Hanine El

Fareeha Batool

Céline Stutz

Abdul Wahid Qureshi

Fatiha El Ghazouani

Olivier Huck

Florence Toti

Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection

Background: Properties and characterization……………………………………...22 I.2 Mechanism of MV generation …………………………………………………….28 I.3 Methods of isolation from tissues, quantification and characterization………………………………………………………………………..31 Chapter II: Tissular and vascular Microvesicles: biomarkers and cellular effectors………………………………………………………………………………33 II.1 Microvesicles and vascular inflammatory response……………………………...35 II.2 Microvesicles in atherosclerosis and thrombosis…………………………………37 Chapter III. Periodontitis: a local infection with cardiovascular issues………….40 III.1 1 Periodontitis and clinical characteristics ………………………………………41 III.2 Porphyromonas gingivalis (P. gingivalis): Role in inflammation and immune response ……………………………………………………………………………….41 III.3 Periodontitis and endothelial dysfunction ………………………………………..47 III.4 Periodontitis and systemic diseases: impact of Porphyromonas Gingivalis on cardiovascular disorders……………………………….........

Aknowledgements

"Through patience, great things are accomplished" Imam Ali (AS) 26 August 2016 was the first day for me in France. My journey started in LaRochelle University for my biotechnology master's degree, where I met Dr. Stéphanie Bordenave-Juchereau, the head of biotechnology department. I would like to thank her for welcoming and supporting me during the whole master program period and honoring me by accepting to be a member of my thesis jury. Please accept my deepest gratitude and my most respectful thanks. I would like also to express my sincere appreciation for Dr Nicolas Bridiau, Dr Michel Goldberg and mme Isabelle Girouy for their kindness and continuous support during my master's program.

Spring 2017 changed my life. I moved to Strasbourg for my first master's training in the laboratory of Pr. Florence Toti who accepted me also for my second master's training and is still supervising my PhD thesis until today. I don't know how to describe this lady. I know she's strong, so strong, and a true human being which is really rare in nowadays. She believed in me since the beginning. I didn't believe in me in the beginning. Beside support, encouragement and freedom to follow my thoughts, she has the supreme art to awake the joy in me in a creative way of her knowledge expression, and I don't think I shall meet another person like her again in my life. Thank you isn't enough for you Mme, I hope I can fit your expectations in my future career. I would like to say thank you to Pr. Olivier Huck, who contributed to the discussions and helped to shape this project. The joy and enthusiasm he has for his research is contagious and so motivational. The respect for timing and his full availability although he's a clinician means a lot to me, without him, the project wouldn't be done, thus I'm honored to share with him already two publications and still working for more. Beyond respect and high appreciation, working with him is a great personal pleasure for me. I am also pleased to thank Dr Eduardo Angles Cano, in Paris, who introduced me to the culture of NETs, and gave me the honor to be trained by him for my experiments. Our meetings and discussions helped to progress in this project and to update the design of the experiments, not mentioning the long phone call conversations that were more than helpful and joyful for me. I really appreciate him for sharing his insightful suggestions.

Undertaking this PhD would not have been possible to do without the support and guidance that I received from many people, hereby, I would like to acknowledge the valuable input of all my lab colleagues in UMR1260, and to express my sincere thanks to our director mme Nadia Benkirane-Jessel, It was great pleasure working with them.

My time at Strasbourg was enjoyable in large part due to the many friends and groups that became a part of my life. I am grateful for the time spent with them. I gratefully acknowledge ADDAL (Association des doctorants et des docteurs en Alsace), who welcomed me as a board member for the last four years, which helped me to get know new people from different cultures, to lead scientific workshops and to enlarge my network.

Finally, to you, although we don't speak today, I will never forget you were the first one to tell me that I'm definitely a PhD fellow, back in 2013, when I didn't even know what PhD is, thank you.

Lastly, I would like to thank my family, for listening to all the boring details of my thesis, hoping that things are much clear now. Thank you to my Grandfather, who left us so early. Your stood next to me in 2009 when you couldn't even walk is the most dominant memory I keep in my mind. Thank you for giving me the strongest father in the world. Thank you to my parents, whom without, I am nothing. Thank you, Dad for teaching me not to be afraid of anything, I'm at my best just because I'm your daughter. Thank you, Mom, for being the best will example in my life. Thank you, Joumana, whom without, I wouldn't be who I am today. Thank you, my brothers and sisters. Thank you, Hassan for being my big brother and big sister during all my life. Thank you Rim, Mahmoud, Maya for everything we lived together. Thank you, Bilal, my brother in law for the joy you added to our family. You're the best family anyone can have, I want you to know that, as always, and despite distances, I shall always be there for you. Thank you to the bravest survivor I know since twenty-two years, my best friend Tania, I'm waiting for you. To my nephew Ali, that I didn't meet yet, I'll make sure I'll be your best aunt.

If I go back thirteen years ago from now, having a bachelor's degree would've been the highest thing I could aim for. Thank you, God, for giving me everything I didn't expect, and still until today, without me asking for.

Hanine El Itawi Strasbourg, France

LIST OF FIGURES

LIST OF TABLES

RESUME DES TRAVAUX

Rate et coeur, un axe fonctionnel ?

La parodontite est une maladie inflammatoire buccale d'origine infectieuse très répandue et la principale cause de perte de dents chez l'adulte. Elle est associée à une destruction progressive des tissus parodontaux conduisant à la formation de poches parodontales.

Étant impliqué dans l'apparition et le développement de nombreuses pathologies systémiques telles que les maladies cardiovasculaires et l'athérothrombose, la parodontite a été associée à une aggravation cardiovasculaire (Sanz M.et al 2020).

La progression des connaissances sur la physiopathologie sous-jacente à ces maladies a permis de mettre en évidence des liens entre eux tant au niveau clinique qu'aux niveaux cellulaires et moléculaires, dont les mécanismes initiateurs et régulateurs restent mal connus.

Les microvésicules (MV) sont des fragments de membrane plasmique libérés en réponse au stress cellulaire qui agissent comme des effecteurs cellulaires.

Elles sont émises de la membrane plasmique des cellules activées ou apoptotiques, initialement caractérisées par leur activité procoagulante et qualifiées de "microparticules" (Ridger et al, 2017). Elles sont émises par tous les types de cellules selon leur capacité de remodelage de la membrane Microvesicles (MVs) are plasma membrane fragments shed in response to cell stress that act as cellular procoagulant effectors. Previous data from the laboratory have shown that MVs may act as pro-inflammatory mediators promoting premature senescence of coronary artery endothelial cells 4,5 leading to endothelial dysfunction and the release of prosenescent MVs and that infection by LPS of Porphyromonas Gingivalis, the main pathogen in periodontitis leads to the emission of proapoptotic microvesicles (MVs) by HUVEC endothelial cells. 6 Previous In vitro data showed that LPS induces the shedding of procoagulant MVs from rat spleen that are able to promote endothelial senescence and a pro-inflammatory and procoagulant responses. 4 The feasibility of a pharmacological control of the release of prosenescent MVs from the spleen was further demonstrated in a rat model. Oral treatment by an optimized omega-3 formula was found protective against both the rate of the release of spleen MVs in middle-aged and aged rats, and their prosenescent noxious properties towards the coronary endothelium. 7 The thesis work aims to study the impact of MVs and NETs (neutrophil extracellular traps) as actors of the remote cell response after periodontitis and a possible cause of thrombotic tendency in patients. Experiments are designed to gain a better knowledge of the interactions between local teeth infection, the spleen response and the cardiovascular tissues. They focus on the endothelial switch from a protective to a proinflammatory and procoagulant and prosenescent surface mediated by P. Gingivalis-induced MVs.

Chapter I Microvesicles: Extracellular vesicles shed from the activated plasma membrane

I.1 Background: Properties and characterization of extracellular vesicles

The International society of extracellular vesicles (ISEV) underwrites extracellular vesicle (EV) as the generic term for particles naturally released from the cell that are delimited by a lipid bilayer, precluding a functional nucleus and asserting they cannot replicate. 8 Extracellular vesicles are designated in the literature as microparticles, microvesicles, or exosomes or apoptotic bodies. 9 All extracellular vesicles are distinguished on the basis of their subcellular origin, their size, their content and the mechanism leading to their formation. 10 (see fig 1)

Exosomes

Exosomes are small, single-membrane, secreted organelles of ∼30 nm to ∼100 nm in diameter having the same form as the cell and enriched in proteins, lipids, nucleic acids, and glycoconjugates. 12 They are present in many biological fluids, including urine, 13 blood, 14 ascites, 15 and cerebrospinal fluid, 16 fractions of body fluids such as serum and plasma, and cultured medium of cell cultures . Exosomes are usually isolated by ultracentrifugation (100,000-200,000g). 17 Regarding their biochemical composition, exosomes are surrounded by a phospholipid membrane containing relatively high levels of cholesterol, sphingomyelin, and ceramide and containing detergent-resistant membrane domains (lipid rafts) 18

Apoptotic bodies

Apoptosis is a highly regulated process of cell death. 19 During apoptosis, starting with the nuclear chromatin condensation, the process is followed by plasma membrane budding, progressing to the breakdown of the cellular content into distinct membrane enclosed vesicles termed apoptotic bodies or apoptosomes. 20 Identified by Kerr, Wyllie in 1972 21 , apoptotic bodies are membrane structures that package cell organelles such as mitochondria, nuclear fragments, granules, cleaved DNA … 22 They expose phosphatidylserine and contain DNA and RNA originating from nuclear membrane fragmentation. 23 (see table 1)

Microvesicles (MVs)

Initially identified by Wolf 1967 as a platelet dust containing phospholipids, and capable of supporting coagulation, 25 microvesicles (MVs), also named microparticles, oncosomes 26 or ectosomes 27 , are submicron fragments of the plasma membrane that spill into the extracellular space of cells under cellular stress conditions. 10 The ectosome nomenclature derives from the definition of ectocytosis introduced in 1991 by Stein and Luzio to describe the shedding of vesicles from the plasma membrane of stimulated neutrophils, [START_REF] Stein | Ectocytosis caused by sublytic autologous complement attack on human neutrophils. The sorting of endogenous plasma-membrane proteins and lipids into shed vesicles[END_REF] while oncosomes are atypical large cancer-cell derived extracellular vesicles.

However, when confirmation of the vesicles identity is achieved according to the recommendations of the ISEV 2018 norms, the most likely term to be used nowadays is microvesicles. 8 MVs are released in biological fluids such as blood, urine, tears and saliva. In the blood, they circulate at low concentration in healthy individuals and their levels are modified in various cardiovascular and chronic diseases including diabetes, chronic renal failure, pre-eclampsia and hypertension. 24 Ranging in size between 100 to 1000 nm in diameter, they are released during cell-surface budding, and considered as physiological liposomes, since formed from the cell plasma membrane lipid bilayer. The MV membrane embeds a content of active proteins, signaling nucleotides, and metabolites as well as RNA, noncoding RNA and bioactive lipids. 9 MVs expose phosphatidylserine, a procoagulant phospholipid.

Plasma membrane phospholipid distribution

Eukaryotic cells are surrounded by a lipid bilayer subdivided into compartments with distinct structural and functional identities termed rafts. Where glycerophospholipids forms almost 70% of the total lipid content of mammalian cells, the other 30% consists of cholesterol, sphingomyelin, and glycosphingolipids. Among the phospholipids, phosphatidylcholine (PC) is the most prevalent and accounts for 40%-50% of the total, [START_REF] Vance | Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells[END_REF] whereas phosphatidylserine (PS) is the commensurate trifling constituent of most biological membranes. [START_REF] Callahan | Phosphatidylserine on HIV envelope is a cofactor for infection of monocytic cells[END_REF] In addition, the content of conventional phospholipids varies among organelles.

Membrane phospholipid asymmetry is characteristic of eukaryote cells, and is regulated by an active process by flipases and floppases. [START_REF] Van Meer | Dynamic transbilayer lipid asymmetry[END_REF] In resting cells phosphatidylserine is sequestered in the inner leaflet of the plasma membrane. [START_REF] Marquardt | Asymmetric lipid membranes: towards more realistic model systems[END_REF]

Phosphatidylserine

Phosphatidylserine (PS) species were first identified by Folch and coworkers in the 1940s. [START_REF] Leventis | The distribution and function of phosphatidylserine in cellular membranes[END_REF] Notwithstanding its low abundance in plasma membrane phospholipids , PS is of a substantial physiological importance owing to its unique physical and biochemical properties. It is the key to the recruitement of vitamin-K dependent coagulation factors [START_REF] Bevers | Generation of prothrombin-converting activity and the exposure of phosphatidylserine at the outer surface of platelets[END_REF] , it also signals important events such as the internalization of viruses by host cells [START_REF] Callahan | Phosphatidylserine on HIV envelope is a cofactor for infection of monocytic cells[END_REF] and the clearance of apoptotic cells via the binding of lactadherin that is secreted by macrophages and bridges PS and  V 3, an integrin exposed by the macrophages. 38

I.2 Mechanism of MV generation

Loss of phospholipid asymmetry is a driving force for the membrane remodeling and generation of MVs. 10 Unlike exosome formation, MV formation involves plasma membrane budding [START_REF] Morel | Cellular mechanisms underlying the formation of circulating microparticles[END_REF] . Reorganization of the cytoskeleton and changes in membrane asymmetry of PS are key for the genesis of MV. In this respect, the dynamics of actin filaments seem crucial for the formation of MVs. 24 For instance, platelet activation leads to an increase in intracellular calcium, which in turn regulates the activity of transporters such as flippases, floppases and scramblases which are responsible for the movement of phospholipids between the two layers of the plasma membrane. [START_REF] Morel | Cellular mechanisms underlying the formation of circulating microparticles[END_REF] Increased intracellular calcium also activates calpains and Rho kinases, responsible for cytoskeleton fragmentation, 10 thus, the membrane tension is not contained by the cytoskeleton and a MV exposing PS is released. 40 41 This process is sometimes considered protective because MV carry stressinduced deleterious signals from the parent cell. Thus, inhibition of apoptotic endothelial cell Rho kinase prevents the release of MP exporting active caspase 3 and promotes detachment and cell death. [START_REF] Hussein | Inhibition of microparticle release triggers endothelial cell apoptosis and detachment[END_REF] MVs are procoagulant because they carry PS which catalyzes the formation of the blood coagulation complexes and cellular effectors because they convey active proteins present in the cell at the time of their emission. [START_REF] Bakouboula | Significance of membrane microparticles in solid graft and cellular transplantation[END_REF] mixing of phsopholipids between the layers) [START_REF] Quazi | Lipid transport by mammalian ABC proteins[END_REF] (Quazi et According to the international society of extracellular vesicles (ISEV), there is no single optimal characterization method neither a recommendation on quantification of MVs, however, characterization of vesicles must be done using two technical complementary approaches 8 .

