Wioleta Marut

Niloufar Kavian

Amélie Servettaz

Carole Nicco

Lalla Ba

Mandy Doering

Christiane Chéreau

Claus Jacob

Bernard Weill

Dr Frédéric Batteux
email: frederic.batteux@cch.aphp.fr

The Organotelluride Catalyst (PHTE)2NQ Prevents HOCl -Induced Systemic Sclerosis in the Mouse

Keywords: Tellurium, oxidative stress, Scleroderma, fibrosis

I would like to especially thank to my PhD supervisor, Frederic Batteux for supporting me during these past three years. Frederic is the funniest advisor and one of the smartest people I know. Also I would like to express my very sincere gratitude to Mr. Bernard Weill for the essential contribution to this work. I am very grateful to Frederic and Mr. Weill for their scientific advice, knowledge, and a lot of useful suggestions.

I also have to thank the members of my PhD committee, Professors Gilbert Kirsch and Philippe Guilpain for reviewing this work. I will forever be thankful to Carole Nicco for her help, suport and friendship. I could always count on her when I had a problems. She is my best role model for a scientist and mentor.

Remecrciemnts également à Christiane Chereau, sans qui ce travail n'aurait pas abouit. Je la remercie pour sa gentillesse permanente et son soutien.

I also thank my best friend Mathilde for providing support and friendship that I needed.

The best outcome from these past three years is finding my best friend, soul-mate, and husband. Grzes has been a true and great supporter and has unconditionally loved me during my good and bad times.

ABBREVIATIONS

Pathogenesis of systemic sclerosis (SSc)

Systemic sclerosis (SSc) is a rare connective tissue disease characterized by fibrosis of skin and internal organs, autoimmunity, and microcirculatory abnormalities 1 [ , 2]. There are two widely recognized subsets of SSc, limited cutaneous (lcSSc) and diffuse SSc (dSSc).

The main difference between these two subsets is the speed of disease progression, and the extent and severity of skin and internal organs. In lcSSc, skin thickening is confined to the face and extremities. In dcSSc, disease progression is very rapid, with the skin changes extending on the trunk, thighs, and upper arms. dcSSc is a more serious subset of the disease, with fatal visceral organ involvement.

SSc affects approximately 75.000-100.000 individuals in the United States. It affect four times more woman than man, and is more frequent in African-Americans than Caucasians [3]. Pathogenesis of SSc is complex and poorly understood. Factors causing dysfunction of fibroblasts, endothelial cells, and cells of the immune system have to be clarified (Figure 1). Among environmental factors, certain viruses, like cytomegalovirus [4] and certain chemicals such as solvents, silica, or pesticides may play a role in triggering the disease. In addition, several studies have shown that reactive oxygen species (ROS) [5][6][7] or TGFβ [8,9] may play a role in the initiation and progression of the disease. These new pathophysiological tracks open new therapeutic perspectives for this disease.

I.1 Fibroblast dysfunction

In SSc the excessive connective tissue fibrosis is the most characteristic pathological manifestation of the disease. In fibrotic tissue, normal architecture is replaced with a largely acellular, collagen-rich, stiff connective tissue, resulting in loss of functional integrity Figure 1. Link between dysfunction of fibroblasts, endothelial cells, and immune cells in systemic sclerosis from J Varga, Nature Reviews Rheumatology, 2012.

. The key cellular moderator of fibrosis are myofibroblasts which are a differentiated and activated form of fibroblasts [10]. Myofibroblasts are responsible for increased collagen synthesis and deposition. Biochemical analysis of SSc skin shows increased accumulation of the main fibrillar collagens (Type I and III). Moreover, lesional skin has abundant fibrillin and elastin fibrils, and elevated levels of enzymes such as lysyl hydroxylase and lysyl oxidase. These enzymes catalyze post-translation collagen modifications [11].

SSc fibroblasts are characterized by abnormal growth, secretion of cytokines and chemokines, expression of cell-surface integrin adhesion molecules, and receptors for PDGF, CCL2, and TGF-β. They differentiate into myofibroblasts which express alpha smooth muscle actin (α-SMA) which is strongly associated with tissue fibrosis [12].

I.1.1 Intrinsic factors implicated in the fibroblast dysfunction

I.1.1.1 Activation of pro-collagen gene via TGF-β signalling

The family of transforming growth factor-β (TGF-β) contain a large number of signaling proteins including TGF-isoforms, the factors that includes bone morphogenic proteins and activins. There are three isoforms of TGF-β encoded by three different genes (TGF-β 1, 2, and 3). TGF-β 1 is synthesized by endothelial cells (EC), hematopoietic cells and connective tissue cells. TGF-β 2 is synthesized by epithelial and neuronal cells, and TGF-β 3 by mesenchymal cells. After secretetion, all isoforms are stored in the extracellular matrix as a complex of TGF-β, the propeptide, and a protein called latent TGF-β-binding protein. TGF-β is released in vivo from the complex by the multifunctional matrix glycoprotein thrombospondin-1, and may also be activated by plasmin-mediated cleavage of the complex [13]. TGF-β can signal through intracellular Smad proteins or through receptor serine/threonine kinases.

• TGF-β signalling through Smad pathway:

The interaction of TGF-β with its specific transmebrane receptors (like TGFβRI)with serine-threonine kinase activity, induces a signal transduction involving intra-cytoplasmic Smad proteins. Ligand-induced phosphorylation of Smad2 and Smad3 allows them to form an heterocomplex with Smad4. This complex migrate into the nucleus where its regulates transcription of certain genes including type I collagen, plasminogen activator inhibitor-1, α-SMA, and CTGF (Figure 2). Ligand-induced signal transduction through the Smad • TGF-β signaling through non Smad pathway

Non-smad molecules activated by TGF-β include protein kinase (the MAPKs p38 and JNK, focal adhesion kinase [FAK], and TGF-β activated kinase 1), lipid kinases (such as PI3K and its downstream target, AKT), and the calcium-dependent phosphatase calcineurin (Figure 2) [15,16]. Non-Smad pathway is involved in regulating cell proliferation, cytoskeletal rearrangement, ECM synthesis, and apoptosis.

• Role of TGF-β in SSc

The expression of TGF-β is elevated in scleroderma dermal fibroblasts, as well as in monocyte/macrophages and other infiltrating inflammatory cells [17]. TGF-β is one of the most potent inducers of synthesis of extracellular matrix, and plays a central role in the process of fibrosis observed in SSc and other fibrosis diseases [18].

The importance of TGF-β in SSc has been confirmed by DNA microarray studies of SSc skin and SSc fibroblasts, in which TGF-β signatures correlated with certain geneexpression subsets (34,35). Moreover Milano et al. (36) have shown that the TGF-βresponsive signature occurred only in a subset of skin biopsies from patients with diffuse Several teams have demonstrated increased expression of type I and type II TGF-β receptors in the fibroblasts from SSc patients [19,20]. Pannu et all have shown that aberrantly expressed TGF-β-RI may drive an autocrine loop involved in the up-regulation of collagen and other matrixrelated genes in SSc fibroblasts. In addition they demonstrated that imatinib mesylate blocks TGF-β-induced activation of Smad1 and ERK-1/2 pathways in control fibroblasts and induces phosphorylation of Smad1 and ERK-1/2 in SSc fibroblasts [16].

SSc. It was not increased in the patients with limited SSc, which suggests that fibrosis in these patients is driven by a different mechanism [21].

I.1.1.2 CTGF -Connective tissue growth factor

Connective tissue growth factor (CTGF) or CCN2 is a member of the CCN early-response gene family. CTGF is involved in essential cellular functions such as the maintenance of homeostasis, wound healing, control of cell growth, as well as the regulation of angiogenesis. Serum levels of CTGF are elevated in SSc patients, and SSc fibroblasts demonstrate overexpression of CTGF [22,23]. Expression of CTGF can be regulated by TGF-β and by many other factors (cAMP, angiotensin II) [24]. In vitro, CTGF can stimulate proliferation, chemotaxis and adhesion of fibroblasts, and increase synthesis of collagen. In animal models of SSc, CTGF level is associated with the development of fibrosis. Some authors have suggested that CTGF is essential to the maintenance of fibrosis, after it has been induced by a signal mediated by TGF-β [23].

I.1.1.3 Early growth response genes

Egr-1 is the prototypical member of a family of zinc finger transcription factors.

Expression of Egr-1 is induced at sites of acute injury and implicated in cell proliferation, differentiation, and survival. Egr-1 regulates gene expression of several pro-fibrotic factors like: TGF-β, platelet-derived growth factor (PDGF), CTGF, vascular endothelial growth factor (VEGF), fibronectin, tissue inhibitor of metalloproteinase (TIMP-1), and osteopontin. In Hep-G2 cells, Egr-1 induces epithelial-mesenchymal transition (EMT) by upregulating the expression of Snail. Abnormal expression of Egr-1 was found in several animal models of fibrosis. In normal skin and lung fibroblasts, forced expression of Egr-1 is sufficient to induce the increase of COL1A2 promoter activity and increase stimulation induce by TGF-β. Subcutaneus injection of bleomycin induces fibrosis into the mice. This leads to the accumulation of Egr-1 in skin fibroblasts and enhances TGF-β signaling.

Transgenic fibroblasts expressing Egr-1 have an eight-fold increase expression of NADPH oxidase-4 (NOX-4), which is involved in the generation of ROS. Egr-1 increase levels of NOX-4onic hypoxia condition. Some authors suggest that in SSc patients, hypoxia and TGF-β might be responsible for overexpression of Egr-1 resulting in a increase activity of NOX-4 and production of ROS, which contribute to the progression of fibrosis. In addition, high levels of Egr-1 in peripheral mononuclear cells were found in SSc patients and is associated with pulmonary hypertension [25].

I.1.1.4 Molecules involved in extracellular matrix degradation

Fibroblasts are able of both synthesis and degradation of extracellular matrix (ECM). The amount of ECM in the tissue is controlled through the balance of ECM degradation enzymes -metaloproteinases (MMPs) and tissue inhibitors of metalloroteinases (TIMPs). TIMPs in SSc patients. In one study, authors have shown that SSc patients have higher serum concentrations of MMP-9 and TIMP-1. Moreover, they demonstrated that serum concentrations of MMP-9 is significantly higher in the diffuse type than the limited type of SSc [30]. Futhermore, expression of TIMP-1 in SSc fibroblasts is elevated and correlated with disease severity in SSc patients. Auto-antibodies blocking anti-MMP1 and -3 were found in SSc patients, and their concentrations are correlated with the extent of cutaneous fibrosis, pulmonary and renal arterioles [28,29,31]. All these data suggest that an imbalance of MMP / TIMP could cause the development of fibrosis in SSc patients.

I.1.1.5 Implication of ERK1/2 in fibrosis

ERK 1/2 (extracellular signal-regulated kinases 1/2) is constitutively activated in SSc lungs and skin fibroblasts. This activation is accompanied by significant production of ROS via NADPH oxidase, and an abnormal accumulation of Ha-Ras protein in the cytoplasm.

Accumulation of Ha-Ras proteins could be the result of a inhibition of their degradation by a proteasome as a result of ERK 1/2 activation. Svegliati et al highlight the importance of ERK 1/2, ROS and Ha-Ras protein in the induction and maintenance of "Scleroderma" phenotype fibroblasts (Figure 3). They have shown that in vitro, "scleroderma" phenotype fibroblasts obtained from SSc patients are rapidly reversible (after 1-2 days). In more recent work, the authors linked ERK1/2 activation to PDGF receptor signaling. They showed that extrinsic activation of PDGF receptor is required to maintain long-term activation of ERK and "scleroderma" phenotype, associated with activation of antibody (Ab) anti-receptor PDGF

. [32,33]. Another studies have shown activation of ERK1/2 by TGF-β in many cells type, and its important role for the CTGF and collagen stimulation [34].

Link between Ras and ROS by Gabrielli, The Journal of biological Chemistry, 2005.

I.1.1.6 Synthesis of free radicals

SSc fibroblasts spontaneously synthesize a large quantities of free radicals (superoxide anions O •- 2 and H 2 O 2 7

), molecules that stimulate fibroblast proliferation and collagen synthesis [ ]. This synthesis appears to be independent from IL4, CTGF and TGF-β, and could be partly dependent on the platelet derived growth factor (PDGF) [33]. Moreover, this increase oxidative stress could results from an inbalance between prooxidants and antioxidants system observed in SSc fibroblasts.

I.1.1.7 Synthesis of cytokines and chemokines

Fibroblasts are involved in the recruitment of leukocytes in the tissues by secreting chemokines and cytokines (IL-1, IL-6, tumor necrosis factor-α (TNF-α), IL-8, monocyte chemo-attractant protein-1 (MCP-1), and PDGF). They promote the migration of circulating lymphocytes through the endothelial barrier and their accumulation in the dermis [35][36][37].

In vitro data suggest that some cytokines secreted by fibroblasts are directly involved in the dysfunction of fibroblasts by the autocrine activation.

-Interleukin-1α: Fibroblasts from scleroderma patients aberrantly synthesize the IL1α active precursor form (pro-IL-1α). The blocking of the synthesis of IL-1 results in a reduction of IL-6 and collagen synthesis, and reduces the transcription of PDGF in these fibroblasts [38].

-MCP 1: This chemokine also appears to have a direct effect in stimulating the synthesis of collagen, according to data obtained in a murine model of bleomycin-induced fibrosis [35].

I.1.2 Extrinsic factors implicated in the fibroblast dysfunction

I.1.2.1 The role of cytokines

Interleukin-4

Interleukin-4 (IL-4) is a cytokine associated with B cell activation and T cell polarization to the Th2 phenotype. IL-4 have distinct roles in the regulation of tissue remodeling and fibrosis. Recent study suggest that IL-4 isa major factor of fibrosis induction [39]. The level of IL-4 is found increased in the bronchoalveolar lavage fluids of patients with idiopathic pulmonary fibrosis (IPF) [40], in the pulmonary interstitium of individulas with cryptogenic fibrosis alveolitis [41], and in peripheral blood mononuclear cells of those suffering from periportal fibrosis [42]. Increased serum levels of IL-4 [43] and IL-4producing T cells were observed in patients with SSc [44] as well as in mice with bleomycin-induced skin sclerosis and lung fibrosis [45]. TSK-1 mice harboring the tight skin (TSK) mutation. TSK-1 spontaneously develop skin fibrosis and express a large quantities of IL-4 receptors. As a consequences, TSK-1 mice have a constitutive activation of IL-4 signaling pathways [46]. This excess of IL-4 seems to have a pathogenic role, since treatment of TSK mice with anti IL-4 prevents collagen deposition in the dermis [47]. In SSc, IL-4 is mostly produced by CD4 + T cells and monocytes. Mice deficient for T-bet, a Th1 initiating factor, show increased and faster development of fibrosis with an exaggerated immune response and a constitutive elevation of IL-13 (45, 46 revue from bleo hocl). In humans, IL-4 stimulates proliferation of fibroblasts, increases deposition of extracellular matrix in the tissues, and is involved in the pathogenesis of SSc [48]. In addition, IL-4 stimulates the synthesis of pro-fibrotic cytokines such as MCP-1 by fibroblasts [39].

Interleukin-13

IL-13 is an immunoregulatory cytokine secreted by activated Th2 cells. IL-13 is involved in the pathogenesis of many fibrotic diseases, including SSc, and appears to be necessary , whereas the maintenance of the type-2 immune response and the activation of proinflammatory mediators are important contributors to the chronic nature of fibrotic disorders (Figure 3).

in
IL-13 stimulate macrophages to produce TGF-β by several different mechanism including the production of latent and up-regulation of MMPs and also through IL-13Rα2 signaling.

The transgenic IL-13 mice have exacerbated pulmonary fibrosis with high levels of TGFβ [50]. Moreover, in SSc-bleomycin mice, mRNA levels of IL-13 were increased in the damaged skin, and expression of IL-13 receptor -IL-13R-2 was increased in mononuclear cells and macrophages infiltratingthe skin. Finally, IL-13 deficient mice did not develop skin fibrosis after daily injection of bleomycin, showing involvement of IL-13 in the development of

Interleukin-17

cutaneous fibrosis [50].

Recent study suggests that patients with SSc have strikingly increased frequencies of Th17 Interferon-gamma is produces by Th1 lymphocytes and is negative regulator of fibroblast activation. IFN-γ inhibit the proliferation of fibroblast and differentiation into myofibroblast. There is a lot of studies showing capability of IFN-γ to selectively inhibit collagen synthesis [53,54]. Another studies in SSc have shown that some fibroblasts from SSc patients are resistant to the inhibitory effect of IFN-γ.

I.1.2.2 Endothelin 1

Endothelin-1 (ET-1) is a potent vasomodulatory peptide produced by endothelial cells, macrophages, fibroblasts, and other type of cells. In addition to its vasomodulatory effects, ET-1 can induce the transformation of fibroblasts into myofibroblasts via the endothelin receptors A (ETRA) and B (ETRB) [START_REF] Xu | Endothelin-1 induces expression of matrix-associated genes in lung fibroblasts through MEK/ERK[END_REF]. ET-1 can induce collagen synthesis by activation of CTGF. The expression of ET-1 and its receptors is elevated in the skin from SSc patients [START_REF] Abraham | Increased levels of endothelin-1 and differential endothelin type A and B receptor expression in scleroderma-associated fibrotic lung disease[END_REF]. Serum concentrations of endothelin-1 is increased in SSc patients 57 [ ].

Moreover, blockade of the endothelin receptors (ET-A and ETB) with bosentan significantly reduces overexpression of α-SMA and ECM concentration by SSc fibroblasts [START_REF] Shi-Wen | Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/phosphoinositide 3-kinase/Aktdependent pathway and is essential for the enhanced contractile phenotype of fibrotic fibroblasts[END_REF].

I.1.2.3 TGF-β

TGF-β is a pleiotropic cytokine, recognized as a master regulator of pathological fibrosis.

This cytokine is clearly involved in the activation and survival of fibroblasts, and synthesis of collagen [START_REF] Cotton | TGF beta--a role in systemic sclerosis?[END_REF]. TGF-β can lead to transformation of normal fibroblasts into activated myofibroblasts and increase the synthesis of extracellular matrix [8]. Moreover TGF-β increases the synthesis of CTGF by fibroblasts and as a consequence the synthesis of collagen [START_REF] Yamakage | Selective upregulation of platelet-derived growth factor alpha receptors by transforming growth factor beta in scleroderma fibroblasts[END_REF]. Nevertheless, SSc fibroblasts do not produce themselves TGF-β [START_REF] Needleman | Secretion and binding of transforming growth factor beta by scleroderma and normal dermal fibroblasts[END_REF] and the level expression of TGF-β is variable [START_REF] Yamane | Epidermal growth factor up-regulates expression of transforming growth factor beta receptor type II in human dermal fibroblasts by phosphoinositide 3-kinase/Akt signaling pathway: Resistance to epidermal growth factor stimulation in scleroderma fibroblasts[END_REF]. TGF-β is originally secreted by endothelial cells, or by inflammatory cells present in the dermis in the first stage of the disease. It has also been shown that the skin of patients with SSc contained varying amounts of three isoforms of TGF-β according the stage of disease [START_REF] Querfeld | Expression of TGF-beta 1,beta 2 and -beta 3 in localized and systemic scleroderma[END_REF]. Isoforms 1 and 2 are found in a large amount in the dermis at an early stage of the disease, a coexistinginfiltration of the dermis by inflammatory cells. In fibrosis stage, these isoforms are absent, as in the skin of control subjects. It seems likely that TGF-β plays a role in the induction of fibrosis at an early stage of the disease, but is less involved in the fibrosis [START_REF] Querfeld | Expression of TGF-beta 1,beta 2 and -beta 3 in localized and systemic scleroderma[END_REF][START_REF] Higley | Immunocytochemical localization and serologic detection of transforming growth factor beta 1. Association with type I procollagen and inflammatory cell markers in diffuse and limited systemic sclerosis, morphea, and Raynaud's phenomenon[END_REF].

I.1.2.4 PDGF

Platelet-derived growth factor (PDGF) is a major mitogen for many cell types of mesenchymal origin, e.g. fibroblasts and smooth muscle cells. PDGF is a dimeric peptide secreted by various cell types such as platelets, fibroblasts, and smooth muscle cells.

PDGF induces migration, differentiation and transformation of various cell types. PDGF participates in the regulation of apoptosis in part through the generation of ROS. Five isoforms of PDGF are described: PDGF AA, AB, BB, CC, DD. Many cells express PDGF, but the degree of expression is variable from one cell type to the other (Figure 3) [START_REF] Fredriksson | The PDGF family: four gene products form five dimeric isoforms[END_REF]. For example, PDGF B is mainly synthesized by endothelial cells, megakaryocytes and neurons [START_REF] Andrae | Role of platelet-derived growth factors in physiology and medicine[END_REF]. PDGF A and PDGF C are mainly secreted by epithelial cells, muscle cells and neural progenitors. PDGF D appears to be mainly synthesized by fibroblasts and smooth muscle cells. The PDGF receptors are formed by the association of two chains with tyrosine kinases: two α chains, two β chains or a heterodimer formed by α and β chain [START_REF] Fredriksson | The PDGF family: four gene products form five dimeric isoforms[END_REF].

Activation of PDGFR induces phosphorylation of MAP kinases (Erk1/2, p38, JNK), Ras activation and the expression of genes such as Egr-1, c-fos, c-jun. Abnormalities of PDGF or their receptors have been identified in tumor process, various vascular diseases (atherosclerosis, pulmonary hypertension) and fibrosis [START_REF] Andrae | Role of platelet-derived growth factors in physiology and medicine[END_REF][START_REF] Bonner | Regulation of PDGF and its receptors in fibrotic diseases[END_REF]. Svegliati et al have shown that stimulatory antibodies to the PDGFR selectively induced the Ha-Ras-ERK1/2 pathway and the stimulation of ROS cascades. This cascade of events increase the production of collagen type I and conversion of normal fibroblasts into activated myofibroblast [32,33].

In mice, activation of PDGFR leads to progressive fibrosis of the skin suggesting the role of this signaling pathway in the development of fibrosis [START_REF] Olson | Increased PDGFRalpha activation disrupts connective tissue development and drives systemic fibrosis[END_REF]. Fibroblasts from SSc patients constitutively synthesize PDGF AA under the autocrine stimulation by IL1α [38], whereas normal fibroblasts do not synthesize PDGF AA, stimulated with mitogens in vitro. PDGF AA concentrations were found increased in the alveolar lavage of SSc patients [START_REF] Ludwicka | Elevated levels of platelet derived growth factor and transforming growth factor-beta 1 in bronchoalveolar lavage fluid from patients with scleroderma[END_REF]. In addition, expression of PDGF receptor α was increased on the surface of fibroblasts from SSc patients. Activation of PDGFR could be inhibited by synthetic molecules inhibiting tyrosine kinases (TKI), including imatinib, axitinib, sunitinib, dasatinib, sorafenib and the nilotinib. These molecules were initially developed for cancer treatment to inhibit signals proliferation via bcr/abl and c-kit and for some of them by inhibiting VEGFR and noangiogenesis. Imatinib has been successfully tested in a murine model of lung hypertension [START_REF] Schermuly | Reversal of experimental pulmonary hypertension by PDGF inhibition[END_REF]. Others have shown that imatinib could reduce the synthesis of collagen and fibronectin in human skin fibroblasts [START_REF] Distler | Imatinib mesylate reduces production of extracellular matrix and prevents development of experimental dermal fibrosis[END_REF][START_REF] Akhmetshina | Treatment with imatinib prevents fibrosis in different preclinical models of systemic sclerosis and induces regression of established fibrosis[END_REF]. Recently, our team provided a new data on the activation of PDGFR in SSc and the effectiveness of certain TKI molecules to conteracts fibrosis, vascular abnormalities and activation of immune system in mouse model of SSc [73].

