Nmur1 signaling promotes impairment of type 2 responses and results in poor clearance of N.Brasiliensis [445]. Natural killer (NK) cells are circulating bone marrow derived lymphocytic cells belonging to the cytotoxic branch of the ILC family and are the innate counterparts of CD8+ T-cells. Their main role in immunity is to protect the host from viral or tumor-infected cells by eliminating unhealthy host cells lacking self-MHC-I molecules [446]. In contrast, ILC1 are mostly resident cells in secondary lymphoid organs and have weak cytotoxic properties [424, 447]. In the presence of intracellular pathogens, NK cells and ILC1 expand in response to IL-12, IL-15 and IL-18 generated by nonhematopoietic cells as well as myeloid cells and produce INF-[448, 449]. IFN-is the main cytokine produced by ILC1 and is involved in many inflammatory diseases. INF-induces macrophage activation and stimulates microbicidal reactive oxygen species production, mediates antibody-mediated cytotoxicity by activating antibody generation, and promotes antigen presentation by MHC class I and class II molecules [450]. Relative to healthy controls, increased ILC1 counts are observed in peripheral blood and bronchial samples of patients with chronic obstructive pulmonary disease (COPD) [451], joint samples of spondyloarthritis patients, tissues and synovial fluids from patients with rheumatoid and inflammatory arthritis [452, 453], inflamed intestinal tissues obtained from patients with Crohn s disease [454], as well as blood samples of patients with systemic lupus erythematous and systemic sclerosis [455, 456]. In comparison to healthy individuals, lymph node biopsies collected from patients at risk of developing rheumatoid arthritis were found to have elevated levels of ILC1 [457]. Increased ILC1 counts and decreased ILC2 and ILC3 counts might be indicative of ILC2 and ILC3 conversion into ILC1s [458]. ILC1 are mainly enriched in the skin, salivary glands, liver, gut, thymus, and uterus [459, 460].1.4.4.2 ILC2ILC2 respond to epithelial injury, parasite infection and allergens by the activation of specific receptors present on their surface that associate with epithelium-derived cytokines like thymic stromal lymphopoietin (TSLP), IL-33 and IL-25, as well as with basophil-derived IL-4 and arachidonic pathway products[301,[461][462][463]. Activation of ILC2 induces the production of type-2 cytokines such as IL-4, IL-5 and IL-13, which are implicated in allergic inflammatory diseases[440]. ILC2s are major players in the pathogenesis of asthma, where they induce eosinophilia and promote epithelial cell and smooth muscle cell contraction following intranasal administration of allergens in mice[464]. Bronchoalveolar lavage collected from asthmatic patients contains elevated 466], which are associated with poor respiratory functions[467]. As early as the first days of life, ILC2 are recruited and help in the development of the lungs of young mice. ILC2 also participate in the development of allergic asthma in the developing lungs of mouse pups by inducing Th2 cytokine skewing[468, 469]. Patients with allergic rhinitis [470] and chronic rhinosinusitis[471] have elevated numbers of ILC2s in their nasal polyps. Increased ILC2 counts are also observed in the peripheral blood and bronchoalveolar lavage of patients with pulmonary fibrosis in comparison with controls[472, 473], and are associated with macrophage differentiation to a profibrotic phenotype and the induction of collagen deposition. Furthermore, suction blisters and dermis samples obtained from patients with atopic dermatitis contain large numbers of ILC2s that produce Th2 cytokines[301, 474]. Allergic sensitization to certain foods in mice is also induced by ILC2s and their associated type 2 cytokines[475, 476]. Following infection with large parasites like helminths, ILC2 produce IL-4, IL-5, and IL-13 that induce vasodilation, mucus,

1. 4 . 4 . 3 ILC3

 443 In response to fungal, microbial and bacterial infection, ILC3 are activated by and produce GM-CSF, lymphotoxins, IL-17 and IL-22. IL-17 is a cytokine crucial for the resistance against fungal infections like Candida albicans in the oral mucosa[482], whereas IL-22 leads to rapid production of antimicrobial peptides like alpha and beta-defensins by acting selectively on stromal and epithelial cells and preventing the dissemination of commensal bacteria[417, 483]. IL-22 is implicated in tissue repair[484] but is also known to play a role in inflammatory diseases[485, 486]. Elevated ILC3 counts associated with increased IL-22 levels are observed in inflamed tissues obtained from the intestines of inflammatory bowel disease patients in comparison with healthy controls [487]. ILC3 are also implicated in skin pathogenesis where they induce the formation of psoriatic plaques and are a major source of IL-17 and IL-22 in a mouse model of psoriasis with subcutaneous IL-23 administration [488]. Furthermore, IL-17, IL-22, and IFN-deriving from ILC-3 produce detrimental effects in a mouse model of ulcerative colitis [486]. Elevated numbers of ILC3 are observed in the peripheral blood of multiple sclerosis patients, and are associated with the initiation and formation of focal aggregates in the cerebrospinal fluid [489]. ILC3 are mainly found in mucosal tissues [490], whereas a small number of ILC3 can be depicted in the liver [491] spleen and lungs [492]. In conclusion, upon homeostatic breach, ILC subsets are activated by signal cytokines produced by local cells or external pathogens and are thereafter capable of producing effector cytokines that direct the immune response based on the original insult (Figure 1-15).

Figure 1 - 15 :

 115 Figure 1-15: Activation of ILC subsets and their corresponding functions:

  to their constitutive presence in many organs such as the skin, lungs, and intestines, ILCs are not only central regulators of murine and human allergic and autoimmune diseases, but also promote defense and homeostasis against different kinds of invading pathogens[494, 495]. NK cells play a role in cardiac remodeling, and begin increasing gradually to culminate 7 days post-MI[161]. In CVD, previous studies have shown that NK cell levels are significantly decreased in non-STEMI patients with stable angina when compared to healthy controls, while the cytokine profile in these patients remained unchanged[496]. In contrast, failure to regain normal NK levels was associated with persistent low-grade inflammation, suggesting a protective role of NK cells in CAD [496], while selective manipulation of NK cells has no effect on atherosclerotic development [497]. Invariant NKT (iNKT) cells are a branch of NK cells and are the most studied and best characterized NK cell population. Upon activation, iNKT cells produce a mixture of Th1 and Th2 cytokines, including TNF-, INF-, IL-4 and IL-10 among other chemokines that help in shaping subsequent adaptive immune responses [498]. In atherogenesis, iNKTcells activate vascular inflammation [499], whereas in obesityinduced glucose intolerance, iNKT cells promote adipose tissue inflammation [500]. In experimental MI, activa ion of iNKT cells b -galactosylseramide, a specific activator of iNKT cells, induce cardioprotective effects by improving mouse survival and attenuating LV remodeling and failure after MI possibly via increased expression of antiinflammatory cytokines such as IL-10 [501]. In atherosclerosis, the role of ILC subsets in the experimental setting has been well established. For instance, mice lacking the transcription factor T-bet [502], or the ILC1 stimulating cytokine IL-12 [503], displayed reduced plaque burden and adoptive transfer of ILC1s was shown to accelerate atherosclerosis [504, 505]. Mice on high fat diet have altered ILC2s numbers and selective ILC2 depletion in Ldlr / mice was shown to accelerate atherosclerosis development [506]. In the MI setting, enhanced ILC levels were found in the circulation of STEMI patients when compared to controls. Increased ILC1 proportions were observed during the first 12 hours of STEMI onset and were associated with poor outcomes. In contrast, ILC2 proportions significantly decreased during the acute phase of STEMI [507]. The presence of ILC3s in human and murine heart, including atherosclerotic lesions remains uncertain, probably due to their scarcity.1.4.6 ILC2 in tissue repair and homeostasisAs mentioned earlier, since ILC2 release type 2 cytokines in response to epithelium and endothelium-derived TSLP, IL-25, and IL-33, they are considered as functionally related to Th2 cells. Due to their presence at barrier surfaces in the respiratory, gastrointestinal and urogenital tracts, ILC2 happen to be the first responders in the fight against virus-induced lung damage, helminth infections and the orchestration of wound healing. Hence, ILC2 are unsurprisingly implicated in reparative processes during tissue and organ injury[17, 427].ILC2 play a major role in preserving tissue integrity by promoting wound healing and tissue repair. In addition to type-2 cytokines such as IL-5 and IL-13, ILC2 express Areg, which is an EGF-like molecule that promotes the restoration of tissue function by regulating cell differentiation and proliferation. As a matter of fact, IL-33 released by damaged cells during intestinal injury and inflammation stimulates ILC2 to promote the repair of intestinal epithelial lesions by expressing Areg[508]. ILC2 activation by IL-9 produces Areg that contributes to lung tissue repair following murine helminth infection[509]. Furthermore, during murine influenza A virus infection, ILC2 also express Areg that promotes the repair of damaged respiratory epithelium[17]. ILC2 are also involved in the pathogenesis of fibrosis, and upon stimulation by IL-33 during chronic hepatocellular stress, produce antiinflammatory and tissue remodeling cytokines that activate profibrotic cascades leading to hepatic fibrosis[33]. ILC2 can also interact with Tregs and promote tissue healing by suppressing inflammation. They also protect against murine cerebral malaria by accumulating Treg cells and M2-like macrophages[510]. Moreover, interactions between ILC2 and Treg cells via maresin 1 (a docosahexaenoic acid-derived lipid mediator) promote resolution of allergic lung inflammation in mice[511]. Apart from supporting tissue repair and suppressing inflammation, ILC2 play a role in maintaining tissue function and regulating metabolic diseases such as obesity, diabetes and atherosclerosis. While type 1 inflammation is directly associated with obesity and metabolic disorders, type 2 cytokines ensure optimized caloric expenditure, leanness and metabolic health[512, 513]. In adipose tissue, binding of IL-33 to the ST2 receptor present on the surface of ILC2 promotes the release of antiinflammatory cytokines that play a role in the improvement of insulin resistance induced during obesity. These cytokines enable the skewing of macrophages present in adipose tissue to an antiinflammatory M2-like phenotype[514, 515].In murine adipose tissue, the regulating role of ILC2 has been well established in local immune and metabolic homeostasis[300]. By producing type-2 cytokines like IL-5 and IL-13, ILC2 can promote the release of bone marrow-derived eosinophils into the bloodstream and their recruitment in adipose tissue. Eosinophils, together with their main cytokine IL-5, once established in the visceral fat tissue, start increasing energy expenditure and total oxygen consumption[300]. In addition, eosinophils and alternatively-activated (M2-like) macrophages promote insulin sensitivity, regulate glucose homeostasis, and prevent adipocytes from acquiring insulin resistance[516, 517]. They also decrease obesity and proinflammatory effects associated with a high-fat diet[518].

[

  200]. In a rodent acute MI model, genetic ablation of IL-13 was shown to aggravate cardiac healing and remodeling post-MI[199]. While IL-13 is mainly derived from Th2 cells, expression of this cytokine was still detectable during total ablation of CD4+ T-cells, indicating that ILC2 might be an alternative source of IL-13 in the myocardium. Genetic deletion of IL-13 results in negative outcomes following MI. IL-13, therefore, contributes to wound healing within the infarct zone by modulating early leukocyte recruitment and monocyte differentiation into an M2-like activating state[199]. In atherosclerosis, IL-13 also plays a major role by preventing lesion development, promoting plaque stability and inducing macrophage skewing towards an alternatively activated phenotype [523]. This ILC2-macrophage regulatory crosstalk is a fundamental feature of innate lymphoid cells. In fact, ILCs are recognized for their involvement in cross-talks with myeloid lineages resulting in reciprocal immune cell phenotype modulation and activation. In cutaneous injury, systemic administration of IL-33 improves wound healing by inducing alternative macrophage activation [427], while deleting ST2 (the IL-33 receptor) promotes inflammatory macrophage phenotype and leads to impaired wound healing [524]. Macrophages can also induce modulation of ILC function, as alveolar macrophages potentiate IL-13 secretion by ILC2 to induce airway hyperreactivity [302], whereas intestinal macrophages control IL-22 expression by ILCs [525]. Therefore, cross-talks between ILCs and macrophages are existent in many organs either at steady state or during injury or infection. This general mechanism happening in several organs suggests that it might also occur in the heart following infarction. ILC effector cytokines also promote dendritic cell (DC) regulation consequently ensuring a cross-talk between ILCs and T cells. For instance, during allergic lung inflammation, IL-13 produced by ILC2 can initiate the migration of activated lung DC to the draining lymph nodes where they promote Th2 cell differentiation from naïve T cells [464]. A direct regulation of T cells is also possible through peptide antigen presentation via type II MHC: ILC2 cells are capable of presenting antigens to T cells by expressing MHC class II. This process stimulates T cells to start producing IL-2, which consequently would induce ILC2 proliferation and IL-13 generation, therefore magnifying type 2 immune responses [526]. Moreover, a cross-talk between ILC2 and T cells has also been reported involving the costimulatory molecule OX40 ligand and ILC2 derived IL-4 [527]. The role of ILC2 and the effect of their signature cytokines on the development of atherosclerosis is beginning to unfold. Recently, a study performed by Newland et al. found that ILC2 are mainly found in fat-associated lymphoid clusters and in the peri-aortic adipose tissue present near the developing lesions. Mice lacking ILC2 developed larger atherosclerotic plaques and had a more inflammatory macrophage phenotype. Moreover, the IL-13 expression by ILC2 was essential for the skewing of the plaque macrophage phenotype into a more favorable wound healing phenotype resulting in limited lesion development and the formation of a more mature collagen cap [506]. In MI, Bracamonte-Baran el al. detected the presence of tissue-resident ILC precursors in the cardiac muscle during homeostatic conditions, which expand and differentiate into ILC2 during inflammatory conditions like ischemic cardiomyopathy and myocarditis. ILC2 presence was also depicted in mouse models of these inflammatory conditions where ILC2 expansion was dependent on the presence of fibroblastderived IL-33 [39]. Choi et al. reported a pathogenic role for cardiac ILC2 in an IL-33-induced model of pericarditis. Following IL-33 administration, ILC2 mainly accumulated in the ventricles near the pericardium, induced cardiac inflammation, and promoted the development of eosinophilic pericarditis [40]. Eosinophils are granulocytes with antimicrobial and antiparasitic immunity, mainly present in the circulation and in certain tissues [528]. Eosinophils measure 12-15 µm in diameter and are recognized by their bilobed nucleus and their numerous acidophilic granules present in their cytoplasm, that stain dark pink with the acid dye eosin [529]. These granules comprise highly charged basic proteins including eosinophilic cationic protein (ECP), major basic protein (MBP), eosinophilic peroxidase (EPO), and many other cytokines, enzymes, and growth factors, which are all released upon cell activation. Eosinophilic granules are capable of promoting tissue damage and dysfunction such as inducing apoptosis and cell necrosis, producing free radicals, stimulating platelets and deactivating the anticoagulant actions of thrombomodulin [530]. Degranulation of eosinophils is a tightly regulated process, where in most cases, instead of a complete degranulation, small amounts of cytotoxins are selectively released from specific granules into the extracellular space, a process commonly known as piecemeal degran la ion [531]. Delayed release of granular content can still be observed up to a whole cell lifespan, where unruptured eosinophil granules, acting as minefields, are found in the extracellular space ready to burst at any inflammatory stimulus [532]. Similar to neutrophils, eosinophils are capable of phagocytosing invading pathogens [533] and eliminating them intracellularly by providing phagosomes with ECP and MPB [534]. Furthermore, eosinophils are equipped with extracellular killing properties, which include extracellular DNA trapping [535], respiratory burst through EPO [536], and degranulation and release of cytotoxins [537].

  in an autocrine/paracrine manner by eosinophils[539, 542]. IL-5 acts as a key regulator in the terminal differentiation of eosinophils and is essential in triggering the migration of eosinophils into the circulation. Hypereosinophilia, characterizing several disease processes, has been associated with elevated levels of including asthma [543], hypereosinophilic syndrome (HES)[544], and eosinophilic esophagitis[545]. Both IL-3 and GM-CSF also act as regulators of eosinophils, but unlike IL-5, their activity is not limited to eosinophils nor their presence required for eosinophil proliferation and differentiation. The respective signaling pathways of all three cytokines is mediated by receptors sharing a common signal-transducing s b ni , which explains few of the overlapping downstream effects of these cytokines on eosinophils [546]. In fact, the activity of all three cytokines can be terminated by the blockade of the binding si e on he s b ni i h an an agoni ing an ibod [547]. Once released from the bone marrow, eosinophils remain in the bloodstream for up to 8-12 hours, a time within which they are recruited to the site of inflammation by a gradient of chemokines produced by endothelial cells [540]. Eotaxin 1, 2, and 3 (CCL11, CCL24, and CCL26 respectively) are chemokines that bind to the chemokine receptor CCR3 and allow the recruitment of eosinophils into the tissues [538]. Evidence suggests that although eosinophils leaving the bone marrow are terminally differentiated cells, progenitors can still be depicted in the circulation and local hematopoiesis can still occur in the tissues [548]. When apoptosis is prevented by GM-CSF, IL-3 or IL-5, tissue eosinophils can survive for up to two weeks [549]. Eosinophils are present in several organs under physiological conditions and exert a vast array of homeostatic properties. When present in tissues, eosinophils act as sentinels of their environment, rapidly recognizing tissue insults and triggering biochemical mechanisms of inflammation and repair. While eosinophils have been associated with detrimental outcomes in allergic diseases, these cells act as protectors of the host against viral [550], helminths [551] and microbial pathogens [552].