Chapter II Tissular and vascular Microvesicles: Biomarkers and cellular effectors

Inflammation is a major actor promoting altered vascular response and often associated with worsen cardiovascular outcome [START_REF] Fioranelli | Stress and Inflammation in Coronary Artery Disease: A Review Psychoneuroendocrineimmunology-Based[END_REF] . Plasma MVs are not only markers of cellular and tissue activation or damage but also actors of major physiological responses such as inflammation [START_REF] Gleizes | β cell membrane remodelling and procoagulant events occur in inflammation-driven insulin impairment: a GLP-1 receptor dependent and independent control[END_REF] , atherosclerosis, 48,49 30 cell survival and apoptosis [START_REF] Guo | Effects of Microvesicles on Cell Apoptosis under Hypoxia[END_REF] , endothelial function, 50 51 plasma membrane remodeling [START_REF] Morel | Mechanisms of microparticle generation: on the trail of the mitochondrion![END_REF][START_REF] Morel | Microparticles: a critical component in the nexus between inflammation, immunity, and thrombosis[END_REF] and angiogenesis. 54 30 In the last decades, many studies explored the role of MVs as bioactive mediators, capable of raising and disseminating the signal responsible for their emission in the vessels and the extracellular space of the tissues in an autocrine or paracrine manner. 55 49 54,56,57 They do exhibit proinflammatory effects and induce the production of cytokines and chemokines while promoting activation of inflammatory cells. 24 Circulating MVs mainly of platelet, leukocyte, and endothelial origin provide an additional phospholipidic surface for the assembly of blood coagulation factors and promote the coagulation cascade and thrombin generation. In the vasculature they constitute the principal reservoir of tissue factor (TF) activity, the celular initiator of blood coagulation. [START_REF] Morel | Procoagulant microparticles: disrupting the vascular homeostasis equation?[END_REF] Emerging as a key marker of global vascular damage, MVs bearing TF mediate noxious cellular responses in diabetic thrombophilia and cardiovascular complications and act as injurious amplifiers of various biological responses such as thrombogenicity and atherothrombotic plaque remodeling. 59

II.1 Microvesicles and vascular inflammatory response

The endothelium is a major player in cardiovascular (CV) physiopathology. [START_REF] Leite | Novel Biomarkers for Evaluation of Endothelial Dysfunction[END_REF] In the healthy state, the endothelium is vasoprotective, mainly by the secretion of vasodilator agents such as nitric oxide (NO), the endothelium-derived relaxing factor [START_REF] Bauer | Nitric oxide--the endothelium-derived relaxing factor and its role in endothelial functions[END_REF] and prostacyclin, [START_REF] Deanfield | Endothelial function and dysfunction: testing and clinical relevance[END_REF] and because the resting endothelium does not promote the assembly of blood coagulation factors (it does not expose PS and very little if any tissue factor). Endothelial dysfunction is an early pathogenic event of atherosclerosis, essential hypertension, and related cardiovascular disorders. [START_REF] Giannotti | Endothelial dysfunction as an early sign of atherosclerosis[END_REF] The loss of cytoprotective NO makes cells susceptible to apoptosis. [START_REF] Zeng | Sustained activation of XBP1 splicing leads to endothelial apoptosis and atherosclerosis development in response to disturbed flow[END_REF] Endothelial MVs are potential biomarkers of cardiovascular disease. In addition, many studies have reported that MVs can directly alter the endothelial function. [START_REF] Liu | Microvesicles: potential markers and mediators of endothelial dysfunction[END_REF] According to the degree of the severity of pathological state associated with vascular dysfunction, the plasma concentrations of MVs are modified.

Numerous clinical data indicate that plasma concentrations of endothelialderived EMVs increase in obesity [START_REF] Esposito | Endothelial microparticles correlate with endothelial dysfunction in obese women[END_REF] , physical inactivity [START_REF] Navasiolava | Enforced physical inactivity increases endothelial microparticle levels in healthy volunteers[END_REF] , type 2 diabetes mellitus [START_REF] Feng | Circulating level of microparticles and their correlation with arterial elasticity and endothelium-dependent dilation in patients with type 2 diabetes mellitus[END_REF] , end-stage renal disease [START_REF] Amabile | Circulating endothelial microparticles are associated with vascular dysfunction in patients with end-stage renal failure[END_REF] , ischemic left ventricular dysfunction [START_REF] Bulut | Circulating endothelial microparticles correlate inversely with endothelial function in patients with ischemic left ventricular dysfunction[END_REF] , and preeclampsia [START_REF] Meziani | Shed membrane particles from preeclamptic women generate vascular wall inflammation and blunt vascular contractility[END_REF] . Furthermore, the phenotype of EMVs varies with the endothelial damage and reflects the initial stress. For instance, in response to platelet MVs, endothelial cells (ECs) release of pro-inflammatory cytokines such as Il-6 or Il-8 cytokines and the expression of adhesion molecules ICAM-1, [START_REF] Barry | Modulation of monocyte-endothelial cell interactions by platelet microparticles[END_REF][START_REF] Nomura | High-shearstress-induced activation of platelets and microparticles enhances expression of cell adhesion molecules in THP-1 and endothelial cells[END_REF] VCAM-1 and E-selectin [START_REF] Nomura | High-shearstress-induced activation of platelets and microparticles enhances expression of cell adhesion molecules in THP-1 and endothelial cells[END_REF] . Interestingly, the majority of (EMVs) isolated from the site of coronary artery plaque during balloon angioplasty express the vascular cell adhesion molecule-1 (VCAM-1) [START_REF] Radecke | Coronary artery endothelial cells and microparticles increase expression of VCAM-1 in myocardial infarction[END_REF] . Of note, VCAM-bearing endothelial cells or derived MVs were in higher amounts (70%-80% ) when isolated from patients with Myocardial infarction as compared to stable coronary artery disease. Because VCAM-1 expression was absent on ECs in the peripheral circulation from these same subjects, authors claimed that VCAM expression at the membrane of cells or EMP appear as unique makers in the plaque tissue. At least they demonstrated that inflammatory markers accumulate in the plaque. Similarly, MVs generated in vitro from endothelial cells human coronary endothelial cells (HCAEC) exposed to high glucose concentration, show increased NADPH oxidase activity and ROS levels, [START_REF] Jansen | High glucose condition increases NADPH oxidase activity in endothelial microparticles that promote vascular inflammation[END_REF] underlining their potential noxious role in diabetes and cardiovascular associated disorders . 76 77 In blood, MVs are most often mainly released from platelets and to a lesser extent from leukocytes and endothelial cells [START_REF] Lovren | Evolving role of microparticles in the pathophysiology of endothelial dysfunction[END_REF] . Plasma MVs are now also studied substantially as vascular effectors [START_REF] Dickhout | Extracellular Vesicles as Biomarkers in Cardiovascular Disease; Chances and Risks[END_REF] , and have been reported to be inflammatory effectors intensifying the secretion of a variety of proinflammatory mediators. [START_REF] Badimon | Microvesicles in Atherosclerosis and Angiogenesis: From Bench to Bedside and Reverse[END_REF] (see table 4)

II.2 Microvesicles in atherosclerosis and thrombosis

Atherosclerosis is a multifaceted pathology combining inflammatory responses as well as certain metabolic imbalance such as elevated LDL-cholesterol and resulting in the formation of thickening of the arterial intima and the formation of atherosclerotic plaque. [START_REF] Hansson | Inflammation, atherosclerosis, and coronary artery disease[END_REF] MVs are crucial actors in atherothrombosis, 48,49 30 . In vitro, plasma MVs foster the release of cytokines and cell adhesion molecules from endothelial cells and prompt the induction of tissue factor, the cellular initiator of the coagulation cascade. [START_REF] Mesri | Leukocyte microparticles stimulate endothelial cell cytokine release and tissue factor induction in a JNK1 signaling pathway[END_REF] Studies have shown that the coagulation capacity of platelet-derived MVs (PMVs) is 50 to100-fold higher than activated platelets. [START_REF] Sinauridze | Platelet microparticle membranes have 50-to 100-fold higher specific procoagulant activity than activated platelets[END_REF] An excessive amount of circulating PMVs has been observed in patients with atherosclerosis. 83 84 In the sequence of events following the initial MV-mediated coagulation, EMVs and leukocyte MVs will later carry the plasminogen activator uPA which enables the transformation of plasminogen into plasmin. MVs bearing uPA will contribute to fibrinolysis cross-talk by docking into the growing thrombus to resorb it and also promote extracellular matrix remodelling during tissue healing. [START_REF] Lacroix | Leukocyte-and endothelialderived microparticles: a circulating source for fibrinolysis[END_REF] Indeed, the cellular uPA borne by EMV and leukocyte-derived MVs promote the plasmin formation [START_REF] Dejouvencel | Fibrinolytic cross-talk: a new mechanism for plasmin formation[END_REF] . (see fig 4) Tissue factor induced specifically at the surface of activated endothelial or leukocytes cells may also activate the coagulation cascade via the FVII/VIIa complex. [START_REF] Voukalis | Microparticles and cardiovascular diseases[END_REF] (Voukalis et al 2019) Many studies focused on the correlation existing between the level of EMVs in the blood and cardiovascular diseases. 5,[START_REF] Amabile | Circulating endothelial microparticles are associated with vascular dysfunction in patients with end-stage renal failure[END_REF][START_REF] Bernal-Mizrachi | High levels of circulating endothelial microparticles in patients with acute coronary syndromes[END_REF] CD31+MVs plasma levels were correlated with a higher risk of major adverse cardiovascular and cerebrovascular events and the need for revascularization [START_REF] Sinning | Circulating CD31+/Annexin V+ microparticles correlate with cardiovascular outcomes[END_REF] .The count of MVs of different origin, including EMV (CD31+, CD51/61+ and CD34+), Platelet MVs (CD42+), monocytes MVs(CD14+) and MV-derived from leukocytes derived (CD45+) enabled the identification of characteristic patterns enabling the discrimination between patients with myocardial infarction (MI) from those with stable coronary artery disease and healthy volunteers. [START_REF] Stępień | Number of microparticles generated during acute myocardial infarction and stable angina correlates with platelet activation[END_REF] Morel et al.

described that patients with ST elevation myocardial infarction (STEMI) and unstable angina had higher circulating levels of procoagulant EMVs detected by CD105 labelling as compared with patients with stable angina [START_REF] Morel | Circulating procoagulant microparticles and soluble GPV in myocardial infarction treated by primary percutaneous transluminal coronary angioplasty. A possible role for GPIIb-IIIa antagonists[END_REF] or acute coronary syndromes. Patients with acute ischemic stroke were found to have increased levels of platelet-derived MPs bearing the integrin GPIIb-IIIa during the acute phase and up to 6 months later, putting forward that thrombotic propensity might last more than the acute phase of the infarct. [START_REF] Cherian | Endothelial and platelet activation in acute ischemic stroke and its etiological subtypes[END_REF] Indeed GPIIb-IIIA is a platelet plasma membrane protein that enables the adhesion of platelets to fibrin and that is overexposed after platelet activation. In carotid, abdominal aorta and femoral arteries from patients with non-symptomatic atherosclerotic disease, elevated levels of circulating leukocyte-derived MVs (CD11a+) were measured [START_REF] Chironi | Circulating leukocyte-derived microparticles predict subclinical atherosclerosis burden in asymptomatic subjects[END_REF] along with endothelial-derived MVs (CD105+).

CD11a+ MVs concentrations correlated with the Framingham index that characterizes patients at cardiovascular risk and with higher carotid intimamedia thickness assessed in 3 different vessels. Altogether, these data indicate the diagnosis value of MVs in the early detection of atherosclerotic disease [START_REF] Chironi | Circulating microparticles may influence early carotid artery remodeling[END_REF] .

Chapter III Periodontitis: a local infection with cardiovascular consequences

III.1 Periodontitis: a clinical overview

Periodontitis, is a chronic inflammatory disease associated with the formation of a dysbiotic biofilm, progressive destruction of the tooth-supporting tissues such as the gingiva and the underlying alveolar bone [START_REF] Darveau | Periodontitis: a polymicrobial disruption of host homeostasis[END_REF] ultimately leading to tooth loss [START_REF] Papapanou | Periodontitis: Consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions[END_REF] and appraised as a silent disease. [START_REF] Fischer | Periodontal disease and its impact on general health in Latin America. Section V: Treatment of periodontitis[END_REF] Having various etiologic and contributory factors, [START_REF] Meyle | Molecular aspects of the pathogenesis of periodontitis[END_REF] severe type of periodontitis affects approximately 11%

of the world's population. [START_REF] Slots | Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia: the "red complex", a prototype polybacterial pathogenic consortium in periodontitis[END_REF] Mostly gram-negative anaerobic bacteria colonize subgingival biofilms. 100 Innate and adaptive immune response are triggered in periodontitis. 101 Porphyromonas gingivalis, Treponema Denticola and Tanerella Forsythia,known as the "red complex" in gingival plaque, are responsible for periodontitis progression.

III.3 Periodontitis and endothelial dysfunction

Endothelial dysfunction occurs early in the pathogenesis of arterial disease, in response to a wide range of factors that have been shown to predict cardiovascular events in epidemiologic studies. 123 Periodontal diseases are associated with major systemic diseases such as cardiovascular diseases, diabetes, or adverse pregnancy outcomes. 124 For more than a decade, the link between systemic inflammation and periodontitis has been studied 125 , in particular by focusing on endothelial dysfunction. (see fig 8) The mechanisms underlying bacterial colonization of plaque worsening atherosclerosis are activation of the immune system, bacteremia, direct involvement of bacterial activated inflammatory mediators, pro-inflammatory cytokine involvement and predisposing factors that can be found in both periodontitis and atherosclerosis. 128 New approaches such as massive sequencing correlated findings from different samples, including atheroma and subgingival/oral samples, 130 thereby confirming that periodontal pathogens can reach and colonize atheroma plaques. Living P.gingivalis and Aggregatibacter actinomycetemcomitans were found in atheromatous tissue. 131 Several in vitro studies indicates also that different periodontal pathogens, in particular, P.gingivalis colonize endothelial cells 6,132,133 .