I.1.2.5 Serotonin

Serotonin (5-Hydroxytryptamine, 5-HT) is a hormone released upon the activation of platelets. Normally, plasma level of 5-HT is very low because circulating 5-HT is stored in platelets, but in SSc patients upon platelets activation, plasma level of 5-HT is significantly increased [START_REF] Biondi | Plasma free and intraplatelet serotonin in patients with Raynaud's phenomenon[END_REF]. Serotonin is known to increase proliferation of fibroblasts and synthesis of collagen I. For example, 5-HT stimulates the proliferation of fibroblasts isolated from pulmonary hypertensive animals [START_REF] Welsh | Proliferation and signaling in fibroblasts: role of 5-hydroxytryptamine2A receptor and transporter[END_REF]. Seven distinct families of 5-HT receptors have been identified (5-HT1-5-HT7). Among them, two receptor subtypes, 5-HT2A and 5-HT2B, dependent mechanism [START_REF] Dees | Platelet-derived serotonin links vascular disease and tissue fibrosis[END_REF]. Thus, serotonin can be a mediator linking vascular injury, platelet activation, and fibrosis in SSc.

I.1.2.6 Angiotensin II

Angiotensin II (ANG II) is a profibrotic component of the renin angiotensin system (RAS)

and plays a critical role in controling of cardiovascular homeostasis. The RAS is a critical regulator of sodium balance, extracellular fluid volume, vascular resistance, and, ultimately, arterial blood pressure.

ANG II is profibrotic moleculr produced by activated fibroblasts and macrophages and exerts its effect by inducing NADPH oxidase activity and by stimulation of TGFβ1 production. This leads to the proliferation of fibroblast and then differentiation into active myofibroblasts. Moreover, ANG II increases TGFβ1 signaling by increasing the level of smad 2/3 [START_REF] Rosenkranz | TGF-beta1 and angiotensin networking in cardiac remodeling[END_REF][START_REF] Bataller | NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis[END_REF]. In addition to its implification in various cardiovascular diseases, ANG II is also involved in cardiac fibrosis and in hypertensive heart disease [START_REF] Watanabe | Angiotensin II and the endothelium: diverse signals and effects[END_REF]. ANG II plays an important role in the development of hepatic, lung, and renal fibrosis [START_REF] Mezzano | Angiotensin II and renal fibrosis[END_REF][START_REF] Kato | Therapeutic effects of angiotensin II type 1 receptor blocker, irbesartan, on nonalcoholic steatohepatitis using FLS-ob/ob male mice[END_REF].

Angiotensin II acts by binding to two receptor subtypes, angiotensin type I (AT1R) and type II (AT2R) receptors. AT1R mediateds the profibrotic effects of ANG II by stimulating fibroblasts proliferation, cardiomycates hypertrophy and by increasing apoptosis of endothelial cells [START_REF] Burstein | Atrial fibrosis: mechanisms and clinical relevance in atrial fibrillation[END_REF]. AT1R can induce fibrosis through the Shc/Grb2/SOS protein complex which activates Ras protein. This phenomenon initiates mitogen-activated protein kinase (ERK 1/2, p38, JNK) phosphorylation cascades which are centrally involved in fibrosis remodeling [START_REF] Hunyady | Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II[END_REF]. The JAK/STAT pathway, activating transcription factors such as activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) are also activated by ANG II(Figure 4). Both pathways lead to the proliferation and differentiation of fibroblasts, and synthesis of collagen.

I.2 Endothelium dysfunction

Vascular injury appears to be one of the earliest clinical manifestations in SSc and can be a crucial initiating event in the pathology of SSc. More than 90% of SSc patients present Raynaud's phenomenon and reversible and transient digital ischemia. This cause changes in the cellular metabolism and the generation of reactive oxygen species (ROS) by ischemia-reperfusion. Early apoptosis of endothelial cells is highlighted in the skin biopsies of patients, resulting in a loss of integrity of the endothelial barrier. Endothelial dysfunction has also consequences for the recruitment of inflammatory cells, changes in the coagulation / fibrinolysis balance, and abnormal vascular tone. In addition, endothelial abnormalities may contribute to the phenomenon of fibrosis in SSc [START_REF] Botzoris | Management of Raynaud's phenomenon and digital ulcers in systemic sclerosis[END_REF].

I.1.3 Vasculogenesis and angiogenesis

In adults, blood vessels formation and repair are mediated via two different processes: vasculogenesis and angiogenesis. Vasculogenesis is describes by the formation of new vessels by circulating EP (bone marrow-derived endothelial progenitor cells) independent from pre-existing vessels [START_REF] Takahashi | Ischemia-and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization[END_REF]. Angiogenesis is a process of sprouting of differentiated EC from pre-existing vessels. It involves the proliferation and migration of mature endothelial cells. Stabilization of vessel wall by pericytes is the final process of angiogenesis and leads to a functional network of new capillary [START_REF] Carmeliet | Mechanisms of angiogenesis and arteriogenesis[END_REF]. [START_REF] Smyth | Digital vascular responses and serum endothelin-1 concentrations in primary and secondary Raynaud's phenomenon[END_REF]. This indicates that increased endothelin levels may be rather a marker of endothelial damage than a reflect of enhanced susceptibility to vasoconstriction .

I.1.4 Dysregulation of vasculogenesis

I.1.4.2 Nitric oxide (NO)

Nitric oxide is an important vasodilator of vascular smooth muscle, constitutively secreted by endothelial cells. NO inhibits vascular smooth muscle cell proliferation via inhibition of mitogenic proteins TGF-β and PDGF, and elevation of cyclic guanosine monophosphate (cGMP) [START_REF] Jeremy | Nitric oxide and the proliferation of vascular smooth muscle cells[END_REF]. The role of NO in the pathogenesis of SSc is complex. Plasma values of NO measured in patients are discordant from one study to another [START_REF] Cerinic | Beauty and the beast. The nitric oxide paradox in systemic sclerosis[END_REF]. Moreover, the role of NO is ambivalent because it may interact with other reactive oxygen forms like superoxide anion to form the higly toxic species peroxynitrite. By contrast, NO can also limits cytokine-induced endothelial cell activation, adhesion of monocyte, and inhibits the release of inflamatory cytokine like IL-6 and IL-8 from endothelial cells. Therefore, NO production in SSc may contribute to the pathogenesis of arteriolar intimal proliferation and may have a prominent role in pathogenesis of the disease.

I.1.5 Dysregulation of angiogenesis

Angiogenesis is a multistep process under the control of angiogenesis inducers and inhibitors. Under normal conditions, the levels of angiogenesis inducers and inhibitors are balanced and angiogenesis does not occur in healthy tissues. Under inflammatory states or in hypoxic environment like Systemic sclerosis, angiogenic growth factors are induced which leads to the initiation of angiogenesis. Tissue ischemia leads to the expression of vascular endothelial growth factor (VEGF), PDGF, and basic fibroblast growth factor (bFGF), which have been characterized as key molecules in angiogenesis (Figure 5).

VEGF is a pro-angiogenic factor, which controls several molecular and cellular steps in the Other pro-angiogenic mediators like PDGF "Placental Growth Factor" (PIGF) and "Fibroblast Growth Factor-2" (FGF-2) also have elevated plasma levels compared to healthy subjects: SDF-1 receptor, and probably has an important role in triggering cell arrest and emigration into the angiogenic niches (Figure 6).

I.1.6 Local recruitment of leukocytes

Several recent studies have evaluated involvement of endothelial progenitor cells in SSc but the results on quantification and properties of these cells are contradictory, probably because the surface markers used in experiments are not the same in each study. Del Papa at al have shown that the rate of endothelial progenitor cells CD45-CD133 + was significantly lower in SSc patients that in healthy subjects [127]. Other team has shown increased angiogenic potency of monocytic endothelial progenitor cells in SSc patients [START_REF] Yamaguchi | Enhanced angiogenic potency of monocytic endothelial progenitor cells in patients with systemic sclerosis[END_REF].

After in vitro activation endothelial cells from scleroderma patients, are capable to secrete MCP-1 and RANTES, two chemokines that attract monocytes and macrophages [START_REF] Anderegg | Chemokine release from activated human dermal microvascular endothelial cells--implications for the pathophysiology of scleroderma?[END_REF]. Innate immunity and adaptive immunity play an important role in the pathogenesis of SSc.

At an early phase of the

Intervention of the immune system

sclerotic process, leukocyte activation is visible in the peripheral blood and damaged tissue, where it is directly involved in the induction of tissue damage.

I.1.8 Innate immunity

Recently accumulating and mounting evidence points toward the importance of immune cells in SSc pathogenesis.

I.1.10.1 Elements contributing to the activation of B cells

The • Fibrillin-1 antibodies

. A recent study revealed alpha-enolase as a primary target antigen of AFA from SSc patients

[133].
Fibrillin is a major component of the extracellular matrix microfibrils. A polymorphism in the fibrillin gene (FBN1) is associated with development of SSc in some populations. Antifibrillin-1 antibody can be detected in patients with SSc most commonly in dcSSc but also in lcSSc or MCTD. The prevalence of these Acts effect varies according to the ethnic background of patients, since they are present in 94% of Choctaw Indians, 87% of Japanese patients and only 4% of subjects of African-American origin. Sera from patients with diffuse SSc or calcinosis, Raynaud's, or mixed connective tissue disease also had significantly higher frequencies of anti-fibrillin-1 Abs than sera from healthy controls or patients with other

• Anti-platelet-derived growth factor receptor antibodies (anti-PDGFR)

connective tissue diseases [134].
PDGFR autoantibodies might have a pathogenic role in SSc patients. PDGFR expression is increased by pathological TGF-β signaling, and binding of anti-PDGFR to the PDGFR ligand results in amplification of the Ras-extra-cellular signal-regulated kinase (ERK) and 1/2-reactive oxygen species (ROS) cascade. Thus, it leads to increased collagen production [135]. Recent study reported autoantibodies against PDGFR in patients with SSc, but not in healthy controls or patients with rheumatoid arthritis, idiopathic pulmonary fibrosis or primary Raynaud's phenomenon [32]. Moreover, h

• Vascular receptor antibodies igher levels of those autoantibodies were detected among those with skin or lung fibrosis.

Angiotensin II and endothelin-1 induce synthesis of collagen through target receptor stimulation in fibroblasts. Anti-angiotensin II type 1 receptor (anti-AT1R) and endothelin- Therefore further studies, with more homogeneous groups of patients should be performed to precise the conclusions about the effectiveness of Rituximab in SSc.

Genetic and environmental factors

I.1.13 Environmental Factors

There is a lot of evidences of the association between systemic sclerosis and certain environmental risk factors, including exposures to vinyl chloride, silicone breast implants, organic solvents, and other agents such as epoxy resins and pesticides [153]. However, a meta-analysis did not find association between particular silicone breast implants and

Animal models of SSc

Fli-1 mouse

Fli-1 ("Friend leukemia integration factor-1") plays an inhibitory role in the expression proteins of the extracellular matrix. Fli-1 is involved in vascular development. Fli-1 -/mice have an increased in vascular permeability, similar to that observed in SSc patients, as well as abnormalities in the formation of collagen fibrils 166 [ ].

•

• Graft Versus Host disease (GVHD) -induced systemic sclerosis

Chronic Graft-versus-Host Disease (GvHD) emerges from allo-reactive processes between donor-derived immune cells and host cell populations. Scl-GvHD can be induced through transfer of splenocytes and-bone marrow cells from B10.D2 donor mice into BALB/c RAG2-/-knockout (RAG2 KO) recipient mice, which are deficient in mature T and B cells. Those mice developed a skin thickness, particularly in the extremities, progressive fibrosis involving internal organs such as kidney and small intestine, but not lungs [START_REF] Ruzek | A modified model of graftversus-host-induced systemic sclerosis (scleroderma) exhibits all major aspects of the human disease[END_REF].

Those changes are observed already after 3-4 weeks. Histological analysis of the skin shows an inflammatory infiltrate of macrophages, monocytes, CD4 + , CD8 + T cells, and fibrosis with accumulation of extracellular matrix.

Role of reactive oxygene species (ROS)

However, the genetic background and autoimmunity make the Tsk1 mice a useful model for SSc which has been extensively studied in the past years.

A radical is an atom or a group of atoms that have one or more unpaired electrons.

II.1 Sources of ROS The superoxide anion O •-

The superoxide anion is formed by adding one electron to an oxygen molecule:

O 2 + 2e -+ 2H + → H 2 O.
However, there is leakage of electrons at each stage of transport and some O 2 molecules will undergo a reduction by one electron, resulting in the formation of superoxide anion O •-

2 : O 2 + 1e -→ O •- 2 -NADPH-Cytochrome P450
. This mitochondrial superoxide anion production is continuous and ubiquitous.

Cytochromes P450 constitute one of the largest superfamilies of enzyme hemoproteins

Hydrogen peroxide (H 2 O 2

The hydrogen peroxide production may result directly from the molecular oxygen reduction, under the action of different enzymes (e.g. glucose oxidase):

)

O 2 + 2H + + 2e - H 2 O 2

177]:

-Cytosolic copper/zinc SOD (Cu/ZnSOD)=SOD1

-Mitochondrial manganese SOD (MnSOD)=SOD2

-Glycosylated extracellular copper/zinc SOD=SOD3 These enzymes catalyze the following reaction: 2O

II.1 Role and consequences of ROS

).

Reactive derivatives of oxygen can interact with each other, but also with all types of cell components: proteins, lipids, carbohydrates, DNA. These effects depend on their own reactivity, their preferential affinity for a particular substrate, their quantity and their ability to diffuse.

I.1.16 Action on proteins

The radical forms can act on the main chain of the protein leading to its fragmentation. The interaction can also be located on the side chain. Particularly sensitive are sulfur residues

I.1.20.1 Examples of enzymatic antioxidants systems

In eukariotic cells following antioxidant systems exist: Superoxide dismutase (SOD), ubiquitous enzymes that catalyze reaction: • Ubiquinone (coenzyme Q10)

2O •- 2 + 2H + → H 2 O 2 + O 2
A vitamin having a significant role in the production of energy at the respiratory chain, and having an antioxidant role in protecting cells against damaging effects of free radicals Furthermore, in mice, several studies emphasize the link between pulmonary fibrosis induced by the inhalation of silica and oxidative stress [192,193]. Subcutaneous injections of bleomycin into the mice induce skin and lung fibrosis [START_REF] Yamamoto | Animal model of sclerotic skin. II. Bleomycin induced scleroderma in genetically mast cell deficient WBB6F1-W/W(V) mice[END_REF]. [ , 198]. In both cell types, the synthesis of ROS is self-sustaining and dependent on a NAPDH oxidase. The same team work suggests that synthesis of collagen in fibroblasts is dependent on the synthesis of ROS [33].

For nitric oxide, several studies attest to quantitative differences in the serum between SS patients and healthy controls, but as stated previously, the results of these studies are contradictory. However that can be explain by differences in diseased subset, inflamatory

Personal work

In this chapter, I will present my work on the effect of ROS/RNS modulators in Systemic sclerosis (SSc).

Several reports have suggested that reactive oxygen and nitrogen species are involved in SSc pathogenesis. SSc fibroblast from skin and internal organs overproduce ROS that trigger the proliferation of fibroblasts and the synthesis of type I collagen leading to the initiation and progresion of SSc [7,206,207]. The role of ROS in the initiation of SSc has been evidenced in our laboratory. Our studies demonstrated that sera from SSc patients can induce not only the production of ROS by endothelial cells but also the proliferation of However, if intracellular ROS can stimulate cell growth, they can also induce cell death beyond a certain threshold, as seen in tumor cells (Laurent et

RESULTS (PHTE) 2 NQ exerted cytotoxic effects on normal and SSc fibroblasts in vitro

Fibroblasts from normal mice and from HOCl-mice were exposed in vitro to increasing amounts of (PHTE) 2 NQ (Suplementary data, Figure 7) to assess the cytotoxic properties of this 'sensor/effector' multifunctional redox catalyst agent on those different types of fibroblasts. In the presence of 1 µM (PHTE) 2 NQ, the rate of viability of normal fibroblasts was 72% (P = 0.0001 vs untreated fibroblasts) and 68% with 4 µM (PHTE) 2 NQ (P = 0.0001 vs untreated fibroblasts; Figure 1a). (PHTE) 2 NQ had a more powerful cytotoxic effect on diseased SSc fibroblasts than on normal fibroblasts. In the presence of 1 µM (PHTE) 2 NQ, the rate of viability of SSc fibroblast was 45% (P = 0.0001 vs untreated fibroblasts and vs normal fibroblasts) and 30% with 4 µM (PHTE) 2 NQ (P = 0.0001 vs untreated fibroblasts and vs normal fibroblasts; Figure 1a).

(PHTE) 2 NQ exerted pro-oxidative effects in vitro

The basal production of H 2 O 2 was increased by 56% in SSc fibroblasts compared to normal fibroblasts (P = 0.001). Production of NO was also higher in SSc fibroblasts than in normal fibroblasts (P = 0.01). Incubation of normal fibroblasts and SSc fibroblasts with increasing amounts of (PHTE) 2 NQ dose-dependently increased the production of H 2 O 2

Glutathione is an essential cofactor of H (P = 0.009 and P = 0.001, respectively) and of NO (P = 0.018 in both cases; Figures 1b andc).

Imbalance of GSH metabolism in fibroblasts

2 O 2 catabolism. The basal level of GSH was decreased by 22% in SSc fibroblasts compared to normal fibroblasts (P = 0.0032). The level of intracellular glutathione was higher (P = 0.0001) in normal fibroblasts following exposure to 1 µM (PHTE) 2 NQ than in untreated normal fibroblasts. By contrast, the treatment of diseased SSc fibroblasts by (PHTE) 2 NQ failed to increase the level of intracellular glutathione (Figure 1d).

Modulations of H 2 O 2 metabolism in SSc fibroblasts

We next investigated the mechanism of action of (PHTE) 2a). On the other hand, specific inhibition of catalase by aminotriazol (ATZ) or addition of exogenous catalase, had no effect on the levels of H 2 O 2 in normal and SSc fibroblasts (Figure 2a). Depleting GSH with BSO significantly decreased the viability of SSc fibroblasts (P = 0.0026) and of normal fibroblasts (P = 0.017). By contrast, specific inhibition of catalase by aminotriazol (ATZ) had no effect on the viability of normal and SSc fibroblasts (Figure 2b). Moreover, addition of suboptimal doses of BSO and of (PHTE) 2 NQ added their effects worked jointly to kill SSc fibroblasts more efficiently than normal fibroblasts alone (P = 0.0168) (Figure 2c).

Determination of cell death pathway in normal and SSc fibroblasts

Fibroblasts from normal and HOCl-mice were incubated with 1, 2 and 4 µM of (PHTE) 2 NQ for 12 h, 24 h, 36 h or 48 h. The highest cytotoxic effect of (PHTE) 2 NQ was observed at a concentration of 4 µM. At this concentration the viability was decreased by 73% for SSc fibroblasts and only by 14.2% for normal fibroblasts (P = 0.001). A kinetic analysis of cell death between 12 h and 48 h indicated that the (PHTE) 2 NQ effects are mainly mediated through a necrotic process (Figure 3).

(PHTE) 2 NQ decreased both skin and lung fibrosis in mice with SSc

Clinically, HOCl-induced SSc in mice (HOCl-mice) is associated with an increase in dermal thickness that's significantly reduced by (PHTE) 2 NQ (P = 0.0001 vs untreated SSc mice; Figure 4a). Histopathological analysis of skin (Figure 4c) and lung (Figure 5a) biopsies stained with hematoxylin and eosin, and lung biopsy stained by Sirius Red (Figure 5c), confirmed the dermal thickness reduction and evidenced a decrease in fibrosis in both tissues (Figure 4c, 5a, 5c). (PHTE) 2 NQ reduced the accumulation of type I collagen induced by HOCl in the skin versus untreated mice (Figures 4b). We also investigated the effects of (PHTE) 2 NQ in the bleomycin model and found the data to yield similar results to those gained by the HOCl model in terms of skin fibrosis. However, no lung fibrosis was observed after 5 weeks of bleomycin injections (Supplementary data, Figure 8).

Furthermore, α-SMA staining in skin sections showed that HOCl-treated SSc animals displayed increased numbers of myofibroblasts (9.53% ± 0.483 of positive area) compared to PBS-injected mice (2.46% ± 0.555 of positive area, P = 0.0001) (Figures 4e and4f). The number of myofibroblasts was reduced by 47.6% after treating HOCl-mice with (PHTE) 2 NQ (P = 0.0005). (PHTE) 2 NQ also reduced the concentration of type I collagen in the lungs of HOCl-mice by 26.7% (P = 0.0106; Figures 5a and5b).

Decrease of dermal thickness in SSc mice through (PHTE) 2 NQ curative treatment

(PHTE) 2 NQ decreased dermal thickness when administered as a curative treatment. Mice with SSc were treated with (PHTE) 2 NQ (10 mg/kg of (PHTE) 2 NQ), which was administered intravenously once a week during 2 weeks. After 2 weeks, the dermal thickness in treated mice was significantly less than in the untreated group (P = 0.0151) (Figure 4d).

(PHTE) 2 NQ reduced the concentration of serum AOPP and nitrite production

Advanced oxidation protein products (AOPP), a marker of systemic oxidative stress, increased in the sera of HOCl-mice compared to PBS control mice (P = 0.0001; Figure 6a).

Treatment of HOCl-mice with (PHTE) 2 NQ reduced the levels of AOPP by 40%, compared to untreated SSc animals (P = 0.0001; Figure 6a). The level of nitrite in the sera, a marker of systemic nitrosative stress, increased in HOCl-mice compared to PBS control mice (P = 0.0006; Figure 6b). Treatment with (PHTE) 2 NQ in HOCl-mice reduced the levels of serum nitrite by 18% compared to untreated SSc mice (P = 0.039 ; Figure 6b).