  Pe er s pa ches presen in the intestines) and assist other leukocytes in their maturation and homing[553]. Eosinophils establish a physiological balance between T-regulatory and Thelper responses in the lungs and in the gut[554, 555], and promote the survival of plasma cells within the gut and bone marrow[556, 557]. Eosinophils can also act as antigen presenting cells following their migration to draining lymph nodes[558]. Apart from performing immunomodulatory functions, eosinophils are producers of several growth factors and are therefore potentially implicated in tissue repair[559]. Following inflammation, increased expression of IL-5 or eotaxins, associated with elevated levels of circulating eosinophils, promotes the nonphysiological homing of eosinophils inside the tissues[537]. Under pathological conditions, ILC2s play a key role in diverting eosinophils to sites of inflammation[38]. For instance, intranasal administration of the protease allergen papain, activates ILC2 and promotes the production of IL-5 and IL-13, which induce mucus hyperproduction and the infiltration of eosinophils into the lungs[305]. In immune-mediated hepatitis, activation of hepatic ILC2 induces the production of IL-5 and the recruitment of eosinophils, which promote liver inflammation and tissue damage[560].

  eosinophils and the duration of eosinophilia. Based on a study performed onGottdiener et al., three phases describe the damage inflicting cardiac tissue following eosinophilia and include: eosinophilic infiltration, eosinophilic thrombosis, and eosinophilic fibrosis[562]. The first phase consists in the infiltration of eosinophils inside the tissues and the subsequent necrosis caused by the release of granular proteins. Biopsies consistently demonstrate deposits of ECP, MBP and EPO. The second stage is described by a thrombus formation and the binding of eosinophilic granules with thrombomodulin. Binding of cationic proteins released from eosinophils with the anion-binding exocite of thrombomodulin, prevents the formation of the complex thrombin-thrombomodulin and the subsequent loss of its anti-thrombic action. The complex thrombin-thrombomodulin is known for its potent role in physiologically inhibiting coagulation[563]. The third and final phase is represented by fibrotic scarring where the cardiac endothelium and valves become thickened and fibrotic, leading to a loss of compliance of the ventricle, a phenomenon ini iall named L ffler s fibroplas ic endocardi is [549].

  eosinophils [564], but the underlying causes of this expansion are not yet fully elucidated. Many mechanisms may be implicated and include: increased egress from the bone marrow, increased recruitment of eosinophils from the blood into the tissues, local maturation of eosinophil progenitors inside the tissues, and proliferation of resident eosinophils. Increasing evidence suggests a role of IL-33 in the basal homoeostasis of eosinophils [565]. Moreover, since the ST2 receptor of IL-33 is expressed on many allergy-related immune cells including mast cells, basophils, ILC2, Th2, and eosinophils [566], stimulation of many of these cells by IL-33 produces type-2 cytokines. Hence, IL-33 was first assumed to promote eosinophilic inflammation by inducing IL-5, the major activator of eosinophils. Previous data has demonstrated that administration of IL-33 in a mouse model of asthma activates the ST2 signaling pathway and promotes lung eosinophilia, which was mainly induced by IL-5 production [260]. It is now well established that IL-33 acts directly on eosinophils to regulate their activation, survival and adhesion [567]. In fact, IL-33 induces the maturation of eosinophils in the bone marrow [567], promotes the activation of mature eosinophils [568] and participates in the development or activation of eosinophil progenitors within the tissue [569]. Furthermore, the IL-33-mediated eosinophilia is in part instigated by the production of IL-5 by ILC2 [570]. While IL-33 is not a chemoattractant of eosinophils, many studies implicate IL-33 in the regulation of eosinophil survival. For instance, transferring selectively depleted ST2 KO eosinophils in naïve recipient mice led to decreased pulmonary eosinophilic infiltration following allergen challenge when compared with WT eosinophils, despite preserved migratory functions, indicating reduced survival [571]. IL-33 also stimulates the secretion of GM-CSF, which acts in an autocrine manner to induce the antiapoptotic protein BCL-XL and promote survival [572]. GM-CSF can also promote the secretion of IL-4 and IL-13 from IL-33-activated eosinophils and lead to the differentiation of macrophages to an alternatively activated (M2-like) phenotype [572]. IL-33 is a stronger eosinophil activator than IL-5 in terms of inducing degranulation and release of superoxide from human eosinophils [336]. Stimulation with IL-33 in mice triggers the alteration of more than 500 genes, which are mostly immune related and include CXCL2, CXCL3, CXCL17, CXCL10, IL-6, and IL-13 [573]. IL-33 can also increase the surface expression of many markers including the eotaxin receptor CCR3 [260], the activation protein CD69 [574], and the adhesion molecule CD11b [575]. While studying the functional nature of IL-33-activated eosinophils, Jacobsen et al. found that transferring eosinophils activated with IL-33, IL-4, and GM-CSF into eosinophil KO asthmatic mice drove the production of IL-13-dependent mucus and accumulation of M2-like macrophages [576]. Similar results were observed following intranasal administration of IL-33 to ST2-deficient mice adoptively inoculated with WT eosinophils, where increased levels of IL-13 and M2-like macrophages were also noted [260]. In the skin, IL-33 promotes IL-4 and IL-13-mediated fibrosis by directly acting on eosinophils [577]. 1.5.5 Eosinophils and metabolic homeostasis In obese mice, monocytes recruited into the visceral adipose tissue (VAT) are from the classically activated M1-like phenotype and secrete proinflammatory cytokines such as TNF-, ha ind ce inflamma ion and ins lin resis ance [578]. By contrast, in lean mice, most of the macrophages present within the VAT are of the alternatively-activated (M2-like) phenotype, which produce anti-inflammatory cytokines including IL-10 and arginase 1, and secrete cytokines that enhance the sensitivity of adipocytes to insulin and contribute to glucose homeostasis [516]. In the absence of eosinophils, high-fat diet fed mice showed increased adiposity and reduced tolerance to glucose and insulin [512]. Obesity and insulin resistance are therefore inversely correlated with the number of eosinophils present in adipose tissue [512]. Upstream of eosinophils, activation of ILC2 by IL-33 in murine adipose tissue promotes the release of tye-2 cytokines including IL-5 and IL-13, and the recruitment of eosinophils into the adipose tissue. Eosinophils, together with their main cytokine IL-5, once established in VAT, start increasing energy expenditure and total oxygen consumption [300]. Eosinophils and alternatively-activated (M2-like) macrophages promote insulin sensitivity, regulate glucose homeostasis, and prevent adipocytes from acquiring insulin resistance [516, 517]. They also decrease obesity and proinflammatory effects associated with a high-fat diet [518]. In the heart, administration of IL-33 was shown to promote eosinophilic pericarditis, and adversely affect heart function even in healthy mice[51]. Others have shown that activation of ILC2 and the egress of eosinophils from the mediastinum to the heart ae the main mediators of eosinophilic pericarditis in mice. Migration of eosinophils was facilitated by the ILC2-mediated expression of eotaxin in cardiac fibroblasts[40].1.5.6 Eosinophils in wound healing and tissue remodelingFollowing tissue damage, eosinophils are activated through pattern-recognition receptors present on their surface, and recognize DAMPs released during necrosis. For instance, binding of the receptor of advanced glycation end products (RAGE) expressed on eosinophils with the necrosis signaling molecule HMGB1 mediates the recruitment of eosinophils to areas of injury and necrosis by chemotactic migration [579]. Similarly following skeletal muscle damage, eosinophils are rapidly recruited to the site of injury and induce the secretion of IL-4 and the proliferation of fibro/adipocyte progenitors to promote tissue regeneration [580]. Eosinophils also act on the vasculature and induce epithelial cell proliferation to promote healing [581]. Furthermore, eosinophils are large producers of fibroblast growth factor, VEGF, and TGF-, hich are all gro h fac ors implica ed in asc lar and tissue repair. In contrast, in diseases where eosinophils mediate an exaggerated healing response, such as eosinophilic esophagitis [582] and bronchial asthma [583], unfavorable profibrogenic processes are often observed. 1.5.7 Eosinophils and their implication in MI Due to their prothrombotic and proinflammatory properties, eosinophils are also implicated in the setting of MI and show contradictory results. For instance, decreased circulating eosinophil counts were observed in patients during the initial phase of MI, indicating a possible infiltration of eosinophils inside the infarcted myocardium and their implication in potentiating the inflammatory response post-MI [584]. In addition, infiltration and degranulation of eosinophils in the infarct zone was shown to affect cardiac structure and lead to cardiac rupture [585]. Patients with STEMI have elevated eosinophil to leukocyte ratio and are at a higher risk of developing adverse cardiac events [586]. Consistently, MI patients undergoing stent implantation have elevated levels of ECP, which corresponded with adverse cardiac events including recurrent MI, cardiac death or clinically driven target lesion revascularization [587, 588]. Similarly, during emergency coronary angiography performed on ACS patients, coronary thrombus aspiration samples depicted elevated levels of eosinophils, which were largely contained within the thrombus area and correlated with larger thrombi. These results indicate that eosinophils participate in the pathophysiology of MI by promoting thrombus growth inside the coronary artery [589, 590]. Conversely, elevated blood eosinophil counts were associated with a lower one-year mortality risk after multivariate adjustments [591]. Patients with severe ACS have lower circulating eosinophil counts in comparison with patients with less severe ACS [590, 592]. In a recent study performed on humans and mice with MI, circulating blood eosinophil counts declined considerably in patients and mice during the early hours of STEMI in humans and MI induction in mice. Eosinophils were subsequently depicted in the heart of patients and mice indicating increased cardiac eosinophil infiltration post-MI. Additionally, induction of MI in mice selectively depleted of eosinophils ( dblGATA) sho ed increased infarc si e, enhanced LV dila ion and decreased cardiac function when compared to their wild type (WT) MI counterparts. These results indicate that eosinophils are recruited to the heart during MI and play a critical role in inducing cardiac repair and preventing adverse cardiac remodeling [593].

  intubated orotracheally and connected to a MiniVent ventilator (type 845)(HSE Harvard, Germany). Ventilation was set at a stroke volume of 150 µl and maintained at 180 strokes/min. Proper visualization of the intubation process was facilitated by a small incision performed at the level of the skin and muscles overlying the trachea. The left front limb of the mouse was taped above the right front limb to allow full access of the left-sided thorax. A parasternal skin incision was performed at the level of the left third and fourth intercostal space, and underneath muscles were transected and retracted with 5-0 prolene threads while making sure not to damage the lungs during retraction. The heart was accessed, and the pericardial sac was cut open to allow clear visibility. The site of ligation of the LAD was determined 2mm below the left atrioventricular border. Using a tapered atraumatic needle, the LAD was isolated, ligated with a 7-0 prolene thread (Ethicon, Norderstedt, Germany) and tied with three knots. Successful ligation and ischemia were confirmed by visual discoloration and cyanosis of the ventricle distal to the occlusion (Figure3-2).

Formalin-fixed heart

  mid-sections were embedded in paraffin blocks, cut into rans erse 5 m sec ions and placed on microscope slides. For staining, tissue sections need to be rehydrated. Most staining compounds are water-soluble and require a hydrophilic medium to achieve appropriate binding. Rehydration consists in dewaxing the tissue sections using xylene (2x3minutes), and hydrating them by slowly replacing the hydrophobic xylene with a gradual amount of alcohol. Hydration is achieved by immersing the sections in a sequential series of decreasing concentrations of ethanol [100% (2X 3 minutes); 95% (2X 3 minutes); 70% (2X 3 minutes)]. Finally, tissue sections are placed in running cold tap water to rinse off ethanol.

  Masson s trichrome (MTC) staining was used to assess collagen deposition and fibrosis. In this staining technique, muscle tissue is stained red, the cytoplasm is stained pink, nuclei are stained blue-black and collagen is stained blue. In brief, formalin-fixed, paraffinembedded sections are deparaffinized and rehydrated through a gradient of ethanol as previously stated. Cardiac sections are hen soaked in Bo in s sol ion (as a secondary fixative)for 1 hour at 56°C, washed in running tap water for 5-10 minutes and rinsed with distilled water. Next, tissues are stained with hematoxylin for 10 minutes, washed and rinsed off again, then differentiated in phosphomolybdic-phosphotungstic acid solution for 10-15 minutes, which prepares sections to easily absorb collagen-staining solution. Finally, sections are transferred to aniline blue solution and stained for 5-10 minutes, dehydrated (as previously stated), mounted with DPX mountant, and protected with a coverslip.For H&E and MTC staining, imaging was performed on an Olympus CX41 microscope, and quantification was assessed in a blinded fashion using ImageJ 1.52 software. A minimum of 10 fields per section and 3 to 4 animals were analyzed per experimental group. The area of myocardial fibrosis was quantitatively measured from the photomicrographs of MTC stained slides by ImageJ 1.52 software. The fibrosis was identified using the differences in color (blue fibrotic area opposed to red myocardium) of MTC stained slides. The results are presented as the ratio of fibrotic area to the whole area of the myocardium using 10X magnification. For cardiomyocyte hypertrophy, a 20X objective was used, and digital images taken from the subendocardial and subepicardial regions of the LV wall were analyzed. The cross-sectional area (CSA) of individual cardiomyocytes was measured by tracing boundaries of each cardiomyocyte on each image. CSAs were defined from an average of 100 cardiomyocytes per mouse.

200

  µl of ice-cold protein extraction RIPA buffer composed of 150 mM NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS (sodium dodecyl sulphate) 50 mM Tris-HCl, pH 8.0 containing a cocktail of protease inhibitors. Samples were then left to incubate overnight on a rotor at 4 o C. The next day, samples were centrifuged at 12,000 rpm for 10 minutes at 4 o C.The supernatants were then transferred to new tubes and quantified using Thermo Scientific NanoDrop 1000 UV-Vis spectrophotometer (Thermo Scientific, Waltham, MA) according to he man fac rer s recommended protocol. Concentrations were estimated based on an average obtained from triplicate measurements of the same sample. The Lowry colorimetric assay and Nanodrop 1000 methods were previously compared in our lab and both gave the same range of protein quantification. An equal amount of protein (100 g) from each sample was heated at 95 o C for 10 minutes and electrophoresed under reducing conditions on a 15% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane. To confirm the success of the transfer, membranes were then incubated in a solution of Ponceau Red composed of (2% Ponceau S in 30% sulfosalicylic acid and 30% trichloroacetic acid) diluted 1:100, on an agitator for 5 minutes. Membranes were then washed in water until the protein bands were well defined.A picture of the membrane was then taken for total protein quantification. Membranes were then completely destained using repeated washings in water or TBST. Membranes were then blocked with 5% nonfat milk and TBST (Tris-buffered saline with 0.1% Tween 20) 1 h at room temperature. At this level, membranes were cut at the appropriate molecular weights to allow for probing of multiple proteins within the same run. Membranes were then incubated with a dilution of the primary antibody in 1% skim milk and 0.1% TBST against TGF-(1/500, cat# ab92486, Abcam); -SMA(1/200, Abcam, cat# ab5694); IL-1 (1/500, Abcam, ca # ab200478); TNF-(1:1000, Cell Signaling Technolog , ca # 11948); and IL-4 (1/1000, Abcam, cat# ab9728); IL-10 (1/1000; Abcam, cat#ab9969), and IL-13 (1/500; Abcam; cat#ab106732) overnight on a rocker at 4 o C. After four washes with TBST (0.02%) (4 X 5 minutes), membranes were incubated for 1 h with species-appropriate peroxidase-conjugated secondary antibody (1/40,000) at room temperature. Finally, membranes were washed twice with TBST (0.02%) (2 X 5 minutes) and TBS (2 X 5 minutes), and immunoreactive bands were then visualized with enhanced chemiluminescence (Clarity Western ECL substrate, Biorad) to detect protein bands using the ChemiDoc MP imaging system. Due to the instability of ho sekeeping pro eins s ch as -actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and -tubulin in animals models of myocardial ischemic infarction [595], total protein levels were used as loading controls. The band densities of each protein of interest were normalized to the density of the total protein bands appearing in the same lane and on the same membrane. All data were then calibrated as fold change to the average of sham-operated vehicle-treated group. Western blot bands and total protein representative figures are shown in (Figure 3-25 A,B).

  concentrations of TNF-, IL-5, IL-33, IL-1 , and GM-CSF were determined in mo se plasma sing ELISA ki s according o he man fac rer s ins r c ions. TNF-(cat# 02030A), and IL-5 (cat# 02024) were purchased from Stem Cell technologies; IL-33 (cat# ab213475) was purchased from Abcam; IL-1 (cat#88-7013-22) and GM-CSF (cat# BMS612) were purchased from Thermofischer. All the ELISA plates were prepared using the sandwich technique which consists in 1-coating the plate wells with a first antibody (known as capture antibody), 2-loading the sample solution, 3-adding a second antibody (known as detection antibody) in order to assess the concentration of the sample. The following is a description of the general sandwich ELISA procedure with minor modifications be een man fac rer s instructions. All ELISA s eps are separa ed b a series of ashings (5X; 300 l/ ell of wash buffer (PBS 1X, 0.05% Tween-20)) and gentle blotting on absorbent paper to remove any remaining buffer that might interfere with the dilution of the reagent being added in the next step.The ELISA plate (Costar 9018, Corning, USA) is first coated with capture antibody (100 l/ ell) prepared in coating buffer (usually 0.1M NaHCO3) and left to incubate overnight at 4°C. This step allows passive binding of the antibody to the solid surface of the polystyrene plate. The next day, to prevent unspecific binding of the utilized antibody, the plate wells are washed and blocked with assay diluent (200 l/ ell; PBS 1X, 10%FBS), and incubated for 1 hour at room temperature. Wells are then washed and plasma testing samples and incremental standards are hen prepared based on he man fac rer s recommenda ions and loaded in duplicates (100 l/well), making sure not to forget to leave two blank wells as control. The position of all samples is recorded on a plate map. The plate is then incubated at room temperature for 2 hours and subsequently washed. Biotin-conjugated detection antibody diluted in assay diluent is then added (100 l/ ell) and left to incubate at room temperature for 1 hour.After washing, avidin-HRP diluted in assay diluent (100 l/well) is added and left to incubate at room temperature for 30 minutes. The plate is then washed and substrate solution (Tetramethylbenzidine; 100 l/well) was added and left to incubate at room temperature for 15 minutes. The reaction is finally stopped by adding stop solution (1M HCl; 100 l/well) and read immediately on an ELISA plate reader (Tecan, Switzerland) at the corresponding wavelength. Optical density (OD) is measured against the reagent blanks, standards and test samples. Relative cytokine concentrations are obtained by interpolating the readings from the standard curve.