The hypothesis of a remote pathogen action is also supported in animal models. Rabbits with experimentally induced periodontal disease, develop fatty streaks in the aorta. 134 In normocholesterolemic pigs, P. gingivalis induced aortic and coronary lesions, 135 while it accelerated atherosclerosis in murine models. 136 Finally, in recent studies, the treatment of animals with patients sera induced endothelial dysfunction in mice, 137 and the up-regulation of ET-1, ICAM-1, Eselectin, IL-1, and sCD14, 138 all surrogate markers of the endothelial activation and inflammatory response. However, data on periodontitis direct outcomes in humans at risk of developing atherosclerosis remain scarce.

III.4 Periodontitis and systemic diseases: impact of Porphyromonas

Gingivalis on cardiovascular disorders

Being involved in different systemic pathologies, including rheumatoid arthritis, cardiovascular pathologies, and neurodegenerative diseases, P.

gingivalis is a clear evidence of correlations existing between oral health and systemic inflammation. 139 (see fig 10) P. gingivalis secretes a peptidyl arginine deiminase (PPAD) with a broad spectrum of action and particular interest in atherosclerotic lesions. 141 In addition, the periodontium of periodontitis patients with no signs of rheumatoid arthritis expresses citrullinated proteins. Furthermore, the serum from these patients contain higher levels of antibodies to citrullinated and non-citrullinated human peptides, suggesting that the overload in citrullination caused by periodontitis leads to a loss of tolerance to citrullinated peptides. This strongly suggests a cross-reaction against citrullinated proteins of the joint, eventually favoring rheumatoid arthritis. 142 (see fig 11)

III.5. P.Gingivalis impact on endothelial response

In vitro, the endothelial response to P. gingivalis has been assessed using HUVECs. LPS of P.Gingivalis or the bacteria itself, lead to the emission of proapoptotic microvesicles (MVs). 6 In addition, It was found that Gramnegative bacteria produce outer membrane vesicles (OMVs) at various stages of their growth, especially in the environment of infected tissues. 150 Bacteria associated with periodontitis can pervade into coronary vessels as well 154 . The major consequence of P. gingivalis infection is the proliferation of smooth muscle cells in the distal aorta and the risk of aneurysms. 155 It was found that P. gingivalis OMVs promote calcification of vascular smooth muscle cells in a concentration-dependent manner through ERK1/2-RUNX2. 156 In vitro and in vivo models showed that P. gingivalis OMVs significantly increase vascular permeability leading to vascular edema and mortality in a gingipaindependent manner. 157 These findings reinforce the hypothesis that P. gingivalis infection is a risk factor in cardiovascular diseases and in the development of atherosclerosis. 158

Nevertheless, the clinical assessment of all pathological aspects potentially supported by chronic gingival infection by P. gingivalis remains to be strengthen.

In addition, the respective contribution of the cellular damages and the sequential occurrence remain to be established.

VI.1 Anatomy and cell composition

The spleen is the largest secondary lymphoid organ in the body and possesses a wide range of immunological functions. 160

VI.2 Splenic Immune response and microvesicles

Spleen microvesicles (SMVs) are representative markers of pathophysiological conditions. Elevated levels of thrombocytes and their MVs were observed in splenectomized zebrafish. 164 In vitro, isolated rats SMV initiate an early proinflammatory, pro-coagulant and a pro-senescent response in endothelial coronary artery cells. 4 The SMV content and their noxious properties vary according to the age of the rat and could be counteracted by oral treatment. 7 In addition, injection of P. gingivalis significantly increased the shedding of procoagulant SMVs. In addition, the important role of neutrophil-derived MVs isolated from the spleen and inducing endothelial senescence was confirmed by immunodepletion. 4

Chapter V: The heart

V. 1 Immune cells in the heart

The heart tissue is not only a muscle submitted to electrical pulses but also the host of a variety of cells. In addition to cardiomyoctes and mesenchymal cells,

V.2 Evidences of inflammation in cardiac tissues

Inflammation has been implicated in the pathophysiology of various cardiac diseases. Heart failure and cardiomyopathy are associated with a 4-6-fold increase in atrial fibrillation prevalence. 165 In the pathophysiology of heart failure, inflammation modulates the cell signaling activation patterns associated with fibrosis, apoptosis and hypertrophy 166 . In addition, serum levels of tumor necrosis factor-α (TNF-α), were significantly in patients with elevation of diastolic heart failure and a history of atrial fibrillation suggesting an inflammatory signature and a possible paracrine effect. 167 The inflammatory response to ischemia and reperfusion is a basic process for cellular debris removal and injured tissues repair. Similarly to the neurohormonal activation observed after acute myocardial infarction, inflammation might as well induce a systemic response that favors adverse remodelling. 168 Through inflammatory processes, the NLRP3 inflammasome in the heart is activated by two independent steps: Initiating and triggereing. (see below)

V.3 Inflammatory signalling pathways in cardiac tissues

The inflammatory pathways in the heart trigger the inflammosome and downstream apoptosis. Activation will be initiated through the TLR-dependent sensing of damage-associated molecular patterns (DAMP) exposed by pathogens or injured cells or tissues at the vicinity of the cardiac cells. This "priming" of the inflamosome pathway initiates the NF-κB-dependent transcription and the up-regulation of various components of the inflamosome cascade like caspase-1, NLRP3 and ASC and the unmature forms of IL-1β, IL-18, (Pro-IL). Priming may also occur directly through NOD2 that also tagets NF-κB. The primary signal is necessary but insufficient to form the inflammasome in cardiomyocytes in the absence of the trigger signal.

Eventually, the response will be amplified through P2X7 receptors activated by Finally, the activation of the inflammosome promotes the secretion of the proinflammatory cytokines IL-1 and IL-18 that are freed from their pro-peptide through the proteolytic cleavage by caspase 1. Gasdermin D (GSDMD) is also essential to interleukin secretion by forming a pore in the plasma cell membrane. Indeed, when cleaved by caspase 1 is able to oligomerize. (TRX) thioredoxin.

VI.1 The different types of senescence

Aging is a universal, ingrained process distinguished by the progressive loss of tissue and organ function over time, resulting in progressive risk of pathologies, such as cardiovascular disorders, cancer, diabetes and neurodegenerative diseases. 169 The theory of aging affirms that human aging is the result of cellular aging, whereupon an enlarged proportion of cells reach senescence. 170 Natural killer cells as well as macrophages are also affected in immunosenescence. NK cells increase in number while their cytotoxicity decreases and macrophages proliferation becomes impaired. 188,189 As for B cells, antibodies diversity is limited, disabling the immune system from high affinity antibodies production. 187 Together, all these age-related changes decrease effector functions and

VII.3 Neutrophils and NETs in sterile Inflammation

Inflammation in the absence of pathogens and their products is referred to as sterile inflammation. 210 Numerous stimuli may lead to sterile inflammation like ischemia reperfusion, macroscopic or particle-induced trauma. In this case, immune cells (DCs or macrophages) or nonhematopoietic cells (endothelial or epithelial cells) sense a variety of stimuli and initiate inflammatory pathways, some of them being shared with pathogen-induced inflammations (TLR, NALP3,) whereas others (RAGE, IL1--R) are distinct to sterile inflammation (see figure 24). In the end of the process, tissue repair relies on macrophages To explore our hypothesis, we chose to establish the proof of concept in a murine model of periondontitis.

The following aims were defined:

1-To characterize the spleen cellular composition and the release of MVs by splenocytes upon periodontal infection using a silk ligature impregnated or not with P. Gingivalis.

2-To identify the pro-senescent effects of MVs on the coronary endothelium using an in vitro model.

3-To identify NETs and MVs as circulating and remote actors of P.gingivalis

by comparing the ligature model and the intraperitoneal injection of P.gingivalis .

Results

I-Background

The majority of clinical studies examine MVs isolated from plasma samples, which typically contain MVs of endothelial, leukocyte, platelet, and erythrocyte origin. [START_REF] Morel | Cellular mechanisms underlying the formation of circulating microparticles[END_REF] Several approaches can be used for the detection and characterization of MVs several requiring a preanalytical procedure.

The most commonly studied tissular MVs are those from atherosclerotic plaques, the cardiac tissue itself, or cancer tissues. 224,225 226 In mice, SMV assessment could be of particular interest in situations where (see second manuscript)

The first objective of this study was to establish a methodological approach for the isolation of splenic MVs SMVs from a periodontal P.gingivalis infection murine model. In this context, we studied the variation of the biochemical parameters of MVs, in order to assess the relevance and correlation between the different methods for the quantification and characterization of these MVs.

To achieve this aim, we first studied the different characteristics of SMVs isolated from young C57BL6 mice to establish a reliable procedure with minimal inter-individual variations in the assessment of SMVs. (see manuscript

N°1 jove)

The first series of experiments also enabled to establish labelling procedures of the spleen tissue for further "en face" investigations (see methods for MV investigation part II)

In both manuscripts, C57BL6 mice were investigated and overall different subsets were assessed and compared either to untreated mice or to mice submitted to oral gavage. Mice were either submitted to local gum injury combined or not with P.gingivalis infection using silk threads eventuallysoabed with the bacterial suspension. Another group of mice consisted of intraperitoneally infected individuals, to mimick an acute and local infection known to recruit neutrophils.

In the laboratory we have established a model for the study of microvesicles isolated from the spleen as an alternative approach to investigate immune and leukocyte responses associated to vascular diseases and procoagulant tendency. Indeed, in small animals, the blood volume is a limiting issue for extensive characterization of MVs as circulating surrogate makers of organ dysfunction. At the opposite, the murine spleen is a larger source of MVs.

II-Methodological approaches for MVs measurement in our laboratory:

Data from the laboratory obtained in rats, had confirmed that the spleen MVs concentration and cell origin profile reflects the physio-pathological conditions and could be an alternate approach to plasma MV assessment. Strikingly,oral treatment by a specific combination of omega 3 was able to modify the baseline level of SMVs generation, thereby adding a new interest to the model since it could be useful to evaluate pro-drugs. 7 Nevertheless, our previous data on the action of pathogens of SMV generation and phenotype profile were only established after in vitro LPS stimulation of isolated rat splenocytes. 4 The approach chosen for the measurement of SMVs in extracts and washed final suspensions of SMVs was to select easy-to handle routine methods, like spectrophotometry, colorimetric assay of the protein content or TPRS (Tunable Resistive Pulse Sensing) that would spare minimal amount of the suspensions.

They were compared to our gold standard prothrombinase assay (see article).

Their respective advantages and limits are given in the table below.To establish the procedure, we compared animals treated by intraperitoneal injections of P.

Gingivalis with untreated littermates, with the perpective of the secondary article dedicated to periodontitis. or tissue enable the ex vivo assessment of their effector properties 6 . The deciphering of the mechanisms governing MV biogenesis and cross-talk abilities is generally achieved in cell culture models to identify active molecules exposed by or encapsulated within the MVs and downstream signaling. The MV interactions with target cells will depend on membrane protein/protein binding, when appropriate counter-ligands are available, and/or lipid fusion 7 .

Under physiological conditions, MVs circulating in the plasma mostly originate from vascular cells, as identified by the lineage cluster differentiation markers (CD) 8 , 9 . However, in pathology, notably in cancer 10 and graft rejection 11 , 12 , MVs are shed from the damaged tissue and bear noxious procoagulant and proinflammatory features. These are detected in the systemic circulation, making them useful probes for monitoring protective or rejuvenation therapies, and possible pharmacological targets 13 . Because MVs circulate as a dynamic storage pool reflecting vascular and tissue cell homeostasis in health and disease, a better understanding of their remote action also needs to be investigated in vivo, after IV injection or nasal instillation in small animals 14 , 15 . Indeed, MVs have been considered major contributors to the intricate pathways coupling exaggerated inflammation and thrombosis 16 .

Periodontitis is an inflammatory disease of infectious origin affecting tooth-supporting tissues 17 , 18 and is associated with thrombotic risk. It is characterized by gingival bleeding, alveolar bone destruction, tooth mobility and can ultimately lead to tooth loss. Periodontitis is highly prevalent worldwide and affects more than 50% of the population, with 11% presenting a severe form 19 . Periodontitis is induced by bacterial dysbiosis of the subgingival biofilms, which sustain an exacerbated inflammatory response that triggers tissue destruction 20 . Over the last decade, periodontitis has been linked to systemic diseases such as cardiovascular disorders, diabetes, and rheumatoid arthritis. The possible explanations are the action of the periodontal pathogens at a distance and/or the increased systemic inflammation mediated by proinflammatory cytokines such as interleukin (IL)-1, IL-6, and TNF-α 21 , 22 .

Among pathogens associated with the periodontitis onset and development, Porphyromonas gingivalis (P. gingivalis) 23 is found in most severe lesions that harvest several virulence factors, including lipopolysaccharide (LPS) 24 inducing Toll-like-receptor (TLR)-mediated inflammatory responses 25 and the recruitment of neutrophils and polymorphonuclear leukocytes (PMNs) at the site of the initial infection 26 . The LPS from P. gingivalis activates TLR-4 or TLR-2, facilitating immune detection and bacterial survival evasion 27 . At the vascular level, activation of TLR2 by LPS is associated with immunothrombosis. The unique ability of P.

gingivalis to prompt TLR-2 signaling while TLR-4-dependent recognition is significantly impaired favors persistent lowgrade infection 28 , 29 .

In vivo procedures to study the MV responses to lowgrade pathogen chronic and sustained infection are scarce.

Methodological approaches for tissular MVs extraction are poorly described in the literature and generally concern the harvest of MVs from pathological tissues like solid tumors, liver steatosis, atherothrombotic plaques, or grafts 11 , 29 , 30 , while the spleen senses bacteria and viruses in the bloodstream. It also stores erythrocytes, platelets, lymphocytes, monocytes, basophils, and eosinophils involved in the immune response. Granulocytes like neutrophils from the red pulp also generate reactive oxygen species (ROS) and proteases that destroy pathogens and prevent infection from spreading 31 , 32 . Amazingly, and to the best of our knowledge, spleen MVs are not investigated in the context of pathogen-induced tissue insults. There is a global unmet need to study the variations of tissular MVs in physiopathology.