(PHTE) 2 NQ reduced serum concentration of anti-DNA topoisomerase 1 Abs

The sera of HOCl-mice contained significantly higher amounts of DNA-topoisomerase I Abs than the sera of mice treated with PBS (P = 0.0008). The level of anti-DNA topoisomerase I Abs significantly reduced in HOCl-mice treated with (PHTE) 2

In this report, inspired by recent results obtained -by us and others -in cancer therapy using selective pro-oxidative molecules, we demonstrates that organotelluride catalysts can NQ compared to untreated HOCl-mice (P = 0.04; Figures 6c). Preliminary data suggest that the molecule is not only efficient as a preventive treatment, but as a curative treatment of SSc as well.

DISCUSSION

MATERIALS & METHODS

Animals and chemicals

our institution. HOCl administration was performed as previously described (Servettaz et al., 2009). Briefly, 300 µl of HOCl were injected subcutaneously (s.c.) into the back of the mice 5 days per week during 6 weeks. One week after the end of the injections, the animals were sacrificed by cervical dislocation. Serum and tissus samples were collected from each mouse and stored at -80°C until used or fixed in 10% acetic acid formol for histopathological analysis. For (PHTE) 2 NQ administration, BALB/c mice treated with HOCl (n=10) or not (n=7) were injected intravenously (i.v.) with (PHTE) 2 NQ (10 mg/kg per injection diluted in PBS once per week for 6 weeks). As the control group, BALB/c mice treated with HOCl (n=10) and those that were not (n=7), were injected intravenously (i.v.) with sterilized PBS once per week for 6 weeks. All cells were cultured as reported previously (Servettaz et al., 2009). All chemicals were from Sigma-Aldrich (France, Saint Quentin Fallavier), except when specified. The detailed synthesis of (PHTE) 2 NQ appears in the supplemental data.

Isolation of fibroblasts from the skin of mice

HOCl or PBS were injected in the same skin area in all the mice. Skin samples were then collected from that area for experimental purposes. The samples were digested with "Liver Digest Medium" (Invitrogen Life Technologies, Grand Island, NY) for 1 h at 37°C. After three washes in complete medium, cells were seeded into sterile flasks, and isolated

fibroblasts

Analysis of ROS metabolism of normal and SSc fibroblasts

Isolated normal or SSc fibroblasts (2×104 cells/well) were seeded in 96-well plates and incubated for 24 h in complete medium alone or with the following molecules: 3.2 mM Nacetylcysteine (NAC), 1.6 mM BSO, 400 µM ATZ or 20 U PEG-catalase. Cells were then washed three times with PBS and incubated with 100 µl per wells of 200 µM H 2 DCFDA for 30 min. Fluorescence intensity was read as described above. Intracellular H 2 O 2 levels were also expressed as described above.

Synergistic effect of BSO and (PHTE) 2 NQ on normal and SSc fibroblasts

Isolated normal or SSc fibroblasts (2×104 cells/well ) were seeded in 96-well plates and incubated for 24 h in complete medium alone or with the following molecules: 0.8 mM BSO, 0.5 µM (PHTE) 2 NQ, alone or in association. The number of viable cells at the end of the experiments were evaluated by the crystal violet assay as described above.

Analysis of cell death by Fluorescence-Activated Cell Sorting

Dermal thickness

Skin thickness was measured one day before sacrifice with a calliper and expressed in millimeters (Servettaz et al., 2010).

Collagen content in skin and lung

Skin was taken from the back region of mice with a punch (6 mm of diameter) and lung pieces, from each mouse, were diced using a sharp scalpel and then put into aseptic tubes, thawed, and mixed with pepsin (1:10 weight ratio) and 0.5 M acetic acid. Collagen content was assayed using the quantitative dye-binding Sircol method (Biocolor, Belfast, N.

Ireland) (Burdick et al., 2005). The concentration values were read at 540 nm on a microplate reader (Fusion, PerkinElmer, Wellesley, MA, USA) versus a standard range of bovine collagen type I concentrations (supplied as a sterile solution in 0.5 M acetic acid).

Curative treatment

BALB/c mice were injected s.c. with 300 µl HOCl five days per week during five weeks to induce mouse SSc. SSc mice were then distributed randomly into two groups. The experimental group of treated mice (n=5) received 10 mg/kg of (PHTE) 2 NQ) administered intravenously once a week during 2 weeks. The control group (n=5) received intravenous saline. Dermal thickness in SSc mice was measured after two weeks of treatment with (PHTE) 2

Fixed lung and skin pieces were embedded in paraffin. A 5-µm-thick tissue section was prepared from the mid-portion of the paraffin-embedded tissue and stained with either hematoxylin eosin or Sirius red. Slides were examined by a pathologist oblivious to the animal group assignment, via standard brightfield microscopy (Olympus BX60, Tokyo, Japan).

NQ.

Histopathological analysis

Analysis of α-SMA expression in mouse skin

Expression of α-SMA in mouse skin was analyzed by immunohistochemistry. Tissue sections were dewaxed then incubated with 200 mg/ml proteinase K for 15 min at 37°C for antigen retrieval. Specimens were then treated with 3% hydrogen peroxide for 20 min at 37°C to inhibit endogenous peroxidases and then blocked with BSA 5% for 30 min at 4°C.

Sections were incubated with a 1:100 dilution of a monoclonal anti-α-smooth muscle Actin, Alkaline phosphatase conjugated (Cat. no. A-5691, Sigma-Aldrich). Antibody binding was visualised using Nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP). The slides were examined by standard brightfield microscopy (Olympus BX60, Tokyo, Japan). Appropriate controls with irrelevant alkaline phosphatase-conjugated Ab were performed as control.

Determination of advanced oxidation protein product (AOPP) concentrations in sera

AOPP were measured by spectrophotometry as previously described (Servettaz et al., 2009). Calibration used chloramine-T within the range of 0 to 100 µmol/l. AOPP concentrations were expressed as µmol/l of chloramine-T equivalents.

Quantification of nitrite in mouse sera

The concentration of nitrite was determined in sera by a spectrophotometric assay using oxidation catalysed by cadmium metal (Oxis, Portland, OR), which converts nitrate into nitrite. The total determined nitrite corresponds to NO production. The detection threshold for nitrite was 0.1 µmol/l (Sastry et al., 2002). In this study we have demonstrated the efficiency of the natural organosulfur compound -dipropyltetrasulfide (DPTTS) in a mouse model of HOCl-induced SSc. DPTTS, naturally found in Allium vegetable, is a pro-oxidative molecule known in the medicine from its beneficial properties.

Detection of serum auto-antibody

To assess the mechanism of action of this compound in our SSc mouse model, the cytotoxic effect of DPTTS was compared in normal and SSc skin fibroblasts. ROS production was higher in SSc fibroblasts than in normal fibroblasts and was still increased by DPTTS to reach a lethal threshold and kill mouse SSc fibroblasts. proliferation of lymphocyte T and B activated with LPS and CD3/CD28, inhibits the production of auto-antibodies, and decreases the levels of main profibrotic cytokines -IL-4 and IL-13 produced by activated T cells. This work demonstrates that DPTTS can combine with endogenous oxidative species overproduced by SSc fibroblasts to selectively kill them. Organosulfur compounds are natural compounds already used in medicine. Therefore, DPTTS could be an interesting compound to use in the treatment of SSc.

Systemic sclerosis is a connective tissue disease characterised by fibrosis of skin and visceral organs, vascular disorders and dysimmunity (1). The pathogenesis of systemic sclerosis is not fully understood yet (2). Nevertheless, lot of evidence suggested that oxidative stress and inflammation play an important role in the initiation and development of this disease (3)(4)(5). In early stage of systemic sclerosis, activated fibroblasts constitutively produce a high amounts of reactive oxygen species (ROS) which caused the synthesis of type I collagen, and in consequence leads to fibrosis (4). Released of highly toxic reactive oxygen species by activated fibroblasts and endothelial cells also induce an inflammatory process that favour the recruitment of inflammatory cells and the production of cytokines that increase the profibrotic process (6). Involvement of the immune system in the pathogenesis of SSc is also reflected by circulating auto-antibodies, like antitopoisomerase I Abs which are presents in many patients with diffuse SSc. Release of ROS by SSc fibroblasts can oxidized DNA-topoisomerase-1 and contributes to the breach of tolerance towards the auto-antigen. Moreover, auto-Abs against platelet-derived growth factor receptor frequently found in SSc patients can also induce the production of ROS.

Altogether these observations demonstrate the key role played by the overproduction of ROS by diseased fibroblasts on SSc pathogenesis. If intracellular ROS can stimulate cell growth and fibrosis, ROS can also lead to cell death at certain level. It has been recently demonstrated that ROS generating molecules like arsenic trioxide can selectively kills diseased fibroblasts and be used to improve signs of fibrosis in two mouse models of SSc.

However, the compounds used so far displayed several side effects that limit their use to severe forms of SSc. Dipropyltertrasulfide (DPTTS) is a natural organosulfur compound (OSC), found in Allium vegetable. This compound has pro-oxidative properties and is used as an antibiotic or anti-mitotic agent (7,8). Polysulfides like DPTTS are already considered as a promising new class of antibiotic for resistant bacteria (9). In this study, we have investigated the effect of DPTTS on the skin fibrosis and immune dysregulations in HOCl-induced SSc in the mouse.

MATERIALS AND METHODS

Animals and chemicals. Six week-old female BALB/c mice were used in all experiments (Harlan, Gannat, France). All mice received humane care according to our institutional guidelines. Mice were divided into control and experimental groups. A total of 300 µl of a solution generating HOCl was injected intra-dermally into the back of the mice everyday during 6 weeks. DPTTS was injected intravenously (i.v.) two times per week (6 mg/kg per injection diluted in PBS, n=8). Controls group received injection of PBS. One week after the end of the injections, the animals were sacrificed by cervical dislocation. Serum and tissue samples were collected from each mouse and stored at -80°C until use.

HOCl generation:

HOCl was produced by adding 166 μl of NaClO solution (2.6% as active chlorine) to 11.1 ml of KH 2 PO4 solution (100 mM (pH 6.2)). HOCl concentration was determined by spectrophotometry at 280 nm. The optical density of the HOCl solution at 280 nm should be between 0.7-0.9, and the amount of Sodium hypochlorite and/or KH 2 PO4 solution must be adjusted for that. All cells were cultured as reported previously (10). All chemicals were from Sigma-Aldrich (France) except specifically specified.

Synthesis of dipropyltetrasulfide. Dipropyltetrasulfide (DPTTS) was synthesized from

propylmercaptan and sulfur chloride (S 2 Cl 2 ). A solution of 10 mM each propylmercaptan and pyridine in 25 ml anhydrous diethyl ether was stirred at -78°C. A solution of 10 mM sulfur monochloride in 50 ml anhydrous diethyl ether was added drop wise over 0.5 h. The reaction mixture was stirred for additional 0.5 h and another solution of 10 mM propylmercaptan and pyridine in 25 ml anhydrous diethyl ether was added drop wise over 0.5 h and reaction mixture was further stirred for 1 h. The reaction was stopped by adding 25 ml of H 2 O and mixture was brought to room temperature. The mixture was washed with 0.5 M NaOH until pH was neutral. The organic phase was dried over MgSO 4 , filtered and evaporated to yield a yellow oil with strong onion smell. The respective compound was purified by column chromatography using petrol ether: chloroform (95:3) as eluent.

Characterization was done by NMR (Bruker (Rheinstetten) type DRX 500 and Avance 500); 1H NMR (500MHz, CDCl 3 ): δ1.02(6H, t, J=7.4Hz), 1.79(m,4H), 2.91(4H,t,J=7.4Hz). The purity of compound was confirmed by GC-MS and HPLC (80%MeOH:H2O) (11).

Isolation of fibroblasts from the skin of mice. At the time of sacrifice, fragments from

HOCl-treated diseased skin and from PBS-treated control skin were collected from HOCland PBS-treated mice, respectively. Fragment of skin were digested with "Liver Digest Medium" (Invitrogen) for 1 h at 37°C. After three washes, cells were seeded into sterile flasks and isolated fibroblasts were cultured in DMEM/Glutamax-I supplemented with 10% heat-inactivated fetal calf serum and antibiotics at 37°C in humidified atmosphere with 5% CO 2 Dermal thickness. Skin thickness was measured on the back of the mice in the are of intra-dermal injection one day before the sacrifice. Dermal thickness was measured with a calliper and expressed in millimeters (10).

ROS production and levels

Measurements of collagen content in skin and lung. Skin was taken with a punch (6 mm of diameter) and lung pieces were diced using a sharp scalpel, mixed with pepsin (1:10 weight ratio) and 0.5 M acetic acid at room temperature. After 3 days, collagen content was assayed using the quantitative dye-binding Sircol method (Biocolor, Belfast, N.

Ireland) (12,13).

Ex vivo skin fibroblasts proliferation. Primary normal and SSc fibroblasts from mice treated or not with DPTTS (4×103 cells/well) (Costar, Corning, Inc., Corning, NY) were incubated in 96-well plates with complete medium, for 48 h at 37°C. Cell proliferation was determined by pulsing the cells with [3H] thymidine (1 µCi/well) during the last 16 hours of culture like described above.

Histopathological analysis.

A 5-µm-thick tissue section was prepared from the midportion of paraffin-embedded skin and lung pieces and stained with hematoxylin eosin.

Slides were examined by standard bright field microscopy (Olympus BX60, Tokyo, Japan) by a pathologist who was blinded to the assignment of the animal.

Analysis of α-SMA and pSmad2/3 expression in mouse skin. Expression of α-SMA and pSmad2/3 in mouse skin was analyzed by immunohistochemistry. Tissue sections were deparaffinized and rehydrated, then incubated with 200 µg/ml proteinase K for 15 min at 37°C for antigen retrieval. Specimens were then treated with 3% hydrogen peroxide for 10 min at 37°C to inhibit endogenous peroxidases and then blocked with BSA 5% for 1h at 4°C. Sections were incubated with a 1:100 dilution of a monoclonal anti-α-smooth muscle Actin, Alkaline phosphatase conjugated antibody (Sigma-Aldrich) and with a 1:100 monoclonal antibody directed to Phospho-Smad2/3 (Cell Signaling Technology, USA) for a 2h at room temperature. Section incubated with pSmad2/3, were then incubated with HRP-conjugated secondary goat anti-rabbit (Rockland, USA) for 1 hour at room temperature. Antibody binding for αSMA staining was visualised using Nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) and for pSmad2/3 using diaminobenzidine tetrahydrochloride (DAB) as a chromogen. The slides were examined by standard brightfield microscopy (Olympus BX60, Tokyo, Japan).

Appropriate controls with irrelevant alkaline phosphatase-conjugated and HRP-conjugated Ab were performed as control.

Determination of advanced oxidation protein product (AOPP) concentrations in sera.

AOPP were measured by spectrophotometry as previously described (10). Calibration used chloramine-T within the range of 0 to 100 µmol/l.

Detection of serum anti-DNA topoisomerase-1 IgG Abs. Serum levels of anti-DNA

topoisomerase-1 IgG Abs were detected by ELISA using coated DNA topoisomerase-1 purified from calf thymus (Immunovision, USA). Optical density was measured at 405 nm using a Dynatech MR 5000 microplate reader (Dynex Technology, USA).

Flow cytometric analysis and splenocytes proliferation. Spleen cells suspensions were prepared after hypotonic lysis of erythrocytes. Splenocyte (106 cells) were incubated with anti-B220-PE antibody diluted 1:200 for 30 minutes at 4°C. Cells were then analyzed with a FACS Canto flow cytometer (BD Biosciences). For spleen cell proliferation, B and T cells were purified from splenocytes by MACS, and were coated 96-well plates. Briefly, splenic B or T cell suspensions (2 x 105 cells) were cultured with 10 µg/ml of LPS (Boehringer, Mannheim) for B cells, or with 2.5 µg/ml precoated anti-CD3 and 1 µg/ml precoated anti-CD28 mAb for T cells. Cell proliferation was determined as described above. 1A) when cells were incubated with 10 µM and 40 µM of DPTTS, respectively. Nevertheless, compared to untreated SSc fibroblasts, the proliferation rate of SSc fibroblasts decreased to 63% (62.9%±4.2, P < 0.01) and 5.1%

Determination of IL

(5.1%±0.5, P < 0.001, Figure 1A) when cells were incubated with 10 µM and 40 µM of DPTTS, respectively.

In the presence of 10 µM DPTTS, the viability of normal fibroblasts was 84% (84.3%±9.5, P < 0.05) and 69% (69.4%±1.1, P < 0.001, Figure 1B) with 40 µM of DPTTS. DPTTS had a more powerful cytotoxic effect on diseased SSc compare to untreated fibroblasts. Indeed, in the presence of 10 µM DPTTS, the viability of SSc fibroblast decresed to 66% (66.1%±2.2, P < 0.001 vs untreated fibroblasts and vs normal fibroblasts) and 8.6% with 40 µM DPTTS (8.6%±4.7, P < 0.001 vs untreated fibroblasts and vs normal fibroblasts, Figure 1B).

DPTTS exerted pro-oxidative effects in vitro.

The basal production of ROS was increased by 39% in SSc fibroblasts compared to normal fibroblasts (4285 A.U.±241 vs 2658 A.U.±235, P < 0.05, Figure 1C). Incubation of normal fibroblasts with DPTTS did not increase significantly the production of ROS. In contrast, SSc fibroblasts with increasing amounts of DPTTS dose-dependently, increased the production of ROS by more than 50% (8595 A.U.±269 vs 4285 A.U.±241, P < 0.001 for 40 µM of DPTTS;

Figure 1C). We also investigate the effect of DPTTS on reduced glutathione levels, which is an essential cofactor of H 2 O 2

Modulations of H

catabolism. The basal level of reduced GSH was decreased by 166% in SSc fibroblasts compared to normal fibroblasts (-132 A.U.±43.8 vs 380 A.U. ±22.6 P < 0.001, Figure 1D). The level of intracellular glutathione was significantly higher (P < 0.001) in normal fibroblasts in the presence of DPTTS in all tested doses than in SSc fibroblasts (Figure 1D). inhibitor DDC had no effect on the levels of H2O2 in normal and SSc fibroblasts (Figure 1E). Depleting GSH by adding BSO to the culture medium with DPTTS significantly decreased the viability of SSc fibroblasts (P < 0.01). By contrast, specific inhibition of catalase by aminotriazol (ATZ) or of superoxide dismutase by DDC had no effect on the viability of normal and SSc fibroblasts (Figure 1F). SSc fibroblasts and only by 14% for normal fibroblasts (P < 0.001, Figure 2A). A kinetic analysis of cell death between 5 h and 24 h indicated that DPTTS effects are mainly mediated through a apoptotic process (Figure 2B).

DPTTS induced apoptosis in normal and

DPTTS decreased skin and lung fibrosis in mice with SSc. HOCl-induced SSc in mice

(HOCl-mice) is associated with an increase in dermal thickness that is significantly reduced by DPTTS (P < 0.001 vs untreated SSc mice, Figure 3A). Those results were confirmed by the histopathological analysis of the skin of control or HOCl-mice treated or not with DPTTS (Figure 3C). In vivo treatment with DPTTS significantly reduced the accumulation of type I collagen induced by HOCl in the skin (P < 0.01, Figures 3B) and in the lung (P < 0.05, Figures 4A) versus untreated mice. Histopathological analysis of lung biopsies stained with hematoxylin & eosin (Figure 4B) confirmed the reduction in lung fibrosis in HOCl-mice treated with DPTTS. Moreover, the high proliferation rate of fibroblasts isolated from HOCl-mice was significantly reduced by in vivo treatment with DPTTS (8729cpm±445 vs 6842cpm±420, P < 0.01, Figure 3D).

DPTTS reduced the expression of αSMA and pSmad2/3 in HOCl mice. The expression of αSMA was significantly higher in fibrotic skin of HOCl-mice compared to PBS mice (22.6%±1.6 positive area vs 10.01%±0.8, P < 0.01, Figure 3C). DPTTS decreased expression of αSMA by 40% compared to HOCl treated mice (13.7%.±0.4 positive area vs 22.6%±1.6 positive area in untreated HOCl mice, P < 0.01, Figure 3C). pSmad 2/3 protein, a key molecule involved in TGFβ-induced fibrogenesis, was overexpressed in HOCl-mice versus PBS controls (24.5%±1.4 positive area in HOCl mice vs 5.8%±0.4 positive area in PBS mice, P < 0.01, Figure 3C). In vivo administration of DPTTS downregulated pSmad2/3 expression in HOCl-mice (7.2%±2.1possitive area vs 24.5%±1.4 positive area in untreated HOCl-mice, P < 0.01, Figure 3C).

DPTTS decreased the serum concentration of AOPP and anti-DNA topoisomerase 1 Abs in SSc mice. Advanced oxidation protein products (AOPP), a marker of systemic oxidative stress, increased in the sera of HOCl-mice compared to PBS control mice (P < 0.001, Figure 5). Treatment of HOCl-mice with DPTTS reduced the levels of AOPP by 28%, compared to untreated SSc mice (P < 0.05, Figure 5B). The sera of SSc-mice contained significantly higher levels of anti-DNA-topoisomerase I Abs than the sera from normal mice (1.96 A.U.±0.1 vs 1.39 A.U. ±0.11, P < 0.05, Figure 5A). DNA-topoisomerase-1 Abs were significantly decreased in the sera from SSc mice treated with DPTTS compared to untreated SSc mice (1.47 A.U.±0.09 vs 1.96 A.U. ±0.1, P < 0.05, Figure 5A).

DPTTS treatment decreased the level of B cells and the proliferation rate of B and T cells in SSc-mice. We next tested the effects of DPTTS on spleen cells population. Intra-dermal injection of HOCl significantly increased the numbers of splenic B cells compared to normal mice (40.3%±0.6 vs 34.1% ±0.61, P < 0.01, Figure 6A). DPTTS decreased the numbers of splenic B cells by 16% in SSc mice compared to untreated HOCl induced SScmice (34.7%±1.1 vs 40.3% ±0.6, P < 0.01, Figure 6A). We also investigated proliferation rate of splenic T cells after stimulation with pre-coated-anti-CD3/CD28 mAb, and B cells after stimulation with LPS. T and B cells isolated from SSc-mice had both higher proliferation rate than T and B cells isolated from normal mice. T cells isolated from SScmice in vivo treated with DPTTS and in vitro stimulated by anti-CD3 mAb, had a lower proliferation rate than T cells obtained from untreated SSc-mice and stimulated under the same conditions (5538cpm±427 vs 10967cpm±786, P < 0.001, Figure 6B). In addition, B cells isolated from SSc-mice terated with DPTTS and stimulated with LPS also displayed significantly lower proliferation rate than B cells obtained from untreated SSc-mice (7625cpm±851 vs 12380cpm±1572, P < 0.05, Figure 6C).

In vivo administration of DPTTS reduced the production of IL-4 and IL-13 in SSc-mice.

Next, we tested the levels of IL-4 and IL-13 produced by splenic T-cells. SSc mice had a higher concentration of those 2 cytokines compared to control mice (1.03±0.09 vs 0.58±0.1, P < 0.01 for IL-4, Figure 6D, 1.2±0.07 vs 0.63±0.15, P < 0.01 for IL-13, Figures 6E). DPTTS decreased the levels of IL-4 in SSc mice by 37% (P < 0.05, Figure 6D), and of IL-13 by 36% (P < 0.05, Figure 6E). Results are expressed in ng/ml.