  Cardiomyocyte apoptosis was evaluated by the TUNEL method using the Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection (Cat# C10619). TUNEL staining was performed on 5 m hick paraffin-embedded heart sections. The TUNEL assay consists in labeling single-stranded DNA breaks nicks , hich are fragmented DNA strands cleaved from genomic DNA during apoptosis. This technique uses a fluophore (Alexa Fluor 647) to identify nicks by labeling the free DNA 3'-hydroxyl (OH) open ends with fluorescently conjugated dUTPs. In brief, paraffinized tissue sections were first deparaffinized and rehydrated. All steps of the procedure were performed in a humidified chamber to avoid samples from running dry. All incubations performed at 37 o C were done in the dark. Tissue sections were primarily permeabilized with proteinase K (30µl/slide) (diluted 1:25 with PBS 1X) for 25 minutes at 37 o C, and rinsed twice with PBS 1X. Sections were then dried and preincubated with terminal deoxynucleotidyl transferase (TdT) reaction buffer (30µl/slide) for 10 min at 37 o C. An incubation with TdT reaction mixture (30µl/slide) for one hour at 37 o C was then required, followed by another with 3% BSA in 0.1% Triton X in PBS (30µl/slide) for 5 minutes at room temperature. Sections were then washed twice with PBS 1X, dried, and incubated with the Click IT Plus reaction cocktail (50 µl/slide) for 30 minutes at 37 o C. After washing twice with PBS 1X, slides were incubated with DAPI (1/1000 in PBS1X) (50 µl/slide) for 5 minutes at room temperature. Finally, anti-fade was added to the tissue, slides were mounted with DPX mountant, and protected with a coverslip. Images were captured using the Zeiss Axio light microscope. Apoptotic nuclei were stained red with Alexa Fluor 647 and couterstained with DAPI (blue) to count the total number of normal nuclei per field. The total number of both TUNEL-positive myocytes and normal cardiomyocyte nuclei was counted using ImageJ (version 1.52) software.

3 . 4 Table 3 - 4 .

 3434 Surface staining was performed in the dark for 30 min at 4 o C. Dead cells were excluded by the use of a viability dye(DAPI 4,). To prepare DAPI, 2.1 µL were first taken from the original stock solution (14.3 mM (5 mg/mL)) and suspended in 100 µL of PBS to make a solution of 300 µM.as an intermediate dilution. Then a 1:1000 dilution in PBS of the latter solution was required to reach a concentration of 300nM suitable for staining. Samples were acquired on a BD FACS ARIA SORP cell sorter flow cytometer (BD Bioscience) and the raw data analyzed using FlowJo software (v10.5.3, Tree Star). All antibodies listed in Table3-2 and Table 3-3 were purchased from Biolegend, eBioscience, or BD Biosciences. Data was analyzed on cells within the live gate and reported as percentage of cell number in CD45+ cells (neutrophils, NK cells, B cells, eosinophils, CD4+ T-cells, and CD8+ T-cells) and as percentage of cell number in live cells (classical monocytes, macrophages, and ILC2). Flow cytometry panels showing immune cell populations and their corresponding fluorochromes are summarized in Table 3-Representative pseudocolor flow cytometric gatings of the population subsets are shown in (Figure 3-27-Figure 3-30).2.13 Gating strategies for the different panels usedFMO controls were used to set all gates. All gating strategies began by plotting the cells in the forward scatter (area) (FSC-A) versus side scatter (area) (SSC-A). Debris were first omitted by excluding clumped cells or doublets from the analysis (Figure3-26A). Then single cells were separated from cell aggregates using (FSC-A) versus (FSC-H) (height) (Figure3-26B). Finally, viable cells were gated using DAPI staining, where viable cells would be negatively stained and appear on the left side of the plot (Figure3-26C).

  -26), plotting CD11b against CD11c allowed DCs (CD11b+ CD11c(hi)) to be distinguished from other CD11b+ subsets . The latter cell population was then plotted against Ly6C (Figure 3-27B). Double positive cells from (Figure 3-27B) were subgated into F4/80 vs Ly6G where double negative cells were defined as monocytes (CD11b+Ly6C(hi)Ly6G-F480-) (Figure 3-27C). Positive CD11b but negative Ly6C cells from (Figure 3-27B) were subgated into F4/80 vs SSC-A. Positive cells for F4/80 were defined as macrophages (CD11b+Ly6C(low)F480+) (Figure 3-27D).

  -26), total leukocytes were gated as CD45+ cells (Figure3-28A). CD45+ cells were subgated into siglecF versus CD11c where positive cells for siglecF and negative cells for CD11c were defined as eosinophils (CD45+SiglecF+CD11c-) (Figure 3-28B). CD45+ cells from (A) were subgated into Ly6G vs CD11b where double positive cells were defined as neutrophils (CD45+CD11b+Ly6G(hi)) (Figure 3-28C). Positive cells for CD11b+ but negative for Ly6G from the latter gate were again subgated with NK1.1 vs SSC-A. Positive cells for NK1.1 with small SSC-A were defined as NK cells (CD45+CD11b+LY6G-NK1.1+) (Figure 3-28D). For B cells, CD45+ cells from (Figure 3-28A) were subgated into B220 vs CD19 (Figure 3-28E). Double positive from the previous gate was again subgated into IgM vs SSC-A where positive for IgM with small SSC-A were defined as B cells (CD45+B220+CD19+IgM+) (Figure 3-28F).

  Induction of MI decreased ejection fraction (EF), fractional shortening (FS), and cardiac o p (CO) significan l a D1 in all MI gro ps (****P 0.0001 s. Baseline; (Figure 3-3A-C) indicating successful ligation of the LAD coronary artery. There was a progressive decline in all hemodynamic parameters (EF, FS, and CO) between days 1 and 4 post-MI, but not to a significant extent (Figure 3-3A-C). Comparing D1 and D7 post-MI, a significant decrease in EF (**P 0.01), FS (*P 0.05) and CO (**P 0.01), as obser ed in he IL-33-treated MI group, but not the vehicle-treated MI group, indicating a worsened adverse LV remodeling with IL-33 treatment (Figure 3-3A-C). Of note, EF decreased significantly at D7 post-MI in the IL-33-treated group compared to the vehicle-treated group at the same time point (Figure 3-3A, * P 0.05). LV end-systolic and diastolic volumes and diameters (LVESV, LVEDV, LVESD, and LVEDD) (normalized to tibia length) increased progressively and significantly as early as day 1 post-MI in all groups regardless of the treatment (Figure 3-4A-F). However, the MI group treated with IL-33 showed substantially higher increases in LVESV, LVEDV, LVESD, and LVEDD at 7 days post-MI compared to the vehicle-treated MI group, indicating worsened cardiac dysfunction (Figure 3-4A-F). Moreover, IL-33-treated MI group experienced a significant decrease in EF (Figure 3-3A, **** P 0.001), and increase in LVESV, LVEDV, LVESD, and LVEDD (Figure 3-4A-D, *** P 0.001-**** P 0.0001) be een da s 4 and 7 post-MI that was not observed in the vehicle-treated MI group. No significant changes in hemodynamic parameters were observed in any sham-operated groups (data not shown).

  5). LV weight/tibia length, an index of hypertrophy, was measured post-mortem (4 and 7 days post-MI). A significant increase in LV hypertrophy was observed in the IL-33-treated MI group when compared to the vehicle-treated MI group at 7 days post-MI (5.56 ± 0.16 mg/mm vs 6.30 0.24 mg/mm; * P 0.05) (Figure3-5A). Moreover, MI mice treated with IL-33 showed a significant increase in pulmonary congestion in comparison with the vehicle-treated 100 MI group treated at 7 days post-MI as evidenced by wet-to-dry water percentage (76.98 ± 0.21% vs 78.66 ± 0.57%, *P 0.05) (Figure3-5B). Despite a progressive decline in cardiac function observed in the vehicle-treated MI group over time, no significant change in pulmonary congestion was observed, which was consistent with previous data showing no difference in lung weight as late as 9 weeks post-MI[598]. Infarct size and expansion was comparable between the MI groups treated with IL-33 or vehicle at 4 days post-MI(28.17 ± 3.78% vs 30.35 ± 1.76%). However, infarct expansion was more pronounced at 7 days in both MI groups, but to a greater extent in the IL-33-treated group (40.58 ± 3.51% vs 59.03 ± 1.69%, *P 0.05), indica ing al ered LV remodeling i h IL-33 treatment following MI (Figure3-5C,D).

3. 3

 3 IL-33 treatment increases mortality rate post-MI Kaplan-Meier survival curve analysis showed a tendency towards decreased survival between days 1 and 4 in MI mice treated with IL-33 compared to vehicle-treated group (Figure 3-5E) (n=17 with 94.12% survival vs n=22 with 81.82% survival). However, a significant increase in mortality rate was observed by 7 days post-MI in the IL-33-treated group in comparison with vehicle-treated MI group (Figure 3-5F) (n=38 with 73.68% survival vs n=46 with 50.0% survival; *P 0.05); most of the mice died between days 3 and 5 post-MI and upon autopsy, cardiac rupture was observed. None of the mice died from sham operation (vehicletreated: n=29; IL-33-treated: n=29).3.4 IL-33 treatment increases hypertrophy and fibrosis 7 days post-MI Histological assessment revealed enlarged cardiomyocyte CSA and increased fibrosis in both MI groups (MI+vehicle and MI+IL-33) compared to the sham-operated groups at 7 days post-MI. Treatment with IL-33, however, further increased the degree of hypertrophy (284.5 ± 21.08µm 2 vs 399.5 ± 13.2µm 2 ; * P 0.05) (Figure 3-6A) and fibrosis (14.35 ± 1.99% vs 30.88 1.85%; ***p 0.0001) (Figure 3-6B) 7 days post-MI compared to the vehicle-treated MI group. of the apoptosis-regulating genes BAX and BCL2 are shown in Figure 3-7 at 4 and 7 days post-MI: BAX (Figure 3-7A,D); BCL2 (Figure 3-7B,E). BAX/BCL-2 ratio increased in both MI groups at 4 days post-MI, but only significantly in the IL-33-treated MI group compared to IL-33-treated sham group (0.90±0.10 vs 1.61±0.13 fold change;*P < 0.05) (Figure 3-7C). At 7 days post-MI, mRNA expression levels of the BAX/BCL2 ratio returned to baseline in the vehicle-treated MI group, but remained significantly higher in IL-33-treated MI group compared to the vehicle-treated MI group and IL-33-treated sham group, respec i el (0.99 0.06 s 1.82 0.38 fold change;*P 0.05; and (0.68± 0.18 vs 1.82±0.38 fold change ** P < 0.01) (Figure 3-7F). To explore whether IL-33 affects cardiomyocyte cell death, a TUNEL assay was performed on myocardial sections harvested 4 and 7 days post-MI. IL-33 significantly increased cardiomyocyte DNA fragmentation in the IL-33-treated MI group compared to all other groups at 4 days post-MI (****P 0.0001) (Figure 3-7G,H). At 7 days, TUNEL stained nuclei were significantly elevated in both MI groups, but to a significantly higher extent in the IL-33-treated MI group in comparison o he MI gro p rea ed i h ehicle (***P 0.001) (Figure 3-7G,I). These results indicate that the administration of IL-33 in the presence of MI increases the susceptibility to cardiomyocyte death, which may contribute to increased infarct size. 3.6 IL-33 treatment increases MMP2 and MM9 mRNA expression 7 days post-MI MMP9 mRNA levels significantly increased in the IL-33-treated MI group compared to all other groups at 4 and 7 days post-MI (*P 0.05) (**P 0.01 and ****P 0.0001) (Figure 3-8Error! Reference source not found.A, B). MMP2, however, did not increase in any group at 4 days (Figure 3-8C), but significantly increased at 7 days (Figure 3-8D) in the IL-33-treated MI group compared to the vehicle-treated sham group (1.00±0.07 vs 4.46±1.25 fold change; *P 0.05). These results suggest that the administration of IL-33 in the presence of MI potentially enhances ECM degradation.

3. 7

 7 Decreased protein and gene expression of profibrotic markers with IL-33 treatment post-MICardiac protein expression of the fibrogenic cytokine TGF-as no al ered b IL-33 or MI at day 4. However, at 7 days, levels of TGF-increased in he sham+IL-33 and MI+vehicle groups without reaching significance. Moreover, TGF-pro ein e pression decreased significantly in the MI group treated with IL-33 when compared with the MI group treated with ehicle a 7 da s (2.29 0.50 s 0.84 0.20 fold change; *P 0.05) (Figure 3-9A,B). The transdifferentiation of fibroblasts into myofibroblasts is characterized by the upregulation of ( -SMA) [599]. -SMA cardiac expression also decreased at 7 days post-MI in the IL-33treated MI group compared to the MI group treated with vehicle (1.63 ± 0.31 vs 0.68 ± 0.11 fold change; *P 0.05) (Figure 3-9C,D). Gene expression of connective tissue growth factor (CTGF), a matrix-associated protein, significantly increased in the MI group treated with vehicle compared to the sham vehicle-treated group at 4 post-MI (1.00 ± 0.03 vs 16.39 ± 2.69 fold change; ***P 0.001). In con ras , IL-33 treatment significantly decreased CTGF gene expression at 4 days post-MI compared to the vehicle-treated MI group (16.39 ± 2.69 vs 6.07 1.22 fold change; **P 0.01) (Figure 3-9E). Of note, CTGF gene expression remained more elevated in the vehicle-treated MI group at 7 days when compared with the sham-operated gro p (1.00 0.21 s 4.87 0.72 fold change; *P 0.05) (Figure 3-9F). These results indicate that administration of IL-33 in MI mice attenuates cardiac profibrotic markers in MI mice.3.8 IL-33 treatment affects cardiac and plasma inflammatory cytokines post-MI Protein expression of representative proinflammatory cytokines (IL-1 and TNF-) was quantified by western blot and ELISA. GM-CSF plasma levels were quantified by ELISA. Four days after MI, cardiac IL-1 pro ein le els increased significan l in he vehicle-treated MI group when compared to the vehicle-treated sham-operated group (1.00 ± 0.18 vs 4.64 ± 1.28 fold change; *P 0.05) (Figure 3-10A). Cardiac IL-1 tended to increase in the sham and MI groups treated with IL-33 without reaching significance (Figure 3-10A). At 7 days post-MI, cardiac IL-1 pro ein le els re rned o baseline in all groups except for the vehicle-treated MI group, which became significantly more elevated than the IL-33-treated MI group (0.69 ± 0.19 s 3.38 1.03 fold change; *P 0.05) (Figure 3-10B). Cardiac TNF-e pression increased significantly in the vehicle-treated MI group compared to the relative sham-operated group at 4 days (1.00 ± 0.05 vs 2.55 ± 0.24 fold change; *P 0.05) (Figure 3-10C). By itself, IL-33 increased cardiac TNF-levels at 4 days (1.00 ± 0.05 (sham+vehicle) vs 2.43 ± 0.56 (sham+IL-33) fold change). However, TNF-pro ein levels were attenuated in the IL-33-treated MI group 4 days post-MI when compared to the MI gro p rea ed i h ehicle (2.55 0.24 s 0.59 0.14 fold change; *P 0.05) (Figure 3-10C). At 7 days post-MI, TNF-pro ein le els in all gro ps ere a baseline i h no significan differences among them (Figure 3-10D). Plasma IL-1 concentrations showed a different trend than the protein levels observed in the heart. No increases in IL-1 plasma concentrations were observed at 4 days in all sham and MI groups. However, treatment with IL-33 for 7 days significantly increased IL-1 plasma concentrations in the sham-operated as well as in the MI treated groups (30.41±0.42 (pg/ml) (sham+vehicle) vs 63.45 ± 6.33 (pg/ml) (sham+IL-33); ***P 0.001) and (34.23±1.36 (pg/ml) (MI+vehicle) vs 53.85 ± 8.24 (pg/ml) (MI+IL-33); *P 0.05), respectively (Figure 3-10E), which is suggestive of possible systemic inflammation. TNF-plasma protein levels showed a similar trend as seen with TNF-cardiac protein expression at 4 and 7 days post-MI (Figure 3-10Error! Reference source not found.F). Plasma GM-CSF levels were significantly elevated in the IL-33-treated MI group at 4 days post-MI compared o all o her gro ps (**P 0.01 -****P 0.0001) (Figure 3-10G). Plasma GM-CSF decreased at 7 days post-MI (128.1±27.75 (pg/ml) (MI+IL-33 D4) vs 51.97±16.78 (pg/ml) (MI+IL-33 D7); **P 0.01), b remained insignifican l more ele a ed than in all other groups at the same time point. The sham-operated group treated with IL-33 showed a slight but insignificant increase in GM-CSF plasma concentrations compared to all vehicle-treated groups at both 4 and 7 days post-MI (Figure 3-10E).