In vitro data from our laboratory showed that LPS induces the shedding of procoagulant MVs from rat splenocytes, which induces the senescence of cultured coronary endothelial cells and promotes a proinflammatory and proinflammatory procoagulant endothelial phenotype 11 . The feasibility of the pharmacological control of the spleen MVs was further demonstrated after treating the animal with an optimized omega-3 formula. The oral gavage of middle-aged and aged rats was found to be protective against both the shedding of procoagulant MVs from splenocytes and their prosenescent noxious properties towards the coronary endothelium [START_REF] Callahan | Phosphatidylserine on HIV envelope is a cofactor for infection of monocytic cells[END_REF] .

Compared to blood, the spleen offers the advantage of being an important source of leukocytes in one individual.

In addition, a splenic-cardiac axis has recently been proposed 3 , 34 , making the spleen a possible contributor to the infection-related cardiovascular risk of beneficial interest for pharmacological control. The assessment of the SMVs properties or release is key in understanding pathogeninduced or inflammatory responses with no alternative methodology to date. Interestingly, it can be achieved in treated animals and in different physiopathological models (age, hypertension, diabetes). Indeed, by comparing middleaged and aged rats [START_REF] Callahan | Phosphatidylserine on HIV envelope is a cofactor for infection of monocytic cells[END_REF] , the differences in spleen MVs properties and release can be evidenced following a simple 24 h splenocyte culture.

Given the above evidence of the alteration of the effector properties of spleen MVs and the feasibility of their pharmacological control in rats, a standardized protocol is described herein for the isolation of murine spleen MVs. The procedure would better fit in-depth investigations of the in vivo mechanisms supporting SMVs-mediated effects, eventually in engineered mice. The procedure was established in C57BL/6 mice using a local intraperitoneal infection by P.

gingivalis, to establish proof of a remote action of the pathogen on spleen MV (SMV) effector properties.

Protocol

All experimental protocols were approved by and followed the relevant guidelines of the local Ethics Committee

Animal preparation

1. Administer the mice with six intraperitoneal injections of P. gingivalis (PG) every 2 days for 2 weeks (three injections of 5 x 10 7 bacteria/week, Supplementary File 1).

2. Anesthetize the mice using a combination of 100 mg/kg of ketamine and 10 mg/kg of xylazine.

Extraction of splenocytes

Transfer the slices to a sieve (see Table of Materials),

which is from beforehand attached to a 50 mL polystyrene tube.

3. Use the plunger of a syringe as a pestle to crush the tissues on the sieve.

4. Rinse the sieve with the RPMI medium (3 mL).

5. Centrifuge the cell suspension eluate at 450 x g for 5 min at room temperature and discard the supernatant with caution by backflow. 2. Dilute by adding 9 mL of RPMI and mix well using aspiration and backflow.

Removal of erythrocytes via an osmotic shock

3. Centrifuge at 450 x g for 5 min at room temperature.

4. Discard the supernatant by aspiration and backflow, and resuspend the pellet in 5 mL of RPMI medium.

Mix gently (aspiration/backflow) and eventually remove

any lipidic debris by pipetting or using a sieve.

Adjustment of isolated splenocyte concentration

1. Fill a 1.5 mL microtube containing 900 µL of RPMI.

2. Using a micropipette, take 100 µL of the cellular suspension, add to the 1.5 mL microtube containing 900 µL of RPMI medium, and mix well. cells/mL (V max = 20 mL; max total capacity is 100 x 10 6 cells/mL).

6. Seed the cells in a 75 mL flask containing RPMI.

7. Incubate the flask vertically in a humidified incubator at 37 °C for 24 h.

CAUTION: Trypan blue is a mutagenic and carcinogenic agent 35 , 36 . Ensure to wear gloves while counting, even

Isolation of splenocyte microvesicles

1. After 24 h, pour the flask content into a 50 mL polypropylene or polystyrene tube.

2. Centrifuge at 450 x g for 15 min at room temperature to remove cells and debris. 13. Resuspend the MV pellet of the first tube by gentle aspiration/backflow cycles (~10 times) using a 1 mL micropipette. Take great care to avoid foaming as this will promote the oxidation of the suspension vesicles and other moieties. 16. Collect all the resuspended MVs in a single (or two) collector microtube (2 mL with conical shape bottom).

Keep 50 µL for the eventual calculation of the yield of this step. Note the suspension volume. 17. Centrifuge the collector tube at 14,000 x g for 1 h at 4 °C.

This step generates SN4.

Representative Results

The data provided give a full representation of the whole procedure, using two main animal conditions: control untreated young C57BL/6 mice and their littermates subjected to six intraperitoneal (IP) PG injections every 2 days, for 2 weeks. They also show the remote action of an intraperitoneal PG injection on the spleen response after 2 weeks. Splenocyte microvesicles were quantified by either prothrombinase enzymatic assay or by measuring their proteins and RNA concentration by spectrophotometric, and the proportion of neutrophils was determined by flow cytometry 33 , 38 in the splenocyte suspension (sections 2-4 of Supplementary File 1 and Supplementary Figure 2).

Spleen weight and cell number increase after 2 weeks of IP injection in P. gingivalis

In young adult mice, the PG injection leads to a significant 2.6-fold increase in spleen weight after 2 weeks, likely due to the immune response. Indeed, the total splenocyte count was also raised by 2.3 folds (Figure 2, n = 5 mice per each condition, p < 0.0001 by Student's T-test).

Splenocyte viability is not impaired by 2 weeks of injection in P. gingivalis

Similar viability rates were measured between treated and untreated mice after splenocyte isolation or 24 h after cell culture. Nevertheless, when the initial percentage of living cells at t0 was below 76%, viability was reduced in both groups by ~50% after 24 h. (see Ctl 1-3, Table 1, as an example). Conversely, and whatever the animal condition, the 24 h recovery was enhanced when the proportion of living cells reached 90% or more at t0, pointing at the crucial role of the first isolation steps (Figure 3).

Splenocyte neutrophil count is enhanced in P. gingivalisinjected mice

After double staining, neutrophils were identified by flow cytometry as Mac1 + and LYG-6-(Gr1) + (see Table of Materials and section 4 in Supplementary File 1). In response to PG, the neutrophil spleen count was significantly enhanced by 40-folds just after cell isolation (t0, 4.8% ± 0.6 vs. 0.86% ± 0.06). Nevertheless, it is worth considering that after 24 h culture, the neutrophil proportion in the splenocyte suspension increases from 4.8% ± 0.6 to 14.67% ± 2.02

(Figure 4).

The shedding of MVs from splenocytes increases in mice infected by P. gingivalis

Following the protocol, we proved that the isolated splenocytes released more MVs when mice were subjected These diameter values are close to those of SMVs isolated from rat splenocytes in response to a 24 h incubation with LPS (5 µg/mL) or Phorbol myristate acetate (25 ng/mL) and ionophore (1 µM), (median diameter, 222 nm vs. control 212 nm) 11 (Supplementary Figure 5). In addition, the protein content of the SMVs can be assessed by western blot, which enables the characterization of specific cytoplasmic or membrane proteins preferentially exported into the SMVs (Supplementary Figure 5).

Discussion

The present study confirms that the spleen is a major and reliable source of MVs with physiopathological relevance compared to other sources like blood, of limited volume in mice. Provided precautions are taken, the method is easy to set up and does not require expensive equipment.

Since no alternate way other than in vivo assessment is available, the current model appears to be a valuable method to study the impact of pro-drugs on MV shedding.

Importantly, the standardized protocol for harvesting murine splenic microvesicles (SMVs) presented here should fit the unmet needs for the in vivo study of the control of SMVmediated effects, eventually in engineered mice after injection or nasal instillation.

The present method also shows that SMVs are relevant preclinical tools to investigate remote effects of local infections, like P. gingivalis IP infection. Indeed, using multiple approaches, significant variations between SMVs released from P. gingivalis-treated vs. untreated animals were measured, suggesting a remote effect of the local IP injection, already detectable after 2 weeks. Preliminary data from our laboratory using other remote P. gingivalis infection sites confirmed that P. gingivalis may initiate a 1.7-fold SMV elevation as early as day-7 post-infection.

It is also observed that the P. gingivalis-induced remote effects were confirmed by spleen analysis with significant variations in weight, cell viability, and total count. Furthermore, P. gingivalis prompted a 6.5-fold increase in the spleen neutrophil count when measured by immunostaining just after splenocyte isolation (t0). Accordingly, variations of the SMV cell origin profile would indicate the shedding of parental cells caused by the initial condition of the animal.

Because SMVs released in the supernatant of 24 h splenocyte cultures were tripled when isolated from IPinjected mice, they appear reliable biomarkers of remote spleen activation by P. gingivalis, making them eventual tools to monitor pharmacological control.

In vitro, the splenocytes culture can be performed using 10% FBS, depleted or not from exosomes. In the current experiments, no differences were observed, and variations between untreated splenocytes remained detectable to the same extent with both types of FBS.

In the present procedure, a 2-week duration did not lead to significant variations of the spleen weight in untreated mice. It is, however, important to keep in mind that the spleen weight may vary with age, as previously reported in a series of young, old, and middle-aged control rats. 1 In such a case, the SMV quantification needs to be expressed as a cell count ratio.

The present procedure was optimized for a murine model and suitable for further transgenic mice investigations. Although the major drawback of the murine model is a small spleen weight compared to rats [START_REF] Callahan | Phosphatidylserine on HIV envelope is a cofactor for infection of monocytic cells[END_REF] , the adapted splenocyte and SMV isolation procedures described herein prevent cell damage and loss, while all contaminating erythrocytes are fully discarded. Washing is crucial for a good splenocyte purification yield and needs to be performed gently with no more than 1 mL of ACK lysis buffer per spleen in 9 mL of RPMI to enable extensive elimination of red blood cells. This step may be repeated until the pellet is white. This procedure does not allow the investigation of monocytes and should be adapted for adherent cells in such eventuality.

After 24 h culture, the SMV concentration measured in cell culture supernatants reflects the balance between MV generation and MV recapture by the cultured splenocytes.

Introduction

Periodontitis is a highly prevalent, oral inflammatory disease of infectious origin and the primary cause of tooth loss in adults. It is associated with a progressive destruction of periodontal tissues leading to the formation of periodontal pockets. Periodontitis has been evoked in the progression of physio-pathological mechanisms both at the clinical, cellular and molecular levels, 2 although the contributive mechanisms and how they interplay remain elusive.

Initially, identified by Wolf 1967 as a platelet dust containing phospholipids, and capable of supporting coagulation, 25 microvesicles (MVs), are submicron fragments of the plasma membrane that shed into the extracellular space in response to a variety of cellular stress conditions. 4 MVs are released in biological fluids such as blood, urine, tears and saliva. In the blood, they circulate at low concentration in healthy individuals and are elevated in various cardiovascular and chronic diseases including diabetes, chronic renal failure, pre-eclampsia and hypertension. 230 Ranging in size between 100 to 1000 nm in diameter, they are released during plasma membrane budding, and considered as physiological shuttles supporting cellular cross-talk. Indeed, the MV membrane embeds proteins, signaling nucleotides, and metabolites as well as RNA, non-coding RNA and bioactive lipids that were present at the site of their emission. 231 MVs are considered pro-coagulant because they expose phosphatidylserine, a phospholipid that catalyzes the assembly of the complexes of blood coagulation . 232 The spleen is the largest secondary lymphoid organ in the body and possesses a wide range of immunological functions 3 . It is an important source of MVs. 7 Previous data from the laboratory have shown that (i) spleen MVs may act as pro-inflammatory mediators promoting premature senescence of coronary artery endothelial cells, 7 4 leading to endothelial dysfunction and the secondary release of pro-senescent endothelial MVs and (ii) that the LPS of Porphyromonas Gingivalis, the main causative pathogen in periodontitis, induces to the shedding of proapoptotic endothelial-derived microvesicles (MVs) from cultured endothelial HUVEC. 6 In vitro and in vivo data from the laboratory also identified LPS as an inducer of the shedding of pro-coagulant, pro-senescent and pro-inflammatory MVs from rat splenocytes. 4 The feasibility of a pharmacological control of the release of prosenescent MVs from the spleen was further demonstrated in a rat model of ageing. Oral treatment by an optimized omega-3 formula was found protective against both the increased release of SMVs isolated from middleaged and aged rats, and their noxious endothelial prosenescent effects assessed in primary coronary endothelial cells . 7 This work aims to study the impact and remoted effects of P.gingivalis local gum infection on the spleen tissue. Experiments are designed to gain a better knowledge of the interactions between local teeth infection and the spleen response.

containing Tissue-Tek optimum cutting temperature (OCT) compound (Sakura 4583, Leiden, Netherlands) and snap-frozen in liquid nitrogen, and cryosectionned (10µm) and mounted on slides. Slides were then incubated with DHE (2.5 µM) for 30 min, at 37°C in a dark slide humidified chamber. After 3 washings with PBS, slides were mounted under coverslip using fluorescence mounting medium (DAKO, USA), dried in dark and analyzed by confocal microscopy (Leica) with a 20X magnification lens. Level of oxidative stress was quantified using the Image J software.

Western blot

Spleen lysates (30 μg) were obtained by incubation in RIPA for 30 min on ice.

SDS-PAGE electrophoresis

5-P.gingivalis enhances the pro-inflammatory response in spleen tissue

The activation of spleen cells was further characterized by assessment of the expression of VCAM-1, an inflammatory marker. In addition, tissue factor (TF) was assessed as an early indicator of leukocyte response to inflammation. As compared to control spleens, a 3-fold over-expression of VCAM-1 was observed in response to gum ligature (LIG), with or without P.

gingivalis. IP injection of the pathogen did not induce higer VCAM-1 expression. Interestingly, TF was also overexpressed after 2 weeks of treatment, the highest value being observed with local gum injury combined to P.gingivalis soaked threads.

Discussion

Neutrophils are key to the multiple spleen cell responses induced by P.

Gingivalis gum insult

The present findings indicate that P. gingivalis is a strong inducer of the spleen response after only 2 weeks of treatment, highest values being observed when the pathogen is given by intraperitoneal injection. (figure 1)

While IP injection led to a drastic elevation after 2 to 4 weeks in spleen weight and cell count, they remained stable in individuals submitted to local gum ligature with soaked threads as compared to control animals. In all animal subsets cell viability did not differ from the baseline, suggesting that the above increase of weight was the consequence of either cell recruitment or cell proliferation. Interestingly, the simultaneous treatment by IP and a PAD 4

inhibitor that blunts histone citrunillation lead to a significant reduction of the spleen weight and total cell count thereby pointing at the possible role of NETosis.