DISCUSSION

In the present study, we have shown the effect of the natural organosulfur compound, DPTTS in the prevention of fibrosis development in a murine model of chemicallyinduced systemic sclerosis.

Several reports have emphasized the involvement of ROS in the pathogenesis of SSc.

An increased oxidative stress occurs in diseased skin and lung fibroblasts during SSc. The reactive oxygen species locally generated favour the synthesis of type I collagen and the proliferation of fibroblasts through the activation of the RAS/MAPkinase pathways. Thus, the overproduction of ROS is one of the major actors of skin and lung fibrosis during SSc.

Skin fibroblasts from the recently described model of SSc induced by daily injection of HOCl in mice skin durably decrease the level of reduced glutathione (GSH) in skin fibroblast and increased their production of ROS as observed in human SSc (3,14). In our study, the pro-oxidant organosulfur compound, DPTTS, (15) additionally increase the level of ROS to generate a lethal oxidative burst in SSc fibroblasts. The cytotoxic effect of DPTTS occurs only in diseased fibroblasts, not in healthy fibroblasts with a normal level of endogenous reduced GSH and low level of ROS. Our results are in agreement with other studies on polysulfides compounds showing the pro-oxidant effect of these molecules. Hosono et al showed that diallyl-polysulfides lead to the ROS formation in cancer cells which also display increased level of ROS, causing ß-tubulin oxidation and the disruption of the microtubule network leading to apoptosis of cancer cells (16). Moreover, the mechanism of action for DPTTS shows the role of the GSH pathway in mediating the cytotoxic effects of DPTTS. NAC, a GSH precursor, has a protective effect against DPTTS in SSc-fibroblasts, while BSO, that depletes intracellular GSH, increases the cytotoxicity of organosulfur compound. The differences between normal and SSc fibroblasts in terms of basal oxidative stress and antioxidant enzyme levels have already been used to selectively kill SSc fibroblasts in vivo (17,18). Another feature in SSc patients is an abnormal activation of immune cells, including T and B cells, infiltration of inflammatory cells, especially CD4+ T cells in the skin and in the lungs along with increased levels of various pro-inflammatory and pro-fibrotic cytokines (6,22). In vivo administration of DPTTS has an immunoregulatory effect in HOCl miceinduced SSc. Indeed, DPTTS significantly limits the expansion of B cells, reduction of the hyper-proliferation of CD3/CD28-activated T cells and the proliferation of LPS-activated B cells in HOCl treated mice. The role of onion-derived organosulfur on leukocytes has been the matter of controversies. Some reports describe their immunostimulatory properties ( 23) while other highlighted their cytotoxic effects on lymphocytes (24).

However, both reports describes that organosulfur induce ROS production on organosulfur-exposed leukocytes (25). In our hand, the immunomodulating properties could be related to the high level of endogeneous ROS produced by activated autoreactive B and T cells that combines to the organosulfur-induced oxidative burst to inhibits lymphocytes proliferation as already described for treatment of SSc mice with the chemotherapeutic and pro-oxidative drug arsenic trioxide (26). The immunomodulatory properties of DPTTS are associated with the decreased in the splenic production of IL-4

and IL-13. The decrease level of those closely linked pro-fibrotic cytokines, elevated in the skin and in the serum of human and mouse SSc (22,27), can, at least in part, explained the DPTTS anti-fibrotic effects observed in SSc mice.

Altogether, organosulfur compounds (OSCs), ubiquitous in Allium plants, are natural compounds with various health beneficial properties (7,8). We demonstrate that organosulfur DPTTS can combine with endogenous oxidative species generated by SSc fibroblasts to selectively kill them by apoptotic process. This observation suggest that natural organosulfur compound could be used as a safe treatment of systemic sclerosis. Our work highlights the important role of angiotensin II in the induction of fibrosis, autoimmunity, and vascular abnormalities in SSc. Our results suggest that AT 1 receptor antagonist could be tested as new therapeutic tools in SSc. On one hand, the vascular dysfunction in SSc triggers the sclerosis of skin and visceral organs (1). On the other hand, vascular complications often determine the rate of survival and the quality of life of patients (2). The levels of angiotensin II (AT) and its type 1 receptor AT 1 R are elevated in SSc (3) and could be pivotal in the development of the disease. This hypothesis has been strengthened by the observation that angiotensin II injected into the back of mice induces skin fibrosis (7). Moreover, angiotensinogen, the precursor of angiotensin II, synthesized in the liver upon inflammation ( 4) is also produced by fibroblasts and myofibroblasts through a ROS- (5) or a TGFβ1-mediated mechanism (6). Blockers of AT 1 R reduce the mortality linked to renal crisis and alleviate the vascular manifestations of SSc in addition to inducing vasodilation (7,8). Therefore, AT which is the main peptide of the renin-angiotensin system (RAS), could be implicated in the pathophysiology of SSc with its functionally pleiotropic AT 1 R (9). AT is involved in the pathogenesis of fibrotic diseases by promoting vasoconstriction, cell proliferation, fibrosis, inflammation, and production of nitric oxide (10)(11)(12)(13)(14). Angiotensin II activates an early Smad signaling via the AT 1 receptor-mediated ERK1/2 MAPK pathway, leading to the accumulation of type I collagen (15).

AUTHOR CONTRIBUTIONS

LEGENDS TO FIGURES

Angiotensin II receptor blockade ameliorates systemic fibrosis in the mouse

Wioleta

The stimulation of AT 1 R by agonistic autoantibodies has been observed in severe vasculopathies associated with allograft rejection and pre-eclampsia (16,17). In SSc, those autoantibodies are associated with a high risk for the diffuse form of the disease and for the development of pulmonary hypertension, lung fibrosis, digital ulcers, and a higher rate of death related to these complications (7). Among the pathways engaged by the stimulation of AT 1 R, the regulation of the oxidative/nitrosative stress plays a key role (18). Particularly, AT and anti-AT 1 R agonistic autoantibodies can induce the production of nitric oxide (NO) that controls the vascular tone. In SSc, NO is overproduced and combines with reactive oxygen species to form the highly cytotoxic peroxynitrite (ONOO-) that contributes to inflammation, fibrosis and endothelial cell apoptosis (19,20).

Those observations have prompted us to test irbesartan, an AT 1 R antagonist (AT 1 RA) used in the treatment of sclerodermal renal crisis (21), in the mouse model of systemic sclerosis induced by intradermal injections of HOCl that produces skin and lung fibrosis, vascular dysfunction and auto-immunity.

MATERIALS AND METHODS

Animals, cells and chemicals. Six week-old female BALB/c mice were used in all experiments (Harlan, Gannat, France). Animals received humane care according to the guidelines of our institution. All mice were housed in autoclaved ventilated cages with sterile food and water ad libitum. All cells were cultured as reported previously (22). All chemicals were supplied from Sigma-Aldrich (Evry, France) except for irbesartan -Aprovel® (Sanofi-Aventis, Le Plessis Robinson, France).

Experimental design. Diffuse SSc was induced according to the protocol of Servettaz et al with minor modifications (23). Four hundred microliters (two injections of 200 μL) of a solution generating HOCl were prepared extemporaneously and injected intra-dermally into the shaved back of the mice, using a 27 gauge needle, everyday for 6 weeks (HOCl-mice). HOCl was produced by adding NaClO solution (9.6 % as active chlorine) to KH2PO4 solution (100 mM, pH 6.2) (24). HOCl concentration was determined by measuring the optical density of the solution at 292 nm, and then adjusted to obtain an OD between 0.7 and 0.9. Control groups received injections of 400 µl sterilized PBS (PBSmice). Irbesartan was administered by oral gavage (50 mg/kg/day) 5 days per week during 6 weeks (n=8 mice per group). Dermal thickness was measured one day before sacrifice with a calliper and expressed in millimeters. The animals were sacrificed by cervical dislocation. Serum and tissue samples were collected from each animal and stored at -80°C until used, or fixed in 10% acetic acid formol for histopathological analysis. In another set of experiments, limited SSc was induced by the injection of a solution generating peroxynitrites (ONOO-) (19) and irbesartan was administered as above.

Histopathological analysis. Fixed skin and lung pieces were embedded in paraffin. A 5µm-thick tissue section was prepared from the mid-portion of the paraffin-embedded tissue and stained with hematoxylin eosin and with picro-sirius red for lung section.

Collagen content in lung and skin. Skin taken from the site of injection with a punch (6 mm of diameter) and lung pieces were diced using a sharp scalpel, put into aseptic tubes, and were then thawed and mixed with pepsin (1:10 weight ratio) and 0.5 M acetic acid.

Collagen content was assayed using the quantitative dye-binding Sircol method (Biocolor, Belfast, N. Ireland) (25,26).

Isolation of skin fibroblasts, rate of proliferation and viability. Primary fibroblasts were isolated from the skin of all mice (n=31) as previously described (27). Cell proliferation was determined by [3H]thymidine incorporation (27) and cell viability by the crystal violet assay as previously described (28).

Flow cytometric analysis of splenocytes. Spleen cells were isolated by disruption of spleens and erythrocytes lysed by hypotonic shock in a potassium acetate solution. Cells (1 x 106) were then incubated with the following antibodies diluted 1:200 for 30 min at 4°C: anti-B220-PE, anti-CD4-APC-Cy7, or anti-CD8-PE-Cy7 mAb (BD Pharmingen, Franklin, NJ). Spleen cell suspensions were subjected to three-color analysis on a FACSCanto II flow cytometer (BD Biosciences). All data were analyzed using Flow-Jo software (Tree Star, Ashland, USA).

Spleen cell proliferation. Spleen cell suspensions were prepared after hypotonic lysis of erythrocytes. Cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS (Invitrogen Life Technologies) as a complete medium. B and T cells were purified from splenocytes by MACS. The proliferation assay was conducted in 96-well flat-bottom plates. Briefly, splenic B or T cell suspensions (2 x 10 5 cells) were cultured in complete medium for 48 hrs in the presence of 10 µg/ml LPS (Boehringer, Mannheim) for B cells, or with precoated anti-CD3 mAb (2.5 µg/ml) for T cells. Cell proliferation was determined by pulsing the cells with [3H]thymidine (1 µCi/well) during the last 16 hrs of culture and by measuring the radioactivity incorporated by liquid scintillation counting.

Western blot analysis of AT 1

Skin and lung samples taken from the injection sites of each mouse were incubated with 250 µl of RIPA. Splenocytes were in vitro stimulated or not with LPS (10 µg/ml) or with CD3/CD28 (2.5 µg/ml) for 48 hours. HPMEC were stimulated with 5 ng/ml mouse TNFα(R&D) for 48 hours. HPMEC and splenocytes were treated with or without 20 and incubated with 50 µl of RIPA. Protein extracts (30 µg total protein) were resolved by 10% SDS-PAGE and were blotted to PVDF membranes. Blots were blocked with 5% milk, and incubated with 1:500 mouse polyclonal anti-AGT 1 Immunohistochemistry of phospho-Smad2/3, angiotensin II, phospho-EGFR, and iNOS. Five micrometer-thick sections were placed onto slides, deparaffinized and then rehydrated. Antigen retrieval was performed by incubating the sections with proteinase K (Dako, France) for 20 min in 37°C for phospho-Smad2/3, angiotensin II, phosphor-EGFR, and with citrate buffer for iNOS. Sections were then treated with 3% H R (mouse skin, Sigma-Aldrich, France), 1:500 mouse monoclonal alpha-SMA (mouse skin, Sigma-Aldrich, France), 1:200 mouse monoclonal anti-nitrotyrosine (mouse skin, clone 1A6, Cell Signaling Technology, USA), 1:200 rabbit polyclonal anti-H-Ras Ab (mouse skin, lung and activated splenocytes, Santa Cruz Biotechnology, France), and 1:50.000 mouse anti-β-actin mAb (Sigma-Aldrich, France), overnight at 4°C. Signals were developed using 1:1000 goat anti-mouse or goat ant-rabbit HRP-conjugated secondary antibody (Santa-Cruz Biotechnology, Paris, France).

Immunoreactivities were revealed using ECL (Amersham) and densitometric analysis performed using ImageJ software (1.36 version). Results are expressed as arbitrary units defined as the densitometric intensity of the bands of the proteins tested divided by the densitometric intensity of the same bands labelled with mouse anti-β-actin mAb.

2 0 2 for a period of 15 min in order to block endogenous peroxidase, and incubated in 5% bovine serum albumin for 30 min to block non-specific binding. Furthermore, they were incubated with 1:100 monoclonal antibody directed to phospho-Smad2/3 (Cell Signaling Technology, USA), with 1:100 iNOS antibody (Santa Cruz Biotechnology, France) for 2 hrs at room temperature or with 1:50 angiotensin II antibody (Novusbio, England), with 1:50 phospho-EGFR antibody (Sigma-Aldrich, France) overnight at 4°C. Sections were then incubated with HRP-labelled secondary goat anti-rabbit (Rockland, USA) for 1 hour at room temperature. Irrelevant HRP-conjugated Abs were used as controls. Staining was finally visualized using diaminobenzidine tetrahydrochloride (DAB) as a chromogen. All slides were analyzed by two independent pathologists blinded with regard to the groups. Pictures were taken with a digital camera on a BX60 microscope (Olympus, France), using DP Manager Software, at x100 magnification. Immunostaining was analyzed using ImageJ ), and dihydroethidium (DHE) respectively, as previously described (28).

Exhaled nitric oxide measurement. The level of nitric oxide exhaled by the mice was measured using a chemiluminescence NO analyzer (Sievers 280 NOATM, Sievers Instruments Inc., Boulder, CO, USA), as previously described (31). The analyzer was connected by 2 unidirectional valves system to a 300ml Plexiglas chamber (Figure 4F).

Each mouse was placed in a Plexiglas chamber and allowed to rest for 5 minutes to accommodate with the chamber's environment. The chamber was flushed with NO-free air (Air Liquide, Puteaux, France) via a rodent ventilator (Harvard, USA) set to a flow rate of 800 ml/min (Vt: 5mL; respiratory rate 160/min) until the level of NO concentration reached zero (NO < 0.2 ppb) to set the baseline NO level. The chamber containing the mouse was then closed for 10 minutes during which exhaled NO produced by the mouse accumulated in the airtight chamber. To assess that mice were not exposed to hypoxia and/or hypercapnia, O2 and CO2 concentrations were measured in the chamber by an offline method using blood gas analyzer (Radiometer S.A.S., France). The CO 2 fraction was constantly less than 47 mmHg (37.7 ± 3.7) and the O2 fraction more than 86 mmHg (100.5 ± 6.9) (data from 6 control mice). After 10 minutes, exhaled NO recording was started and continuously maintained for 20 seconds, until a stable plateau of NO was obtained for at least 5 seconds. Values were then recorded to calculate mean value of exhaled NO (ppb). We noted a small range (max -min) of exhaled NO used as an internal control quality (≤ 0.5 ppb and < 10% of the mean). The mean of two measurement values was reported.

Statistical Analysis. All quantitative data are expressed as means ± SEM. Data were compared using one-way ANOVA plus the Tukey post-hoc multiple-comparisons test for comparisons of means among multiple groups. An unpaired Student t test was used to compare two groups. All analyses were carried out using the GraphPad Prism 5.0 statistical software package, and a p value of less than 0.05 was accepted as statistically significant.

RESULTS

Irbesartan reduced skin fibrosis in mice with SSc. In HOCl-mice, Irbesartan reduced dermal thickness by 60% (P < 0.001, Figure 1A) and skin collagen content by 20% (P < 0.05, Figure 1B) versus untreated HOCl-mice. Histopathological analysis confirmed the reduction in skin fibrosis in HOCl-mice (Figure 1C) and in ONOO-mice (Supplemental Figure 7).

Smad 2/3 protein was overexpressed in HOCl-mice versus PBS controls (19.52%±0.77 positive areas in HOCl mice versus 5.89%±0.15 in PBS mice, P < 0.001, Figure 1C).

Irbesartan reduced Smad 2/3 expression in the skin (13.41%±0.57 positive areas) of HOCl-mice (versus 19.57%±0.77 in untreated HOCl-mice, P < 0.001, Figure 1C). The expression of αSmooth Muscle Actin (α-SMA) (Figure 1E) in fibrotic skin of HOCl-mice was higher than in PBS mice (24.12 A.U.±3.2 versus 8.15 A.U.±1.7, P < 0.05). Irbesartan downregulated α-SMA expression by 77% compared to untreated HOCl-mice (5.66 A.U.±0.5 versus 24.12 A.U.±3.2, P < 0.05).

Irbesartan reduced lung fibrosis in mice with SSc. We next evaluated whether irbersartan could alter the systemic spreading of fibrosis in HOCl-mice. Irbesartan significantly reduced collagen content in the lungs (P < 0.05, Figure 2A) compared to untreated HOCl-mice. Histopathological analysis of lung biopsies stained with hematoxylin & eosin (Figure 2B) or Sirius Red (Figure 2C) confirmed the reduction in lung fibrosis in HOCl-mice treated with Irbesartan. As in the skin, HOCl induced H-Ras expression in the lungs compared to PBS-mice (P < 0.001, Figure 2D), but H-Ras expression was almost completely abolished in irbesartan-treated animals (P < 0.001, Figure 2D).

Irbesartan exerted immunomodulating properties in mice with SSc. The levels of anti-DNA-topoisomerase I Abs were higher in HOCl-mice than in PBS-mice (P < 0.05) and reduced following treatment by irbesartan (P < 0.05 versus HOCl-mice, Figure 3A).

We next tested the effects of irbesartan on splenic cell populations. Subcutaneous injections of HOCl increased the numbers of splenic B cells by 17% (P < 0.05), of CD4+ T cells by 21% (P < 0.05), and CD8+ T cells by 31% (P < 0.05) compared to PBS-mice.

Irbesartan decreased the numbers of splenic B cells by 14% (P < 0.05), CD4+ T cells by 27% (P < 0.01), and CD8+ T cells by 30% (P < 0.05) in HOCl-mice versus untreated HOCl-mice (Figure 3B, 3C, 3D).

We also investigated the ex-vivo proliferation rate of splenic T and B cells upon stimulation with coated-anti-CD3 mAb or LPS. B cells isolated from HOCl-mice treated with irbesartan and stimulated with LPS displayed a lower proliferation rate than B cells obtained from untreated HOCl-mice (7047±663 versus 9541±588, P < 0.05, Figure 3F). T cells isolated from HOCl-mice treated with irbesartan, then stimulated by anti-CD3 mAb, had a lower proliferation rate than T cells obtained from untreated HOCl-mice and stimulated under the same conditions (25277±1736 vs 34181±1740, P < 0.01, Figure 3E).

H-Ras expression was dose-dependently decreased in anti-CD3 mAb-and in LPSactivated splenocytes incubated with irbesartan compared to untreated activated cells (Figures 3G). Irbesartan decreased the nitrosative stress in HOCl-mice. HOCl-mice displayed higher levels of serum nitrites, a systemic marker of nitrosative stress, than PBS-mice (2.25-fold increase, P < 0.001, Figure 4A). Irbesartan reduced those levels by 30% (P < 0.05 versus untreated HOCl-mice, Figure 4A). The tissular level of 3 nitrotyrosine (3NT) is a surrogate marker of the in vivo nitrosative stress (34). 3NT was significantly higher in the skin (8.7-fold increase, P < 0.01, Figure 4B) and in the lungs (6.05-fold increase, P < 0.01, Figure 4C) of HOCl-mice than in PBS-mice. In HOCl-mice, Irbesartan reduced the expression of 3NT in the skin (2.4-fold decrease, P < 0.05, Figure 4B) and in the lungs (2.4-fold decrease, P < 0.05, Figure 4C) of HOCl-mice. Immunohistochemistry showed that the number of iNOS-positive cells in the lungs was higher in HOCl-mice than in PBS mice (6.87±0.3 versus 0.068±0.009, P < 0.001, Figure 4D). HOCl-mice treated with irbesartan showed a decreased number of iNOS-positive cells in the lungs (1.83±0.42 versus 6.87±0.3, P < 0.001 versus untreated HOCl-mice, Figure 4D). Exhaled NO is a relevant marker of in vivo lung inflammation and fibrosis in SSc patients (35). As in human, exhaled nitric oxide (NO) was significantly higher in HOCl-mice than in PBSmice (3.22±0.18 versus 1.87±0.2, P < 0.01, Figure 4E). Irbesartan significantly reduced the levels of exhaled NO in HOCl-mice (2.4±0.05 versus 3.22±0.18, P <0.05 versus untreated HOCl-mice, Figure 4E). To gain further insight into the mechanism of inhibition of NO production by irbesartan, the anti-oxidative effects of irbesartan were tested on Human Pulmonary Microvascular Endothelial Cells (HPMEC) in vitro. Incubation of HPMEC with increasing concentrations of irbesartan dose-dependently decreased the production of NO (P < 0.01) (Figure 5A), of O2°-(P < 0.05) (Figure 5B), and of H202 (P < 0.001) (Figure 5C), and decreased iNOS expression in TNF-α-stimulated HPMEC (Figure 5D).

Irbesartan decreased serum levels of soluble-E-selectin/sCD62E in HOCl-mice.

Soluble-E-selectin is a relevant marker of in vivo vascular damages in SSc (36). HOClmice displayed higher levels (1.32-fold) of serum s-E-selectin (1.56±0.06 pg/ml) than PBS-mice (1.19±0.04 pg/ml, P < 0.01) (Figure 5E). Irbesartan significantly reduced s-Eselectin levels in the sera of treated-HOCl-mice (1.28±0.06 pg/ml) versus untreated HOClmice (1.56 ±0.06 pg/mL, P < 0.05, Figure 5E).

DISCUSSION

This report shows that Irbesartan, an angiotensin II type 1 receptor antagonist, prevents fibrosis in murine models of systemic sclerosis induced by reactive oxygen species, an observation consistent with the upregulation of AT and AT 1

Irbesartan reduces fibrosis through inhibiting the accumulation of type I collagen in the skin and in the lung. This improvement is consecutive to the inhibition of fibroblast proliferation and differentiation into myofibroblasts, and to the decreased phosphorilation of smad3, a transcription factor implicated in the activation of the type I collagen gene. In addition, the phosphorilation of the epidermal growth factor receptor (EGFR) which occurs in the skin of HOCl-mice, is inhibited by Irbesartan. These data are in line with the observation that AT R observed in HOCl-mice.

1 R antagonists abrogate angiotensin II-induced connective tissue growth factor, collagen type-I and αSMA expressions via a TGFβ-dependent Smad3 signalling pathway (15,37,38). This effect is reinforced by the activation through the AT /AT 1 R pathway of the NADPH oxidase to produce ROS and to trigger collagen synthesis and fibrosis (39,40). Similarly, the transactivation of EGFR by angiotensin II triggers the proliferation, the migration of fibroblasts and is abrogated by AT 1 R antagonist (41).