3. 9

 9 IL-33 treatment promotes type 2 cytokines release IL-33 administration in the MI group for 4 days resulted in a significant increase in IL-4, IL-13, and IL-10 cardiac pro ein e pression (*P 0.05 and **P 0.01 s. sham+ ehicle) (Figure 3-11A, C and E). Moreover, IL-13 and IL-10 protein expression at 4 days was significantly more elevated in IL-33-treated MI group compared with the vehicle-treated MI group (1.83 0.30 s 4.61 0.89 fold change; **P 0.01) and (1.65±0.27 vs 4.38±1.05 fold change; *P 0.05) (Figure 3-11C and E) respectively. At 7 days, IL-4, IL-13, and IL-10 protein expression returned to baseline in all groups (Figure 3-11B, D, and F). IL-33 significantly increased IL-5 mRNA levels in the heart of both sham and MI groups at 4 days (1.00±0.08 vs 47.47±4.26 fold change; (sham+vehicle vs sham+IL-33); *P 0.05) and (1.85±0.40 vs 85.02±29.14 fold change; (MI+vehicle vs MI+IL-33); **P 0.01) (Figure 3-11G). IL-5 mRNA levels decreased between day 4 and day 7 with IL-33 treatment in both sham and MI mice, but remained significantly more elevated than the levels observed in the vehicle treated groups at 7 days (1.00±0.12 vs 13.29±1.46 fold change; (sham+vehicle vs sham+IL-33); **P 0.01) and (1.49±0.29 vs 7.86±2.27 fold change; (MI+vehicle vs MI+IL-33); *P 0.05) (Figure 3-11H). Similarly, IL-33 treatment increased IL-5 plasma concentrations in comparison to the vehicle-treated groups at 4 days in MI and sham groups (0.44±0.21 (pg/ml) MI+vehicle vs 97.56±4.94 (pg/ml) MI+IL-33) (****P 0.0001) and (0.51±0.29 (pg/ml) sham+vehicle vs 95.10±0.55 (pg/ml) sham+IL-33) (****P 0.0001) respectively (Figure 3-11I). 7 days post-MI, IL-5 plasma concentrations decreased in the IL-33-treated groups compared to their counterparts at 4 days (97.56±4.94 (pg/ml) (MI+IL-33 D4) vs 23.92±6.73 (pg/ml) (MI+IL-33 D7) (****P 0.0001) and sham gro ps (95.10±0.55 (pg/ml) (sham+IL-33 D4) vs 56.19±6.74 (pg/ml) (sham+IL-33 D7) (***P 0.001)), b remained significantly more elevated compared to the vehicle-treated MI and sham groups at 7 days post-MI (0.45±0.06 (pg/ml) (MI+vehicle) vs 23.92±6.73 (pg/ml) (MI+IL-33); (*P 0.05)) and (1.55±0.60 (pg/ml) (sham+vehicle) vs 56.19±6.74 (pg/ml) (sham+IL-33); (***P 0.001)) (Figure 3-11I). No plasma IL-5 levels were detected in vehicle-treated MI and sham groups. Our results are in agreement with a previous study showing elevated IL-5 levels in mouse serum as early as 3h following IL-33 administration. IL-5 levels decreased but remained detectable after 7 days of IL-33 (i.p.) administration [600].

  monocytes were identified as CD45+CD11b+Ly6G(hi) and CD11b+Ly6C(hi)Ly6G-F480-cells respectively. Compared to day1, neutrophil and Ly6C(hi) monocyte production in the bone marrow peaked at 3 days in all experimental groups including the vehicle-treated sham-operated group (Figure3-12A and Figure 3-13A). At 7 days, bone marrow neutrophil and Ly6C(hi) levels decreased significantly in the IL-33-treated MI group compared to the levels observed at day 3 (neutrophils: 46.37±1.58% (MI+IL33 D3) vs 29.85 1.94% (MI+IL33 D7) *P 0.05) (Ly6C(hi): 16.80±1.75% (MI+IL-33 D3) vs 5.59±0.99% (MI+IL-33 D7); ****P 0.0001) (Figure 3-12A and Figure 3-13A). Moreover, bone marrow neutrophils at 7 days were significantly lower in the IL-33-treated MI group in comparison to the vehicle-treated MI group at the same time point (46.13±2.28% vs 29.85±1.94%; *P 0.05) (Figure 3-12A). In the spleen, compared to day 1, neutrophils peaked at 3 days post-MI in the IL-33-treated group (4.73±0.83% D1 vs 10.72±1.47% D3; *P 0.05) where levels were significantly higher than in the vehicle-treated MI group at the same time point (5.01±0.93% (MI+vehicle D3) vs 10.72±1.47% (MI+IL-33 D3); *P 0.05, Figure 3-12B). Neutrophil circulation in the blood culminated at day 1 in all experimental groups and reached its lowest point at 7 days where levels in the IL-33-treated MI group were significantly lower than in the vehicle-treated group at the same time point (26.62±6.54% vs 7.07±2.95%; *P 0.05) (Figure 3-12C). At 7 days, Ly6C(hi) levels in the spleen and in the blood were significantly lower in the IL-33-treated MI group when compared to the levels observed in the vehicle-treated MI group (Figure 3-13B and C; *P 0.05). Of note, neutrophils and monocytes were already present in the heart of both experimental groups (MI+vehicle and MI+IL-33) on day 1 post-MI, and decreased significantly and concomitantly on day 7 post-MI (Figure 3-12D and Figure 3-13D). Macrophages were identified as CD11b+F480+Ly6C-. Compared to day 1, macrophages significantly peaked with IL-33 treatment in the bone marrow as early as 3 days post-MI in the IL-33-treated sham and MI groups, and remained elevated when compared to the vehicletreated groups at 7 days post-MI (Figure 3-14A). In the spleen and blood, macrophages increased significantly with IL-33 treatment and culminated at 7 days post-MI (Figure 3-14B and C). In the heart, MI induced a 6-7 fold increase in macrophage levels in the vehicle-treated group at 3 days post-MI when compared to day 1 (Figure 3-14D). These results suggest a possible local monocyte to macrophage differentiation, a process that can take place as early as day 1 post-MI [161]. In parallel, a gradual and significant increase in macrophage levels was observed in the heart of the IL-33-treated MI group between day 1 and day 7 post-MI (Figure 3-14D). Furthermore, macrophage levels in the IL-33-treated MI group at 7 days were two folds higher than the vehicle-treated MI group at the same time point (Figure 3-14D).

[

  567]. IL-33 treatment for 7 days induced a drastic increase in eosinophils in all organs (bone marrow, spleen, blood and heart). Percentage of eosinophils out of CD45+ cells ranged between (8.81±1.23% and 42.37±5.89%) in the IL-33-treated sham-operated group and (10.02±1.74% and 62.26±5.56%) in the IL-33-treated MI group in all organs at 7 days (Figure3-15A-D). No significant increase in eosinophil counts was observed in the vehicle-treated sham and MI groups in any organ nor at any timepoint.

day 1 ,

 1 a significant increase in ILC2s was observed at 3 days in the IL-33-treated shamoperated group (0.14±0.01% (D1) vs 0.46±0.11% (D3); ***P 0.001) (Figure 3-16A). Moreover, treatment of the sham-operated group with IL-33 increased ILC2 circulation in the blood at 7 days when compared to the vehicle-treated sham-operated group at the same time point (0.014±0.014% vs 0.30±0.06%; *P 0.05) (Figure 3-16C). In the spleen and in the heart, a tendency towards an increase in ILC2 percentage was observed in the IL-33-treated shamoperated group but without reaching significance. ILC2 significantly increased in the IL-33treated MI group and ranged between (0.20-1.26)% of the total number of cells in all organs at 7 days (Figure 3-16A-D).

3. 13

 13 NK levels are decreased in the heart and spleen at day 7 post-MI with IL-33 treatment NK cells were identified as CD45+CD11b+Ly6G-NK1.1+ cells. NK cells were present in the bone marrow in very small numbers in both IL-33-treated groups at day 1 post-surgery and decreased significantly by day 7 (sham+IL-33: 0.36±0.04% (D1) vs 0.020±0.004% (D7); **P 0.01) and (MI+IL-33: 0.48±0.07% (D1) vs 0.04±0.01% (D7); **P 0.01). Although NK cells were also present in the bone marrow at day 1 in the vehicle-treated MI group, their numbers did not change by day 7 (Figure3-17A). In the spleen, NK cells constituted 1-2% of total leukocytes in all experimental groups at day 1. NK levels increased in the vehicle-treated MI group in the spleen in a time dependent manner to peak at day 7 post-MI but without reaching significance. In the IL-33-treated MI group, NK levels tended to decrease over time and were significantly lower than the vehicle-treated MI group at 7 days (MI+vehicle:1.94±0.19% vs MI+IL-33: 0.72±0.12%;*P 0.05) (Figure3-17B). In the blood as in the heart, elevated NK levels were observed at day 7 in the vehicle-treated MI group, which were significantly higher than the levels observed in the IL-33-treated MI group at the same timepoint (Figure3-17C, D). Elevated levels of NK cells observed in the vehicle-treated MI group at day 7 were consistent with the literature [161].

7 ( 4 .

 74 -18A). In the spleen, no remarkable change in DC numbers was observed at any timepoint (Figure3-18B), whereas IL-33 administration significantly increased DC circulation in the blood in the sham-operated but not in the MI-treated mice at 7 days (MI+IL-33: 1.94±0.29% vs sham+IL-33: 11.39±1.73%; ****P 0.0001) (Figure 3-18C). In the heart, DC are known to infiltrate the heart at 5-7 days post-MI [161]. DC infiltration increased in all groups at D3 but only significantly in the IL-33treated MI group (Figure 3-18D).CD4+ T-cells were identified as (CD45+CD3+CD4+CD8-CD4+). T-cells were present in the bone marrow at day 1 post-surgery in all experimental groups and decreased significantly at day 7, suggesting a possible migration of CD4+ T cells towards the site of injury (Figure3-19A). In the spleen, a significant decrease in CD4+ T-cells was also observed between day 1 and day 7 in the IL-33-treated MI group (18.50±2.30% vs 8.61±0.39%; **P 0.01) (Figure 3-19B). Circulating CD4+ T-cells increased gradually between day 1 and day 7 post-MI in the vehicle-treated group but without reaching significance (4.63±0.71% vs 7.28±0.56%). In addition, CD4+ T-cells in the blood were significantly more elevated at day 7 in the vehicletreated MI group when compared to the IL-33-treated MI group at the same timepoint (1.59±0.25% vs 7.28±0.56%; ****P 0.0001) (Figure 3-19C). CD4+ T-cells were barely present in the heart at day 1 but increased gradually to reach their highest levels at 7 days in both MI groups [161] (MI+vehicle: 0.05±0.03% D1 vs 2.96±0.39% D7; ****P 0.0001) and (MI+IL-33 0.04±0.01% D1 vs 1.30±0.36% D7). Of note, CD4+ T-cells were more than 2 folds higher in the vehicle-treated MI group at day 7 when compared to the IL-33-treated MI group at the same timepoint (Figure 3-19D).CD8+ T-cells were identified as (CD45+CD3+CD4+CD4-CD8+). Being the adaptive counterparts of NK cells, CD8+ T-cells displayed a similar pattern to that observed with NK cells in all organs but only with IL-33 treatment. In the bone marrow, CD8+Tcells constituted ~1% of total leukocytes at day 1 post-surgery and decreased between day 1 and day 7 in all experimental groups (Figure3-20A). In the spleen, CD8+ T-cells tended to decrease between day 1 and day 7 in the IL-33-treated MI group without reaching significance (11.13±0.82% vs 4.44±0.45%). In addition, CD8+ T-cell levels were more than 2 folds higher in the vehicle-treated MI group when compared with IL-33-treated MI group at day 44±0.45% vs 10.32±%0.33; *P 0.05) (Figure3-20B). In the blood, CD8+ T-cells increased by ~3 folds between day 1 and day 7 post-MI in the vehicle-treated group (2.57±%0.45 vs 7.26±0.61% ; **P 0.01), which was in agreement with a previous study stating that circulating CD8+T lymphocytes increase one week following MI [252] . Of note, CD8+ Tcells in the vehicle-treated MI group were 5-6 folds higher than the IL-33-treated MI group at 7 days (1.26±0.21% vs 7.26±0.61%; **P 0.01) (Figure 3-20C). In the heart, although CD8+ T-cells are known to increase significantly in the heart at day 7 post-MI [161], no significant change was observed despite a tendency towards an increase between day 3 and day 7, which was observed in both MI groups (Figure 3-20D).

  -21A). In the spleen, B cells constituted between 30-45% of total leukocytes and no remarkable change in B cell numbers was observed at any timepoint (Figure 3-21B). In the heart, increased B cell levels observed in the present study are consistent with previous data showing an increased infiltration of mature B lymphocytes in the myocardium after the onset of MI and peaking 5-7 days post-MI [141, 161]. A significant increase in B cells was observed between day 1 and day 7 in both MI groups (MI+vehicle: 8.60±1.37% vs 20.80±3.87% ; *P 0.05) and (MI+IL-33: 10.89±2.86% vs 25.86±1.24% ; **P 0.01) (Figure 3-21D).

Figure 3 -

 3 23 is a representative figure showing the size and complexity of immune cell populations present in the spleen of all experimental groups at different timepoints. Leukocytes were mainly separated into three main populations based on their size and granularity: 1-small sized cells with low granularity or complexity, 2-cells with intermediate size and granularity (intermediate complexity) and 3-cells with larger size and high granularity (high complexity). While small sized-cells were mainly lymphocytes, the last two populations were indicative of myeloid cells. Following IL-33 administration, both sham and MI mice showed a decrease in the percentage of lymphocytes and an increase in the percentage of cells with high complexity in the spleen (Figure 3-23). In fact, both splenic CD4+ (Figure 3-19B) and CD8+ (Figure 3-20B) T-lymphocytes as well as NK cells (Figure 3-17B) decreased over time in the spleen with IL-33 treatment, while remained unchanged in the vehicle-treated groups. Of note, B-cell populations were not expanded in any group in the spleen (Figure 3-21B). In contrast, lineage negative cells such as ILC2 were significantly increased with IL-33 treatment at 7 days in the spleen (Figure 3-16B). In addition, cells with high complexity such as macrophages (Figure 3-14B) and eosinophils (Figure 3-15B) both increased significantly in the spleen with IL-33 treatment while remained unchanged in the vehicle-treated groups. As for splenic neutrophils, they were only increased at 3 days post-MI in the IL-33 treated group (Figure 3-12B). Monocytes are cells with intermediate complexity and were shown to decrease at 7 days with IL-33 treatment when compared with the vehicletreated MI group at the same timepoint (Figure 3-13B).

  adverse outcomes in acute decompensated heart failure[601]. Moreover, in stable, well-treated congestive heart failure patients, elevated sST2 levels were associated with disease-specific end-points such as heart failure hospitalization and cardiovascular death[602]. mRNA levels of cardiac sST2 were measured at 4 and 7 days in all groups treated with IL-33 or vehicle. sST2 levels were not changed at 4 days in all sham and MI groups (Figure3-24A). However, at 7 days, sST2 mRNA levels increased significantly in the sham and MI groups treated with IL33 compared with the sham group treated with vehicle (**P 0.01 and ***P 0.001 respec i el ). MI increased sST2 levels in the vehicle-treated group 7 days post-MI with no significance when compared to the relative sham group. Administration of IL-33 significantly upregulated sST2 cardiac expression in the MI group in comparison with vehicle-treated MI group (***P 0.001) (Figure 3-24B). The receptor for IL-33 (ST2) is found primarily on many allerg associa ed arge cells, incl ding, Th2 cells, eosinophils, basophils, mas cells and type 2 innate lymphoid cells (ILC2) [566]. The dynamic expression of plasma IL-33 was measured by ELISA at 4 and 7 days post-MI (Figure 3-24B). Our results are in agreement with a previous study showing increased serum levels of IL-33 following daily (i.p.) administration of 1µg of IL-33 for 7 days when compared with the basal levels observed in the saline-treated controls [603]. Plasma IL-33 concentrations peaked at 4 days in the sham and MI groups treated with IL-33 (Figure 3-24C),and decreased significantly at 7 days in both groups but remained more elevated than the vehicle-treated sham and MI groups (Figure3-24C). It is possible that sST2 levels are increased in the plasma at 7 days, therefore acting as a decoy receptor to IL-33 and decreasing its availability in the plasma at 7 days.

FiguresFigure 3 - 1

 31 Figures

Figure 3 - 2

 32 Figure 3-2 LAD ligation procedure.

Figure 3 - 3 :

 33 Figure 3-3: Functional cardiac parameters 4 and 7 days post-MI in vehicletreated and IL33-treated mice. (A) Ejection fraction (EF), (B) fractional shortening (FS), (C) cardiac output (CO). Bar graph data are mean ± SEM. Sham-operated animals (n=8-11); MI-operated animals (n=12-17). Two-way ANOVA followed by T ke s pos -hoc test. *p 0.05, **p 0.01, ***p 0.001****p 0.0001.

Figure 3 - 4 :

 34 Figure 3-4: Increased left ventricular volume and diameter with IL-33 treatment 7 days post-MI. Analysis of LV end-systolic and diastolic volume and diameter normalized to tibia length. (A) left ventricle end-systolic volume (LVESV); (B) left ventricle end-diastolic volume (LVEDV); (C) left ventricle end-systolic diameter (LVESD); (D) left ventricle end-diastolic volume (LVEDD); (E) and (F) Representative B-mode and M-mode echocardiographic images obtained in mice before undergoing LAD ligation (baseline (BL)), at day 1(D1), day 4 (D4), and day 7 post MI (D7) for mice treated with IL-33 or its vehicle (PBS). Echocardiographic analyses revealed impaired contractile function with IL-33 treatment 7 days post-MI when compared to MI+vehicle at 7days. Sham-operated animals (n=8-11); MI-operated animals (n=12-17). Two-way ANOVA follo ed b T ke s pos -hoc test. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001.

Figure 3 - 5 :

 35 Figure 3-5: Increased adverse left ventricular remodeling and death with IL-33 treatment post-MI. (A) Variation of LV weight normalized to tibia length at 4 and 7 days post-LAD ligation or sham-operation. Sham-operated animals (n=8-11); MI-operated animals (n=12-17). Two-a ANOVA follo ed b T ke s post-hoc test. (B) Percentage of retained water in the lungs represented as degree of pulmonary congestion at 4 and 7 days. Sham-operated animals (n=8-11); MI-operated animals (n=12-17). Two-way ANOVA followed by T ke s pos -hoc test. (C) Infarct size and (D) representative images at 4 and 7 days post-MI, evaluated by TTC staining (n=8 per group). Two-way ANOVA follo ed b T ke s pos -hoc test. Kaplan-Meier survival curves for mice subjected to MI at (E) 4 and (F) 7 days. Bar graph data are mean ± SEM. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001.