Analysis of the cell composition revealed that the gum insult, eventually combined to P. gingivalis local infection, was also able to significantly modify the spleen cell composition, mainly cells involved into naïve immunity. Although to a lesser extent than IP infection, increased neutrophil and monocyte counts were found in LIG and LIG-PG subsets while granulocytes were decreased after 2 weeks. (see figure 4) When treatment was extented to 4 weeks only monocytes and granulocytes were strongly elevated in LIG-PG thereby underlining the different kinetics in cell proliferation and recruitment.

Conversely to the IP infection, gum insult did not induce any count modification of the cell involved in adaptative immunity after 2 or 4 weeks.

Gum insult early promotes spleen responses driven by naïve immunity

To confirm the spleen cell modification after 2-weeks gum insults, we measured by western blot the expression of VCAM-1, an endothelial inflammatory marker and TF, induced at the plasma membrane of activated monocytes, neutrophils and endothelial cells in response to inflammatory and procoagulant mediators.

Conclusion

Altogether data are highly suggestive of the strong and early contribution of the cells of naïve immunity in periodontitis and of a key role of neutrophils and of Netosis in the initiation and progression of periodontitis-driven spleen responses after 2 weeks of infection.

To further confirm the remote effects of gum insults described in the article, measurements have been initiated to detect

1) The properties of SMVs on coronary endothelial cells. The aim was to assess SMVs as possible mediators of a thrombotic tendency known to be associated with periodontitis in humans. For this purpose, we used a MVmediated endothelial crosstalk model. The endpoint was the induction of endothelial senescence.

2) The heart inflammatory responses eventually driven by periodontitis by immunolabelling and western blot using heart lysates sampled from the different individuals that were analyzed in the article. We more particularly investigated markers of the endothelial inflammation (VCAM-I), the upregulation of pro and anti-inflammatory cytokines and the expression of Gasdermin D, a recently identified contributor to the inflammosome.

Methods

Primary coronary endothelial cell culture

Primary coronary artery endothelial cells (ECs) were prepared from left coronary arteries of pig hearts, collected from local slaughterhouse (COPVIAL, Holtzheim, France) as described previously. Thereafter, first passage ECs (P1ECs) were grown for 72 hours with medium changed every 48 hours.

Treatment of endothelial cells with SMVs

Following trypsinization (Trypsin, Gibco, Life Technologies SAS, St Aubin, France), P1 endothelial cells were seeded in 6-well plate at 65-75% confluency and incubated with identical amounts of washed SMVs (30nM PhtdSer eq.) for 48 hours, P3 cells was our standard for senescence.

Measurement of Senescence-associated β -galactosidase activity

SAβ -gal activity was measured by flow cytometry using the C12FDG

Results

1) Complementary data to establish the proof of concept of endothelial damage induced by SMVs

SMVs induce the senescence of primary coronary endothelial cells

Conclusion

Taken together, all data confirm the hypothesis of a remote effect of P.gingivalis contributing to the gum insult. They also bring new evidences in the relationships between the spleen and the heart also termed the cardiac splenic axis. Responses.

Future experiments

To further confirm the remote effects of MVs and of Netosis in the initiation and progression of P.gingivalis-mediated effects, the plasma concentration of NETs could be measured as well as the variations of cytokines and of circulating microvesicles of leukocyte origin. For this purpose, pre-analytical sampling has been achieved using individuals of the seven animal subsets experimental procedures were established according to the following methods.

The aims were to examine and confirm the eventual Netosis effects in the IP treated mice, to identify remote mediators in the plasma. In addition, lymph nodes of the abdomen were also harvested to examine early remote effects by RT-PCR.

Preparation of preanalytical samples for anticipated experiments

Intraperitoneal (IP) liquid collection

After mice anesthesia using a combination of 100mg/kg ketamine and 10mg/kg xylazine, injection of 2 ml of salt solution (NaCl) into the peritoneal sac was performed followed by a massage to the hall abdominal cavity using a 2ml syringe. Then, peritoneum, intraperitoneal liquid was collected slowly liquid (approximately 1,7 ml might be collected) and stored at -80°C for long storage.

Plasma preparation

After IP liquid collection, mice were sacrificed. Rapidly the blood was collected from the mice's heart and harvested on 0.129 M Na citrate ratio: 9 volume blood per 1vol anticoagulant. The tube was well mixed by inverting then centrifuged rapidly the at 13000g for 3 min at room temperature. Supernatant was collected in a new Eppendorf and centrifuged again at 13000g for 3 min at room temperature.

Platelet free plasma (PFP) was then collected by rapid upside-down inverting in a 1.5 ml Eppendorf and stored at -80 °C.

After IP liquid and blood collection, the left part of the heart was isolated and stored at -80°C for long storage and the spleen was harvested after laparotomy.

Detection and quantification of NETs in the mice intraperitoneal liquid (IPL)

For comparative purposes, NETs will be characterized from the IPL of the different subsets and the and the activity of histones measured as a ratio of the NET length. After thawing, samples are centrifuged at 10,000g for 10 minutes. visualized using chemiluminescent detection reagents. The signal produced will be proportional to the amount of analyte bound.

In our study, we established the proof of concept of the impact of periodontitis on spleen and confirmed the interest of spleen in a C57BL6 murine model as a reliable and relevant source of MVs with no alternative method available for the procurement of large quantities of MVs. In addition, we confirmed that even after 2 weeks, the physio-pathological impact of any injury in the gum could be remotely sensed by the spleen, at much lower thresholds than those observed by intraperitoneal injection of the pathogen. (El Itawi et al 2022)

Interestingly, our standardized protocol for harvesting murine splenic microvesicles (SMVs) fits the unmet needs for the in vivo study of the control of SMV-mediated effects in engineered and transgenic mice.

Our findings indicate that P. gingivalis is a strong trigger for the shedding of pro-senescent spleen-derived microvesicles (SMVs) that were demonstrated to prompt senescence in primary coronary endothelial cells, on-line with our previous observation in the rat. 4,7 Our data are in accordance with the cardiac splenic axis that has been proposed to explain the link between inflammation and athero-thrombogenicity and in particular the pericardiac inflammation occurring post myocardial infarction. 227 229 However, the role of cardiac or splenic Tregs lymphocytes and their constitution remains to be evaluated. 228 Nevertheless, measurement of IL-17 and of its receptor has been undertaken by western blot and by antibody chips using cardiac extracts from individuals of all subsets. Detailed analysis will be necessary to compare plasma and tissue expression levels of all cytokines and more specifically of those involved in the control of the innate and adaptative immune response.

Used as relevant preclinical tools in our investigation, SMVs showed remote effects of local infection by P. gingivalis using multiple approaches. We showed significant variations between SMVs released from P. gingivalis-treated vs.

untreated animals, already detectable after 2 weeks. We observed that these remote effects were associated with significant variations in weight, cell viability, and most importantly of the cell composition of the spleen. Indeed, P.

gingivalis prompted neutrophil count raise as assessed by immunostaining just after splenocyte isolation (t0) in mice submitted to gum injury and suggested a remote action caused by chronic infection.

Interestingly, ROS accumulation during P. gingivalis infection was reversed by a Netosis inhibitor after reaching it highest raise in response to IP infection.

In the supernatant of 24 h splenocyte cultures, released SMVs were tripled when isolated from IP-injected mice only. However, the SMVs shed from all the conditions were able to alter primary endothelial cells to various degree, indicating on-going effects potentially noxious after a prolonged exposure.

Indeed, the prosenescent features of SMVs shed from LIG-PG, LP4w, IP and gvg mice observed in coronary artery primary endothelial cells confirms their authentic role as biomarkers of a remote spleen activation by P. gingivalis.

Fig 1 :Fig 2 :

 12 Fig 1: Schematic representation of the types of extracellular vesicles released by a cell Fig 2: Shematic content and membrane of extracellular microvesicle released from platelet.

Fig 3 :Fig 4 :

 34 Fig 3: Mechanisms of generation of MVs in response to cell stimulation and membrane transporters Fig 4: Mechanisms and molecules related with MVs-induced coagulation.

Fig 5 :

 5 Fig 5: P. gingivalis leads the inflammatory disease Fig 6: P. gingivalis employs both complement and TLR signaling to induce bacterial persistence Fig 7: P. gingivalis-induced C5a receptor-1/toll-like receptor-2 crosstalk in neutrophils and macrophages.

Fig 8 :

 8 Fig 8: Link between periodontitis and atherosclerotic cardiovascular diseases induced by endothelial dysfunction

Fig 9 :

 9 Fig 9: Periodontitis leads to hypertension and vascular dysfunction Fig 10: P. gingivalis emprises the progression of multiple chronic inflammatory diseases.

Fig 11 :

 11 Fig 11: P. gingivalis in rheumatoid arthritis.

Fig 12 :

 12 Fig 12: Possible inflammatory mechanisms linking periodontitis to Cardiovascular diseases (CVDs).

Fig 13 :

 13 Fig 13: Production of P. gingivalis outer microvesicles (OMVs).

Fig 14 :

 14 Fig 14: P.gingivalis-induced stimulation of accelerated atherosclerosis Fig 15: Mouse and human splenic immune cellular architecture.

Fig 16 :

 16 Fig 16: Cellular composition of the heart.

Fig 17 :

 17 Fig 17: The formation of the inflammasome in the heart.

Fig 18 :

 18 Fig 18: Signs of cellular senescence.

Fig 19 :

 19 Fig 19: Mediators of senescence, SCAPs, the SASP, and effects of senescent cells.

Fig 20 :

 20 Fig 20: VSMC senescence in atherosclerosis.

Fig 21 :

 21 Fig 21: SMVs become prosenescent with ageing as measured in coronary endothelial primary cells.

Fig 22 :

 22 Fig 22: Neutrophils in response to sterile inflammation.

Fig 23 :

 23 Fig 23: NET formation in thrombosis by lytic and non-lytic NETosis

2 - 4 - 3 - 4 - 5 - 6 -

 243456 plasmique. Dans le système vasculaire,d'origine endothéliale, plaquettaire, leucocytaire et de cellules musculaires lisses (Boulanger et al, 2010) Initialement étudiées pour leurs propriétés procoagulantes sanguine, plus récemment, les microvésicules ont été reconnues comme ayant un rôle dans la communication cellule-cellule. (Guillaume van Niel et al, 2018) Elles sont actuellement reconnues comme de véritables effecteurs cellulaires car elles sont dotées d'une activité biologique intrinsèque. Elles sont capables de moduler de nombreuses réponses vasculaires telles que l'hémostase, l'inflammation, la vasomotricité, l'angiogenèse et la croissance vasculaire. Ces propriétés dépendent de l'origine cellulaire de la MV et du stress initiant leur libération et de leur capacité à se lier à la cellule cible. (Cocucci E et al, 2009) Ainsi, dans le choc septique, les taux de MVs procoagulantes circulantes de neutrophiles sont corrélées à une hypercoagulabilité et à la présence de Neutrophil Extra cellular Traps (NETs) circulants. (Steil et al 2016, DeLabranche et al 2017). Les NETs sont composés de chromatine décondensée qui portent certains constituants cytoplasmiques et des granules des neutrophiles qui les émettent. La Nétose des neutrophiles est probablement un mécanisme évolutif de défense contre la déssimination des agents pathogènes envahisseurs qui sont piégés dans les NETs (Brinkmann et al 2016). En effet, les NETs ont été associés à la parodontite chez les patients à risque cardiovasculaire, ainsi un axe splénocytaire avec invasion par les neutrophiles de la rate dans le coeur post-infarctus a été proposé (Moonen et al 2019) comme un nouveau mécanisme liant infection chronique et atteinte cardiovasculaire (Tian et al 2016). Dans notre laboratoire, nous avons établi des méthodes d'extraction des MVs d'aorte de rat et démontré que ces MVs sont sensibles au microenvironnement vasculaire. Nous avons aussi montré que les MVs de rat d'âge moyen (48 semaines) ou âgé (92 semaines) isolées à partir de la rate sont prosénescentes et procoagulantes vis-à-vis de l'endothélium uniquement lorsqu'elles sont émises dans un microenvironnement proinflammatoire ( El Habhab et al 2020) et (Qureishi et al 2019). Par ailleurs, d'autres données antérieures du laboratoire ont montré que le LPS induit l'excrétion de MV procoagulants de la rate de rat capables de favoriser la sénescence endothéliale et une action pro-inflammatoire et réponses procoagulantes. (El Habhab et al 2020). L'infection par LPS du Porphyromonas Gingivalis ou par la bactérie inactivée conduit à l'émission de MVs proapoptotiques par les cellules endothéliales HUVEC ( Bugueno et al 2020). La faisabilité d'un contrôle pharmacologique de la libération de MV prosénescentes de la rate a été démontrée dans un modèle de rat. Le traitement oral par une formule optimisée d'oméga-3 s'est avéré protecteur à la fois contre le taux de libération des MV de la rate chez les rats d'âge moyen et âgés, et contre leurs propriétés nocives prosénescentes envers l'endothélium coronaire. (Qureshi et al 2020) Le travail de thèse vise à étudier l'impact des MVs et des NETs (neutrophil extracellular traps) comme acteurs de la réponse cellulaire induite par la parodontite et cause possible de la tendance thrombotique chez les patients. Des expériences sont conçues pour acquérir une meilleure connaissance des interactions entre l'infection locale des dents, la réponse de la rate et les tissus cardiovasculaires. Elles se concentrent sur le changement du phénotypage de l'endothélium qui est initialement une surface protectrice et devient proinflammatoire, procoagulant et prosénescent après contact avec les MVs émises localement en réponse à P. gingivalis. Les hypothèses émises après examen de la littérature et en s'appuyant sur les données du laboratoires sont : 1-Les MVs excrétées en réponse à une infection par des agents pathogènes parodontaux sont des acteurs à distance expliquant l'impact de la parodontite sur l'aggravation des conditions vasculaires perpétuent l'inflammation vasculaire chronique. La libération des MVs spléniques et leurs propriétés varient selon la sévérité de l'infection et / ou le type de lésion tissulaire 3-Les MV libérées par les splénocytes en réponse à Porphyromonas Gingivalis sont délétères pour les cellules endothéliales et agissent comme des médiateurs cardiovasculaires pro-inflammatoires et prosénescents Les microvésicules splénocytaires et les NETs libérés en réponse à Porphyromonas Gingivalis favorisent l'athérothrombose à des sites éloignés de l'infection dentaire Les objectifs généraux du travail sont en conséquence : 1-Établir la preuve de concept dans un modèle murin de parodontite. 2-Caractériser la composition cellulaire de la rate et la libération des MVs par les splénocytes lors d'une infection parodontale par ligature combinée ou non à une infection locale par Porphyromonas.gingivalis. Comparer les propriétés endothéliales proinflammatoires et procoagulantes des MVs tissulaires de souris dans ce modèle d'infection dentaire en utilisant la rate comme source majeure de MVs inflammatoires libérées en réponse à P. gingivalis. Identifier les effets pro-sénescents des MVs sur l'endothélium coronaire en utilisant des cellules primaires dans un modèle de communication intercellulaire. Identifier les NETs et les MVs comme acteurs circulants et distants de P.Gingivalis en comparant le modèle de ligature et l'injection intrapéritonéale de P.gingivalis. Evaluer les répercussions de la parodontite sur le profil inflammatoire et procoagulant du tissu cardiaque. Prolog Spleen and heart contributing to pathological response in periodontitis. Periodontitis is a highly prevalent, oral inflammatory disease of infectious origin and the primary cause of tooth loss in adults. It is associated with a progressive destruction of periodontal tissues leading to the formation of periodontal pockets. Amazingly, periodontitis has been involved as a inducer in pathologies such as cardiovascular diseases and atherothrombosis, associated with cardiovascular worsened outcome 1 .The evolution of the knowledge of physio pathological mechanisms underlying both diseases has made it possible to highlight cellular and molecular links , although these mechanisms remain to be deciphered. The thesis work is investigating relationships between different effectors possibly contributing to a remote action of the local periodontitis. Recently, Neutrophil Extracellular Traps (NETs), a mesh of decondensed DNA shed by neutrophils in response to local infection, and already known to contribute to hypercoagulability, have been associated with periodontitis in patients at cardiovascular risk. 2 Finally, leukocytes invasion of the spleen in the post-ischemic heart reperfusion has been proposed as a new mechanism linking chronic infection and cardiovascular damage 3 possibly constituting a new spleen-cardiac deleterious axis.