Another study has connected skin fibrosis in SSc with the stabilization of the oncogene H-Ras following the stimulation of ROS production by autoantibodies. Ras proteins are members of a large family of small membrane-bound GTPases which control profibrotic pathways engaged by TGFβ, angiotensin II, platelet-derived growth factor (PDGF) or ROS (42,43). Recent reports have shown that increased levels of H-Ras protein can directly stimulate Smad2/3 signalling and collagen I accumulation (32). We found that irbesartan reduced the expression of H-Ras in the skin and lungs of SSc mice, which can also explain the reduction in fibrosis in HOCl-mice compared to untreated HOCl-mice.

Another feature in SSc patients is an abnormal activation of immune cells, including T and B cells, infiltration of inflammatory cells, especially CD4+ T cells in the skin and in the lungs along with increased levels of various pro-inflammatory cytokines (44,45). In In addition to its immunomodulating properties, the antioxidant properties of irbesartan can explain its beneficial effect in HOCl-mice. Indeed, the hight amounts of ROS produced by endothelial cells and fibroblasts lead to the oxidation of proteins, in particular of DNA-topoisomerase I. The release of oxidized DNA-topoisomerase I into the bloodstream is a key event in the breach of immune tolerance in both mouse and human SSc (22). Therefore, the in vivo abrogation of the auto-immune response by irbesartan in HOCl-mice can be explained by its antioxidant properties that limits the oxidation of DNA-topoisomerase I and thus prevents the production of autoantigen.

Several reports have suggested that, in addition to reactive oxygen species, reactive nitrogen species are also involved in the pathogenesis of SSc (9,15,(47)(48)(49)(50). In patients, the serum levels of nitric oxide (NO) are significantly elevated, as evidenced by increased nitrate levels, increased expression of 3-nitrotyrosine in diseased tissue, and increased exhaled NO (49). NO can be either beneficial because of its vasodilating properties or deleterious when it combines with superoxide anions in an inflammatory context, to form highly toxic peroxynitrites responsible for vascular damages, fibrosis, and selfmaintenance of reactive species production (50).

Inducible nitric oxide synthase (iNOS), an enzyme that allows the production of NO, is activated by cytokines, ligands of toll-like receptors or angiotensin II in various cell types, including endothelial cells and fibroblasts (51,52). In contrast, AT 1 R antagonists prevent the nitrosative stress (53). In our hands, irbesartan decreases iNOS expression in the lung of HOCl-mice and the generation of ROS, resulting in a drop in the production of peroxynitrites and in a decrease in exhaled nitric oxide which is a highly relevant marker of the extent of lung involvement in systemic sclerosis (54). Nitric oxide is largely

Discussion

Systemic sclerosis is a severe, immune-mediated disorder, the main features beeing microvascular damage, autoantibodies, and fibroblasts activation with fibrosis of the skin and visceral organs [213]. Despite significant efforts, the pathogenesis of SSc is still unclear, which is probably related to the complexity of factors involved in the pathophysiology of disease, but also its heterogeneity. Indeed, it is likely that the different clinical forms of the disease and its complications result from different mechanisms more or less distinct, making it difficult to understand. This gap in knowledge impairs the development of novel therapeutics able to modify the natural history of this disease.

Several years ago the pathogenesis of SSc was linked to the presence of a large excess of reactive oxygen species. This hypothesis, over the years, has been supported by several reports which documented the presence of an abnormal redox state in SSc patients [5,6].

Oxidative stress appears to play a major role in SSc because it is able to modify the

Conclusions and perspective

Our work confirms an important role of reactive oxygen / nitrogen species in the pathogenesis of SSc. Many studies confirmed that there is an increased oxidative stress in SSc patients [6,7]. This phenomenon contributes directly to the deregulation of various

2 RNS 8 SCIDTh2 lymphocyte T helper 2 TIMPFigure 1 . 11 Figure 2 .

 2821112 FIGURE LISTFigure 1. Link between dysfunction of fibroblasts, endothelial cells, and immune cells in systemic sclerosis from J Varga, Nature Reviews Rheumatology, 2012. ................ 11 Figure 2. TGFβ Smad and non-Smad signaling pathways from initiation to nucleus. (a) Signal initiation (b) Smad-dependent pathway (c) Smad-independent pathway from Souchelnytskyi, Experimental Oncology, 2012. ...................................................... 13

Figure 3 . 16 Figure 4 . 19 Figure 5 . 25 Figure 6 . 29 Figure 7 . 30 Figure 8 . 47 Figure 9 .

 3164195256297308479 Figure 3. Link between Ras and ROS by Gabrielli, The Journal of biological Chemistry, 2005. ......................................................................................................................... 16 Figure 4. IL-13 binding to the IL-13 receptor induces the activation of fibroblasts, differentiation into myo fibroblasts, increased ECM deposition, and fibros is from Fuschiotti, Cytokine, 2011. ...................................................................................... 19 Figure 5. Possible profibrotic pathways by angiotensin II by Dobrev, Circulation: Arrythmia and Electrophysiology, 1995. ................................................................................... 25 Figure 6. Endothelial receptor tyrosine kinases involved in angiogenesis adapted from Alitalo, The Journal of Cell Biology, 1995. ............................................................. 29 Figure 7. Angiogenesis in systemic sclerosis from Giacomelli, Autoimmunity Review, 2011. .................................................................................................................................. 30 Figure 8. Animal models of inducible and spontaneous SSc by Batteux, Current Opinion in Rheumatology, 2011. ............................................................................................... 47 Figure 9.

Figure 10 . 58 Figure 11 .

 105811 Figure 10. Regulation of cancer cells and SSc fibroblasts proliferation adapted from P. Huang, Nature Reviews, Drug Discovery, 2009.................................................58

  proteins is tightly controlled by endogenous inhibitors such as Smad7. Compared to healthy fibroblasts, fibroblasts from SSc patients are at the site of an abnormal nuclear accumulation of phosphorylated smad3 and a lack of smad7 expression. The lack of Smad7 helps to keep an abnormal production of collagen in SSc fibroblasts 14 [ ]. The imbalance of Smad 3 and Smad 7 in SSc fibroblasts might contribute to the initiation of the abnormal fibrogenic response. Smad proteins are the most potent mediators of COL1A2 gene activation in SSc fibroblasts. Overexpression of Smad3 and Smad4 leads to the activation of COL1A2 promoter in normal fibroblasts via interaction with SBE (Smad Binding Element), and thus transcription of the COL1A2 gene.

Figure 2 .

 2 Figure 2. TGFβ Smad and non-Smad signaling pathways from initiation to nucleus. (a) Signal initiation (b) Smad-dependent pathway (c) Smad-independent pathway from Souchelnytskyi, Experimental Oncology, 2012.

  MMPs and TIMPs are produced by mesenchymal cells (fibroblasts, myofibroblasts, endothelial cells), innate immunity cells (macrophages, monocytes, neutrophils), and metastatic cancer cells. MMPs 26-28 are a family of at least 23 calcium-activated and zincdependent endopeptidases. They are essential for various biological processes such as embryonic development, morphogenesis, and tissue reproduction [ ]. MMP-1 to 8 are the collagenases and play a major role in the digestion of type 1 collagen 29 [ ]. Too low or too high accumulation of ECM may results in different diseases such as connective tissue disorders or organ fibrosis. Numerous studies have shown overexpression of MMPs and

Figure 4 .

 4 Figure 4. IL-13 binding to the IL-13 receptor induces the activation of fibroblasts, differentiation into myo fibroblasts, increased ECM deposition, and fibrosis from Fuschiotti, Cytokine, 2011.

  cells. Th17 cells are cells that secrete Interleukin-17. IL-17 is cytokine family of six members (IL-17 A-F). IL-17A and IL-17F are implicated in a variety of autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, asthma, and psoriasis. IL-17A and IL-17F have similarities in their amino acid sequence, and both bind to the same receptor: IL-17R type A. The effect of IL-17 in SSc is still poorly understood. It was recently shown that expression of IL-17A, but not IL-17F was significantly increased in the skin and serum of SSc patients[START_REF] Fredriksson | The PDGF family: four gene products form five dimeric isoforms[END_REF]. On the contrary, the IL-17R type A is under-expressed in skin fibroblasts from SSc patients compared to normal fibroblasts, which is caused by activation of the intrinsic TGF-β pathway in these cells.According to Nakashima study, IL-17A has an anti-fibrotic effect via down-regulation of type I collagen and CTGF through the microRNAs. IL-17A receptor signaling is suppressed by the intrinsic activation of TGF-β1 in SSc fibroblasts, which may amplify the increased collagen accumulation and fibrosis in SSc[51]. By contrast, Yoshizaki et al have shown that IL-17 has a pro-fibrotic effect Interferon γ . They demonstrated in bleomycine-induced fibrosis mice that L-selectin and ICAM-1 regulate Th17 cell accumulation into the skin and lung, leading to the development of fibrosis[52].

  have been shown to play a role in the lung fibrosis. Large amounts of serotonin 5-HT2B receptor is expressed in the lung of patients with pulmonary fibrosis, and 5-HT2A and B receptors are induced in the lungs after intratracheal instillation of bleomycin, a molecule inducing pulmonary fibrosis. Inhibition of these receptors decreases fibrosis induced by bleomycin. Moreover, inhibition of the serotonin receptors on human fibroblasts reduce synthesis of collagen by inhibiting one of TGF-β dependent pathways involving Smad proteins. In 2011, studies conducted in different murine models of SSc demonstrated that serotonin induced platelet synthesis of extracellular matrix via activation of 5-HT2B by a TGF-β

Figure 5 .I. 1 . 2 . 7

 5127 Figure 5. Possible profibrotic pathways by angiotensin II by Dobrev, Circulation: Arrythmia and Electrophysiology, 1995.

  angiogenesis process. It causes proliferation and migration of endothelial cells, and stabilization of the lumina to form new vessels. Patients with early SSc have elevated level of VEGF in the serum [123]. VEGF and its receptors VEGFR-1 and VEGFR-2 are overexpressed in the skin of SSc patients [124]. However, the role of VEGF in the pathogenesis of SSc is unknown, although some authors have suggested that local hypoxia in the skin could be a contributing factor to increase of VEGF in some patients [125].

Figure 6 .

 6 Figure 6. Endothelial receptor tyrosine kinases involved in angiogenesis adapted from Alitalo, The Journal of Cell Biology, 1995.

I. 1 . 7

 17 The coagulation/fibrinolysis balance in An increase of coagulability is observed in SSc patients. Many clotting factors are present in excess in plasma of SSc patients like: dermatan sulfate, thrombin, antithrombin, Meanwhile, there is a deficiency in D-dimer, suggesting a defect in fibrinolysis in these patients [ ].

Figure 7 .

 7 Figure 7. Angiogenesis in systemic sclerosis from Giacomelli, Autoimmunity Review, 2011.

6 andI. 1 . 9

 619 Monocytes and macrophages have been demonstrated in the skin of patients in the first stage of SSc 95 [ ]. These cells were also observed in a mouse model of SSc secondary to Graft versus Host Disease 96 [ ]. Monocytes and macrophages express on their surface a large amounts of antigen presenting molecules and they can induce the activation of LT. Mast cells are increased in the skin of patients at an early phase of the sclerotic process compared to healthy skin 97 [ ]. These cells release IL-4 and histamine, two compound involved in the fibrotic process. In murine models of fibrosis induced by bleomycin, a mast cell deficiency delayed the appearance of skin fibrosis [98]. Overexpression of CD163, a marker for M2 macrophages found on TLR4 activated monocytes in SSc patients with interstital lung disease along with the increase production of CCL18 and IL-10 [99]. Interestingly, the expression of CD163 is also increase in diseased SSc skin [100]. Another study identifided a circulating TLR4 ligand in SSc sera [101]. They have shown that ligation of TLR4 induces an incresed production of IL-10 in SSc monocyte-derived dendritic cells [102]. Moreover, overproduction of IL-10 is partly related to the increased level of profibrotic CCL-18. In SSc patients, the expression of TLR3 in the skin is higher that in healthy controls. In vivo administration of several TLR ligands, especially TLR3, throught a transcutaneous pump, induces fibrosis progression in mouse model of SSc [103]. Activation of TLR stimulates several inflammatory mediators, such as IL-1, IL-TNFα in macrophages and dendritic cells. Activation of these and other inflammatory mediators, through TLR sensors, may colaborate to upregulate fibrotic mediators such as TGFβ and IL-13. Cellular adaptive immunity Several arguments favor the role of T lymphocyte in the pathogenesis of SSc, especially in the initial phase of the disease. Infiltrating T cells, predominately CD4+, are the major lymphocytes seen in the involved skin of Systsemic sclerosis patient [104]. The importance of this cells is correlated with a short diseased period and high inflamatory skin score. In fact, skin biopsies performed at the late stage of the disease with advanced fibrosis showed only moderate inflammatory infiltrates [63]. The perivascular infiltrates are predominantly CD3 + and CD4 + 105 T cells that express the activation markers -HLA-DR and CD25, and secrete fibrogenic cytokines and chemokines [ ]. T cells found in the affected tissues have restricted TCR specificities, suggesting an oligoclonal T cells expansion [106]. However, it is unclear whether these clones are activated nonspecificaly by cytokines or chemokines or specifically by unknown antigens. In the skin, the recruitment of lymphocytes occurs through adhesion molecules. In 2010, Sato's team studied the role of various adhesion molecules in mice trated with bleomycin induced-SSc [52]. They shown that the L-selectin and ICAM-1 regulates accumulation of Th2 and Th17 cells in the skin and lungs. Both, Th2 and Th17 had a pro-fibrotic properties. In contrast, Th1 infiltrate appears to be regulated by P-selectin and PSGL-1, and have the property of inhibiting the development of fibrosis in this model. Th1/Th2 cytokine balance in SSc patients is still unclear. Several studies have shown conflicting results. On the one side, high levels of IL-4 were found in the serum of SSc patients [107]. In addition, a microarray analysis of the transcriptome of circulating lymphocytes of patients with scleroderma in 2006 showed high levels of GATA-3, a factor involved in the Th2 polarization. On the other side a profile of Th1 gene expression was observed in peripheral blood mononuclear cells of SSc patients [108]. Finally, proteomic analysis of bronchoalveolar lavage of patients confirms the predominance of Th2-type cytokines. Moreover, mice deficient in T-bet, a transcription factor, specific for the activation of Th1 lymphocytes, develop exacerbated fibrosis in response to bleomycin injections [109]. Inducible costimulator ICOS, expressed on activated T Cells, and its ligand ICOSL on antigen-presenting cells, has recently been implicated in the pathogenesis of SSc. Indeed, ICOS deficiency reduces lung and skin fibrosis induced by bleomycin injection. In contrast, the ICOS-ligand deficiency increases the fibrotic process. The authors have shown that the severity of fibrosis was correlated with the expression levels of ICOSligand in B lymphocytes and macrophages [110]. Thus, ICOS-ligand plays a regulatory role in the activation of antigen-presenting cells in bleomycin-induced fibrosis. Other studies have shown the role of activated T lymphocyte upon contact with fibroblast, in the production of collagen type I by cultured dermal fibroblasts from patients with diffuse systemic sclerosis (SSc) and healthy controls. They found that production of type I collagen by skin fibroblasts is inhibited by membranes from activated T cells 111 [ ]. This inhibitory effect is mediated by a direct contact between LT and fibroblasts, and dependent on a prior in vitro activation of T Cells. The same team has recently shown that Th-1 cells inhibit collagen production by skin fibroblasts, through the membrane associated tumor necrosis factor alpha [112]. Only anti-CD3 activated T Cells can reduce collagen synthesis by fibroblasts in SSc patients. These two last works shown the complexity of cellular interactions in SSc. I.1.10 Humoral adaptive immunity Abnormalities of B lymphocytes in SSc have been known for several years. They are characterized by the hypergammaglobulinemia, the production of autoantibodies, and the hyperactivity of B lymphocytes. More recently, anomalies on various B cells populations were identified.

1

  type A receptor (anti-ETAR) autoantibodies were detected in the sera of SSc patients. Higher levels of both autoantibodies 136 were associated with more severe disease manifestations and predicted mortality [ ]. I.1.11 Targeted therapy directed against B lymphocytes Recently, four clinical trials and four case studies suggest the possible use of the chimeric monoclonal anti-CD20 (Rituximab) in SSc patients [137-140]. This study included patients with the diffuse form of SSc and most of them were positive for anti-Scl 70 autoantibodies (anti-DNA-topoisomerase-1). In 3 of 4 trials, a significant improvement of Rodnan score in histological (reduction of B cell infiltrate in the skin, myofibroblasts and collagen deposition) and biological changes (serum concentration of IL-6 and BAFF ) has been reported. Those patients had relatively heterogeneous clinical forms (disease duration, severity, lung / heart involvment). They have received different immunosuppressive treatments in the past, and Rituximab doses used in those trials were highly variable.

I. 1 . 12

 112 Genetic factorsSSc is not a disease determined only by genetic factors. Nevertheless, it has been suggested that genetic risk factors predispose individuals to the onset of SSc[141, 142].Many genes might interact to promote or influence on the clinical and serological manifestations of the disease (diffuse or limited cutaneous, vascular or fibrotic, anticentromere Ab or anti-DNA topoisomerase-1). Fibrillin 1 is one of candidate genes that may promote fibrosis. Some polymorphisms in this gene were identified in the North American Indians Choctaw population, where the prevalence of SSc is very high[143].Moreover, this gene is duplicated in TSK-1/+ mice which spontaneously develop cutaneous fibrosis[144]. However, this polymorphism was not found in other populations.Gene mutations encoding type II receptor of "bone morphogenetic proteins" (BMP) have been identified in familial forms of idiopathic pulmonary arterial hypertension. Gene mutations encoding "activin receptor-like-kinase-1 "(ALK-1) have been identified in pulmonary hypertension associated with Rendu-Osler Syndrome[145]. So far, these mutations have not been demonstrated in the SSc. There is no gene of the major histocompatibility complex (MHC) which confers increased susceptibility to develop SSc. However, many studies have shown associations between certain alleles of MHC genes and certain subgroups of SSc patients. In 2009, GENISOS (Genetic versus Environment In Scleroderma Outcome Study) demonstrated that the HLA DRB1*0802 and DQA1*0501 allels were death predictive factors in SSc patients. A polymorphism at position 945 of the CTGF gene promoter has recently been demonstrated in two different groups in patients with SSc [146]. Study from few years, shows several single nucleotide polymorphisms (SNPs) enriched in SSc patients compared with healthy controls. The SNPs were usually selected based on their locus in a gene involved in SSc pathogenesis. Using these strategies, several novel genetic risk factors were identified: STAT4, CD247, IRF5, Hepatocyte Growth Factor ( CD247 play an important role in the immune system by encoding the T-cell coreceptor CD3 zeta, a component of the T-cell receptor complex. Low expression of CD247 may lead to defect in the immune response [ HGF), and OX40L [147]. . STAT4 is a transcription factor playing a role in the differentiation of T lymphocyte. It also induces the transcription of type I IFN in activated monocytes. Its role as a mediator of inflammation in SSc was confirmed by development a moderate fibrosis after induction of SSc through the injections of bleomycin into STAT4 -/-mouse. 148]. IRF5 is a member of the IFN regulatory factors, which are critical components of virusinduced immune activation and type I IFN regulation. IRF5 was identified as a susceptibility gene for SSc. IRF5 rs4728142 [149]. IRF5 rs2004640 T allele was found to be associated with SS in the French Caucasian population [150, 151]. HGF is an antifibrotic factor involved in regenerating damaged tissue by promoting angiogenesis and by inhibiting fibrosis. A recent Japanese study have shown that the HGF -OX40L is tumor necrosis factor superfamily-4, natural ligand of OX40 present on T cells and influences T-cell survival and expansion. OX40L binding partner OX40 is overexpressed in the serum of patients with SSc than in healthy controls [ 1652 TT genotype occurs significantly more frequently in SSc patients with end-stage lung disease compared with healthy controls (41% vs 8%). 147]. In addition, analysis of clinical phenotypes demonstrated associations of OX40L variants with lcSSc and dcSSc, antitopoisomerase antibody and anticentromere antibody profiles [152].

  attracted particular attention in SSc and are the subjects of several works: the Parvovirus B19 and the cytomegalovirus (CMV). DNA from parvovirus B19 was detected in the skin and bone marrow of SSc patients. However this finding was made in a small number of patients and no viral protein was found in those patients 154 [ , 155]. Nevertheless, this virus remains a major candidate due to its capacity to fibroblasts activation 156 [ ]. More convincing arguments suggest a possible involvement of CMV in SSc. There is a structural homology between the viral protein UL94 and NAG 2, a membrane proteins expressed by human EC and normal fibroblasts [4]. It has been shown that anti-UL94 Ab were able to bind to EC and fibroblasts and induce their activation with transcription of adhesion molecules, cytokines, TGF-β, and CTGF [157]. In addition, these Abs induced apoptosis of EC. Structural homology was also observed between another protein of CMV (UL 70) and the DNA topoisomerase I 158 [ ].