Figure 3 - 6 :Figure 3

 363 Figure 3-6: Administration of IL-33 in mice significantly enhances cardiac remodeling (fibrosis and hypertrophy) in vivo at 7 days. (A) Quantification of myocyte CSA and representative H&E stained cross-sections at the mid-ventricle level of LAD-ligated or sham-operated mice treated with IL-33 or vehicle for 7 days. A total of 10 images per section were taken from the subendocardial and subepicardial regions and were analyzed. CSAs were defined from an average of 100 cardiomyocytes per mouse. (B) Quantification of fibrosis and representative MTC stained cross-sections at the mid-ventricle level of LAD ligated and sham-operated mice treated with IL-33 or vehicle for 7 days. 10 fields per section were analyzed from each animal. IL-33 treatment exacerbates cardiac remodeling (fibrosis and hypertrophy) post-MI when compared with the MI mice treated with vehicle (n=4 per group). One-a ANOVA follo ed b T ke s pos -hoc test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 3 - 8 :

 38 Figure 3-8: Upregulation of matrix metalloproteinases with IL-33 treatment at 7 days. (A,B) Normalized MMP9 gene expression by quantitative RT-PCR at 4 and 7 days showing up-regulation of MMP9 with IL-33 treatment at 4 and 7 days (P<0.05). (C,D) Normalized MMP2 gene expression by quantitative RT-PCR at 4 and 7 days showing up-regulation of MMP2 with IL-33 treatment at 7 days post-MI (P<0.05) (n=4 6 per group); one-a ANOVA follo ed b T ke s pos -hoc test. *P<0.05, **P<0.01, ****P<0.0001.

Figure 3 - 9

 39 Figure 3-9 Decreased profibrotic protein and gene expression with IL-33 treatment. (A, B) TGF-pro ein e pression i h represen a i e bands at 4 and 7 days showing decreased TGFcytokine expression in the MI group treated with IL-33 for 7 da s (p 0.05). (C, D) -SMA protein expression with representative bands a 4 and 7 da s sho ing decreased -SMA cytokine expression in the MI group treated with IL-33 for 7 da s (p 0.05). (E, F) normalized connective tissue growth factor (CTGF) gene expression by quantitative RT-PCR at 4 and 7 days post-surgery showing a significant decline in CTGF mRNA expression at 4 days in the MI group treated with IL-33 when compared with the MI group treated i h ehicle (p 0.001). (n=4 to 6 per group); one-a ANOVA follo ed b T ke s pos -hoc test.

Figure 3

 3 Figure 3-10: IL-33 treatment affects inflammatory cytokine protein expression. (A, B). IL-1 pro ein e pression i h represen a i e bands a 4 and 7 da s showing an increase in IL-1 cytokine expression in the MI group treated with vehicle a 4 da s (p 0.05) and a significant decrease in the MI group treated with IL-33 in comparison with the vehicle-treated MI group at 7days. (C, D). TNF-pro ein expression with representative bands at 4 and 7 days showing decreased TNF-c okine e pression in he MI group treated with IL-33 for 4 days (p 0.05) (n=4 or 5 per gro p); one-a ANOVA follo ed b T ke s post-hoc test. ELISA results for (E) IL-1 , (F) TNF-, and (G) GM-CSF plasma protein concentrations at 4 and 7 days post sham or MI operation. Increased plasma concentrations of GM-CSF at 4 days with IL-33 treatment (n=4 or 5 per group; two-a ANOVA follo ed b T ke s pos -hoc test). *P <0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3 - 11 :

 311 Figure 3-11: Increased type 2 cytokine production with IL-33 treatment. (A, B) IL-4 protein expression with representative bands at 4 and 7 days showing increased IL-4 cytokine expression in the MI group treated with IL-33 for 4 da s (p 0.05) (n=3-4 per group; one-way ANOVA followed b T ke s pos -hoc test). (C, D) IL-13 protein expression with representative bands at 4 and 7 days showing increased IL-13 cytokine expression in the MI group treated with IL-33 for 4 da s (p 0.05). (E, F) IL-10 protein expression with representative bands at 4 and 7 days showing increased IL-10 cytokine expression in the MI group treated with IL-33 for 4 da s (p 0.05). (G, H) IL-5 mRNA expression at 4 and 7 days showing increased IL-5 levels with IL-33 treatment. (I) Increased plasma concentrations of IL-5 at 4 and 7 days with IL-33 treatment. Plasma concentrations of IL-5 were examined by ELISA at 4 and 7 days post-MI (sham animals n=3 per group; MI animals n=5 per group; two-way ANOVA followed by Tuke s pos -hoc test). *P <0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3 - 12 :

 312 Figure 3-12: Comparable neutrophil resolution in the heart between IL-33-treated and vehicle-treated MI groups at 7 days post-MI. Time course variation (day 1 (D1), day 3 (D3) and day 7 (D7) post-surgery) of neutrophil percentage calculated out of CD45+ cells from, (A) the bone marrow (BM), neutrophil production in the bone marrow peaks at day 3 in all experimental groups when compared with day 1. (B) Spleen, neutrophil infiltration peaks at day 3 in the spleen in the MI group treated with IL-33. (C) Blood, neutrophils are at their highest levels at day 1 and decrease at day 7 in all experimental groups, and (D) heart, neutrophil infiltration drops significantly at day 7 in both MI groups. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Each dot represents one mouse (n=3in sham-operated groups and n=5-6 in LAD-ligated groups; two-a ANOVA follo ed b T ke s pos -hoc test).

Figure 3 - 13 :

 313 Figure 3-13: Dynamics of proinflammatory monocytes at different timepoints with and without IL-33 treatment. Time course variation (day 1 (D1), day 3 (D3) and day 7 (D7) post-surgery) of monocytes percentage calculated out of total live cells from (A) the bone marrow (BM), monocyte production in the bone marrow peaks at day 3 in all experimental groups when compared with day 1. (B) Spleen, a significant decrease in monocyte infiltration in the spleen is observed in the MI group treated with IL-33 when compared with the MI group treated with vehicle at day 7. (C) Blood, circulating monocytes in the blood decrease significantly at day 7 in the MI group treated with IL-33 when compared with the MI group treated with vehicle, and (D) heart, monocyte levels are at their highest in both MI groups at day 1 and decrease gradually over time. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Each dot represents one mouse (n=3 in sham-operated groups and n=5 6 in LAD-ligated groups; two-a ANOVA follo ed b T ke s pos -hoc test).

Figure 3 - 14 :

 314 Figure 3-14: Increased macrophage levels with IL-33 treatment at 7 days post-MI. Time course variation (day 1 (D1), day 3 (D3) and day 7 (D7) post-surgery) of macrophage percentage calculated out of total live cells from, (A) the bone marrow (BM) macrophages in the bone marrow increase significantly with IL-33 treatment at day 3, (B) spleen, macrophage levels start increasing with IL-33 treatment as early as day 3 and become significantly more elevated in the MI group treated with IL-33 at day 7, (C) blood, a significant increase in macrophage circulation in the blood is observed with IL-33 treatment at day 7, and (D) heart, macrophage levels start increasing in the heart at day 3 and become significantly more pronounced in the MI group treated with IL-33 at day 7. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Each dot represents one mouse (n=3 in sham-operated groups and n=5 6 in LAD-ligated groups; two-way ANOVA followed by T ke s pos -hoc test).

Figure 3 - 15 :

 315 Figure 3-15: IL-33 induces eosinophilia at day 7 regardless of the surgical intervention. Time course variation (day 1 (D1), day 3 (D3) and day 7 (D7) post-surgery) of eosinophil percentage calculated out of CD45+ cells, (A) the bone marrow (BM), (B) spleen, (C) blood, and (D) heart. Eosinophil levels start increasing as early as day 3 in the sham and MI groups treated with IL-33 and become significantly more increased at day 7 in these two groups in all tissues.*P<0.05, ****P<0.0001. Each dot represents one mouse (n=3 4 in sham-operated groups and n=5 6 in LAD-ligated groups; two-a ANOVA follo ed b T ke s post-hoc test).

Figure 3 - 16 :

 316 Figure 3-16: Treatment with IL-33 increases ILC2 levels at 7 days post-MI. Time course variation (day 1 (D1), day 3 (D3) and day 7 (D7) post-surgery) of ILC2 percentage calculated out of total live cells from, (A) the bone marrow (BM), ILC2 levels start increasing as early as day 3 with IL-33 treatment and become significantly more elevated in the MI group treated with IL-33 at day 7, (B) spleen, ILC2 levels peak in the spleen at day 7 in the MI group treated with IL-33, (C) blood, ILC2 circulation in the blood increases significantly at day 7 in the sham and MI groups treated with IL-33, and (D) heart, ILC2s increase significantly in the heart at day 7 in the MI group treated with IL-33. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Each dot represents one mouse. (n=3 in sham-operated groups and n=5 6 in LAD-ligated groups; two-a ANOVA follo ed b T ke s pos -hoc test).

Figure 3 -

 3 Figure 3-17 NK cells are not increased in the heart at 7 days post-MI with IL-33 treatment. Time course variation (day 1 (D1), day 3 (D3) and day 7 (D7) post-surgery) of NK cell percentage calculated out of CD45+ cells from, (A) the bone marrow (BM) NK cells are present in the BM at day 1 and decrease significantly with IL-33 treatment. (B) Increased infiltration of NK cells is observed in the spleen in the vehicletreated MI group and is significantly more elevated than the IL-33 treated MI group at D7. (C) In the blood, a significant increase in NK cells is observed at D7 in the vehicle-treated MI group and is significantly more pronounced that the IL-33-treated MI group at the same timepoint. (D) In the heart, increased infiltration of NK cell is observed in the vehicle-treated MI group at D7 and is significantly more pronounced that the IL-33treated MI group at the same timepoint. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. Each do represents one mouse. (n=3 in sham-operated groups and n=5-6 in LAD-ligated groups; two-way ANOVA follo ed b T ke s pos -hoc test).

Figure 3 -

 3 Figure 3-18 Dynamics of dendritic cells at different timepoints with and without IL-33 treatment. Time course variation (day 1 (D1), day 3 (D3) and day 7 (D7) post-surgery) of DC percentage calculated out of total live cells from, (A) the bone marrow (BM) IL-33 seems to increase DC production in the bone marrow in a time-dependent fashion to culminate at 7days. (B) In the spleen, no remarkable change in DC numbers is observed at any timepoint. (C) In the blood, IL-33 administration increases DC circulation in the sham-operated but not in the MI-treated mice. (D) In the heart, DCs infiltration increases in all groups at D3 but only significantly in the IL-33-treated MI group. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. Each do represents one mouse. (n=3 in sham-operated groups and n=5-6 in LAD-ligated groups; two-way ANOVA follo ed b T ke s post-hoc test).

Figure 3 -

 3 Figure 3-19 Dynamics of CD4+ T-cells at different timepoints with and without IL-33 treatment. Time course variation (day 1 (D1), day 3 (D3) and day 7 (D7) post-surgery) of CD4+ T-cell percentage calculated out of total CD45+ cells from, (A) the bone marrow (BM) CD4+ T-cells are present in the BM at D1 and decrease significantly at D7 in all experimental groups. (B) In the spleen, a significant decrease in CD4+ T-cell levels is observed between D1 and D7 in the IL-33-treated MI mice. (C) In the blood, significantly lower levels of CD4+ T-cells are observed at D7 between vehicle-treated and IL-33-treated MI mice. (D) In the heart, no activation of CD4+T-cells is observed in any group at D1. CD4+ T-cells increase gradually in both MI groups but only significantly in the vehicle-treated MI group at D7, and are significantly higher than the IL-33-treated MI mice at D7.*p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. Each do represents one mouse. (n=1-2 in sham-operated groups and n=4-6 in LAD-ligated groups; two-way ANOVA followed by T ke s pos -hoc test).

Figure 3 -

 3 Figure 3-20 Dynamics of CD8+ T-cells at different timepoints with and without IL-33 treatment. Time course variation (day 1 (D1), day 3 (D3) and day 7 (D7) post-surgery) of CD8+ T-cell percentage calculated out of total CD45+ cells from, (A) the bone marrow (BM), CD8+ T-cells constitute more than 1% of total leukocytes in the BM at D1 and decrease in all experimental groups at D7. (B) In the spleen, a tendency towards a decrease between day 1 and day7is observed in the IL-33-treated MI group. CD8+ T-cell levels were more than 2 folds higher in vehicle-treated MI group when compared with the IL-33-treated MI group at D7. (C) In the blood, a significant increase in CD8+ T-cell circulation is observed between D1 and D7 in the vehicletreated MI group, which is significantly higher than the IL-33-treated MI group at the same timepoint. (D) In the heart, no significant change is observed despite a tendency towards an increase which was observed between D3 and D7 in both MI groups.*p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. Each do represents one mouse. (n=2-3 in sham-operated groups and n=5-6 in LAD-ligated groups two-way ANOVA followed by T ke s pos -hoc test).

Figure 3 -

 3 Figure 3-21 Dynamics of B cells at different timepoints with and without IL-33 treatment. Time course variation (day 1 (D1), day 3 (D3) and day 7 (D7) post-surgery) of B cell percentage calculated out of CD45+ cells from, (A) the bone marrow (BM), B cells 7-14% of total leukocytes at day 1 in all experimental groups. At day 7, a decrease in B cell levels is observed between D1 and D7 in all studied groups. (B) In the spleen, B cells are between 30-45% of total leukocytes and no remarkable change in B cell numbers is observed at any timepoint. (C) In the blood, B cell circulation gradually increases in the vehicle-treated groups but not in the IL-33-treated groups. (D) In the heart, all groups have increased B cell numbers between D1 and D7. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. Each do represents one mouse. (n=3-4 in sham-operated groups and n=5-6 in LAD-ligated groups; two-a ANOVA follo ed b T ke s pos -hoc test).

Figure 3 -

 3 Figure 3-22 Variation of spleen weight normalized to tibia length at 4 and 7 days post-LAD ligation or sham-operation. At 4 days, sham and MI mice treated with IL-33 had a significantly increased spleen weight, which was further increased at 7 days with IL-33 treatment. Sham-operated animals (n=8-11); MI-operated animals (n=12-17). Two-a ANOVA follo ed b T ke s pos -hoc test. **P < 0.01, ****P < 0.0001.

Figure 3 -

 3 Figure 3-23 Effect of IL-33 administration on the size and complexity of immune cell populations observed in the spleen. Variation in leukocyte populations observed in the spleen of vehicle and IL-33-treated animals at 1, 3, and 7 days post LAD ligation or sham operation. Leukocytes are mainly separated into three main populations based on their size and granularity: 1-small sized cells with low granularity or complexity, 2-cells with intermediate size and granularity (intermediate complexity) and 3-cells with larger size and high granularity (high complexity). While small sized-cells are mainly lymphocytes, the last two populations are indicative of myeloid cells. Following IL-33 administration, both sham and MI-operated mice show a decrease in the percentage of lymphocytes and an increase in the percentage of cells with high complexity in the spleen

Figure 3 -

 3 Figure 3-24 Cardiac sST2 and plasma IL-33 levels. Upregulation in cardiac sST2 mRNA levels at 7 days in the sham and MI group treated with IL-33 and increased IL-33 plasma concentrations with IL-33 administration. Normalized sST2 gene expression by quantitative RT-PCR at (A) 4 and (B) 7 days post-surgery. (n=5 or 6 per group; one-a ANOVA follo ed b T ke s poshoc test). (C) Plasma concentrations of IL-33 were examined by ELISA at 4 and 7 days. (n=4 or 5 per group; two-a ANOVA follo ed b T ke s pos -hoc test).

Figure 3 -

 3 Figure 3-25 Western total protein membranes.Western bands with their corresponding total protein membranes stained with ponceau red (A) at 4 and (B) 7 days post-surgery.

Figure 3 -

 3 Figure 3-26 Primary gating. All cells are visible in the forward scatter (area) (FSC-A) versus side scatter (area) (SSC-A) plot. (A) Debris are omitted from the analysis by excluding the left lower corner of the plot. (B) To exclude clumped cells or doublets, single cells are separated from cell aggregates using (FSC-A) versus (FSC-H) (height). (C) Viable cells are gated using DAPI staining, where viable cells would be negatively stained and appear on the left side of the plot.

Figure 3 -

 3 Figure 3-27 Gating strategy for panel #1. After the primary gating (Figure 3-26), (A) Plotting CD11b against CD11c allows the differentiation of DCs from other CD11b+ subsets (CD11b+ CD11c(hi)). (B) Then CD11b vs Ly6C are plotted. (C) Double positive cells from (B) are subgated into F480 vs Ly6G where double negative cells are defined as monocytes (CD11b+Ly6C(hi)Ly6G-F480-). (D) Positive cells for CD11b but negative for Ly6C cells from (B) are subgated into F480 vs SSC-A. Positive cells for F480 are defined as macrophages (CD11b+Ly6C(low)F480+).

Figure 3 -

 3 Figure 3-28 Gating strategy for panel #2. After the primary gating (Figure 3-26), (A) total leukocytes are gated as CD45+cells. (B) CD45+cells from (A) are subgated into siglecF versus CD11c where positive cells for siglecF and negative cells for CD11c are defined as eosinophils (CD45+SiglecF+CD11c-). (C) CD45+cells from (A) are subgated into Ly6G vs CD11b where double positive cells are defined as neutrophils (CD45+CD11b+Ly6G(hi)). (D) Positive cells for CD11b+ but negative for Ly6G are again subgated with NK1.1 vs SSC-A. Positive cells for NK1.1 with small SSC-A are defined as NK cells (CD45+CD11b+LY6G-NK1.1+). (E) For B cells, CD45+cells from (A) are subgated into B220 vs CD19. (F) Double positive cells from the previous gate were again subgated into IgM vs SSC-A where positive for IgM with small SSC-A are defined as B cells (CD45+B220+CD19+IgM+).

Figure 3 -

 3 Figure 3-29 Gating strategy for panel #3. After the primary gating (Figure 3-26), (A) lineage markers (CD3, CD4, CD8, CD19, Cd11b, Cd11c, Gr1 NK1.1, Fc Ri and Ter119) are d mped in he FITC channel and e cl ded from he anal sis. (B) ICOS positive cells but negative for Lin are subgated into CD25 vs SSC-A where CD25+ cells are defined as ILC2 (Lin-ICOS+CD25+).