Fig 1 :

 1 Fig 1: Schematic representation of the types of extracellular vesicles released by a cell. Exososomes, microvesicles and apoptotic bodies are secreted by the cell into the extracellular environment. Their biogenesis are different, exosomes are secreted by endocytosis,

Fig 2 :

 2 Fig 2: Shematic content and membrane of extracellular microvesicle released from platelet.(Zaldivia MTK et al 2017)[START_REF] Zaldivia | Platelet-Derived Microvesicles in Cardiovascular Diseases[END_REF]

  These proteins maintain the phospholipid asymmetry of the membrane at cell rest (fig 5). Activation of floppases and scramblases and inhibition of flippases in response to calcium influx induces exposure of PS at the outer leaflet of a cell membrane, whereas PS is sequestered in the inner leaflet of the plasma membrane at cell rest. The increase in cytoplasmic calcium concentrations causes the translocation of phosphatidylserine (PS), from the inner layer to the outer layer under the effect of a net floppase activity. At the same time, reverse anionic phospholipid transporters (flippases) are inhibited by calcium. The transport of PS by floppases is very rapid while the reverse transport of other non-anionic phospholipids such as phosphatidylcholine is slower. The translocation of the PS creates a transient overload of the outer leaflet which leads to budding.

Fig 3 :

 3 Fig 3: Mechanisms of generation of MVs in response to cell stimulation and membrane transporters A: cell response to stimuli: the activators bind to their specific receptor and induce a calcium influx. The increase in cytoplasmic calcium concentrations causes the translocation of phosphatidylserine (PS), from the inner layer to the outer layer under the effect of a net floppase activity. Proteases such as calpains and ROCK kinases are activated by high calcium concentrations and cause controlled proteolysis of the cytoskeleton, which promotes the release of MVs. B: Different types of phospholipid transporters in the membrane of eukaryotic cells. Flippase (repatriation of PC, floppases: externalization of PS, Scramblases:

Fig 4 :

 4 Fig 4: Mechanisms and molecules related with MVs-induced coagulation. PS: phosphatidylserine; GLA:c-carboxyglutamic acid; clotting proteins factors VII, IX, X and prothrombin. PS electrostatically catalyzes the assembly of coagulation enzyme complexes by interacting with the GLA domain of the clotting factors called vitamin-K dependent factors.

102 103 III. 2 Fig 5 :

 10325 Fig 5: P. gingivalis leads the inflammatory disease. The bacterium's gingipain, a C5 convertase-like enzyme cleaves C5 giving rise to high levels of C5a locally. C5a-induced activation of C5aR triggers inflammation and also is censoriously involved in a subversive crosstalk with TLR2 that impairs leukocyte killing. 105 (by Hajishengallis et al 2012)

Fig 6 :

 6 Fig 6: P. gingivalis employs both complement and TLR signaling to induce bacterial persistence. C5a: receptor for C5a.Mal adaptator protein; PI3K: phosphatidylinositol 3kinase. .RhoA: small GTPase Rho. 115 (by Cortes-Vieyra et al 2016)

Fig 7 :

 7 Fig 7: P. gingivalis-induced C5a receptor-1/toll-like receptor-2 crosstalk in neutrophils and macrophages. GSK, glycogen synthase kinase; iNOS, inducible nitric oxide synthase; Mal, myeloid differentiation primary response protein 88 adaptor-like; MyD88, myeloid differentiation primary response protein 88; NF-κB, nuclear factor-kappaB; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; TGF, transforming growth factor; TLR, toll-like receptor. 122 (by Hajishengallis et al 2020)

Fig 8 :

 8 Fig 8: Link between periodontitis and atherosclerotic cardiovascular diseases induced by endothelial dysfunction 126 (by Zardawi et al 2021)

Fig 9 :

 9 Fig 9: Periodontitis leads to hypertension and vascular dysfunction. Prospective mechanisms causally linking periodontitis with hypertension and vascular dysfunction. NO, nitric oxide; O-2, S, superoxide; Th, T-helper cells; ROS, reactive oxygen species 129 (by Aguilera et al 2020)

Fig 10 :

 10 Fig 10 : P. gingivalis emprises the progression of multiple chronic inflammatory diseases. Through the cross-reactive mediators in atherothrombosis, rheumatoid arthritis, the increased levels of systemic inflammation, gut microbiome dysbiosis, metabolic disorders. (↑ = increase ↓= decrease). 140 (by Mulhall et al 2020 & authorized by co-author Olivier Huck)

Fig 11 :

 11 Fig 11: P. gingivalis in rheumatoid arthritis. P. gingivalis breaking immune tolerance to citrullinated proteins in rheumatoid arthritis (RA) by bacterial citrullination of proteins through PPAD, initiating an immune response leading to loss of tolerance for citrullinated joint proteins in the joints. Abbreviations : PPAD: peptidyl arginine deiminase from P. gingivalis; APC: antigen presenting cell; TNF: tumour necrosis factor; IL: interleukin; GMCSF: granulocytemacrophage colony-stimulating factor 143 (by Lopez-Oliva et al 2019)

Fig 12 :

 12 Fig 12: Possible inflammatory mechanisms linking periodontitis to Cardiovascular diseases (CVDs). (1) The imbalance in pathogens of the biofilm leads to gingival epithelium inflammation that releases inflammatory mediators, such as interleukins (ILs), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α) and MMPs, that recruit immune cells. This inflammatory response induces alveolar bone reabsorption, by osteoclasts. (2) At a chronic

Fig 13 :

 13 Fig 13: Production of P. gingivalis outer microvesicles (OMVs). 1. After the cell wall disruption, phospholipids accumulation in the outer membrane leaflets, and the expansion pressure continues to produce, which intensifies the further enrichment of outer membrane components. The linkage between the peptidoglycan and the outer membrane layer is disrupted, and finally P. gingivalis OMVs are formed. 2. The fim locus and GalE mutant strains reduced or even eliminated the production of P. gingivalis OMVs, while the OmpA mutant strain overproduce P. gingivalis OMVs. 153 (By Zhang et al 2021)

Fig 14 :

 14 Fig 14: P.gingivalis-induced stimulation of accelerated atherosclerosis. Possible mechanisms: inflammation , endothelial injury, lipid peroxidation, and molecular mimicry. ox-LDL: oxidized low-density lipoprotein. 159 (by Mai et al 2020)

  It is divided by function and structure into the red and white pulp separated by the marginal zone (MZ) in rodents and the perifollicular zone in humans. Lacking afferent lymphatic vessels, all cells and antigens enter the spleen via the blood flow. The White pulp (WP) the primary immunologic region of the spleen in both species, and the red pulp (RP) constitutes the majority of the tissue with a distinct immune function.161

Fig 15 :

 15 Fig 15: Mouse and human splenic immune cellular architecture.

  the cardiac tissue contains immune cells and all the cellular equipment of a vessel like endothelial cells, pericytes and circulating blood cells. The macrophages are the major immune cell population in the resting heart. They are found in the interstitium and around endothelial cells. Inflammatory monocytes and neutrophils are not present in the myocardial tissue but can be observed upon tissue damage. Mast cells, dendritic cells, B cells, NK cells and regulatory T cells are found sporadically in cardiac tissue.

Fig 16 :

 16 Fig 16: Cellular composition of the heart. The heart tissue contains different sub-types of immune cells depending on the physiopathological condition.

  high concentrations of extracellular ATP released by activated or damaged cells. P2X7 receptors activation leads to K+ efflux, a step that triggers NLRP3 activation. Another mechanism triggering the inflamasome, is the lysosomal destabilization by indigestible material that leads to the leakage of the cathepsin B and the induction of K+ efflux favoring NLRP3 activation. Upon binding to the serine threonine kinase Nek7 that senses the K+ efflux, NLRP3 is activated by phosphorylation. In cardiomyocytes, the mitochondria count is high and their damage causes important oxidative stress through the release of ROS leading to the unfolded protein response also targetting NLRP3 via the capture of ROS by TXN further enabling the dissociation of the complex TXN/TXNIP. Mitophagy and autophagy maintain low levels of NLRP3 activation by effective clearance of mitochondrial debris that otherwise would favor lysosome destabilization.After ischaemia, the activation of the inflammosome occurs upon binding of the tyrosine-protein kinase BTK and of apoptosis-associated speck-like protein containing a CARD (ASC). The active NLRP3 oligomer functions as a platform for the polymerization of ASC into filaments, and in turn for the branching of caspase 1 filaments branches.

Fig 17 :

 17 Fig 17: The formation of the inflammasome in the heart. 168 (by S. Toldo and A. Abbate 2018) TLRs: Toll-like receptors; MyD88 myeloid differentiation primary response protein, and IRAKs: interleukin-1 receptor-associated kinases). NOD2: nucleotide-binding oligomerization domain-containing protein 2. TXNIP): Thioredoxin-interacting protein;

Fig 18 :

 18 Fig 18: Signs of cellular senescence. 170 (by Kamal et al 2020)

Fig 19 :VI. 2 1 (

 1921 Fig 19: Mediators of senescence, SCAPs, the SASP, and effects of senescent cells. 177 (by J. N. Farr and S. Khosla 2020)

Fig 20 :VI. 3

 203 Fig 20: VSMC senescence in atherosclerosis. Through the establishment of the SASP, senescent VSMCs that are Unable to proliferate, put on the line plaque instability and inflammation. 182 (by Gorenne et al 2006)

Fig 21 :VI. 4 Immunosenescence

 214 Fig 21: SMVs become prosenescent with ageing as measured in coronary endothelial primary cells. ECs show a pro-inflammatory and procaogulant phenotype with overexpression of VCAm-1, TF, ICAM-1, ACE.and down-regulation of eNOS and over-expression of COS-2. MVy: SMVs isolated from young individuals, MVMA from middle-aged rats, MVO from old rats Intake of a specific formulation of omega-3 abolishes the noxious properties of SMVs (by Qureshi et al 2020 & authorized by the first author Qureshi AbdulWahid)

190 VII. 1

 1901 increase the susceptibility to infections and various pathological cell responses like dysfunctional mitochondria, oxidative stress and NLRP3 inflammosomme activation. Inflamm-ageing is also associated with central obesity and increased gut permeability and contributes to the initiation and progression of atherosclerosis , diabetes, dementia peripheral vascular and chronic diseases. Neutrophils: a first line of defense against pathogens and actors of innate Immune response Neutrophils are main actors in the innate immune system, they are heterogeneous in morphology, phenotype and function.191 They are incessantly generated in the bone marrow from myeloid precursors and can reach up to 2×10 11 cells produced daily.192 During inflammation, neutrophils are recruited in tissues, and with time the cells undergo apoptosis and are removed by macrophages and dendritic cells.193 In humans, 50-70% of circulating leukocytes are neutrophils, whereas only 10-25% are in mice169,170. Three types of neutrophil granules are formed consecutively during their maturation, and they are filled with pro-inflammatory and degradation proteins.192,194,195 They are azurophilic (primary) granules, containing myeloperoxidase (MPO), specific (secondary) granules, containing lactoferrin, and gelatinase (tertiary) granules, containing matrix metalloproteinase 9 (MMP9).196 Different intra and extracellular mechanisms are implicated in pathogens elimination by neutrophils. First, neutrophils have a phagocytosis ability. After they are encapsulated in their phagosomes, pathogens are destroyed by ROS produced by NADPH oxygenase and/or cleaved by proteolytic proteins released by the granules (cathepsins, defensins, lactoferrin and lysozyme).