98 Bleomycin

 98 Animal models of systemic sclerosis (SSc) have provided valuable insights into the causative mechanisms and the pathogenesis of SSc[159]. Several mouse models are available to study different aspects of the SSc disease related to fibrosis, vasculopathy, inflammation and autoimmunity (Figure8). Since none of these models can reproduce all features of human SSc, a critical selection of animal models is very important for successful in vivo studies.I.1.14Inducible models of SScBleomycin was originally used as a cancer antibiotic that has established itself as a potent initiator of tissue injury and fibrosis in mice. Daily subcutaneous injection of bleomycine during 4 weeks induces skin and lung fibrosis[START_REF] Yamamoto | Animal model of sclerotic skin. II. Bleomycin induced scleroderma in genetically mast cell deficient WBB6F1-W/W(V) mice[END_REF]. Nevertheless, in contrast to the human SSc, in bleomycin-treated mice there is a few signs of autoimmunity and no evidence of microvasculopathy. Cutaneous changes are localized to the injected skin area and remain at least for 6 weeks after the last bleomycin treatment. Histological analysis of the skin and lungs from those mice shows an inflammatory infiltrate composed mainly of CD4 + T cells and mast cells. The extracellular matrix contains large amounts of TGFβ which is produced by fibroblasts and infiltrating cells like macrophages [160, 161]. The mechanisms involved in fibrosis induced by bleomycin seems to be complex and is probably caused by direct toxicity of the molecule, as well as by the effect of secondary mediators. Indeed, bleomycin induces the synthesis of ROS, especially superoxide anion and hydroxyl radical which also participate in its toxicity. Indeed, injections of SOD, an enzyme that converts superoxide anions into the less toxic H 2 O 2 , reduce the accumulation of collagen in the dermis if those injections are made at the same time as the bleomycin injection. model was developed 10 years ago by Yamamoto et all, and since that time, was extensively used by several groups, providing substantial and valuable information about the pathogenesis of human SSc [ ]. However, this molecule acts by a complex mechanisms still poorly understood and probably acts through different pathways at least for the initial phase -to those involved in human SSc • Hypochlorous acid (HOCl) diseases. There is a lot of studies showing an important role of ROS in pathogenesis of SSc. Diseased fibrosblasts from SSc patients produces a large amounts of ROS which activate collagen synthesis. The study conducted in our laboratory reproduced diffuse subset of SSc by subcutaneous injections of HOCl to BALB/c mice during around 6 weeks. Those daily injections induce localized cutaneous and lung fibrosis along with the production of antitopoisomerase-I antibodies in the serum, and numerous features which characterize diffuse cutaneous SSc in humans [162]. Moreover injections of HOCl increase number of B and CD4+ T cells in the spleen and total levels of IgG and IgM antibody in the serum. Inflammatory changes observed after HOCl injections are also observed in early stages of SSc patients. In the skin, HOCl induce myofibroblasts which increase proliferation rate and collagen production. High amounts of endothelial microparticles, sVCAM-1 and sEselectin in HOCl mice shows the involvement of endothelial cells demage in this model. Subcutaneous HOCl stimulates fibroblasts to produce ROS which increase phosphorylation of ERK1/2 and activate Ras pathway. Moreover ROS production also induces the differentiation of fibroblast into α-SMA-expressing myofibroblasts which stimulate the production of type-I collagen, leading to skin fibrosis [163]. This model leads to developmment of skin and lung fibrosis and production of antitopoisomerase I antibodies, which characterized general feautures of SSc in humans. • MRL / lpr mice deficient in IFN receptor-gamma MRL / lpr mice, deficient in IFN receptor-gamma, develop erythema and fibrosis with collagen accumulation in the skin, kidneys, liver, lungs, and salivary glands compared to the mice expressing IFN receptor 164 [ ]. Histological analysis also shows extensive thrombosis of small vessels. • FRA-2 transgenic mice Overexpression of FRA-2 was observed in the skin of SSc patients, suggesting that this transcription factor exerted an important role in vascular abnormalities and fibrosis. Maurer et al has developed a model based on overexpression of this transcription factor. FRA-2 mice (Fos-related antigen-2) have a microangiopathy and progressive cutaneous fibrosis 165 [ ]. These events are preceded by apoptosis of endothelial cells. FRA-2 is a transcription factor belonging to the family of AP-1 (activation factor-1) and its expression is independent of TGF-and • PDGF.

•

  Wnt-10b mouse Wnt proteins are involved in the tissue remodeling processes and fibrosis. Wnt-10b plays a role in adipogenesis and osteoblastogenesis. A new transgenic mouse model -Wnt-10b was recently described by J. Varga team. These mice develop dermal fibrosis, large deposits of collagen, fibroblast and myofibroblast accumulation 167 [ ]. Mice exposed to Vinyl Chloride Injection of vinyl chloride, an organic solvent into BALB / cJ mice retired breeder. Those mice developed skin fibrosis and inflammatory infiltrate. Exposure to vinyl chloride increases quantitatively circulating cells of fetal origin in the mice. This substance may have a role in the activation of fetal cells, which in turn are involved in the development of fibrosis. This model is interesting because it is based on a cascade of events • similar to humans [168]. The autosomal-dominant Tsk-2 mutants are developed by administration of the mutagenic agent ethylnitrosourea (ENU) [ Tight skin-2 mouse model 169]. The mutation is located in chromosome 1. Only heterozygous animals survive and develop the Tsk2 phenotype characterized by several biochemical and histological abnormalities similar to those in the skin of SSc patients. Moreover, increased expression and accumulation of collagen were found in the skin of those mice. The molecular mechanisms, leading to fibrosis of the skin and internal organs are still unknown. In addition, Tsk-2 mice display inflammatory cell infiltrates in the affected tissue as well as autoimmunity including the presence of several autoantibodies against nuclear antigens including antitopoisomerase-I, anticentromere and anti-dsDNA antibodies [170].

Figure 8 .

 8 Figure 8. Animal models of inducible and spontaneous SSc by Batteux, Current Opinion in Rheumatology, 2011.

  However, this state is transient and ends either by accepting another electron by the molecule or by the transfer of the free electron on another molecule. Radicals have extremely high chemical reactivity, which explains their anormal biological activities that inflicts destructive impact on cells. The term "reactive oxygen species" includes oxygen free radicals itself (superoxide anion O •- 2 , hydroxyl radical OH • ), but also some nonradical reactive oxygen species with high toxicity (hydrogen peroxide H 2 O 2 173 , peroxynitrite ONOO) [ , 174]. Figure 9 shows different ROS generated by the cells.

Figure 9 .

 9 Figure 9. Sources of ROS by Dawes, Trends in Cell Biology, 2005.

2 O 2 2 -

 222 + e -→ O •- 2 . This radical has a short half-life by spontaneous dismutation accelerated by superoxide dismutase. It is not very toxic by itself, but can react with hydrogen peroxide or nitric oxide and result in the generation of strong oxidants (hydroxyl radical and peroxynitrite). Four cellular enzyme systems can lead to the formation of O •- Mitochondrial respiratory chain:The mitochondrial respiratory chain is made up of several enzymes grouped inside of the inner membrane of the mitochondrion. It allows to oxidize NADH and succinate generated in Krebs cycle. In the respiratory chain, electrons accepted from substrates are passed through the chain of electron carriers. This drives proton pumping through the inner mitochondrial membrane to intermembrane space. Those events results in the transmembrane potential gradient generation. The return of protons to mitochondrial matrix is coupled to the regeneration of ATP. Electrons flow through respiratory chain terminates on molecular oxygen leading to the formation of H 2 O:

[ 2 (- 2 - 6 .

 226 175]. Some cytochromes are located in the endoplasmic reticulum (microsomal cytochromes class), and play a role in the metabolism of xenobiotics. The others are located in the mitochondria (mitochondrial class) and some participate in steroid metabolism. In all cases, cytochromes are associated with the electron transfer chain using NADPH as an electron source. These reactions may generate superoxide anions O •- Family of NADPH oxidases 2 This family is also called NOX family ("superoxide-producing NADPH oxidase ") and constitutes seven members: NOX 1-5, and two DUOX1. Certain enzymes are ubiquitous (NOX4), while the others are only expressed preferentially in certain cell types such as phagocytes (NOX2)[176]. The phagocytic NADPH oxidase is a complex enzyme composed of membrane proteins (gp91phox (or NOX2) and p22phox) and cytosolic proteins (p47 phox, p67phox, p40phox). Activation of the membrane complex requires a presence of phosphorylated cytosolic proteins and a RAC -small G protein. These events occur during the activation of phagocytes (monocytes, macrophages, neutrophils and eosinophils) and finally allow the following reaction ("oxidative burst"), which is essential in innate immunity: 2O 2 + NADPH NADP + H + + 2O •- This enzyme is the main enzyme of purine degradation pathway. There are two forms of these enzymes: xanthine dehydrogenase reduces NAD Xanthine oxidoreductase + and xanthine oxidase which reduces the molecular oxygen and form preferably superoxide anions O •- 2 Xanthine dehydrogenase is converted into oxidase through sulfhydryl oxidation especially during the ischemia-reperfusion phenomenon [ ].

H 2 O

 2 2 can also be formed from O• - 2 Three types of SOD have been described in eukaryotic cells [ after spontaneous dismutation or dismutation accelerated by superoxide dismutase (SOD).

Finally

  , myeloperoxidase, the enzyme located in the azurophilic granules of neutrophils and monocytes, catalyzes a reaction leading to the OH • Nitrogen oxide (NO) production. NO is synthesized from L-arginine by NO synthase (NOS). It is an important physiological signalling molecule, potent vasodilator, and oxidative stress mediator. In mammals, there are three isoforms of NO synthase enzyme: -NO synthase-1 or neuronal -NO synthase 2 or inducible -NO synthase 3 or endothelial Unlike the other two isoforms, NO synthase 2 is not constitutively expressed but is induced by IFN-γ, TNF-α, IL-1, IL-6, including LPS. NO production is physiological and plays a major role in vascular tone. However, at high concentrations, NO becomes deleterious to the cells, especially by reaction with superoxide anions (O •- 2 ) to form the strong oxidant, peroxynitrite (ONOO -

  of methionine and cysteine. Reaction with aromatic amino acids (tyrosine, tryptophan, phenylalanine) leads to the formation of dityrosine or nitrotyrosine. Other amino acids are less sensitive and only strong ROS like HOCl may interact with lysine or arginine and lead to the formation of carbonyl groups [178]. These changes cause conformational changes, fragmentation or bonds between proteins and are thus likely to alter the activity of proteins. I.1.17 Effect on lipids ROS interactions with lipids lead to lipid peroxidation. The biological implications are of high importance for cells since all membranes are mainly composed of lipids. Peroxidation of the mitochondrial membrane can lead to such severe processes like apoptosis. Lipid peroxidation can also result in the formation of toxic byproducts such as malone dialdehyde (MDA) [179]. I.1.18 Action on DNA ROS can cause oxidation of bases in deoxsyribonucleotides, resulting in changes in the structure of base rings (for example oxidation of guanine leading to the formation of 8oxo-guanine) or the oxidation of the sugar backbone resulting in the breakage of a single or double stranded DNA. Changes can also cause DNA-lipid or DNA-protein cross-links [180]. I.1.19 Roles and effects of ROS in cellular metabolism Neutrophils and macrophages produce large amounts of O •- 2 and H 2 O 2 181 via activation of NADPH oxidase complex [ , 182]. This enzyme is a key component of the antimicrobial arsenal. NADPH oxidase deficiency is responsible for immune deficiency in humans, the chronic granulomatous disease. ROS, especially H 2 O 2 183 , are also involved in cell cycle regulation [ ]. Thus, low levels of ROS promote cell proliferation via the activation of ERK [184]. A higher level of ROS, other "mitogen-activated protein kinases" (MAP kinases) and "stress activating protein kinases" (SAPK) are activated and promote apoptosis [185]. The proliferation of tumor cells is also regulated by H 2 O 2 183 (see Figure 10 below) [ ].

Figure 10 .

 10 Figure 10. Regulation of cancer cells and SSc fibroblasts proliferation adapted from P. Huang, Nature Reviews, Drug Discovery, 2009.

21

 21 The role of ROS/RNS in SScMany studies show an important role of ROS in the development of SSc. Stress induced on the endothelium by reactive nitrogen species or oxygen free radicals is locally generated by the inflammatory process and by frequent episodes of reperfusion injury (Raynaud's phenomenon). These stress may contributes to the inflammation and activation of the endothelium favoring the fibrotic process in SSc. ROS have been demonstrated to be cell transducers of collagen-gene expression, fibroblast proliferation, and myofibroblast phenotype conversion in SSc[5][6][7]. Recently, a number of studies have suggested that ROS plays a significant role in the progression of fibrosis in SSc. The biological consequences of free radicals overproduction was investigated by Avouac J et al. They found a higher levels of 8-idroxy-2'dedeoxyguanosine (8-oxodG), a biomarker of endogenous oxidative damage to DNA in the blood of a large cohort of SSc patients[191]. Svegliati at all had shown that PDGF, by stimulating ROS and Erk1/2 induced post-transcriptionally Ha-Ras protein. High level of ROS, Ha-Ras protein and activation of Erk1/2 stimulate collagen synthesis[33]. Another study, has shown that Silica can exert their effects in part through the oxidative stress. Silica can directly induce ROS formation on its surface, especially through the Fenton reaction, and indirectly through phagocytic cells activation.

  disorders, and different patients treatment. In most biological condition NO is oxidized to nitrate (NO 3 -) and nitrite (NO 2 -). Many studies shows overproduction of NO in SSc patients, increased plasma levels of NO 3 -/NO 2 -199 , and elevated nitration of plasma proteins, a marker of ONOO-production [ ]. Abnormality in NO metabolism was demonstrated in the tight-skin (TSK-1/+) mouse model of fibrosis. Decreased activity of NOS was detected in TSK-1/+ mouse tissue compared to wild type mice, suggesting an abnormal NO metabolism. Also the expression of endothelial NOS was significantly reduced in TSK-1/+ mice [200]. Immunohistological studies of SSc skin also shown that as the disease progresses to the later fibrotic stages, the production of eNOS is downregulated, while iNOS is upregulated. Nitrated proteins are also found in the SSc skin associated with sites of inflammation [3, 4]. Another studies are showing NO acting as an antifibrotic effector in animal models of experimental fibrosis [201, 202]. NO has been shown to inhibit adhesion molecule expression, adhesion of platelet, and smooth muscle cell proliferation. Release of NO is dependent on the integrity of vascular endothelium. Under pathological states, endothelium dysfunction leads to constitutive NO synthesis and release. In SSc, the basal release of NO might be a potent chemical barrier that protects the vascular endothelium from oxidation injury. For example, in a mouse model of pulmonary fibrosis, a loss of NO bioactivity in eNOS knock-out mice, resulted in prolonged fibrosis [201]. Moreover, other study also shows reduced fibrotic content using transgenic mice overexpressing eNOS after bleomycin-induced fibrosis [202]. In contrary, other studies show harmful role of NO in SSc. A UK team have shown how the progression of the disease can change the NO synthase from the endothelial to the inducible form [203]. Moreover, cells in the damage skin express nitrotyrosine which indicate free radical injury [203, 204]. During the initial proinflammatory state of SSc NO and O •- 2 is increased by the presence of macrophages or activated fibroblasts. Free radicals produced by inflammatory cells can react with NO produced by iNOS to form peroxynitrite ONOO - 205 that may favour endothelial cells apoptosis [ ]. In SSc, low concentration of NO contribute to the survival of endothelial cells whereas high level of NO induce the apoptosis of endothelial cells.

III.1 Article 1

 1 fibroblasts [208]. Moreover, by studying the involvement of ROS in the pathogenesis of SSc, a new chemically induced murine model of SSc was developed by injection of HOCl. This model presents all features characterizing SSc in human, among others skin and visceral fibrosis, vascular disease and autoimmunity diseases [162]. These are strong arguments confirming an important role of ROS in the pathophysiology of SSc. Thus, we decided to test new therapeutic approaches in the HOCl induced SSc murine model. As in human SSc, skin fibroblasts from SSc mice constitutively produce large amounts of ROS. We have used this property to selectively induce apoptosis in the diseased fibroblast of SSc mice (Article 1, 2). Indeed, the organotelluride catalyst-(PHTE) 2 Many studies have also proved an importance of nitrogen species in the pathogenesis of SSc [ NQ and natural organosulfur compound-DPTTS are able of increasing ROS production by fibroblasts and inducing a lethal oxidative stress specificaly in SSc fibroblasts. This phenomenon has no impact on normal fibroblasts that present normal levels of ROS and a normal oxidant/antioxidant status. 206, 207]. In patients with SSc, the serum level of nitric oxide is significantly increased [206]. Furthermore, NO can combine with other free radicals like superoxide anion (O •- 2 ) to form the highly cytotoxic peroxynitrite (ONOO - 205 ) that contributes to inflammation, fibrosis and apoptosis of endothelial cells. Production of NO by endothelial cells or by fibroblasts can be stimulated by angiotensin II, the main peptide of the reninangiotensin system (RAS) [ ]. The level of angiotensin II is increased in SSc patients as well as in our HOCl mouse model and can promote proliferation of fibroblasts, fibrosis, and inflammation [209]. These observations led us to test irbesartan, an angiotensin II type I receptor antagonist (AT 1 RA) in the murine model of SSc (Article 3). ROS/RNS modulators investigated during my thesis affect not only fibrosis process but also vascular tone (Article 3) and immune system (Articles 2 and 3), which are important in the initiation of SSc. In conclusion, a new animal model based on chronic exposure to ROS and with many similarities to the human disease, allowed me to study new therapeutic approaches in SSc based on the cytotoxic action of pro-oxidative compounds -(PHTE) 2 NQ and DPTTS -and on the anti-nitrosative effect of irbesartan. These new therapeutic strategies open interesting perspectives in the treatment of SSc, where the therapeutic arsenal is currently still limited. The organotelluride catalyst (PHTE) 2 NQ prevents HOCl -induced systemic sclerosis in the mouse Wioleta Marut 1 , Niloufar Kavian 1 , Amélie Servettaz 1,2 , Carole Nicco 1 , Lalla Ba 3 , Christiane Chéreau 1 , Claus Jacob 3 , Bernard Weill 1 , Frédéric Batteux Cancer cells, like SSc fibroblasts also overproduce ROS that may participate to the proproliferative phenotype of those cells. Indeed, chronic exposure to low levels of ROS leads to carcinogenesis and tumor progression. However, when the oxidative burst reach a certain level, for example upon treatment with cytotoxic drugs, this burst can leads to the apoptosis of cancer cells [ 1 Journal of invegistative dermatology, 2012 (Published) SSc fibroblasts from HOCl mice produce a large amounts of ROS that stimulate their proliferation and synthesis of type I collagen. 183]. By analogy to cancer cells which can be killed by prooxidadnt molecules through increased level of ROS above a lethal threshold, we tested the effectiveness of pro-oxidative organotellurium compound (PHTE) 2 NQ on SSc fibroblasts. (PHTE) 2 NQ selectively kills activated SSc skin fibroblasts in vitro obtained from from HOCl treated mice but have no effect on normal skin fibroblasts from healthy mice. (PHTE) 2 NQ amplifies the production of ROS by SSc fibroblasts which can not be detoxified due to low concentration of glutathione (GSH) observed in the diseased fibroblasts. By contrast, normal fibroblasts with a normal oxidative/antioxidant status resist to the action of (PHTE) 2 NQ. In vivo HOCl treated mice were injected intravenously with (PHTE) 2 NQ once per week during six weeks. We observed the reduction of dermal thickness in the SSc mice treated with (PHTE) 2 NQ. Disease improvement was confirmed by histological analysis of skin and lung. (PHTE) 2 NQ reduces the collagen content in the skin and in the lung and inhibits differentiation of fibroblasts into myofibroblasts in SSc mice. Furthermore, in vivo treatment with (PHTE) 2 NQ resulted in a reduction of vascular abnormalities through the reduction of nitrite and also the level of oxidized protein-AOPP generated by SSc fibroblasts which can induce the production of reactive oxygen species by fibroblasts and endothelial cells, and thus 162 enhance the vascular damage by increase the apoptosis of endothelial cells [ ]. In addition, overproduction of ROS by SSc fibroblasts allows the production of oxidized intracellular proteins like oxidized DNA-topoisomerase-1 and then favour the breach of tolerance to DNA topoisomerase I and favour autoimmunity induction. As a consequence of the reduction of AOPP in the sera, HOCl mice treated with (PHTE) 2 NQ have a significantl decreased in the anti-DNA topoismerase I Ab concentration in the sera. In conclusion, our results show that the organotelluride catalyst (PHTE) 2 NQ can interact with intracellular ROS generated in SSc fibroblasts to selectively kill them. This property lead to the clinical and biological improvement in HOCl treated SSc mice administrated with (PHTE) 2 NQ. clinical manifestations of the disease remain unclear (Gabrielli and Avvedimento, 2009). Several reports have shown that reactive oxygen species (ROS) are involved in the pathogenesis of SSc (Simonini et al., 1999; Sambo et al., 2001; Allanore et al., 2004; Ogawa et al., 2006; Servettaz et al., 2007; Avouac et al., 2010). Indeed, skin fibroblasts in SSc patients spontaneously produce large amounts of ROS that trigger collagen synthesis (Sambo et al., 1999; Sambo et al., 2001). Intradermal injection of specific ROS-generating substances can reproduce the main features of local and systemic mouse SSc, depending on the type of ROS generated (Servettaz et al., 2009). In addition, anti-PDGFR autoantibodies (AAbs) found in SSc patients also trigger the production of ROS (Svegliati Baroni et al., 2006). Recently, we have reported that sera from patients with SSc can induce not only the production of ROS by endothelial cells but also by the proliferation of fibroblasts (Servettaz et al., 2007). The release of ROS by fibroblasts and endothelial cells can activate several processes that trigger the development of SSc. In fact, ROS can oxidize specific SSc antigens such as DNA-topoisomerase-1 and lead to this autoantigen's breach of tolerance (Casciola-Rosen et al., 1997; Servettaz et al., 2009). The autoimmune phenomenon can be amplified by the inflammatory reaction that results from the overproduction of ROS. This fibrotic process may also be related to the imbalance of the oxidant/antioxidant status in SSc. For instance, in vitro studies have shown that ROS triggers the differentiation of fibroblasts into myofibroblasts, either directly or through the activation of the ADAM17/NOTCH pathway (Kavian et al., 2010), and also through the release of TGF-ß from activated cells (Bellocq et al., 1999). Finally, ROS can stimulate the growth of dermal fibroblasts through the activation of the RAS pathway, which explains the hyperproliferative properties of SSc skin fibroblasts (Svegliati Baroni et al., 2005).

  [START_REF] Coriat | The organotelluride catalyst LAB027 prevents colon cancer growth in the mice[END_REF]. Several reports have linked the intracellular oxidative burst and the initiation of the immune response in human or in murine models of SSc(Simonini et al., 1999;[START_REF] Harðardo | Exercise in systemic sclerosis intensifies systemic inflammation and oxidative stress[END_REF]. Indeed, local generation of HOCl or hydroxyl radicals triggers the production of anti-DNA-topoisomerase-1 autoantibodies, and consequently, the generation of peroxynitrite triggers the production of anti-centromeric protein B Abs.The mechanism through which the breach of tolerance to DNA-topoisomerase-1 occurs following exposure to HOCl or hydroxyl radicals, is related to the oxidation and cleavage of DNA-topoisomerase 1 within the cells(Servettaz et al., 2009). The selective destruction of diseased fibroblasts that chronically produce high levels of intracellular ROS by (PHTE) 2 NQ, probably explains the decreased concentration of circulating oxidized proteins in the sera, and especially of oxidized DNA-topoisomerase-1 anti-DNAtopoisomerase-1 Abs. Furthermore, oxidized topoisomerase-1 exerts a proproliferative effect on fibroblasts and increases the intracellular level of H 2 O 2 in fibroblasts and endothelial cells. The decrease of circulating oxidized proteins linked to the selective destruction of diseased fibroblasts also explains the decrease in fibroblast proliferation.One of the major consequences of the oxidative stress in diseased fibroblasts is their transformation into myofibroblasts and their increased production of type I collagen(Sambo et al., 2001;[START_REF] Toullec | Oxidative stress promotes myofibroblast differentiation and tumour spreading[END_REF]. As for autoantibodies and fibroblast proliferation, the selective destruction of diseased mouse SSc fibroblasts by (PHTE) 2 NQ is followed by the decrease in the numbers of myofibroblasts. These phenomena were associated with the inhibition of the development of mouse SSc because they were correlated with a decrease in the concentration of type I collagen in the skin of treated animals.Altogether, our results show for that the organotelluride catalyst (PHTE) 2 NQ can interact with intracellular ROS generated in SSc fibroblasts to selectively kill the cells. This observation suggests a new therapeutic strategy in SSc, based on the modulation of the intracellular redox state of the activated fibroblasts. Indeed, new molecules such as (PHTE) 2 Specific pathogen-free, 6 week-old female BALB/c mice were used in all experiments (Harlan, Gannat, France). All mice were housed in autoclaved ventilated cages with sterile food and water ad libitum. They were given humane care according to the guidelines of NQ, could help selectively eliminate the activated cells responsible for the development of skin and visceral fibrosis and, indirectly, be used for autoimmunity.