Figure 3 -

 3 Figure 3-30 Gating strategy for panel #4.

  and antiinflammatory IL-10, and reduced levels of cardiac inflammatory TNF-(a 4 and 7 days) and IL-1 (7 da s). Nonetheless, IL-33 was associated with a pronounced decrease in systolic function at day 7 with ventricular dilation and infarct expansion, along with increased apoptosis (increased BAX/BCL2 and TUNEL+ nuclei). The overall results of the present study reveal that IL-33 is associated with worsened cardiac function and increased adverse cardiac remodeling in the MI mouse model. The significant increase in LV dilatation (LVESD and LVEDD) observed in the IL-33-treated MI group at day 7 reflected the increased LV volumes (LVESV and LVEDV) observed. Increased chamber dilation and volumes might be considered as a compensatory mechanism to recover stroke volume at an early phase post-MI [598]. Administration of IL-33 increased mortality due to cardiac rupture, induced pulmonary congestion and cardiomyocyte apoptosis, and promoted LV hypertrophy and fibrosis. During the early phase post-MI, infarct expansion as well as regional dilatation both participate in the enlargement of the ventricular chamber [604].

  and to positively affect eosinophil development in vivo [567]. Mice lacking IL-33 have decreased eosinophil counts and protection against asthma when compared to wild type mice [612]. IL-33 has also been implicated in activating mature eosinophils. For instance, IL-33 was reported to play a role in either the survival or death of human eosinophils depending upon context [567] and to stimulate NF-B-mediated inflammatory gene expression in murine eosinophils by both IL-4-independent and IL-4dependent/autocrine mechanisms [573]. An increase in plasma type 2 cytokine release, as well as an amplified expansion of eosinophils and ILC2 in the spleen and the bone marrow were also promoted by IL-33 administration in a mouse model of collagen-induced arthritis [613].

IL- 33

 33 induced a delayed increase in eosinophils in the heart in both the absence and presence of MI. Increased plasma concentrations of IL-5 and GM-CSF (which are potent cytokines associated with eosinophil development and increased viability [546]) at 4 days may have facilitated this recruitment and survival. Eosinophils are known to spontaneously die by apoptosis upon withdrawal of certain growth factors (mainly IL-5) by mechanisms that are not completely understood [616]. Previous studies have shown that IL-33 indirectly promotes the survival of eosinophils by inducing the secretion of GM-CSF in an autocrine manner [572].

  Although IL-33 was shown to favor the expansion of NK cells during viral infections [625], the results of the present study indicate that NK levels were significantly decreased in the bone marrow and spleen of IL-33-treated MI mice at 7 days. This decrease in NK cells might be the result of a possible local differentiation of NK cells into ILC2 or their mobilization to other lymphoid organs. Moreover, a significant increase in NK cells was observed in the circulation as well as in the spleen and heart of the vehicle-treated group at day 7 post-MI, an increase that was totally absent in the IL-33-treated MI group at the same timepoint. Previous studies have shown that NK cells induce the apoptosis of eosinophils in vitro. Moreover, NK cells play a protective role in myocarditis where their presence was associated with reduced cardiac eosinophilc infiltration in mice [626]. Therefore, decreased cardiac levels of NK cells observed in the present study might have participated in the increase of eosinophil numbers observed in the heart of IL-33-treated MI mice at 7 days. In the present study, elevated numbers of DC were observed at day 7 in the bone marrow of IL-33-treated mice. Previous data has shown that IL-33 promotes in vitro generation of dendritic cells in bone marrow cultures in a GM-CSF-dependent manner [627]. Activation of DC by IL-33 can directly polarize naïve T cells into a Th2 phenotype or enhance Treg proliferation [628, 629]. Whether GM-CSF participated in the increased levels of DC observed in the bone marrow at day 7 with IL-33 treatment and whether those DC promoted the polarization of naïve T cells into Th2 or Treg cells need further investigation. In experimental MI animal models, increased infiltration of both T and B-lymphocytes has been observed into the infarct zone following MI [141, 161, 189]. While previous data suggests that CD4+ T-cells are detrimental and increase infarct size following MI [630], others suggest that their presence is essential for proper wound healing after MI and participate in collagen deposition, which is an essential mechanism against LV dilation and rupture. Mice deficient in CD4+ T-cell showed disrupted ECM formation in the infarct zone and died from cardiac rupture a result probably due to Treg deficiency [240, 631]. In agreement with previous data, both vehicle and IL-33-treated MI groups showed increased CD4+T-cell infiltration in the heart at 7 days post-MI [161]. The IL-33-treated MI mice however showed significantly lower CD4 lymphocyte levels at 7 days when compared with the vehicle-treated group at the same timepoint. This insufficient infiltration of CD4+ T-cells in the heart might have contributed in the increased incidence of cardiac rupture observed in our IL-33-treated MI mice.

  Despite similarities with IL-33 administration modality of this study,Yin et al. study differs in several aspects: 1) In the current study, hemodynamic parameters were collected at baseline, day 1, day 4, and day 7 post-MI. The results at day 1 clearly show that the hemodynamic parameters (EF, FS, CO, LVESV, LVEDV, LVESD, LVEDD) were in the same range in all MI groups, reflecting similar MI induction in all groups at day 1. Although no significant hemodynamic changes were observed at day 4, animals in the IL-33-treated MI group exhibited significantly decreased EF and increased LVESV, LVEDV, LVESD, LVEDD at day 7 post-MI in comparison with the vehicle-treated MI group. IL-33 administration and follow up was ethically limited to 7 days post-MI due to the very high mortality rate in the IL-33-treated MI animals (~50%). In the Yin et al. study, LV function was assessed at day 14 and day 28 post-MI and no data was shown at day 1 post-MI to prove that both MI groups (treated with vehicle and IL-33) underwent the same degree of LAD ligation. Additionally, IL-33-treated MI groups showed extraordinarily improved cardiac function in comparison with the vehicle-treated MI group at 14 and 28 days post-MI. 2). In the present study, DNA fragmentation was assessed at 4 and 7 days post-MI and was shown to be significantly increased with IL-33 treatment post-MI. Yin et al. observed DNA fragmentation at an earlier phase (3 days post-MI only) and was shown to be decreased with IL-33 treatment. 3) The flow cytometry data in this study showed that macrophages increased in the heart 3 days post-MI in the vehicle-treated group, without being affected in any other organ such as the bone marrow, spleen and blood. No significant difference in macrophage levels in the heart were observed between the IL-33 and vehicletreated MI groups at 3 days post-MI. However at 7 days post-MI, a significant increase in macrophage infiltration in the heart was observed in the IL-33-treated MI group in comparison with the vehicle-treated group at the same time point. In contrast, in the Yin et al study, immunofluorescent-staining showed a significantly decreased F4/80+ macrophage infiltration in the heart in the IL-33-treated MI animals at 3 days post-MI. Macrophage levels might have changed in the heart post-MI with IL-33 treatment if they were assessed at later stages. 4) Both GAPDH and -actin are likely to display protein expression variability in the MI setting and therefore are not recommended to be used as western blot loading controls post-MI [595].

Figure 1 .

 1 Figure 1. Functional cardiac parameters 4 and 7 days post-MI in vehicle-treated and IL-33 treated mice (A) EF, (B) FS, (C) CO. Bar graph data are mean + -SEM. *P<0.05, **P<0.01, ****P<0.0001.

  ). Comparing D1 and D7 post-MI, a significant decrease in EF (**P<0.01), FS (*P<0.05), and CO (**P<0.01), was observed in the IL-33 treated MI group, but not the vehicle-treated MI group, indicating a worsened adverse LV remodeling with IL-33 treatment (Figure1A-C). Of note, EF decreased significantly at D7 post-MI in the IL-33 treated group compared with the vehicle-treated group at the same time point (Figure1A, *P<0.05). LV end-systolic and diastolic volumes and diameters (LVESV, LVEDV, LVESD, and LV end-diastolic diameter (LVEDD)) (normalized to tibia length) increased progressively and significantly as early as day 1 post-MI in all groups regardless of the treatment (Figure2A-F). However, the MI group treated with IL-33 showed substantially higher increases in LVESV, LVEDV, LVESD, and LVEDD at 7 days post-MI compared with the vehicle-treated MI group, indicating worsened cardiac dysfunction (Figure2A-F). Moreover, IL-33 treated MI group experienced a significant decrease in EF (Figure1A, ****P<0.0001), and increase in LVESV, LVEDV, LVESD, and LVEDD (Figure 2A-D, ***P<0.001 to ****P<0.0001) between days 4 and 7 post-MI that was

Figure 2 .

 2 Figure 2. Increased left ventricular volume and diameter with IL-33 treatment 7 days post-MI: analysis of LV end-systolic and diastolic volume and diameter (A) LVESV; (B) LVEDV; (C) LVESD; (D) LVEDD. Representative B-mode and M-mode (E,F) echocardiographic images obtained in mice before undergoing LAD ligation (baseline (BL)), at day 1 post-MI (D1), and at day 4 or day 7 post MI (D4), (D7) for mice treated with IL-33 or its vehicle (PBS). Echocardiographic analyses revealed impaired contractile function with IL-33 treatment 7 days post-MI when compared with MI+vehicle at 7days. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

88 + - 1 .

 881 85 vs 14.35 + -1.99%; ***P<0.0001) (Figure 4B) 7 days post-MI compared with the vehicletreated MI group. IL-33 treatment increases cardiac BAX/BCL2 mRNA levels and DNA fragmentation mRNA expression levels of the apoptosis-regulating genes BAX and BCL2 are shown in Figure 5 at 4 and 7 days post-MI: BAX (Figure 5A,D); BCL2 (Figure5B,E). BAX/BCL-2 ratio increased in both MI groups at 4 days post-MI, but only significantly in the IL-33 treated MI group compared with IL-33 treated sham group (1.61 + -0.13 vs 0.90 + -0.10 fold change; *P<0.05) (Figure5C). At 7 days post-MI, mRNA expression levels of the BAX/BCL2 ratio returned to baseline in the vehicle-treated MI group, but remained significantly higher in IL-33 treated MI group compared with the vehicle-treated MI group and IL-33 treated sham group, respectively (1.82 + -0.38 vs 0.99 + -0.06 fold change; *P<0.05 and 1.82 + -0.38 vs 0.68 + -0.18 fold change; **P<0.01) (Figure5F). To explore whether IL-33 affects cardiomyocyte cell death, a TUNEL assay was performed on myocardial sections harvested 4 and 7 days post-MI. IL-33 significantly increased cardiomyocyte DNA fragmentation in the IL-33 treated MI group compared with all other groups at 4 days post-MI (****P<0.0001) (Figure5G,H). At 7 days, TUNEL-stained nuclei were significantly elevated in both MI groups, but to a significantly higher extent in the IL-33 treated MI group in comparison with the MI group treated with vehicle (***P<0.001) (Figure5G,I). These results indicate that the administration of IL-33 in the presence of MI increases the susceptibility to cardiomyocyte death which may contribute to increased infarct size.

Figure 3 .

 3 Figure 3. Increased adverse left ventricular remodeling and death with IL-33 treatment post-MI (A) Variation of LV weight normalized to tibia length at 4 and 7 days post-LAD ligation or sham-operation. (B) Percentage of retained water in the lungs represented as degree of pulmonary congestion at 4 and 7 days. (C,D) Infarct size and representative images at 4 and 7 days post-MI, evaluated by TTC staining. (E,F) Kaplan-Meier survival curves for mice subjected to MI at 4 and 7 days. Bar graph data are mean + -SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 4 .

 4 Figure 4. Administration of IL-33 in mice significantly enhances cardiac remodeling (fibrosis and hypertrophy) in vivo at 7 days (A) Quanti cation of myocyte CSA and representative H&E stained cross-sections at the mid-ventricle level of LAD-ligated mice treated with IL-33 or vehicle for 7 days. (B) Quanti cation of brosis and representative MTC stained cross-sections at the mid-ventricle level of LAD ligated mice treated with IL-33 or vehicle for 7 days. IL-33 treatment exacerbates cardiac remodeling ( brosis, and hypertrophy) post-MI when compared with the MI mice treated with vehicle (n=4 per group). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 5 .

 5 Figure 5. IL-33 administration increases cardiomyocyte apoptosis at 4 and 7 days post-MI Normalized BAX and BCL2 gene expression and BAX/BCL2 ratio by quantitative RT-PCR at 4 (A-C) and 7 days (D-F) post-MI (n=4-6 per group) showing increased BAX/BCL2 ratio at both time points. Number of TUNEL positive cardiomyocytes at 4 and 7 days post MI with representative gures (G-I). DNA fragmentation increases with IL-33 treatment at 4 and 7 days post-MI. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ## P<0.01 (MI+vehicle 7d vs MI+vehicle 4d) (n=3-4 per group).

Figure 6 .

 6 Figure 6. IL-33 treatment affects inflammatory cytokine protein expression (A,B) IL-1β protein expression with representative bands at 4 and 7 days showing an increase in IL-1β cytokine expression in the MI group treated with vehicle at 4 days (P<0.05) and a signi cant decrease in the MI group treated with IL-33 in comparison with the vehicle-treated group at 7 days. (C,D) TNF-α protein expression with representative bands at 4 and 7 days showing decreased TNF-α cytokine expression in the MI group treated with IL-33 for 4 days (P<0.05) (n=4 or 5 per group). (E) ELISA results for GM-CSF plasma protein concentrations at 4 and 7 days post sham or MI operation. Increased plasma concentrations of GM-CSF at 4 days with IL-33 treatment (n=4 or 5 per group). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

IL- 33

 33 administration in the MI group for 4 days resulted in a significant increase in IL-4, IL-13, and IL-10 cardiac protein expression (*P<0.05 and **P<0.01 vs. sham+vehicle) (Figure 8A,C,E). Moreover, IL-13 and IL-10 protein expression at 4 days was significantly more elevated in IL-33 treated MI group compared with the vehicle-treated MI group (4.61 + -0.89 vs 1.83 + -0.30 fold change; **P<0.01) and (4.38 + -1.56 vs 1.65 + -1.56 fold change; *P<0.05) (Figure 8C,E), respectively. At 7 days, IL-4, IL-13, and IL-10 protein expression returned to baseline in all groups (Figure 8B,D,F). Treatment with IL-33 increased IL-5 plasma concentrations in comparison with the vehicle-treated groups at 4 days in MI and sham groups (97.56 + -4.94 (pg/ml) MI+IL-33 vs 0.44 + -0.21 (pg/ml) MI+vehicle) (****P<0.0001) and (

Figure 7 .

 7 Figure 7. Up-regulation of matrix metalloproteinases with IL-33 treatment at 7 days (A,B) Normalized MMP9 gene expression by quantitative RT-PCR at 4 and 7 days showing up-regulation of MMP9 with IL-33 treatment at 4 and 7 days (P<0.05). (C,D) Normalized MMP2 gene expression by quantitative RT-PCR at 4 and 7 days showing up-regulation of MMP2 with IL-33 treatment at 7 days post-MI (P<0.05) (n=4-6 per group). *P<0.05, **P<0.01, ****P<0.0001.

Figure 8 .

 8 Figure 8. Increased type 2 cytokine production with IL-33 treatment (A,B) IL-4 protein expression with representative bands at 4 and 7 days showing increased IL-4 cytokine expression in the MI group treated with IL-33 for 4 days (P<0.05) (n=3-4 per group). (C,D) IL-13 protein expression with representative bands at 4 and 7 days showing increased IL-13 cytokine expression in the MI group treated with IL-33 for 4 days (P<0.05). (E,F) IL-10 protein expression with representative bands at 4 and 7 days showing increased IL-10 cytokine expression in the MI group treated with IL-33 for 4 days (P<0.05). (G) Increased plasma concentrations of IL-5 at 4 days with IL-33 treatment. Plasma concentrations of IL-5 were examined by ELISA at 4 and 7 days post-MI (sham animals n=3 per group; MI animals n=5 per group). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 9 .

 9 Figure 9. Comparable neutrophil resolution in the heart between IL-33-treated and vehicle-treated MI groups at 7 days post-MI Time course variation (day 1 (D1), day 3 (D3) and day 7 (D7) post-surgery) of neutrophil percentage calculated out of CD45+ cells from, (A) the bone marrow (BM), neutrophil production in the bone marrow peaks at day 3 in all experimental groups when compared with day 1. (B) Spleen, neutrophil in ltration peaks at day 3 in the spleen in the MI group treated with IL-33. (C) Blood, neutrophils are at their highest levels at day 1 and decrease at day 7 in all experimental groups, and (D) heart, neutrophil in ltration drops signi cantly at day 7 in both MI groups. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Each dot represents one mouse (n=3 in sham-operated groups and n=5-6 in LAD-ligated groups).

Figure 10 .

 10 Figure 10. Dynamics of proinflammatory monocytes at different timepoints with and without IL-33 treatment Time course variation (day 1 (D1), day 3 (D3) and day 7 (D7) post-surgery) of monocytes percentage calculated out of total live cells from (A) the bone marrow (BM), monocyte production in the bone marrow peaks at day 3 in all experimental groups when compared with day 1. (B) Spleen, a signi cant decrease in monocyte in ltration in the spleen is observed in the MI group treated with IL-33 when compared with the MI group treated with vehicle at day 7. (C) Blood, circulating monocytes in the blood decrease signi cantly at day 7 in the MI group treated with IL-33 when compared with the MI group treated with vehicle, and (D) heart, monocyte levels are at their highest in both MI groups at day 1 and decrease gradually over time. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Each dot represents one mouse (n=3 in sham-operated groups and n=5-6 in LAD-ligated groups).

Figure 11 .

 11 Figure 11. Increased macrophage levels with IL-33 treatment at 7 days post-MI Time course variation (day 1 (D1), day 3 (D3) and day 7 (D7) post-surgery) of macrophage percentage calculated out of total live cells from, (A) the bone marrow (BM) macrophages in the bone marrow increase signi cantly with IL-33 treatment at day 3, (B) spleen, macrophage levels start increasing with IL-33 treatment as early as day 3 and become signi cantly more elevated in the MI group treated with IL-33 at day 7, (C) blood, a signi cant increase in macrophage circulation in the blood is observed with IL-33 treatment at day 7, and (D) heart, macrophage levels start increasing in the heart at day 3 and become signi cantly more pronounced in the MI group treated with IL-33 at day 7. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Each dot represents one mouse (n=3 in sham-operated groups and n=5-6 in LAD-ligated groups).

Figure 12 .