VII. 2

 2 antibacterial proteins, are also released from the neutrophil granules into the extracellular milieu via a specific process termed degranulation, thereby enabling the destruction of extracellular pathogens. Highly activated neutrophils can eliminate extracellular microorganisms by releasing neutrophil extracellular traps (NETs). 198 Netosis and formation of NETs Netosis is the mechanism of neutrophil extracellular traps (NETs) formation. 199 NETs are made of decondensed chromatin that forms meshes of DNA fibers with approximately 200-nm pores 200 . NETs are coated with nuclear proteins (histones), granule proteins, like neutrophil elastase and myeloperoxidase, and cytosolic proteins such as S100 calcium-binding proteins A8, A9, and A12, as well as actin and α-actinin 201-203 . NET formation occurs via two processes: non-lytic or vital NETosis and lytic NETosis. Both lytic and non-lytic NETosis can cause thrombosis. In lytic fibrosis, PAD4 causes hypercitrullination of histones driving nuclear swelling due to chromatin decondensation, followed by release of NETs containing DNA and histones. When Occurring without the necessity of cell lysis, the release of extracellular traps is mediated by vital NETosis. 204 Upon stimulation, eosinophils 205 , basophils 206 , mast cells 207 , and macrophages 208 were also described to release extracellular DNA. Initiation of the NETosis signaling occurs through the ERK MAPKinase pathway that triggers nuclear activation of chromatin decondensation and the shattering of the nuclear membrane. The shedding chromatin fibers by passing through the cytoplasma embark bactericidal proteins like myeloperoxidase, neutrophil elastase and also bear citrullinated histones. Histone citrullination is mediated by peptidylarginine deiminase (PAD) and is mandatory for NET release.

  that clear necrotic or apoptotic neutrophils and produce factors that accelerate the healing process (resolvins, TGF-β). Growth factors released in the healing area by vascular cells (e.g., VEGF) also contribute to the restauration of the tissue. For instance, IL-4 produced by eosinophils is sensed by FAPs to mediate repair after acute skeletal muscle necrosis. Induced sterile inflammation may trigger the adaptive immune responses, as in the case of organ transplantation.Neutrophil recruitment in response to sterile inflammatory stimuli contributes to the immunological damages observed in many diseases, such as ischemic injuries/infarction, trauma, autoimmunity, drug-induced liver injury. As an ultimate neutrophil activation, Netosis takes place from 2-3 h up to 8 h from activation. About 20%-60% of isolated human neutrophils typically release NETs 2-4 h after contact with microbes or chemicals, however, they are able to respond within minutes when activated by LPS-stimulated platelets under conditions of flow.212 It is worth noting that neutrophil stimulated in vitro by fMLP (N-formyl-Met-Leu-Phe)213 release MVs but do not extrude NETs, suggesting that the two mechanisms are not directly dependent one from the other. The mechanisms by which neutrophils respond to sterile tissue injury and cell death are of both homeostatic innate immune responses and pathogenic immune responses in disease. 214 Neutrophils are leukocytes exposing CD11b and able to roll on the endothelium through interaction with different endothelial membrane proteins like E-Selectin, VCAM, ICAM . Tethered neutrophils are able to secrete chemokines (CXCLs) and interleukins (IL-8), and contribute to maintain the inflamed endothelial phenotype. In addition neutrophils also interacting with naïve platelets via P-Selectin and Mac-1 binding promote platelet activation. Altogether, these interactions at the endothelial layer favor a procoagulant state at the vicinity of the site where neutrophils and maintain local inflammatory mediator production. Such mediators nurture cell activation and consecutive exposure of procoagulant phosphatidylserine as well as the induction of tissue factor (TF) finally exposed at the surface of both the inflamed endothelium and activated neutrophils. Importantly, TNF, IL-1 and ROS produced by the neutrophils extruding NETs are also inflammatory and oxidative mediators triggering ECs.

Fig 22 :

 22 Fig 22: Neutrophils in response to sterile inflammation. Neutrophils produce NETs after activation by CD11b, integrin alpha M, ICAM, intercellular adhesion molecule; IL-8, interleukin-8 (chemokine receptor ligand 8); CXCLs, chemokine ligands; CXCR, chemokine receptor; DAMP, damage associated molecular pattern; TLR, Toll like receptor;IL-1, interleukin 1; PY2R, purinergic receptor; FPR, formyl peptide receptors; and TNFα, tumor necrosis factor-alpha. 212 (by Edwards et al 2020)

Fig 23 :VII. 4

 234 Fig 23: NET formation in thrombosis by lytic and non lytic NETosis APC= activated protein C, DNAse= deoxyribonuclease, NET= neutrophil extracellular traps, PAD4= peptidylarginine deiminase, PSGL= P-selectin ligand 1, RBC= red blood cell, TF= tissue factor, TFPI= tissue factor pathway inhibitor, TM= thrombomodulin, vWF= von willebrand factor, WP= weibel palade bodies. 218 (by S. Kapoor, A. Opneja and L. Nayak 2018)

Fig 24 : 2 - 3 -

 2423 Fig 24: Role of neutrophils in atherosclerosis. CCL5, CC-chemokine ligand (CCL)5; IFN, interferon; IL, interleukin; LDL, low-density lipoprotein cholesterol; NET, neutrophil extracellular trap; Ox-LDL, oxidized low-density lipoprotein cholesterol; pDCs, plasmacytoid dendritic cells; ROS, reactive oxygen species. 221 (by Bonaventura et al 2019)

  active molecuiles or therapeutical devices are precious and limited in quantity, or for comparative purposes of different genetic modification in Knock-in or Knock out mice. The study of spleen derived MVs (SMVs) in the context of cardiovascular dysfunction appears consistent with the recently evoked cardio splenic axis, as a new response driven by pericardiac inflammation post myocardial infarction. 227 228 229Indeed, based on the proposed link between periodontitis and enhanced cardiovascular risk, a part of the work explores the possibility of an elevated procoagulant and pro-inflammatory activity in spleen, pertaining to the remote action of microvesicles released after local gum infection by P.gingivalis.

IntroductionFigure 1 ).

 1 Figure1). MVs have been extensively studied as circulating biomarkers of thrombotic risk in chronic diseases associated with cardiovascular disorders such as diabetes, chronic kidney disease, preeclampsia, and hypertension3 , 4 . They are currently recognized as true cellular effectors in fluids or organ tissues like the myocardium1 . Because they convey active proteins, lipids, and miRNA, they remotely modulate vascular responses such as hemostasis, inflammation, vascular angiogenesis, and vascular growth or tissue remodeling5 .

(

  APAFIS#28745-2020121815051557) and animal care of the home University and of the INSERM. Male Young C57BL/6 mice, 6-8 weeks of age, were used for the experiments. Mice were regularly examined to evaluate pain and stress, and their weights were monitored daily. Unless otherwise stated, all extracting buffers and solutions must be sterile and at room temperature.

3 .Supplementary File 1 .

 31 Sacrifice the anesthetized mice and harvest the spleen after laparotomy. NOTE: The specific conditions for P. gingivalis infection and the sacrifice of the animals for spleen harvest are detailed in the "animal experiment" section of The schematic representation of the protocol steps is shown in Figure 1.

3 . 4 .

 34 Transfer 10 µL of the diluted cells into a microtube, add 10 µL of the trypan blue exclusion dye, and mix well. Place 10 µL of the solution (step 3.3) into the counting slide of the automatic cell counter (see Table of

Materials) and determine 5 .

 5 the cell count. NOTE: The device software gives two values: the total cell number and the living cell number and percentage. Adjust the concentration of living cells to 3 or 5 x 10 6

  outside of the hood. Immediately discard tips and fluids into containers and wash the bench surface. NOTE: If the collected cells are less than 50 x 10 6 , use a 25 mL culture flask. The cell number should not vary much from one individual to the other. The percentage of living cells is generally superior to 80% at this step.

3 .

 3 After centrifugation, keep 100 µL of the 1st supernatant (SN1) for an eventual measurement of the initial MV concentration to calculate the isolation yield. Note the SN1 volume.

4 . 5 . 9 .

 459 Centrifuge the SN1 at 450 x g for 15 min at room temperature. Discard pellet and centrifuge again at 450 x g for 15 min at room temperature. Rapidly withdraw and collect SN2 (note the volume). 6. Transfer SN2 into 1.8 mL microtubes with conical shape bottoms. Keep 100 µL of SN2 for an eventual measurement of the initial MV concentration to calculate the isolation yield in case too much debris eventually impairs MV measurement in SN1. 7. Add 5 mL of RPMI medium to the pellet (cells) and count the splenocytes. NOTE: The cell suspension can also be fixed for flow cytometry characterization in 0.1% paraformaldehyde (final concentration) 37 . 8. Centrifuge SN2 at 14,000 x g for 1 h at 4 °C and eventually refrigerate the rotor in advance by running the empty rotor at 4 °C. This step generates SN3. Immediately withdraw SN3 from each microtube by inverting over a single sterile 50 mL tube (under the hood). Note the SN3 volume. 10. Gently mix SN3 using a pipette under the hood. Keep aliquots for eventual measurements in the suspension. NOTE: The solution is now depleted of MVs but contains the soluble mediators and exosomes. 11. Scrape each microtube (closed) on a spiked rack to mechanically favor the resuspension of the MVs in the pellet. 12. Add 200 µL of Hank's balanced saline solution without Ca 2+ and Mg 2+ (HBSS) in the first microtube (seeTable of Materials).

14 .

 14 Harvest the first 200 µL of resuspended MVs and pour them into the second tube over the pellet. Gently mix as above, and then pour again into the third tube and mix gently. The suspension becomes more and more turbid from tube to tube. Close the third tube. 15. Initiate the collection of the next three microtubes as above (step 4.14).

18 . 1 .

 181 Immediately discard the SN4 (pour over a supernatant collecting tube). Keep an aliquot of SN4 to calculate the yield of the purification step and gently resuspend the pellet in 500 µL of HBBS (without Ca 2+ and Mg 2+ ). Keep a 50 µL aliquot of the resuspended pellet to calculate the purification yield and note its volume. 19. Store the resuspended pellet at 4 °C for up to one month for functional assays under sterile conditions or at -20 °C for structural assays. 20. Calculate the purification yield (see NOTE below) of each step or the whole procedure by measuring the concentration of MVs in supernatants and/or pellet suspensions. NOTE: Purification Yield (%): ([MV1] x SN1 volume) / ([MV4] x final suspension volume) x 100. This yield needs to be rationalized to the number of splenocytes isolated from the culture flask and counted in step 4.4. The present experiment's purification yield is ~70% starting from 111 x 10 6 cells and 60 mL of supernatant. Evaluate the loss of SMVs caused by the procedure by measuring the SMVs remaining in the supernatants depleted of SMVs and mainly containing exosomes (SN2 and SN4).

2 .

 2 Measure the MV concentration by TRPS or prothrombinase assay 38 for high sensitivity and specificity. CAUTION: Harvest the MVs after centrifugation by the rapid upside-down pouring of the supernatant. Waiting or using U-shape microtubes will prompt MV loss on the microtube wall. Never use a micropipette to discard the supernatant; the pellet can be aspirated instead. Never delay the process after the centrifuge is stopped. Overspeed centrifugation will result in contamination of the MV pellet by exosomes. NOTE: The centrifugation speeds and duration are optimized for an MV enrichment with minimal contamination by exosomes. This protocol is not reliable to study spleen monocyte/macrophage MVs because these cells can phagocyte and rapidly recapture their own MVs 39 . The protocol cannot be paused and restarted later because of loss of cells due to cell viability.

Figure 4 )

 4 Figure 4) comparing SMVs from PG-stimulated and

Figure 1 :

 1 Figure 1: Schematic representation of the protocol steps. This consists of tissue dissection, red blood cell (RBC) lysis, splenocyte counting, seeding, microvesicle harvest from supernatant, washing and concentration determination. Please click here to view a larger version of this figure.

Figure 2 :

 2 Figure 2: IP injection of P. gingivalis significantly enhances spleen weight (A) and splenocyte count (B). CTL: untreated mice, IP: mice subjected to PG injection for 2 weeks. Data are expressed as mean ± SEM. n = 5: ***: p < 0.0001 and 0.0005, respectively. Please click here to view a larger version of this figure.

Figure 3 :

 3 Figure 3: Splenocyte viability is reduced 24 h after splenocyte culture, whereas IP injection of P. gingivalis does not alter cell viability. CTL: untreated mice, IP: mice subjected to PG injection for 2 weeks, t0: viability measured immediately after isolation, t24: after 24 h culture. Data are expressed as mean ± SEM. n = 5, ***: p < 0.0001 by Student's T-test. Please click here to view a larger version of this figure.

Figure 4 :

 4 Figure 4: P. gingivalis injection increases the neutrophil count at t0 and after 24 h of cell culture. Cells were freshly isolated from the harvested spleen and labeled with fluorescent antibodies directed against granulocytes (LyG-6 Gr1) or neutrophils (LY6-C) and identified in the CD11b + gate that delineates monocytes, granulocytes, and neutrophils. Neutrophils were identified as LY6-C + , CD11b + , and LYG-6 Gr1 low. Representative plots showing SSC/FSC and CD11b + , and LYG-6 Gr1 double staining for each condition at t0 (A,B) or 24h after seeding (C,D). A,C: untreated mice splenocytes; B,D: splenocytes from mice subjected to IP injection of P. gingivalis. FSC: Forward Scatter; SSC: Side Scatter; CTL: unexposed mice. n = 5, ***: p < 0.0001, 10000 events acquired per sample. Please click here to view a larger version of this figure.

Figure 5 :Supplementary Figure 5 :Supplementary File 1 : Details on the experimental procedures and explanations for Supplementary Figures 1 , 2 , 5 .

 551125 Figure 5: P. gingivalis favors enhanced MV splenocyte shedding after 24 h culture. Splenocytes were isolated from P. gingivalis infected and control mice and cultured for 24 h and SMVs measured by different assays. Black bars: untreated mice (CTL), grey bars (IP): mice subjected to P. gingivalis injection for 2 weeks. A: Quantification by prothrombinase assay, B: protein concentration of the intact SMVs determined by spectrophotometry, C: RNA content of SMVs determined by spectrophotometry. Data are expressed as mean ± SEM. n = 5, **: p < 0.0001, by Student's T-test. Please click here to view a larger version of this figure.

Results 1 -Fig 1 :

 11 Fig 1: IP injection of P. gingivalis significantly enhances spleen weight (A) and splenocyte count (B) that is reduced by the PAD4 inhibitor.

Fig 2 : 3 .

 23 Fig 2: IP injection of P. gingivalis favors enhanced splenocyte shedding in vitro.

Fig 3 :

 3 Fig 3: Spleen cell composition varies with the type of induction.

Fig 4 :

 4 Fig 4: Upon gum injury and infection ROS accumulation increases in spleen tissue.A: representative photographies of immuno-histological staining with the red fluorescent probe with DHE. CTL: untreated mice, IP: mice submitted to PG injection for 2 weeks, LIG: mice submitted to gum ligation for 2 weeks, LIG-PG : mice submitted to gum ligation for 2 weeks with a thread impregnated with P.gingivalis, IP+I: mice submitted to simultaneous P.gingivalis and PAD4 inhibitor

Fig5:

  Fig5: Enhanced expression of VCAM-1 (A) and tissue factor (TF) in response to gum injury.