  Cell death was analyzed by fluorescence-activated cell sorting (FACS)Canto™ II flow cytometer (Becton Dickinson, Franklin Lakes, NJ), using the Membrane Permeability/Dead Cell Apoptosis Kit with YO-PRO®-1 and Propidium iodide (PI) for Flow Cytometry (Invitrogen Life Technologies), under the manufacturer's recommendations. Briefly, 1.2x104 normal or SSc fibroblasts isolated from the skin were incubated with 1, 2 or 4 µM (PHTE) 2 NQ at 12 h, 24 h, 36 h, or 48 h. After the incubation period, the cells were collected, washed twice with PBS, stained on ice for 10 min with 1.5 µM PI and 0.1 µM YO-PRO®-1, and analyzed by flow cytometry.

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Figure 4 . 2 NQ

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  Figure 3.

  of intracellular reduced glutathione. 4×104 cells/well of isolated normal and SSc fibroblasts were coated in 96-well plates (Costar) and incubated . In vitro cells proliferation and viability assays. Isolated normal and SSc fibroblasts (4×103 cells/well) (Costar, Corning, Inc., Corning, NY) were incubated in 96-well plates with complete medium, and various dose of DPTTS (10 to 40 µM) for 48 h at 37°C. Cell proliferation was determined by pulsing the cells with [3H] thymidine (1 µCi/well) during the last 16 hours of culture. Cell viability was evaluated by the crystal violet assay (Alexandre et al., 2006). Results are expressed as percentage of viable treated cells compared to untreated cells. for 48 hours at 37°C with either medium alone or with 2.5, 5, 10, 20 or 40 µM of DPTTS. Levels of ROS and GSH were assessed spectrofluorometrically (Fusion, PerkinElmer, Wellesley, MA) using 2 ′,7′ -dichlorodihydrofluorescein diacetate (H 2 DCFDA) and monochlorobimane, respectively. Cells were incubated with 200 μM of H 2 DCFDA for 1h or 50 μM monochlorobimane in PBS for 15 minutes at 37°C. The intracellular ROS and the intracellular glutathione level were expressed as arbitrary units of fluorescence intensity normalized by the number of viable cells. The number of viable cells at the end of the experiments was evaluated by the crystal violet assay as described above. Modulation of ROS metabolism in normal and SSc fibroblasts. Isolated primary fibroblasts (2 × 10 4 cells/well) from normal and SSc mice were seeded in 96-well plates and incubated for 12 hours in complete medium alone or with the following molecules: 3.2 mM N-acetylcysteine (NAC), 1.6 mM BSO, 20 U PEG-catalase, 400 μM AT, or 8 μM DDC. DPTTS (30 μM) was added in the last 16 hours. Cells were then washed three times with PBS and incubated with 100 μl per wells of 200 μM H2DCFDA for 30 min. Intracellular H2O2 levels were expressed as described above. Fluorescence-Activated Cell Sorting Analysis of cell death. Apoptosis and necrosis were analyzed by the fluorescence-activated cell sorting (FACS)Canto™ II flow cytometer (Becton Dickinson, Franklin Lakes, NJ), using the Membrane Permeability/Dead Cell Apoptosis Kit with YO-PRO®-1 and Propidium iodide (PI) for Flow Cytometry (Invitrogen) under the manufacturer's recommendations. Briefly, of Isolated normal and SSc fibroblast (1.2x104) were incubated with 40 µM of DPTTS for various times (5 h, 10 h, 15 h and 24h). After the incubation period, cells were collected, washed two times with PBS, stained for 10 minutes on ice with 1.5 µM PI and 0.1 µM YO-PRO®-1 and analyzed by flow cytometry.

  SSc fibroblasts. Fibroblasts extracted from the skin of normal and of SSc mice were incubated with 10, 20, and 40 µM of DPTTS for 5 h, 10 h, 15 h or 24h. The highest cytotoxic effect of DPTTS was observed after incubation with 40 µM. At this concentration and after 15h the viability was decreased by 38% for

  Moreover, DPTTS decrease the level of oxidized protein-AOPP generated by SSc fibroblasts. AOPP could enhance the vascular damage by increase the apoptosis of endothelial cells(10). Moreover, ROS produced by SSc fibroblasts can oxidized different proteins, like anti-topoisomerase I, favouring the breach of tolerance towards this autoantigen in SSc and the production of anti-DNA Topoisomerase I Abs(10). Selective destruction of SSc fibroblasts which chronically produce high levels of intracellular ROS by DPTTS explain decrease serum level of AOPP and of oxidized DNA topoisomerase I. Therefore, DPTTS could stop the induction of auto-immune reaction by the selective and early destruction of diseased fibroblasts. Smad2 and Smad3 are transcription factors that are overexpressed in human SSc fibroblasts as well as in fibroblasts from HOCl-mice. Phosphorylated Smad2/3 activate genes coding for type I collagen which lead to fibrosis in several organs (19, 20). In vivo administration of DPTTS down-regulates the phosporylation of Smad2/3 and contributes to decrease the accumulation of type I collagen in the skin of mice with HOCl-induced SSc compared to untreated HOCl mice. In addition, TGFβ which induced Smad2/3 phosphorylation is inhibited by a thiol antioxidant-NAC, GSH, and L-cysteine, which highlight the role of ROS in the activation of the Smad2/3 pathway (21). Therefore, in HOCl induced SSc in the mice, the selective deletion of fibroblast overexpressing ROS by DPTTS treatment decreased the number of cells with high phosphorylation levels of Smad2/3.

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F 3 AngiotensinFrédéric Batteux 1 1 210)

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Frédéric Batteux 1 . 1

 11 Marut 1 *, Niloufar Kavian 1 *, Amélie Servettaz 1,2 , Thong Hua-Huy 3 Carole Nicco 1 , Christiane Chéreau 1 , Bernard Weill 1 , Anh Tuan Dinh-Xuan 3 Université Paris Descartes, Faculté de Médecine, EA 1833 et Laboratoire d'immunologie biologique, Hôpital Cochin, AP-HP, 75679 Paris cedex 14, France. 2 Faculté de Médecine de Reims, Service de Médecine Interne, Maladies Infectieuses, Immunologie Clinique, Hôpital Robert Debré, 51092 Reims cedex, France.

3 1833, 8 rue

 38 Méchain 75679 Paris cedex 14, France. Tel: +33 (0) 1.58.41.20.09; Fax: +33 (0) 1.58.41.20.08; e-mail: Département de Physiologie, Université Paris Descartes, Faculté de Médecine, Hôpital Cochin, AP-HP, 75679 Paris cedex 14, France. This work was supported by European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement 215009 RedCat for financial support. * Equal contribution Address correspondence to: Dr. Frédéric Batteux, Laboratoire d'Immunologie, EA frederic.batteux@cch.aphp.fr Key words: angiotensin II; irbesartan; fibrosis; systemic sclerosis; reactive oxygen species; nitric oxide. Systemic sclerosis (SSc) is a connective tissue disorder characterized by vascular alterations, extensive fibrosis and immunological dysregulations associated with specific autoantibodies (AAbs). The involvement of visceral organs determines the prognosis of the disease. Despite progresses in the treatment of some visceral complications, no treatment has been designed to date that can cure SSc, in part because the mechanisms underlying the disease remain unclear.

  40 µM of irbesartan (Sigma-Aldrich, France). Then, cells were washed three times, then R, αSMA, 3-Nitrotyrosine (3-NT), iNOS and H-Ras proteins in the skin and lungs from mice and in activated splenocytes and HPMEC cells.

Software ( 1 .

 1 36 version; National Institute of Health) for quantitative assessment of staining positivity. Results are expressed as percents of positive skin area analysed with the ImageJ Software. Detection of serum autoantibodies. Serum anti-DNA topoisomerase-1 IgG Abs were assayed by ELISA using purified calf thymus DNA topoisomerase-1 bound to the wells of a microtiter plate (Immunovision, USA) as previously described (29). Determination of IL13. Splenic T cells purified by MACS (2 x 10 5 Quantification of soluble E-selectin and nitrites in mouse sera. Concentrations of serum sE-selectin were measured by ELISA using the Mouse sE-selectin/sCD62E Duo Set kit (R&D Systems, USA) following the manufacturer's instructions. The concentration of nitrites was determined in sera by a spectrophotometric assay using oxidation catalysed by cadmium metal (Oxis, Bioxytzch, USA), which converts nitrates into nitrites ( cells/well) were seeded in 96-well flat bottom plates and cultured in complete medium for 48 hrs in the presence of 5 µg/ml concanavalin A. IL13 concentrations from the collected supernatants were measured by ELISA (E-bioscience, San Diego, USA). The concentrations were calculated from a standard curve according to the manufacturer's protocol. Results are expressed in pg/ml. 30). The total nitrites determined correspond to NO production. The detection threshold for nitrites was 0.1 µmol/l. Cellular Production of H 2 O 2 , NO, and O 2 °-. Human Pulmonary Microvascular Endothelial Cells (HPMEC, PromoCell, Heidelberg, Germany) (1x10 4 cells per well) were seeded in 96-well plates (Costar) and incubated for 48 hours with various concentrations of irbesartan or with culture medium alone. Levels of H 2 O 2 , NO, and O 2 were assessed spectrofluorometrically (Fusion, PerkinElmer, Wellesley, MA) using 2 ′,7′ dichlorodihydrofluorescein diacetate (H2DCFDA), diaminofluorescein-2 diacetate (DAF-2DA

  Next, we tested the level of IL13 produced by splenic T-cells, since IL13 is involved in the development of fibrosis in HOCl mice. SSc mice had higher serum concentrations of those cytokines than control mice (641±78.2 versus 318±36.3, P < 0.01) Irbesartan decreased the level of IL13 in HOCl mice by 40% (P < 0.01, data not shown).

  HOCl-mice, irbesartan reduces the number of splenic B cells, of CD4+ and CD8+ T cells, the hyperproliferation of CD3/CD28-activated T cells and of LPS-activated B cells. These effects on B and T cells are in agreement with our observation that H-Ras expression is dose-dependently decreased in vitro by irbesartan in LPS-and in anti-CD3 mAb-activated splenocytes. Indeed, H-Ras transmits TCR signals that prime CD4 (+) T cells and trigger B cell activation(46), thus suggesting how irbesartan decreases the levels of antitopoisomerase I Abs in the serum of HOCl-mice.

  metabolized into nitrites, which are increased in the serum of patients with systemic sclerosis and also in HOCl-mice. The nitrosative stress induces vascular damages reflected by elevated serum levels of soluble E-selectine. Administration of irbesartan reduces the level of both serum nitrite and E-selectine in HOCl-mice, confirming the efficacy of an AT 1 R blocker in the prevention of the systemic nitrosative stress in vivo and of its consequences on the vascular damages in HOCl-mice. We also tested irbesartan in the model of peroxynitrite-induced SSc and found that the molecule is as effective in this model as in HOCl-mice on dermal thickness and collagen deposition in the skin. In conclusion, in addition to its use in the treatment of sclerodermal renal crisis, irbesartan and other AT 1 REFERENCES R blockers should be evaluated as anti fibrotic and immunomodulating drugs in SSc patients with diffuse fibrosis. disease in systemic sclerosis. Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society 2009;20(3):200-6.

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  structure and antigenicity of proteins. Several clinical, epidemiological and biological arguments excuse the role of oxidative stress in early SSc. It is noteworthy that more than 90% of clinical patients have repeated ischemia-reperfusion phenomenon during the years preceding the development of SSc (approximately six years before the development of limited cutaneous SSc and approximately two years and a half before the development of diffuse SSc). This phenomenon generates a large amount of ROS, including O •- . Epidemiologically, several toxics involved in triggering SSc exert their action through the generation of ROS. This is the case of silica, which directly induces ROS formation through the Fenton reaction, and indirectly through the activation of phagocytic cells. Syndrome toxic oils, which shares similarities with SSc, also seems to involve free radicals. Finally, on a biological level, it has been shown that monocytes and fibroblasts of SSc patients produced large amounts of O •- 2 In a previous work conducted in our laboratory, it was observed that the sera of SSc patients are able to induce oxidative stress. This stress is mediated by serum oxidized protein products (AOPP) which both induce fibroblasts proliferation and inhibit endothelial cells proliferation. In addition, sera from patients with lung fibrosis induce a higher proliferation rate of SSc fibroblasts and increased synthesis of H . Therapeutic options are limited. Therefore advances in medical understanding for the development of novel treatments are critical. The aim of this thesis is to update the role of oxidative stress in SS as a paradigm of fibrotic disorders. of SSc patients without pulmonary involvement. Sera from patients with serious vascular complications additionaly induces NO release by endothelial cells, and strongly inhibit their growth. At the opposite, sera of patients with pulmonary fibrosis, they do not stimulate fibroblasts proliferation [ ]. The correlation between ROS production induced by serum of SSc patients and clinical complications in these patients is a strong argument which favors the role of ROS in the pathogenesis of SSc. Therefore, in another study conducted in our laboratory, we decided to assess the extent, nature and consequences of oxidative stress in SSc. To study SSc on animal models, mice were exposed to different types of agents generating various types of ROS. We demonstrated a direct role for ROS in SSc and showed that the nature of the ROS dictates the form of SSc. Indeed, administration of peroxynitrites induced skin fibrosis and serum anti-CENP-B Abs that characterize limited SSc, whereas hypochlorite or hydroxyl radicals induced cutaneous and lung fibrosis and anti-DNA topoisomerase 1 autoantibodies that are characteristic of human diffuse SSc. Moreover, this work demonstrated that oxidized proteins, and especially oxidized DNA topoisomerase I, generated in high amounts after exposition to agents generating ROS are directly involved in the spreading of the fibrosis from the skin to visceral organs through the systemic circulation. Thus leads to the increase release of oxidized DNA topoisomerase I, the production of anti-DNA-topoisomerase I Abs and the development of the diffuse form of SSc [162]. These observations are in agreement with previous studies, which demonstrated a direct link between the concentration of AOPP in the serum of SSc patients and the development of lung fibrosis.

Figure 11 .

 11 Figure 11. Induction of murine SSc through daily intradermal injections of HOCl generating agents, by Batteux et al, Curr. Op Rheumatol, 2011.

  compared to untreated SSc mice. AOPP is generated in the skin by SSc fibroblasts that overproduce ROS, and are transported by the circulation into the tissues [ ]. Since AOPP are released by SSc fibroblasts, the selective killing of those cells, early in the course of the disease, explain the decrease of AOPP level in the serum of SSc-mice treated with (PHTE) 2 NQ or DPTTS. Furthermore, in HOCl-induced SSc, AOPP can elicit the apoptosis of endothelial cells and cause vascular damages. In vivo treatment of SSc-mice with (PHTE) 2 NQ or DPTTS reduces the levels of AOPP. In addition, (PHTE) 2 NQ treatment reduces the concentration of nitrates, a marker of vascular activation and injury in murine and human SSc. Those results reflect the beneficial effect of (PHTE) 2 One of the major consequences of the oxidative stress in diseased fibroblasts is their transformation into myofibroblasts expressing α-SMA and their increased production of type I collagen [ NQ and DPTTS on the vascular part of the disease. 7]. The skin and lungs of patients with SSc contain myofibroblasts that displays an excessive rate of fibroblasts proliferation. The same observation has been made in the mouse model of HOCl induced SSc [162]. Selective destruction of SSc fibroblasts by (PHTE) 2 Several reports have linked the intracellular oxidative burst and the initiation of the immune response in human or in murine models of SSc . Indeed, the hight amounts of ROS lead to the oxidation of proteins, in particular of DNA-topoisomerase I. The oxidation of DNA-topoisomerase I is a key event in the breach of immune tolerance in both mice and human SSc and the released of high amounts of oxidized topoisomerase-1 favour the production of anti Topoisomerase I Abs. The selective destruction of SSc fibroblats that chronically produce high levels of intracellular ROS by (PHTE) NQ or DPTTS is followed by the decrease in the numbers of myofibroblasts that correlate with down-regulation of α-SMA, and decrease in the concentration of type I collagen in the skin and lung of treated SSc mice compared to untreated SSc mice. 2 NQ or DPTTS, explains the decreased release of oxidized DNA-topoisomerase-1, and in consequence the decrease production of anti-DNA-topoisomerase-1 Abs. In addition, DPTTS treatment exerts an immuno-regulating effect in SSc mice, reflected by the reduction in B cell numbers in the spleens of treated mice and by the reduction in hyper-proliferation of CD3/CD28-activated T cells and the proliferation of LPS-activated B cells. The beneficial effect of DPTTS on the immune system also results in the decrease in spleen production of IL-4 and IL-13, two cytokines that trigger the synthesis of collagen in human skin fibroblasts, and whose concentrations are elevated in the skin and serum of SSc patients. Article 1 and 2 shows that organotelluride catalyst (PHTE) 2 Various studies indicate an important role of nitrosative stress in SSc patients [ NQ and natural organosulfur DPTTS, can interact with intracellular ROS generated in SSc fibroblasts to selectively kill the cells. This observation suggests a new therapeutic strategy in SSc, which is based on the modulation of the intracellular redox state of the activated fibroblasts. 7, 206, 207]. Nitric oxide (NO) is one of the most important mediators of the nitrosative stress and a relevant physiological signaling molecule. In SSc patients, the serum levels of NO are significantly elevated, as evidenced by increased nitrate levels, increased expression of 3nitrotyrosine in diseased tissue, and increased exhaled NO [206]. NO can be either beneficial because of its vasodilating properties, or deleterious when combining with superoxide anions in an inflammatory context, to form the highly toxic molecule peroxynitrite that is responsible for vascular damages and fibrosis [207]. NO is synthesized from L-arginine by nitric oxide synthase (NOS). Increased levels of NO in SSc patients can be caused by excessive expression of NOS, especially of inducible nitric oxide synthase (iNOS). In various cell types, this enzyme is known to be activated by a variety of inflammatory signals such as cytokines and ligands of toll-like receptors [236]. In the Article 3, we shows that Irbesartan, a blocker of the angiotensin II type 1 receptor (AT 1 R), prevents fibrosis in a mice model of SSc, mostly through the inhibition of the nitrosative stress. Angiotensin II is able to stimulate the production of nitric oxide by various cells, including endothelial cells and fibroblasts [237]. Increasing doses of irbesartan progressively decrease iNOS expression in cytokine-activated human pulmonary microvascular endothelial cells [236]. As a consequence, the level of NO but also of hydrogen peroxide and of superoxide anions in cytokine-activated human pulmonary microvascular endothelial cells is also reduced by irbesartan. The peroxynitrites generated following the hyperproduction of NO can induce changes in the structure and function of normal proteins and lead to cell death and tissue damages [238]. 3-nitrotyrosine is a product of tyrosine nitration mediated by peroxynitrites and reflects the extent of tissular damages caused by the nitrosative stress [228]. 3-nitrotyrosine is increased in HOCl-mice, but down regulated in the skin and lungs by irbesartan. Additionally, in vivo administration of irbesartan abrogates the expression of iNOS in the lungs of SSc mice. As a consequence, exhaled nitric oxide, which is a highly relevant marker of the extent of lung involvement in systemic sclerosis, is reduced in HOCl-mice treated with irbesartan [239]. Nitric oxide is largely metabolized into nitrites, which are increased in the serum of SSc patients and also in HOCl-mice. The nitrosative stress induces vascular damages and elevated serum levels of soluble E-selectine. Administration of irbesartan reduces the level of both serum nitrite and E-selectine in HOCl-mice, confirming the efficacy of an AT 1 Moreover, administration of irbesartan in vivo down-regulates the phosporylation of Smad2/3 and secondarily decreases the accumulation of type I collagen in the skin of HOCl-mice compared to untreated HOCl mice [ R blocker in the prevention of the systemic nitrosative stress in vivo and of its consequences on the vascular damages in HOCl-mice. 242]. Phosphorylated Smad2 or Smad3 are transcription factors overexpressed in fibroblasts from HOCl-mice that activate genes coding for type I collagen, thus leading to fibrosis in several organs. Our results are in agreement with a recent study showing that angiotensin II activates Smad signalling through the AT 1 210 receptor-mediated ERK1/2 MAPK pathway, inducing the accumulation of type I collagen [ ]. Another study has connected skin fibrosis in SSc with the stabilization of the oncogene H-Ras following the stimulation of ROS production by autoantibodies. Ras proteins are members of a large family of small membrane-bound GTPases which control profibrotic pathways engaged by TGFβ, angiotensin II, plateletderived growth factor (PDGF) or ROS [212]. Recent reports have shown that increased levels of H-Ras protein can directly stimulate Smad2/3 signalling and type I collagen accumulation [212, 243]. We found that irbesartan reduced the expression of H-Ras in the skin and lungs of SSc mice, which can also explain the reduction in fibrosis in HOCl-mice compared to untreated HOCl-mice. Irbesartan reduces an abnormal activation of immune cells through the reduction of number of splenic B cells, of CD4+ and CD8+ T cells [233, 234]. In addition, irbesartan reduces the hyper-proliferation of CD3/CD28-activated T cells and the proliferation of LPS-activated B cells observed in HOCl-mice. These effects on B T cells are in agreement with our observation that H-Ras expression is dose-dependently decreased in vitro by irbesartan in LPS-and in anti-CD3 mAb-activated splenocytes. Indeed, H-Ras acts as a critical controller of T helper cell responses mostly by transmitting TCR signals that prime CD4 (+) T cells and trigger B cell activation [244]. This immunosupresing effect of irbesartan can explained why the increased levels of antitopoisomerase I Abs in the serum of the HOCl-mice are reduced by irbesartan. Moreover, the in vivo abrogation of the auto-immune response by irbesartan in HOCl-mice can also be explained by its antioxidant properties that limits the oxidation of DNA-topoisomerase I and thus prevents the production of autoantigen. Article 3 shows that in addition to the effectiveness of Irbesartan in the treatment of the renal crisis of SSc, irbesartan and other AT 1 R blockers should be evaluated as anti fibrotic and immunomodulating drugs in SSc patients with diffuse fibrosis.

  signalling pathways leading to hyper-proliferative phenotype of fibroblasts and as a consequence to synthesis of collagen I and fibrosis. The importance of ROS in SSc has first been highlighted in our laboratory by showing that sera form SSc patients do not only induce ROS production by endothelial cells but also increase the proliferation of fibroblasts [245]. SSc is characterized by an abnormal proliferation of fibroblast which differentiates into activated myofibroblastes that express alpha smooth muscle actin (α-SMA). To better understand the role of ROS in the SSc process we have conducted in vivo experiments. Injections of various types of ROS-inducing agents were performed on normal and immunodeficient mice. Those studies led us to develop new and more representative mice models of human SSc [162]. The importance of ROS encouraged us to test new therapeutic approaches using ROS modulators. SSc fibroblasts produce large amounts of ROS. We used this properties to selectivly kill SSc fibroblasts with prooxodative compounds. We have demonstrated that cytotoxic molecules like organotellurium catalyst-(PHTE) 2 NQ and natural organosulfur-DPTTS can increase the level of ROS leading to a lethal oxidative stress in SSc fibroblasts but not in normal fibroblasts. Moreover, various NO production may contribute to the development of SSc vascular disease by enhancing studies indicate an important role of nitrosative stress in SSc patients. platelet aggregation, upregulating endothelial and leucocyte adhesion molecules, and increasing wall thickness. Further, NO can enhance proliferation of fibroblasts, collagen synthesis, and production and activity of extracellular matrix. NO production is modulated by Angiotensin II. Our study have confirmed an important role NO production of in SSc. We have demonstrated that irbesartan, an angiotensin II receptor I antagonist, can decrease nitrosative stress which contributes to the decline of vascular disease, and skin and lung fibrosis in murine fibrosis model. The current therapies arsenal does not propose molecules acting on ROS level. Our results open new perspectives for the SSc treatment.