 12 Figure 12. IL-33 induces eosinophilia at day 7 regardless of the surgical intervention Time course variation (day 1 (D1), day 3 (D3) and day 7 (D7) post-surgery) of eosinophil percentage calculated out of CD45+ cells, (A) the bone marrow (BM), (B) spleen, (C) blood, and (D) heart. Eosinophil levels start increasing as early as day 3 in the sham and MI groups treated with IL-33 and become signi cantly more increased at day 7 in these two groups in all tissues.*P<0.05, ****P<0.0001. Each dot represents one mouse (n=3-4 in sham-operated groups and n=5-6 in LAD-ligated groups).

Figure 13 .

 13 Figure 13. Treatment with IL-33 increases ILC2 levels at 7 days post-MI Time course variation (day 1 (D1), day 3 (D3) and day 7 (D7) post-surgery) of ILC2 percentage calculated out of total live cells from, (A) the bone marrow (BM), ILC2 levels start increasing as early as day 3 with IL-33 treatment and become signi cantly more elevated in the MI group treated with IL-33 at day 7, (B) spleen, ILC2 levels peak in the spleen at day 7 in the MI group treated with IL-33, (C) blood, ILC2 circulation in the blood increases signi cantly at day 7 in the sham and MI groups treated with IL-33, and (D) heart, ILC2s increase signi cantly in the heart at day 7 in the MI group treated with IL-33. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Each dot represents one mouse. (n=3 in sham-operated groups and n=5-6 in LAD-ligated groups).

Figure 1 .

 1 Figure 1. Principal actions of IL (interleukin)-33 on certain immune cells that are relevant for heart function and remodeling. 1 CCL17 indicates chemokine (C-C motif) ligand 17; ILC2, group 2 innate lymphoid cells; IFN, interferon; LPS, lipopolysaccharides; MCP, monocyte chemoattractant protein; TNF, tumor necrosis factor; and Treg, tissue-resident regulatory T cells.

Figure 2 .

 2 Figure 2. Controversies surrounding the IL (interleukin)-33/sST2 axis in the heart. Although multiple studies highlight the use of circulating sST2 levels in cardiac diseases, there are many unresolved issues concerning the sources and stimuli for release of IL-33 and sST2, as well as the cellular targets of IL-33.Whether IL-33 is beneficial or harmful is also not known. In the (human) heart, endothelial cells are likely the major source for IL-33 and sST2 release, with a lesser contribution from fibroblasts and myocytes. Although undoubtedly important, the contribution of immune cells is unexplored. The primary target of IL-33 in the heart would seem to be endothelial cells. A direct effect via ST2L on fibroblasts and myocytes in the adult human heart is less certain. However, in these cells, loss or increase in nuclear IL-33 levels may impact on inflammatory gene expression (inset). Although likely targeted, the role of immune cells in IL-33 actions on the heart awaits investigation. Dendritic cells and possibly other immune cells may internalize IL-33, but the cytoplasmic or nuclear consequences of this are not known. Finally, the contribution of extracardiac sources of sST2 and IL-33, such as adipose tissue, liver, large vessels, and lungs, remains a possibility. CBM indicates chromatin binding motif; H2A and H2B, core histones; IL1AcP, IL-1 receptor accessory protein; ILC2, group 2 innate lymphoid cells; Treg, tissue-resident regulatory T cells; and VSMC, vascular smooth muscle cells.

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Figure 1

 1 Figure1Pro-IL-33 processing: Pro-IL-33 possesses three major domains including nuclear domain, activation domain, and interleukin-1 like cytokine domain. Following expression, pro-IL-33 may be processed into three major forms: (1) inactive forms, following cleavage by caspases 3 and 7 at interleukin-1 like cytokine domain if the cell undergoes apoptosis, (2) regulator of transcription, following localization to the nucleus due to the presence of two bipartite nuclear localization sequences in the nuclear domain, ubiquitination of IL-33 as well as its association with chromatin via protein-protein interaction is implicated in its activation/repression of transcription, and (3) active forms, also known as cytokine or alarmin, following cleavage by extracellular proteases including cathepsin G and elastase at the activation domain after being released extracellularly in response to cellular necrosis or stress. CBM, chromatinbinding motif; Ub, ubiquitination.

Figure 2

 2 Figure2IL-33 effects post-activation and release: active IL-33 binds to sST2 and ST2L. Upon binding to the decoy receptor sST2, the effects of IL-33 on the cardiovascular system are neutralized or diminished, promoting use of sST2 as a prognostic biomarker. Binding to ST2L receptor which together with the co-receptor IL-1R accessory protein (IL-1RAcP) recruits MYD88, IRAK1, IRAK4, and TRAF6, followed by activation of multiple signalling pathways, including ERK1/2, JNK, p38 MAPK, and NF-jB and subsequent activation and regulation of transcription. Cytokines secretion, immunomodulation, cell proliferation, activation, and survival contribute to observed effects of IL-33 on the cardiovascular system. IL-33 effects, although mostly cardioprotective, vary depending on the disease state and cell type. IR, insulin resistance; WAT, white adipose tissue; I/R, ischaemia/reperfusion; T2D, type II diabetes; CAD, coronary artery diseases; HF, heart failure; AS, aortic stenosis; ROS, reactive oxygen species; IBD, inflammatory bowel disease; COPD, chronic obstructive pulmonary disease.

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Figure 3

 3 Figure 3 Conflicting actions of IL-33 in atherosclerosis. IL-33 has a number of actions on endothelial and immune cells that promote inflammation and atherosclerosis. In contrast, evidence indicates that IL-33 can act on T cells, macrophages, and B cells to attenuate plaque development and progression. A better understanding of the temporal and spatial/cellular factors involved in regulating the actions of IL-33 is needed to reconcile its opposing actions in atherosclerosis.

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Figure 4

 4 Figure4Cell-type specific pro-and anti-inflammatory actions of IL-33. IL-33 also increases generation of sST2 by certain cells, which serves as a decoy receptor. Note that generalized responses are highlighted, and in some cases an opposite response may be elicited such as mast cell-induced bronchodilation in small airways. See text for additional details.

1.1.4.1 Cellular death of cardiomyocytes

  Death of cardiomyocytes begins within the first hour following coronary obstruction and marks the onset of the inflammatory response. Necrosis of cardiomyocytes triggers the

release of a variety of endogenous molecules, termed danger-associated molecular patterns (DAMPS) or alarmins, hich serve as danger signals that activate local proinflammatory

1.2.6 Mast cells

	rise to mature mast cells in a stem cell factor-dependent fashion [223]. In the heart, mast cells
	Following infarction, and due to the dynamic changes in growth factors and cytokine are mainly located near the vessels [224] and are capable of secreting a vast array of
	milieu, monocytes infiltrating the heart are also capable of acquiring a macrophage phenotype, inflammatory and profibrotic mediators. Following infarction, resident and infiltrating mast
	a process that can take place as early as day 1 post-MI [161]. Local upregulation of growth cells peak in the heart 7 days post-MI [223] and start degranulating and releasing large
	factors, such as macrophage colony stimulating factor (MCSF/CSF1), promotes monocyte quantities of pro-and antiinflammatory mediators including histamine, TNF-, pro eases,
	survival and induces monocyte-to-macrophage proliferation and differentiation into distinct lysosomal enzymes, cytokines and growth factors. While TNF-deri ing from mas cells
	macrophage subsets [218]. In a mouse model of experimental MI, evidence suggests that stimulates the expression of proinflammatory cytokines infiltrating the heart [165], histamine
	Ly6C(hi) monocytes expressing the orphan nuclear hormone receptor Nr4a1 [219] can give induces the expression of adhesion molecules such as P-selectin on the surface of endothelial
	rise locally to reparative Ly6C(lo)F480+(hi) macrophages, which exhibit significant cells. Furthermore, histamine released from mast cells post-MI promotes collagen synthesis
	proliferative activity [219]. In the infarcted myocardium, macrophages are highly dynamic and fibroblast growth in vitro. In addition, mast cells play a role in the fibrotic process where
	cells and are capable of acquiring unique phenotypic features in response to micro-increased mast cell accumulation was observed in areas of collagen deposition during the
	environmental cues. During the early phase of inflammation (1-3 days post-MI), classical proliferative phase of infarct healing [225]. In response to MI, mast cells play a key role in
	Tissue-residing cardiac macrophages, originating from the yolk sac and liver during proinflammatory M1-like macrophages dominate and are mainly activated by IFN-. M1-like preserving cardiac function by regulating postischemic cardiomyocyte contractility. Selective
	embryonic development, harbor the heart and are sustained by local proliferation [213]. macrophages promote the secretion of proinflammatory cytokines (IL-1 , TNF-), and MMPs depletion of mast cells post-MI leads to decreased cardiac function and reduced cardiomyocyte
	Moncoyte-derived macrophages can also enter the myocardium and become tissue-residing that induce inflammation, clearance of cell debris, and ECM degradation. Between 4 and 7 contractility, mainly due to the myofilament calcium desensitization [223] .
	macrophages [214]. The post-natal and embryonic resident cardiac macrophages can be days post-MI, macrophages convert to an antiinflammatory M2-like phenotype, and become
	distinguished from each other by their expression of the chemokine receptor CCR2 [215]. This the main effector cell type responsible for inflammation resolution. M2-like macrophages
	receptor and its matching ligand CCL2 play an essential role in the migration of monocytes. produce antiinflammatory, proreparative and proangiogenic factors such as IL-10, TGF-and Previous data has shown that while CCR2 -resident macrophages originate from embryonic VEGF that facilitate the engulfment of apoptotic cells and promote resolution of inflammation development, CCR2 + macrophages derive from monocytes [215, 216]. Additionally, CCR2 -and ECM deposition. TGF-prod ced b M2-like macrophages is associated with increased macrophages are self-replenished by local proliferation, whereas CCR2 + are replenished by production of type I and type III collagen [220, 221]. Shifting macrophages to the M2-like myocardial extravasation of monocyte-derived macrophages [215]. Both CCR2 -and CCR2 + phenotype improves cardiac function and tissue repair post-MI. Macrophages play therefore
	macrophage populations orchestrate several responses following cardiac injury such as MI. three major roles in tissue remodeling post-MI. First, they are able to clear the infarct from CCR2 + cells enable myocardial monocyte recruitment via trafficking mediated by CCR2-apoptotic cells and matrix debris. Second, they can secrete cytokines and growth factors that
	MCP1 and produce elevated levels of proinflammatory cytokines such as TNF-, IL-1 and regulate inflammation, promote tissue growth and fibroblast activation, and induce neovessel
	IL-6 [216]. In a murine MI model, reduced infarct size is observed following the depletion of formation and maturation. Third they can produce MMPs and their inhibitors, therefore this cell population [216] . In contrast, CCR2 -macrophages seem to play a pro-regenerative contributing to the remodeling process of the ECM [222].
	imuunomodulatory role by expressing high levels of the growth factors platelet-derived growth
	factor C (PDGF-C) and insulin-like growth factor 1 (IGF1) [216]. Depleting this CCR2 -
	population enhances cardiac monocyte/macrophage infiltration suggesting that these cells can
	have a potential immunomodulatory role during MI [216, 217]. Furthermore, CCR2 -
	macrophages highly express the sodium channel (SCN4) and sodium channel modulator
	(FGFI3) implicating these cells in the modulation of the cardiomyocyte electrical activity [147, Cardiac mast cells are immune cells originating from mast cell progenitors settled in white
	215]. adipose tissue and are recruited into the heart, where they proliferate and differentiate giving

1.2.7 Dendritic cells

	groups. Functions and gene-expression profiles of human CD141+DCs and CD1c+DCs mirror MI, the release of autoantigens associated with proinflammatory cytokine release (IL-1 , IL-
	those seen in mouse cDC1s and cDC2c respectively. Hence as a unifying nomenclature, human 1 , HMGB1, and IL-6), promotes the maturation and activation of DCs that consequently
	CD141+DCs are denoted cDC1s and CD1c+DC are denoted cDC2c [226]. prime cardiac m osin hea chain -specific CD4+-T cells to shift to a pathogenic (IL-17/IFN-
	) effec or pheno pe [233]. While this sequence might set the ground for increased
	DCs are antigen-presenting cells that play a central role in bridging innate and adaptive predisposition to autoimmune attacks post-MI, tolerance can still remain functional, even in
	immunity. Following tissue injury or pathogen invasion, splenic and bone marrow-derived the absence of this pathogenic autoimmune-prone background, and favor a protective Treg
	precursors as well as circulating monocytes differentiate into DC and accumulate in the local autoimmune response [234, 235].
	injured or infected tissue, exerting various functions on the immune system. In the context of
	MI, mature and activated DCs originating from the BM infiltrate the infarcted heart and
	culminate 5-7 days post-MI [161]. DC internalize and process antigens released from necrotic
	cardiomyocytes by lysosomal degradation and migrate to lymphoid organs such as the lymph
	nodes and spleen, where they present processed peptides on both class I and class II major
	histocompatibility complex (MHC) molecules to naïve T lymphocytes [227].
	In experimental MI, both detrimental and beneficial effects of DCs were observed
	during post-infarct healing. While deterioration in LV remodeling was associated with
	increased numbers of mature DCs in the infarcted heart [228], selective depletion of DCs
	showed enhanced inflammation, decreased survival and worsened LV function and remodeling
	28 days post-MI [229]. Furthermore, depletion of DCs affected infarct healing by increasing
	on one hand myocardial matrix degradation by MMPs (mainly by increased MMP9 and MMP2
	activity) and promoting prominent collagen deposition in the peri-infarct area on the other. In
	Dendritic cells (DCs) arise from adult HSC-derived progenitors, which are different human infarcted myocardial tissue, decreased numbers of DC was associated with impaired
	from the progenitors of monocytes and macrophages, and are therefore classified as a distinct reparative fibrosis and the development of cardiac rupture soon post-MI [230]. The roles of
	lineage of mononuclear phagocytes. DCs are subdivided into three major subtypes: classical cDc and pDC have also been investigated in MI. While cDC depletion improved systolic
	pe 1 DCs (for CD103+ and CD8 + DCs), classical pe 2 DCs (for CD172a+CD11b+ DCs), function and decreased infarct size, pDC depletion had no impact on cardiac function [231].
	and plasmacytoid DCs, also known as interferon-producing cells (IPCs). Differentiation of DC Tolerogenic DCs (tol-DCs) are another subset of DCs involved in the MI pathophysiology
	into distinct subtypes is governed by specific sets of transcription factors. While the where treatment of MI mice with tol-DCs improved cardiac function and prolonged survival
	differentiation of cDC1 and cDC2 depends on the transcription factor interferon regulatory mainly via MI-targeted Treg cell production. Activation of Tregs enhanced M2-like
	factor (IRF)-8 and IRF-4 respectively, pDCs are regulated by the transcription factor E-E2 macrophage polarization, promoted myocardial neovascularization and consequently limited
	[226]. Furthermore, monocyte-derived (Mo) DCs or myeloid-derived suppressor cells cardiac remodeling post-MI [232]. During homeostasis, DCs are capable of governing
	(MDSCs) are cells derived from Ly6C(hi) monocytes based on restricted functional properties tolerance to autoantigens, where IRF8-dependent cDC1s present cardiac myosin heavy chain
	that are not easily assessed in vivo. In humans, DCs present in lymphoid and non-lymphoid to CD4+T-cells in heart-draining mediastinal lymph nodes. DCs drive the differentiation and
	tissues are subdivided into two main subsets: myeloid (or classical) DCs and pDCs. Based on expansion of these CD4+T-cells into a cardioprotective Treg phenotype [233]. In the setting of
	their expression of CD141 and CD1c, classical DCs can be subdivided into two separate

1.3.1 History of IL-33

	Elevated serum levels of sST2 can predict negative outcomes in heart failure [274], pulmonary
	arterial hypertension [275] and various respiratory diseases [276].
	IL-33 was first discovered in 1995, during a search for the ligand that associates with
	the ST2 receptor [277]. Kumar and his colleagues depicted the production of two proteins by
	quiescent 3T3 cells that were precipitated by the fusion protein ST2-Fc. Having a mass of
	~32kD and 18kD, these proteins represented the full length and mature forms of IL-33
	respectively, but at that time, IL-33 was still not yet identified [277]. In 1999, IL-33 was
	originally named DVS-27, a messenger RNA (mRNA) highly upregulated during subarachnoid
	hemorrhage in the canine vasospastic cerebral vasculature [278]. In 2003, endogenous IL-33
	protein and mRNA were detected for the first time in human and mouse tissues. The IL-33
	Moreover, IL-33 can protect against allograft rejection by promoting Th17 to
	Th2 cytokine switch, inducing Treg expansion and reducing antibody-mediated rejection [273].

2 Materials and Methods 2.1 Experimental design

	Animals were housed in sterilized cages and maintained on a 12 h (light:dark) cycle
	with access to autoclaved rodent chow and water ad libitum. Operating procedures were
	performed in the animal care facility of the American University of Beirut Medical Center
	(AUBMC). All experimental protocols were reviewed and approved by the Institutional
	Animal Care and Use Committee of the AUB (IACUC # 18-2-RN560). Adult WT male
	C57/BL6J mice (12-14 weeks of age) were randomized into four different groups:
	sham+vehicle, sham+IL-33, MI+vehicle, and MI+IL33. In the sham and MI groups treated
	with vehicle, a single daily injection of PBS (200µl/day) was intraperitoneally (i.p.)

administered right after sham operation or left anterior descending (LAD) ligation, starting day 0 and continued for 4 or 7 days thereafter. IL-33 dose and route of administration were based on previous studies

[46, 47]

. In the sham and MI groups treated with IL-33, a single daily injection of mouse recombinant IL-33 (BioLegend, San Diego, CA, USA) (1µg suspended in 200µl PBS) was administered i.p. right after sham operation or LAD ligation (starting day 0)

Table 3 -2 Flow cytometry panels showing antibodies and their conjugated fluorochromes.