  Interestingly, gum insult combined or not to P. gingivalis let to a drastic and similar over-expression of both VCAM-1 and TF, that nearly reached the values observed after IP injection of P. gingivalis. (figure5) In addition, ROS accumulation in the spleen also indirectly confirmed the active contribution of neutrophils and granulocytes after gum insults. Altogether, data from LIG and LIG-PG subsets strongly suggest a key role of monocytes and neutrophils in accordance with the flow cytometry cell analysis. (see figures3 & 5).Nevertheless, the significant induction of splenocyte shedding assessed by prothrombinase assay or protein content was only detected in response to intraperitoneal P. Gingivalis infection. Interestingly, PAD4 was not efficient in the limitation of IP-driven SMV shedding,thereby suggesting that the release of MVs in the spleen in response to IP injection was not dependent on NET extrusion after 2 weeks. Therefore, SMVs are most likely from another cell origin than neutrophilsor released by non-extruding neutrophils in response to IP. The characterization of SMV cell origin would confirm the degree of activation of each contributing cell line.

Fig 1 : 2 )

 12 Fig 1: SMV generated in the presence of P.gingivalis induce endothelial senescence.

  50 µL of SN is recovered from washing. (SNL) and the pellet is suspended in 50 µL of PBS pH 7.4 or physiological solution. The obtained volumes are the separated into four 25 µL aliquots. 25 µl aliquot from the pellet and (SNL) are placed on slides, stained with 25 µL of DAPI or Hoetsch, incubated 10 min and then visualized under a fluorescence microscope. The remaining aliquot of each sample is used for elastase detection using the chromogenic substrate: MeOSuc-AAPV-pNA (Km 0.52 mM).

1 - 2 -

 12 At the heart level, P.gingivalis mediated pro-atherothrombotic and proinflammatory markers in left ventricular heart extracts. IP induced elevation of all inflammatory markers in heart tissue including VCAM, Gasdermin,TNFalpha, IL-6, IL-10. The Netosis inhibitor PAD-4 only down-regulated IL-10 suggesting the impact of netosis on anti-inflammatory responses. After 2 weeks-ligature, P.gingivalis soaked thread significantly upregulated VCAM and Gasdermin while LIG alone had no effect. A 4-weeks sustained treatment (LP4w) raised the expression of VCAM, TNF-alpha, IL-6. Strikingly, only the IL-10 expression reached the peak values induced by IP, strongly suggesting a delayed anti-inflammatory effect prompted by P.gingivalis while gavage led to elevated IL-10 already after 2 weeks.This confirms that our model as suitable for the study of remote cardiac effects by P.gingivalis local infection. Moreover, low but sustained pathogen-induced periodontitis alter the balance of heart inflammatory mediators where the control of Netosis appears valuable to preserve or delay heart tissue inflammation.In the present procedure, a 2-week duration induced variations in the splenocytes population, ROS accumulation, prosenescent MVs shedding and heart inflammation. It is important to keep in mind that although the major drawback of the murine model is a small spleen weight as compared to rats, the present procedure was optimized for a murine model and suitable for further transgenic mice investigations.Nevertheless, data confirm the model as suitable for the study of remote spleen and cardiac effects by P.gingivalis local or intraperitoneal infection.Confirmation of our hypothesis of a remote action of periodontitis on spleen immune responses and noxious cardiac impact was obtained by examining spleen and cardiac tissues and the prosenescent effect of SMVs on endothelial cells. Owing to pandemia, samples remain to be assessed. In particular, the plasma circulating mediators, like circulating fragments of NETs and their enzyme activities remain to be measured. To confirm the early impact on the cardiac outcome, other proteins like MMP or NLRP3 associated to cardiac inflamosome could be of interest as markers of heart tissue remodeling. Finally, the role of the periodontitis in the recently proposed spleen-cardiac axis could be explored in the lymph nodes. Indeed, IL-17 related regulatory events could not be explored at the protein level in spleen or MV extracts, but remain accessible by RT-PCR. P.gingivalis-associated periodontitis induces spleen activation that seems to be an important source of MVs, eventually associated with heart inflammation and the shedding of deleterious procoagulant SMVs. Our data, although preliminary, show the feasability of the evaluation of the noxious potency of circulating mediators such as MVs or NETs in individuals with periodontitis.Such results emphasize the need of periodontal management of patient at-risk.Moreover, the control of Netosis could be considered as an interesting strategy to preserve or delay the spleen inflammation. 2331-Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection. H. EL Itawi, Fareeha Batool, Céline Stutz, Abdul Wahid Qureshi, Fatiha El Zobairi, Olivier Huck, Florence Toti. (Journal of visualized experiments Jove. Accepted manuscript 18 March 2022) DOI: 10.3791/63480) Peridontal injury prompts remote spleen activation and the generation of spleen procoagulant microvesicles in a periodontitis murine model (International society for thrombosis and hemostasis ISTH -International online congress 17-23 rd July 2021, granted a travel fellowship) H. EL Itawi, S. Amissi, F. Batool, C. Stutz, F. El Ghazouani, A.B. Chaker, A.W. Qureshi, F. Moschovaki-Filippidou, C. Auger, E. Anglès-Cano, O. Huck, F. Toti Remote spleen activation in murine periodontitis prompts the generation of procoagulant and pro-senescent spleen microvesicles associated with early heart inflammation (European molecular biology lab congress : engineering in vascular biology. Barcelona, Spain 9-11 May 2022)

Table 1 :

 1 Classification of different types of extracellular vesicles and methods

	for their detection

Table 2 :

 2 Table of bioactive molecules of extracellular microvesicle MVs

Table 3 :

 3 Different methods for MVs quantification

Table 4 :

 4 Microvesicles and inflammation

Table 5 :

 5 Comparison of different methods for MVs characterization

		LIST OF ABBREVIATIONS
	ANO6	Anoctamine 6
	ATP	Adenosine Tri-Phosphate
	ADP	Adenosine di-phosphate
	C12FDG	5-dodecanoylaminoFluoresein Di-β-D Galactopyranoside

Table 1 :

 1

Classification of different types of extracellular vesicles and methods for their detection

24

(Burger et al 2013)

  10

Table 2 :

 2 Table of bioactive molecules of extracellular microvesicle MVs.

[START_REF] Voukalis | Microparticles and cardiovascular diseases[END_REF]

(Voukalis et al 2019)

3 Methods of MV isolation from tissues, quantification and characterization

	al 2011 & authorized by co-other of MV proteins and RNA concentration 7 and other proposed methods listed in
	Florence Toti).
	There are different strategies currently available to characterize EVs,
	including electron microscopy-based approaches and flow cytometry
	combined with antibodies against surface markers. Other strategies for
	studying EVs molecular signatures are based on molecular analysis of their
	cargo (mRNA, protein, or bioactive lipid content) by employing omics
	technologies aimed at the universal detection of mRNA species
	(transcriptomics), proteins (proteomics), lipids (lipidomics), and metabolites
	(metabolomics) 9 . Bulk analysis techniques, such as Western blots, ELISAs,
	mass spectrometry, and sequencing, and bead capture assays, have been
	widely used and instrumental in the field to date, associating EV phenotype
	(molecular cargo) with function 45 .
	As for MVs, the majority of clinical studies examine MVs isolated from plasma
	samples, which typically contain endothelial, leukocyte, platelet, and
	erythrocyte MVs. 39 Several approaches can be used for the detection and
	characterization of MVs with sometimes a pre-analytical isolation phase by
	high speed centrifugation and washing, before analysis by various methods
	such as prothrombinase enzymatic assay, spectrophotometric measurement

I.

table 3 .

 3

Table 3 :

 3 Different methods for MVs quantification24 (Dylan Burger et al 2013)

Table 4 :

 4 Microvesicles and inflammation[START_REF] Voukalis | Microparticles and cardiovascular diseases[END_REF] (Voukalis et al 2019)

injury and P. gingivalis infection favor ROS accumulation in spleen tissue

	Compared to control spleens, gum ligature alone was able to trigger a 5-
	fold ROS accumulation as assessed by histological staining with
	dihydroethidium. Addition of P. gingivalis in threads further doubled
	values of ROS (LIG-PG), up to 75% of the highest ROS value observed
	after IP treatment. Interestigly, PAD4 drastically prevented the effect of
	intraperitoneal P. gingivalis, suggesting that NETosis is key to ROS
	accumulation in the spleen.

4-Gum

  7 Left coronary arteries were dissected and cleaned of any adhesive conjunctive tissues then flushed with PBS. After debris removal and blood excess, cleaned coronary arteries were treated with Collagenase type I (Gibco, life technologies corporation, USA)

	solution at 1 mg/ml prepared in MCDB-131 medium (Gibco, life technologies
	limited, UK), supplemented with streptomycin (100 U/ml), penicillin (100 U/ml),
	fungizone (250 mg/ml), and L-glutamine (1 mM, all from Lonza,St Quentin en
	Yvelines, France) for 15 min at 37°C. Endothelial cells were then extracted into
	50-mL falcon tubes by circular massage of arteries with frequent flushing with
	medium. The collected medium containing endothelial cells was then
	centrifuged (450g, 5min, at room temperature), supernatant was discarded and
	cells were re-suspended with complete MCDB-131 medium supplemented with
	15% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 U/ml),
	fungizone (250 mg/ml), and L-glutamine (1 mM). Endothelial cells of 4-5
	different coronary arteries were cultured in adherent T25 flask in humidified
	incubator at 37 °C, with 5% CO2. After 6 hours, cells were washed with PBS
	non-adherent cells removal and fresh complete medium was added.

March 2022 • • e63480 • Page 8 of 17

March 2022 • • e63480 • Page 12 of 17

Complementary data

Acknowledgments

Authors are indebted to Claudine Ebel from the Service commun de cytométrie en flux (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg) for expert assistance and formation to complex flow cytometry analysis of the spleen and Ali El Habhab for initial training on rat spleen cells labelling. Deniz Karagyoz helped in digging gathering literature. This work was partly supported by two ANR grants COCERP (N°A17R417B), ENDOPAROMP(N °ANR-17-CE17-0024-01).

1 st place prize winner for the best presentation of the Doctoral school ED414 days -University of Strasbourg 2020-2021

Disclosures

The authors have nothing to disclose. (APAFIS#28745-2020121815051557). Mice were examined to evaluate pain and stress, and their weight monitored daily. All infection procedures were repeated 3 times per week)

Mouse splenocyte isolation and culture

Splenocytes were obtained as detailed elsewhere (see manuscript 1)

After sacrifice, fresh mouse spleens were weighed, washed in PBS (phosphate-buffered saline, Lonza, USA) and cleaned from any fat debris.

Under sterile conditions, spleens were cut using a scalpel into 1mm slices before homogenization by the plunger of a 2ml syringe used as a pestle and further filtered through 100-μm sterile strainers (Sarstedt, Germany). Following 450g centrifugation for 5 min, the cell pellet was re-suspended in a 1-ml ammonium-chloride-potassium erythrocyte lysis buffer for 5min, then completed with 9ml RPMI-1640 medium (PAN biotech) supplemented with Lglutamate (2mM), penicillin (100 U/mL), streptomycin (100 U/mL), fungizone (250 mg/mL) and 10% fetal bovine serum (FBS) (Dutscher). Cells were counted, seeded at 5 x 10 6 cells/mL in a 75 mL culture flask for adherent cells and incubated in humidified incubator at 37°C, with CO2 for 24 hours.

Characterization of splenocytes

Isolated splenocytes were characterized by flow cytometry (FACS 543 FORTESSA), according to a standardized protocol using APH7, BV786, PerCP-Cy5.5, Alexa Fluor700, BUV395 and eFluor 450 labelled monoclonal antibodies against mouse CD4, CD8, CD19, LY-6C, LYG-6 (GR-1), (BD Biosciences) and CD11b (Mac-1) (e-Biosciences). Data were analyzed by FlowJo.

Isolation and quantification of splenocytes microvesicles

After 24 hours culture, supernatants were collected under sterile conditions.

Splenocytes and cell debris were discarded by centrifugation at 450 g, 15 min at room temperature. Supernatants were further centrifuged twice at 14000g, 60 min, 4°C, washed pelleted SMVs were concentrated in Hanks Balanced Salt Solution (HBSS, without phenol red, without calcium and magnesium, Lonza, Belgium) and stored at 4°C for a maximum of 1 month. Prothrombinase assay was performed to quantify SMVs after their capture on Annexin-A5 coated micro-wells using a microplate spectrophotometer in kinetic mode. Annexin-5

has high affinity for phosphatidylserine (PhtdSer) exposed at the surface of MVs. In this assay, PhtdSer is the rate-limiting factor of the generation of thrombin from prothrombin detected at 405 nm using a chromogenic substrate (PNAPEP-0216, cryopep, Montpellier, France) [START_REF] Jy | Measuring circulating cell-derived microparticles[END_REF] . SMV concentration was referred to as nanomolar PhtdSer and was obtained by subtracting the OD values measured using isotype control biotynylatyed IgGs.

Protein content of splenic microvesicles

MVs protein content was measured by spectrophotometry using a Nanodrop (Thermo Scientific)

Characterization of oxidative stress in spleen tissues

Oxidative stress was determined in spleen tissue using DiHyroEthidium (DHE), a redox-sensitive fluorescent probe.Tissues were embedded in molds

2-P. gingivalis increases the splenocyte ability to shed SMVs independently of histone citrullination

After 24h culture, only splenocytes extracted from individuals treated by intraperitoneal injection of P. Gingivalis showed enhanced ability to shed SMVs as measured by prothrombinase assay or protein content culture medium. SMVs concentration doubled as compared to the SMVs from splenocytes of control spleens (IP vs CTL, p< 0.01). Only the IP treatment enhanced the shedding of SMVs and the PAD4 inhibitor had no effect, suggesting that histone citrullination is not causative. Interestingly, when the P.

Gingivalis treatment was extended from 2 weeks to 4 weeks, SMV shedding returned to the baseline levels of control individuals. Noteworthy, the delivery of P. Gingivalis by oral gavage was ineffective indicating that P. Gingivalis woud not activate spleen cells via a digestive route.

Discussion

Dosage of cytokines in blood plasma

Cytokine assay was performed from blood samples collected during animal sacrifices. The assay of these cytokines was carried out using the Proteome The samples will be mixed with a cocktail of biotinylated detection antibodies, then incubated with the array membrane which is spotted in duplicate with capture antibodies to specific target proteins. After, captured proteins will be