I.1.10.2 Role of autoantibodies in SSc

  role of B lymphocyte in the development of fibrosis has been explored by studies in

	development of tissue fibrosis. The activation of B lymphocyte and humoral response are activity, incuding skin involvement, internal organ manifestations and prognosis. Anti-
	facilitated by the Th2 envirronnement characterized by overproduction of IL-4, IL-5, IL-6, topoisomerase I antibodies (anti-topo I) and anti-centromere antibodies (ACA) are the
	several animal model of SSc. Those mice produce autoantibodies against SSc-specific IL-10 and IL-13. These cytokines promote the production of antibodies by the B classic autoantibodies associated with SSc [121]. Futhermore, antibodies against several
	target autoantigens including topoisomerase I (scl70), fibrillin 1, type I collagen, and Fc-lymphocyte, and as we have already seen, they stimulate synthesis of collagen by other structures (including U3RNP, PM/Scl, snRNP, mitochondrial components, antigens
	receptors [40]. Saito et al have shown that in bleomycin-induced fibrosis mice, deficiency fibroblasts. Moreover, B cells might influence the Th1⁄Th2 balance by regulating dendritic present on endothelial cells and fibroblasts, and extracellular antigens) can also be detected
	of CD19 suppresses fibrosis and autoantibodies production by inhibiting TLR4 signals. cells function. Activated B cells produces IL-10 which inhibits the production of IL-12 by in SSc serum [122]. However, the contribution of SSc-specific autoantibodies in the
	Constitutive CD19 tyrosine phosphorylation is increased in TSK B cells compared with dendritic cells, and promotes Th2 differentiation. That suggest a critical role of B cells for pathogenesis of SSc is not clearly demonstrated.
	wild-type B cells . Chronically activated TSK B cells in vivo , and the cytokines produced the development of Th2 response. IL-6 produced by B lymphocyte can also induce the
	I.1
	by those cells may participate in the induction of skin fibrosis in TSK mice. In this animal production of collagen and glycosaminoglycans by dermal fibroblasts. Several studies have
	113 model, the down-regulation of B-cell function decreased skin thickness [ , 114]. Saito et shown a high level of IL-6 in the skin and in the serum of SSc patients. This results
	al. shown that CD19 expressed by B lymphocytes is abnormally activated and overexpress indicate that this cytokine may plays a role in the development of fibrosis via its pro-
	in SSc patients [115]. inflammatory activity [119]. Fibroblasts from scleroderma patients produce four times
	Furthermore, memory B cells from SSc patients show overexpression of CD80 and CD86, more IL-6 than fibroblasts from healthy controls. In Tsk1 mice, B lymphocytes stimulated
	that are critical co-stimulatory molecules of B cells activation. To up-regulate the with anti-IgM Ig and CD40-Ig produced significantly higher levels of IL-6 than B
	expression of CD80 and CD86, activation of B cells is required. This results indicate, that lymphocytes from control mice [113]. The B lymphocytes also secretes activated TGF-β,
	SSc memory B cells are chronically activated in vivo, possibly due to overexpression of which amplify the excessive production of extracellular matrix proteins by fibroblasts
	CD19. [120].
	Another molecule, BAFF, is also involved in B cell homeostasis. BAFF is overexpressed
	in the blood and skin of SSc patients. Indeed, as patients with systemic lupus
	Autoimmune disorders in SSc patients are characterized by activation of the immune
	erythematosus or Sjögren's syndrome, SSc patients have elevated serum level of soluble
	system. This activation leads to lymphocyte activation and cytokines secretion, and to the
	BAFF compared to healthy control [116-118]. The level of BAFF correlate with the
	production of specific autoantibodies. SSc patients have specific autoantibodies against a
	severity of skin disease. Further, expression of BAFF receptor on B cells is increased in
	variety of self antigens which are not associated with other autoimmune diseases.
	SSc patients. SSc B cells overexpressing BAFF receptor, have a higher ability to produce
	Antinuclear autoantibodies are observed at the first diagnosis in more than 95% of SSc
	IgG and IL-6, showing that BAFF signaling might contribute to the development of SSc.
	patients. Some of the autoantibodies are associated with distinct disease severity and
	The evidence of T cell polarization toward a Th2 response play an important role in the

.10.3 Autoantibodies relatively specific for SSc • Anti-centromere antibodies

	Anti-centromere antibodies (ACA) were described in 1980 by Moroi et al. ACA are
	detected in the serum of 20 to 30% of scleroderma patients. The presence of these
	antibodies is correlated with certain clinical signs: limited cutaneous involvment,
	calcinosis, and Raynaud's syndrome. The presence of ACA can develop pulmonary
	hypertension but not pulmonary fibrosis. ACA-positive SSc patients have a more
	promising prognosis than patients with autoantibodies related to diffuse form of the
	disease. So far, no pathogenic role of ACA has been demonstrated [123, 124]

• Anti-topoisomerase I antibodies .

	developed Raynaud's phenomenon syndrome and have approximately double rate of lung
	fibrosis compared with anti-CENP patients [126]. Another recent studies have shown that
	anti-topoisomerase I is able to bind to the fibroblasts membrane, and induce adhesion of
	monocyte to fibroblasts and their activation 121 [	].
	• Anti-RNA polymerase III antibodies
	These antibodies are detected approximately in 15% of patients with SSc. They are
	associated with the diffuse form of the disease, especially with renal involvement, and a
	poor prognosis. Their presence may be associated with the anti-DNA topoisomerase-1.
	Their determination in the serum of patients may have a prognostic value in the context of
	scleroderma renal crisis. Their pathogenicity has not	been demonstrated yet [127].
	• Anti-Th ⁄To antibodies
	Antibodies to Th/To are directed against subunits of the ribonuclease mitochondrial RNA
	processing and ribonuclease P complexes. Those antibodies were recently detected in
	patients with localized scleroderma [128]. Anti-Th ⁄To are present in 2 to 5% of SSc
	patients and are relatively specific for SSc. They are associated with limited skin
	involvement but high risk o organ involvement and therefore a worse prognosis.
	Autoantibodies against a chromatin-associated protein were described for the first time in
	SSc patients in 1979. Anti-topoisomerase I antibodies (ATA) are highly associated with
	the dcSSc disease subset. ATA have been found in approximately 40% of patients with
	dSSc, and in less than 10% of lcSSc patients [125]. ATA are associated with pulmonary
	fibrosis, cardiac and musculoskeletal involvement, increased mortality, and with higher
	risk of developing malignancies. SSc patients with undetectable ATA have milder disease
	and better survival. Recent studies have shown that ATA positive patients earlier

• Anti-endothelial cell antibodies (AECA)

	• Fibroblasts antibodies (AFA)
	AFA were identified in the serum of SSc patients in the 1980s [130]. Since then, AFA
	have been well characterized. They can be detected by ELISA in the serum from 46 to
	58% of SSc patients [131]. They are more common in patients with diffuse form. They can
	be also detected by indirect immunofluorescence and flow cytometry. After binding to
	their ligands, some of AFA are internalized. In vitro, they induce the expression of
	adhesion molecules such as ICAM-1, and cytokines (IL1, IL6) by fibroblasts [132]
	Autoantibodies against endothelial cell antigens were found in 25-85% of patients with
	SSc, but were also detected in other connective tissue diseases. They are more frequently
	found in patients with a diffuse form of SSc, digital ulcers, severe Raynaud's phenomenon,
	pulmonary fibrosis and / or cardiac disease. Their antigenic targets are being identified.
	Several studies have demonstrated a pathogenic role of AECA. In SSc patients, AECA can
	induce EC activation in vitro, resulting in the expression of adhesion molecules and release
	of cytokines and chemokines 129 [ ].

  GSH can also directly detoxify ROS. Over 95% of GSH are under the reduced form. A change in the GSH/GSSG ratio can lead to serious disruption of cell homeostasis. Maintaining the reduced form of GSH depends on glutathione reductase,

	which depends on a balance of NADPH / NADP under the influence of G6PD (glyco-6-
	phosphate dehydrogenase).
	I.1.20.2 Examples of non-enzymatic antioxidant systems
	• Vitamin C and E
	Vitamin E at the membrane (lipophilic) and vitamins C predominantly cytosolic
	(hydrophilic) eliminate the hydroxyl radicals.
	Catalase, an enzyme detoxifying H 2 O 2	is located mainly in the liver and kidney
	189 peroxysomes [ ], whose synthesis is proportional to the H 2 O 2 concentration:
	H 2 O 2 + Fe 3 + catalase → H 2 O + O = Fe 5 + catalase
	H 2 O 2 + O = Fe 5 + catalase → Fe 3 + catalase + H 2 O + O 2
	Glutathione peroxidase, catalyzes the detoxification of H 2 O 2 and lipid hydroperoxides
	(ROOH), and is using glutathione (GSH) as a cofactor.
	H 2 O 2 + 2GSH → H 2 O + GSSG
	ROOH + 2GSH → ROH→ H 2	O + GSSG
	The GSH system is the most powerful antioxidant systems of aerobic organisms. GSH is
	present in almost all eukaryotic cells. It is synthesized from L-glutamate, L-cysteine and L-
	glycine by the glutamylcysteine synthetase and GSH [ 190]. The antioxidant functions of

GSH are related to its thiol group (-SH) which can be oxidized (GSSG). GSH serves to cofactor glutathione peroxidase, but also to dehydroascorbate reductase that regenerates the antioxidant ascorbate.

  al., 2005; Lonkar and Dedon, 2010; Morgan and Liu, 2010,[START_REF] Coriat | The organotelluride catalyst LAB027 prevents colon cancer growth in the mice[END_REF]. Interestingly, some catalytic agents called 'sensor/effector' multifunctional redox catalyst, can modulate the intracellular redox balance by generating more reactive oxidants or by targeting cysteine residues in proteins.Those molecules seem to be particularly effective and selective[START_REF] Fry | Sensor/effector drug design with potential relevance to cancer[END_REF]).Among the 'sensor/effector' multifunctional redox catalyst agents, quinone-and chalcogen (selenium, tellurium)-containing agents have shown an in vitro cytotoxic activity against various type of tumor cells (Doering et al., 2010; Lilienthal et al., 2011, Coriat et al., 2011). Those observations have prompted us to try these new cytotoxic organotelluride catalysts in the treatment of mouse SSc. 2 O 2 that are treated with (PHTE) 2 NQ are likely to significantly increase their intracellular concentration of ROS over its cytotoxic level, to ultimately get pushed over a critical "life and death (redox) threshold" (Fry and Jacob, 2006; Mecklenburg et al., 2009). Since mouse SSc fibroblasts produce high amounts of H 2 O 2 , as opposed to normal fibroblasts, we hypothesize that (PHTE) 2 NQ could selectively kill disease fibroblasts in murine HOCl-induced SSc (Servettaz et al., 2009) and inhibit the development of the disease.

The organotelluride catalyst, (PHTE) 2 NQ, is a prototypical member of this newly described family

[START_REF] Fry | Sensor/effector drug design with potential relevance to cancer[END_REF]

; Shabaan et al., 2009; Ba et al., 2010; Jamier et al., 2010). Within this family of compounds, (PHTE) 2 NQ has been the most widely studied compound in vitro on tumor cells (Doering et al., 2010; Jamier et al., 2010) and it has also been the only one used in vivo on mice (Coriat et al., 2011). Intracellular oxygen radicals and H 2 O 2 associate with the quinone moiety of the (PHTE) 2 NQ and strongly oxidized cellular thiols in a reaction catalyzed by the tellurium part of the molecule. This reaction significantly increases the intracellular level of ROS. Therefore, cells already overproducing H

  2 NQ using specific modulators of the enzymatic and non enzymatic systems involved in H 2 O 2 catabolism. Hydrogen peroxide is converted into H 2 O by two sets of enzymes, catalase and the GSH/GPx system.

	The latter uses reduced glutathione as cofactor. Depleting GSH with BSO significantly
	increased H 2 O 2 production in SSc fibroblasts (P = 0.0007) and in normal fibroblasts (P =
	0.0029; Figure 2a). Moreover, incubation with (PHTE) 2 NQ in association with BSO
	increased H 2 O

2 production by 10.9% in normal fibroblasts and by 22% in SSc fibroblasts versus BSO alone (P = 0.001, data not shown). By contrast, adding NAC, a precursor of GSH, decreased H 2 O 2 production in normal fibroblasts (P = 0.04) but had no effect on SSc fibroblasts (Figure

  combine with endogenous oxidative species generated 'naturally' in mouse SSc fibroblasts to selectively kill cells through a necrotic process.Primary fibroblasts extracted from areas diseased skin areas, display an elevated basal level of ROS that is amplified by (PHTE) 2 NQ to generate a lethal oxidative burst in contrast with healthy fibroblasts that display a normal level of endogenous ROS and resist to the action of (PHTE) 2 NQ. The cytotoxic effects of ROS result from an imbalance between their rate of production and the activity of various detoxification systems(Laurent et al., 2005). Indeed, in SSc fibroblasts, the level of GSH is low and is not modified by (PHTE) 2 NQ, whereas, in normal fibroblasts, this level is higher and is raised by (PHTE) 2 NQ. Thus, when normal fibroblasts are exposed to (PHTE) SSc and normal fibroblasts, confirms the role of the GSH pathway in mediating the cytotoxic effects of (PHTE) 2 NQ. In contrast, blocking the catalase pathway or adding PEG catalase, showed that this pathway has no effect on cell survival whatever the considered cell type is. Thus, H 2 O 2 -mediated (PHTE) 2 NQ toxicity is controlled by the glutathione pathway and not by the catalase pathway, as already observed with several cytotoxic molecules[START_REF] Jing | Arsenic trioxide selectively induces acute promyelocytic leukemia cell apoptosis via a hydrogen peroxide-dependent pathway[END_REF][START_REF] Hussain | p53-induced up-regulation of MnSOD and GPx but not catalase increases oxidative stress and apoptosis[END_REF] Laurent et al., 2005;[START_REF] Alexandre | Accumulation of hydrogen peroxide is an early and crucial step for paclitaxel-induced cancer cell death both in vitro and in vivo[END_REF]. These findings are significant, since they seem to identify disturbances in the GSH household (and subsequent increases in ROS levels), rather than ROS generation per se, as the prime activity of such redox modulating agents.In SSc, the local oxidative stress is associated with a systemic oxidative state, as demonstrated by the significant increase in the serum concentrations of advanced oxidation protein products, and of nitrite, in HOCl-treated SSc mice that are compared to PBS injected animals. Therefore, (PHTE) 2 NQ induces a lethal oxidative burst in cells with

2 NQ, pro-oxidative activity of the compound is counterbalanced by the rise of GSH and no oxidative stress can be induced. In contrast, in diseased fibroblasts, (PHTE) 2 NQ induces a huge increase in ROS that is not counterbalanced by the low level of induced GSH and thus, reaches a lethal threshold. Because the level of endogenous ROS in SSc fibroblast is close to that threshold, the exposure to ROS-generating, redox modulating agents such as (PHTE) 2 NQ, enhances the rate of cell death. Since normal fibroblasts are not targeted by (PHTE) 2

The depletion of GSH by BSO, and the consecutive increase of (PHTE) NQ, that data could have important clinical implications

[START_REF] Amstad | The balance between Cu,Zn-superoxide dismutase and catalase affects the sensitivity of mouse epidermal cells to oxidative stress[END_REF][START_REF] Rodriguez | Mitochondrial or cytosolic catalase reverses the MnSOD-dependent inhibition of proliferation by enhancing respiratory chain activity, net ATP production, and decreasing the steady state levels of H(2)O(2)[END_REF][START_REF] Hussain | p53-induced up-regulation of MnSOD and GPx but not catalase increases oxidative stress and apoptosis[END_REF], Laurent et al., 2005)

.

2 NQ toxicity in increased levels of intracellular ROS, but it is not a systemic pro-oxidative molecule, as already observed in various cancer models

[START_REF] Fry | Sensor/effector drug design with potential relevance to cancer[END_REF][START_REF] Doering | Synthesis and selective anticancer activity of organochalcogen based redox catalysts[END_REF]

ACKNOWLEDGEMENTS

I would like to thank all those people who made this thesis possible and an unforgettable experience for me.

ACKNOWLEDGMENTS

This work was supported by European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement 215009 RedCat for financial support.

ACKNOWLEDGMENTS

The authors are grateful to Ms Agnes Colle for her excellent typing and editing of the manuscript.

ABSTRACT

Systemic sclerosis (SSc) is a connective tissue disorder characterized by skin and visceral fibrosis, microvascular damages and autoimmunity. HOCl-induced mouse SSc is a murine model that mimics the main features of the human disease, in particular the activation and the hyperproliferation rate of skin fibroblasts. We demonstrate here the efficiency of a tellurium-based catalyst 2,3-bis(phenyltellanyl)naphthoquinone ((PHTE)2NQ) in the treatment of murine SSc, through its selective cytotoxic effects on activated SSc skin fibroblasts.

SSc mice treated by (PHTE)2NQ displayed a significant decrease in lung and skin fibrosis and in alpha-Smooth Muscle Actin (α-SMA) expression in the skin compared to untreated mouse SSc animals. Serum concentrations of advanced oxidation protein products, nitrate, and anti-DNA topoisomerase-I autoantibodies were increased in SSc mice but were significantly reduced in SSc mice treated by (PHTE)2NQ . To assess the mechanism of action of (PHTE) 2 NQ, the cytotoxic effect of (PHTE) 2 NQ was compared in normal and in mouse SSc skin fibroblasts. ROS production is higher in mouse SSc fibroblasts than in normal fibroblasts and was still increased by (PHTE) 2 NQ to reach a lethal threshold and kill mouse SSc fibroblasts. Therefore, the effectiveness of (PHTE)2NQ in the treatment of mouse SSc seems to be linked to the selective prooxidative and cytotoxic effects of (PHTE) 2 Systemic sclerosis (SSc) is an autoimmune disease of unknown aetiology, characterized by fibrosis of the skin and of internal organs, vascular dysfunction, and dysimmunity (LeRoy and Medsger, 2001). However, the mechanisms that underlie the NQ on hyperproliferative fibroblasts.

INTRODUCTION

Serum anti-DNA topoisomerase-1 IgG Abs were assayed by ELISA using purified calf thymus DNA topoisomerase-1 bound to the wells of a microtiter plate (Immunovision, Miami, FL). Plates were blocked with PBS-1% BSA and 100 µl of 1:20 mouse serum were added and allowed to react for 1 h at room temperature. Bound Abs was detected with alkaline phosphatase-conjugated goat anti-mouse IgGAb and the reaction was developed by adding p-nitrophenyl phosphate. Optical density was measured at 405 nm using a Dynatech MR 5000 microplate reader (Dynex Technologies, Chantilly, VA).

Statistical Analysis

All quantitative data was expressed as means ± SEM. Data was compared using the Mann-Whitney nonparametric test or the Student's t paired test.

CONFLICT OF INTEREST

The authors state no conflict of interest. Conclusions. Irbesartan, an AT 1 R antagonist, prevents fibrosis, inflammation and inhibits nitric oxide production in HOCl induced models of systemic fibrosis. This report extends the indication of an AT 1 R antagonist to SSc patients with diffuse fibrosis,especially those with lung involvement.

The natural organosulfur Dipropyltetrasulfide Prevents Systemic Sclerosis in the mice

Wioleta

Irbesartan reduced the expression of angiotensin II, AT 1 R, and phospho-EGFR in

HOCl-mice. The expression of AT (percent of positive areas) was significantly higher in fibrotic skin of HOCl-mice than in PBS mice (16.79%±0.53 versus 1.62%±0.47, P < 0.01, Figure 1C). Irbesartan decreased AT expression by more than 80% in HOCl-mice (16.79%.±0.53 versus 2.35%±1.1, P < 0.01, Figure 1C). AT 1 R was overexpressed in HOCl-mice versus control PBS mice (24.1 A.U.±4.8 versus 5.8 A.U.±0.4, P < 0.05, Figure 1E). In vivo administration of irbesartan downregulated AT 1 Thus, irbesartan counteracted the hyperproliferative phenotype of HOCl-SSc fibroblasts.

Ras GTPases (H-Ras) are activated in SSc skin fibroblasts and play a key role in regulating their proliferation rates ( R expression in HOCl-mice (7.6 A.U.±0.4 versus 24.1 A.U.±4.8 versus untreated HOCl-mice, P < 0.05, Figure 1E). Furthermore, the level of pEGFR was significantly higher in HOCl-mice (21.49%±2.5) than in PBS mice (7.3%±0.66, P < 0.01, Figure 1C). Irbesartan downregulated the expression of pEGFR in HOCl-treated mice versus untreated HOCl-mice (8.39%±1.05 versus 21.49%± 2.5, P < 0.01, Figure 1C).

Irbesartan reduced the hyperproliferative phenotype of fibroblasts from the skin of

HOCl-mice. Skin fibroblasts isolated from HOCl-mice displayed a higher proliferation rate than fibroblasts obtained from mice injected with PBS (8557±774.3 vs 5218±536.7, P < 0.01, Figure 1D). The proliferation rate of fibroblasts isolated from HOCl-mice treated with irbesartan was lower than that of fibroblasts isolated from mice injected only with HOCl (5996±378.7 vs 8557±774.3, P < 0.05). 32,33). By western blot analysis, we found that H-Ras was overexpressed in the skin of HOCl-mice compared to PBS-mice (Figure 1E) (28.6 A.U.±11 versus 3.64 ±0.5, P < 0.01). In HOCl-mice, H-Ras expression was reduced by 54% in irbesartan-treated mice compared to untreated animals (Figure 1E) (13.2 A.U.±2.2 versus 28.6 A.U.±11, P < 0.01, ).

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for intellectual content and all authors approved the final version to be published. Dr Batteux had full access to all of the data in the study and takes the responsibility for the integrity of the data and the accuracy of the data analysis.

Appendices

In this chapter I will present the rest of publications in which I am co-author. development of fibrosis and autoimmunity in a mouse model of systemic sclerosis. The American journal of pathology, 177(1):187-196.