 3

	Pane l #	Antibody with conjugated fluorochrome	Clone	Catalog #	volume/test (µl)
		L/D DAPI		1306	0.5
		CD11b-efluor450	M1/70	48-0112-82	0.625
		CD11c-PECy7	HL3	558079	1
	1
		Ly6G-PE	1A8	551461	1
		F480 APC	BM8	123116	1
		Ly6C FITC	AL-21	553104	1
		L/D DAPI		1306	0.5
		CD11b-efluor450	M1/70	553104	0.625
		CD11c-PECy7	HL3	558079	1
		Ly6G PE	1A8	551461	1
		NK1.1 APC	PK-136	17-5941-82	0.625
	2
		CD45 APC-efluor 780	30-F11	47-0451-82	0.625
		B220 (CD45R) V500	RA3-6B2 (RUO)	561226	1
		IgM PerCP efluor 710	II/41	46-5790-82	2
		CD19 PEefluor 610	1D3	61-0193-82	1
		SiglecF FITC	S17007L	155504	2
		L/D DAPI		1306	0.5
		ICOS APC	C398.4A	17-9949-82	0.625
	3
		CD25 PE	PC61.5	12-0251-81	0.625
		Lin**-FITC			2.5
		DAPI		1306	0.5
		CD45 BV510	30-F11	103137	1
	4	CD3 PerCP5.5	17A2	100218	1
		CD8 PeCy7	53-6.7	100722	1
		CD4 APC Cy7	RM4-5	100526	1
	For staining, 50µl of a PBS mix containing (50µl of PBS + the volume in (µl) of each antibody listed from each
	panel (volume /test (µl))) were added to 100µl of cells collected from each organ ((heart, spleen, blood, or bone
	marrow).

** For panel# 3, lineage antibodies are listed in

Table 3 -3 Lineage cocktail for Panel#3.

 3

	Panel #3	Antibody with labelled fluorochrome	Clone	Catalog #	volume/test (µl)
		CD3e FITC	145-2C11	100305	0.6
		CD4 FITC	RM4-5	100509	0.5
		CD19 FITC	1D3	152404	0.25
		CD11b FITC	M1/70	101205	0.5
	**Lineage	Gr1 FITC NK1.1 FITC FCeRI FITC	RB6-8C5 PK136 MAR-1	108405 108705 134305	0.5 0.5 0.5
		Ter119 FITC	TER 119	116205	0.5
		CD11c FITC	N418	117305	0.5
		CD8 FITC	53-6.7	100705	0.5

Lineage antibodies were mixed together based on the volumes required in the table and 2.5 µl were taken from this mix and added to the antibody mix of panel#3.

Table 3 -4 Characterization of immune cell populations and their corresponding conjugated fluorochromes.

 3

	Panel#	Immune Cells	Characterization	Fluorochromes
		Monocytes	CD11b+Ly6C(hi)F4/80(lo)	e450+ FITC(hi) APC (lo)
	1	Macrophages	CD11b+Ly6C(lo)F4/80+	e450+ FITC (lo) APC+
		Dendritic cells	CD11b+CD11c(hi)	e450+ PeCy7(hi)
		Natural Killers	CD45+CD11b+Ly6G-NK1.1+	APC Cy7+ e450+ PE-APC+
		Neutrophils	CD45+CD11b+Ly6G(hi)	APC Cy7+ e450+ PE(hi)
	2	B lymphocytes	CD45+B220+IgM+CD19+	APC Cy7+ V500+ PerCP Cy5.5+ PE texas red+
		Eosinophils	CD45+SiglecF(hi)CD11c-	APC Cy7+ FITC (hi) PeCy7-
	3	ILC2	Lin-ICOS+CD25+	FITC-APC+ PE+
		T helper cells	CD45+CD3+CD4+CD8-	V500+ PerCPCy5.5+ APC Cy7+ PeCy7-
	4
		Cytotoxic T cells	CD45+CD3+CD8+CD4-	V500+ PerCPCy5.5+ PeCy7+ APC Cy7-

Table 3 -5 Variation of weight over time with and without IL-33 treatment.

 3

	Days post-surgery	Sham+Vehicle	Sham+IL-33	MI+Vehicle	MI+IL-33
	4	26.1±0.77	27.18±0.65	24.75±0.90	24.8±0.45
	7	27.32±0.67	28.39±0.58	24.21±0.47 **	23.79±0.44 α , ß
	Values are Mean ±SEM. is significance vs sham+vehicle; ß is significance vs sham+IL-33. Sham-operated
	animals (n=8-11); MI-operated animals (n=12-17). One-a ANOVA follo ed b T ke s pos -hoc test. **p
	0.01, ****p 0.0001.

  association study in Han Chinese identifies four new susceptibility loci for coronary artery disease. Nat Genet, 2012. 44(8): p. 890-4. 396. Schunkert, H., et al., Large-scale association analysis identifies 13 new susceptibility loci for coronary artery disease. Nat Genet, 2011. 43(4): p. 333-8. 397. Wang, F., et al., Genome-wide association identifies a susceptibility locus for coronary artery disease in the Chinese Han population. Nat Genet, 2011. 43(4): p. 345-9. 398. Consortium, C.A.D., et al., Large-scale association analysis identifies new risk loci for coronary artery disease. Nat Genet, 2013. 45(1): p. 25-33. 399. Peden, J.F. and M. Farrall, Thirty-five common variants for coronary artery disease: the fruits of much collaborative labour. Hum Mol Genet, 2011. 20(R2): p. R198-205. 400. Prins, B.P., et al., Genetics of coronary artery disease: genome-wide association studies and beyond. Atherosclerosis, 2012. 225(1): p. 1-10. 401. Chen, Y., et al., IL-16 rs11556218 gene polymorphism is associated with coronary artery disease in the Chinese Han population. Clin Biochem, 2011. 44(13): p. 1041-4. 402. Blagodatskikh, K.A., et al., [Gene IL6 G(-174)C and gene IL10 G(-1082)A polymorphisms are associated with unfavourable outcomes in patients with acute coronary

	652-60.
	405. Yang, J.H., et al., Association of IL33/ST2 signal pathway gene polymorphisms with
	myocardial infarction in a Chinese Han population. J Huazhong Univ Sci Technolog Med
	Sci, 2015. 35(1): p. 16-20.
	406. Tsapaki, A., et al., Genetic variability of the distal promoter of the ST2 gene is
	associated with angiographic severity of coronary artery disease. J Thromb
	Thrombolysis, 2010. 30(3): p. 365-71.
	407. Ho, J.E., et al., Common genetic variation at the IL1RL1 locus regulates IL-33/ST2
	signaling. J Clin Invest, 2013. 123(10): p. 4208-18.
	408. Akhabir, L. and A. Sandford, Genetics of interleukin 1 receptor-like 1 in immune and
	inflammatory diseases. Curr Genomics, 2010. 11(8): p. 591-606.
	409. Wu, F., et al., A functional variant in ST2 gene is associated with risk of hypertension
	via interfering MiR-202-3p. J Cell Mol Med, 2017.
	410. Wu, F., et al., Associations between variants in IL-33/ST2 signaling pathway genes and
	coronary heart disease risk. Int J Mol Sci, 2014. 15(12): p. 23227-39.
	411. Addison, W., Experimental and Practical Researches on the Blood: Second Series. Prov
	Med J Retrosp Med Sci, 1843. 6(152): p. 444-5.

syndrome]. Mol Biol (Mosk), 2010. 44(5): p. 839-46. 403. Zhang, X., et al., Interleukin-17A gene variants and risk of coronary artery disease: a large angiography-based study. Clin Chim Acta, 2011. 412(3-4): p. 327-31. 404. Tu, X., et al., The IL-33-ST2L pathway is associated with coronary artery disease in a Chinese Han population. Am J Hum Genet, 2013. 93(4): p.

Table 1

 1 Primers used for RT-PCR analysis

	Mouse	mRNA forward primer (5 -3 )	R e v e r s e p r i m e r ( 5 -3 )
	HPRT	GTTGGGCTTACCTCACTGCT	TAATCACGACGCTGGGACTG
	sST2	ACGCTCGACTTATCCTGTGG	CAGGTCAATTGTTGGACACG
	BAX	ATCCAAGACCAGGGTGGCT	CCTTCCCCCATTCATCCCAG
	BCL2	AGTACCTGAACCGGCATCTG	TATGCACCCAGAGTGATGCAG
	IL-5	ATGAGGCTTCCTGTCCCTACT	TACCCCCACGGACAGTTTGA
	MMP2	AGATGCAGAAGTTCTTTGGGCTGC	AGTTGTAGTTGGCCACATCTGGGT
	MMP9	ACCACAGCCAACTATGACCAGGAT	AAGAGTACTGCTTGCCCAGGAAGA
	CTGF	ACCCAACTATGATGCGAGCC	GGTAACTCGGGTGGAGATGC

Table 2

 2 Flow cytometry panels showing antibodies and their corresponding fluorochromes and the targeted cell populations

	Panel
	number	Antibodies

Table . Diagnostic or Prognostic Use of sST2 for Various Cardiomyopathies

 .

	Cardiomyopathy	n	Outcome
	Acute Kawasaki disease 113	55 acute Kawasaki disease (KD)	Elevated sST2 in acute KD vs convalescent KD and HC subjects.
		subjects (30 with paired convalescent
		samples), 54 FC, 50 HC children
			sST2 levels negatively correlated with illness days and deceleration time.
			NT-proBNP and sST2 levels correlated with impaired myocardial relaxation and
			increased oxidative stress in acute KD.
	Arrhythmogenic right ventricular	44 subjects (41±15 y old, 23 males, 21	sST2 associated with RV and LV function and ventricular arrhythmias.
	cardiomyopathy 114	females)
			sST2 more elevated in males than in females.
			sST2 associated with weight and BMI.
			No correlation between elevated sST2 and history of ventricular arrhythmias.
	HIV 115	332 HIV-infected controls and 50 age-	Increased levels of all biomarkers in HIV-infected patients except for sST2, IL-6
		and sex-matched controls	(after controlling for age, sex and estimated glomerular filtration rate/GFR).
			Association between sST2 levels and DD: each doubling of sST2 corresponded
			to 43% increased risk.
			sST2 remained independently related to DD after adjustment for cardiovascular
			disease and HIV-related risk factors.
			Strong association between sST2 and all-cause mortality: each doubling of
			ST2 corresponded to a 104% increased risk of death.
	Idiopathic dilated cardiomyopathy 116,117	107 DCM patients (50.3±17.5 y) 116	Correlation between sST2 and all-cause mortality with stable chronic heart
	(nonischemic/nonhypertensive)		failure because of nonischemic DCM. 116
		102 patients (≥18 y old) with LVEF of	Association between sST2 and severity of heart failure in patients admitted for
		26±10% and diagnosis of ICM 117	assessment of DCM. 117
			Patients with markedly elevated sST2 concentration at baseline prone to
			subsequent death from worsening heart failure or transplantation. 117
			Increased sST2 levels associated with signs of hemodynamic decompensation,
			including elevated heart rate and RAP in nonischemic heart failure patients. 117
			Attenuation of hemodynamic parameters with clinical improvement while sST2
			distribution narrows. 117
	TTC 118,119	64 patients: 21 with TTC and 43 with	Elevated ST2 within first 12 h of hospitalization correlated with increased mortality,
		AMI (either ST or non-ST elevation) 118	cardiovascular complication, and worse outcome in TTC and CAD patients. 118
		76 patients (64±17 y old; 46 males, 30
		females) admitted to noncardiac MICU 119

Table 1

 1 Principal immune cells responsive to IL-33 Increases circulating IL-10-producing B cells15 • Enhances proliferation capacity of B1 B cells and IgM, IL-5, and IL-13 production16

	Immune cell type	Action
	B cells • Basophils • Promotes secretion of type 2 cytokines (e.g. IL-4 and IL-13) and IL-8 in synergy with IL-3 and/or FcRI-activation and enhances
		FcRI-induced mediator release

  17

  19

  • IL-33-activated murine DCs required for in vitro and in vivo expansion of ST2þ Tregs due to IL-2 production21 • IL-33-activated DCs prime naive lymphocytes to produce the Th2 cytokines IL-5 and IL-13 but not IL-4 and IFN-c18,20 Eosinophils• Regulates homeostatic development and activation during disease22 • Enhances adhesion, CD11b expression, and survival23 • Induces superoxide anion production, degranulation, and IL-8 production24

  • Exacerbates eosinophil-mediated airway inflammation (increases IL-13, TGF-b, CCL3, CCL17, and CCL24)

  25

  • Enhances Siglec-8 mediated apoptosis26

  80,81

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Acknowledgments

I would like to express my most profound respect and appreciation to my principal supervisor,

Acknowledgements

The authors thank Mr. Ali Mroueh for helping with the IL-33 ELISA and TUNEL assay and Miss Maria Esmerian for ow data acquisition.

Sources of Funding

This work was supported by grants to F.A. Zouein from the American University of Beirut Faculty of Medicine (MPP-320145). R. Altara is supported by the South-Eastern Norway Regional Health Authority (HSØ-RHF), project no. 2016089. G.W. Booz acknowledges the support of the University of Mississippi Medical Center Department of Pharmacology and Toxicology.

Funding

This work was supported by the American University of Beirut Faculty of Medicine [MPP -320145 (to F.A.Z.)]; the British Heart Foundation [grant numbers CH/10/001/27642 (to Z.M.), 1659].

Funding

Grants to FAZ from the American University of Beirut Faculty of Medicine (MPP -320145); South-Eastern Norway Regional Health Authority (HSØ-RHF), project #2016089 to R.A.

source for type 2 cytokine production. A recent study has shown that IL-33 markedly increased the secretion of IL-4 and IL-13 from eosinophils in a GM-CSF-dependent fashion [572]. It would be interesting to explore in future studies whether the signaling pathways downstream the IL-33 and GM-CSF receptors could converge to induce type-2 cytokine secretion from eosinophils. For instance, STAT5 could be an interesting signaling pathway to explore since GM-CSF and IL-5 are both potent STAT5 activators and eosinophils deficient in STAT5 fail to acquire IL-4 producing capacity [666].

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Author Contribution

F.A.Z. and Z.M. contributed to the study design and conception. R.G., N.J.H., A.K., C.T., E.A., A.B., R.F., H.I., A.J., G.W.B., Z.M., and F.A.Z. participated in the acquisition, analysis or interpretation of the data. R.G. performed and supervised all the experiments. F.A.Z. and R.G. drafted the manuscript. All authors judiciously revised the manuscript and gave their nal approval for all the data presented, ensuring reliability and integrity.

Abbreviations

ANOVA, analysis of variance; CO, cardiac output; CSA, cross-sectional area; CTGF, connective tissue growth factor; DAPI, 4,6-diamino-2-phenylindole; ECM, extracellular matrix; EF, ejection fraction; ELISA, enzyme-linked immunosorbent assay; FS, fractional shortening; FSC, forward scatter; IL, interleukin; ILC2, type 2 innate lymphoid cell; i.p., intraperitoneally; LAD, left anterior descending; LV, left ventricle; LVEDD, LV end-diastolic diameter; LVEDV, LV end-diastolic volume; LVESD, LV end-systolic diameter; LVESV, LV end-systolic volume; MI, myocardial infarction; MMP, matrix metalloproteinase; MTC, Masson's Trichrome; NF-κB, nuclear factor-κB; PI3K, phosphoinositide 3-kinase; RIPA, Radioimmunoprecipitation assay; RNA, ribonucleic acid; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SSC, side scatter; ST2, serum stimulation 2; ST2L, long or transmembrane isoform of ST2; sST2, soluble ST2; TBST, Tris-buffered saline-Tween; TGF-β, transforming growth factor β; Th2, helper T cell; TNF-α, tumor necrosis factor-α; TTC, 2,3,5 triphenyltetrazolium chloride; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; α-SMA, α smooth muscle actin. Raffaele Altara 1,2,3 ,Rana Ghali 4 ,Ziad Mallat 5,6 ,Alessandro Cataliotti 1,2 sST2 not correlated with stable CAD severity, but higher levels independent predictor for all-cause mortality and cardiovascular death 7 Only sST2 and hs-CRP predicted cardiac death or heart failure hospitalization in both diabetics and non-diabetics with CAD 89 Higher levels independently predicted short-and long-term risks for total mortality, and short-term risk for fatal cardiovascular events 90 Higher levels associated with increased all-cause and cardiovascular mortality 91

Disclosures

Conflicting vascular and metabolic impact of the IL-33/sST2 axis

STEMI sST2

Levels correlated positively with heart damage 92,93 Positively correlated with infarct size and expansion, as well as greater infarct transmurality and endocardial extent, microvascular obstruction, and plasma aldosterone 94 Early values predict increased mortality and heart failure over subsequent 30 days, independent of baseline characteristics or NT-proBNP levels; improved risk stratification in combination with NT-proBNP 93,95 Predictor of heart failure or short-term cardiovascular death along with troponin T and MPO 96 Greater prognostic value than hs-cTnI for 30 day cardiac mortality in both STEMI and NSTEMI patients 97 Elevated levels associated with increased all-cause mortality out to 1 year and improved risk stratification in multimarker approach 98 NSTEMI sST2 Elevated sST2 predicated long-term major adverse event but did not improve risk stratification for established markers 99 Elevated sST2 associated with increased risk of death and heart failure over next 5 years, independent of other prognostic indicators; higher values associated with age, female sex, hypertension, and diabetes 100

Higher levels associated with adverse outcomes at 30 days and 1 year; improved risk stratification in CVD and heart failure at 30 days and 1 year when levels added to established clinical biomarkers 101 Elevated levels predict mortality at 1 year; independent of cardiovascular (CV) Higher levels in type 2 diabetes 4,10,110-112 Positive association of levels with risk factors for diabetes after adjusting for age and sex; highest increases associated with increased risk for diabetes 9 Association of hs-TnT and sST2 with cardiovascular and all-cause mortality during 5 year follow-up among diabetics 113 Levels among diabetics increased further by LV diastolic dysfunction 111,114 Association of severe obesity with increased expression in endothelial cells of human adipose tissue 109

Il-33

Levels lower in non-lean vs. lean individuals, and negatively correlated with BMI and body weight in those lean and overweight, but not obese 115 ; negatively correlated with HbA1c in non-diabetic persons, and associated with protective lipid profile Severe obesity associated with increased expression in endothelial cells of human adipose tissue 109

Conflict of interest: none declared.