Several beneficial effects of green tea polyphenols (GTPs) have been shown in both in vitro and animal studies. This pilot study tested their effects on relief of dry mouth and quality of life (QoL) in Sjögren’s syndrome (SS) patients. After one month of green tea consumption in 18 SS patients, and three months in 7 patients, there was an improvement in the patients’ oral health and QoL. In addition there was an increase in the unstimulated salivary flow rate and a decrease in the viscosity of stimulated saliva, although not statistically significant. Furthermore, 83% of the patients reported that they would continue to drink green tea. Although the mechanism of action of the GTPs is unknown in this patient population, we have shown that regular green tea consumption by SS patients is a simple yet effective and enjoyable means of dry mouth relief.

Several beneficial effects of green tea polyphenols (GTPs) have been shown in both in vitro and animal studies. This pilot study tested their effects on relief of dry mouth and quality of life (QoL) in Sjögren’s syndrome (SS) patients. After one month of green tea consumption in 18 SS patients, and three months in 7 patients, there was an improvement in the patients’ oral health and QoL. In addition there was an increase in the unstimulated salivary flow rate and a decrease in the viscosity of stimulated saliva, although not statistically significant. Furthermore, 83% of the patients reported that they would continue to drink green tea. Although the mechanism of action of the GTPs is unknown in this patient population, we have shown that regular green tea consumption by SS patients is a simple yet effective and enjoyable means of dry mouth relief.

Autoimmune diseases are clinical manifestations of a loss-of-tolerance of the immune system against the body’s own substances and healthy tissues. Primary Sjögren’s syndrome (pSS) and systemic lupus erythematosus (SLE) are two chronic inflammatory autoimmune diseases characterized by autoantibody production and an activated type I interferon system. Although the precise mechanisms leading to autoimmune processes are not well defined, recent studies suggest that aberrant DNA methylation and gene expression patterns may play a central role in the pathogenesis of these disorders. The aim of this thesis was to investigate DNA methylation and gene expression in pSS and SLE on a genome-wide scale to advance our understanding of how these factors contribute to the diseases and to identify potential biomarkers and novel treatment targets. In study I, differential DNA methylation was analyzed in multiple tissues from pSS patients and healthy controls. We identified thousands of CpG sites with perturbed methylation; the most prominent finding was a profound hypomethylation at regulatory regions of type I interferon induced genes in pSS. In study II, a cases-case study comparing DNA methylation in pSS patients with high fatigue to patients with low fatigue, we found methylation patterns associated to the degree of fatigue. In study III, RNA-sequencing was applied to investigate the transcriptome of B cells in pSS in comparison to controls. Increased expression of type I and type II interferon regulated genes in pSS was observed, indicating ongoing immune activation in B cells. In study IV, the impact of DNA methylation on disease susceptibility and phenotypic variability in SLE was investigated. We identified DNA methylation patterns associated to disease susceptibility, SLE manifestations and different treatments. In addition, we mapped methylation quantitative trait loci and observed evidence for genetic regulation of DNA methylation in SLE.   In conclusion, the results presented in this thesis provide new insights into the molecular mechanisms underlying autoimmunity in pSS and SLE. The studies confirm the central role of the interferon system in pSS and SLE and further suggest novel genes and mechanisms to be involved in the pathogenesis these autoimmune diseases.

This thesis comprised patients with chronic rheumatic diseases. The studies aimed to elucidate food sensitivity by measuring mucosal inflammatory reactivity and thereby a possible link between the gut and joints. In all the studies, the mucosal path technique was used to evaluate the rectal mucosal response to rectal challenge with gluten and/or cow’s milk protein (CM). In some patients with primary Sjögren’s syndrome (pSS) and the genetic susceptibility genes HLA DQ2, mucosal reactivity measured with nitric oxide (NO) was found after rectal gluten challenge without detectable serum antibodies to gluten or transglutaminase. This gluten sensitivity was not linked to coeliac disease. After rectal CM challenge, a rectal mucosal inflammatory response measured with NO and myeloperoxidase (MPO) was detected in 38% of pSS patients, all of whom fulfilled the criteria for irritable bowel syndrome. In a questionnaire study of self-experienced adverse reactions to food, 27% of patients with rheumatoid arthritis (RA) reported intolerance to various foods and CM in particular. After rectal CM challenge performed in RA patients (n=27), strong mucosal reactivity to CM was observed in a few patients and a moderate increase in 23%. After gluten challenge, a moderate increase in mucosal reactivity was found in 35% of patients. No correlation to self-perceived intolerance and mucosal reactivity measured with NO and MPO was seen. Inflammation of the gut is a prominent feature of spondyloarthropathies (SpA). After rectal challenges with CM protein and gluten, an increase in rectal NO production was seen in 26% and 19% respectively (p<0.001). An increase in the mucosal release of MPO as a sign of neutrophil activation was seen in the CM- and gluten-sensitive patients. NO production in SpA patients was more enhanced compared with RA and pSS patients and could contribute to the increased barrier permeability described in SpA patients.

Le syndrome de Gougerot-Sjögren (SGS) est une épithélite auto-immune caractérisée par des lésions des glandes exocrines et manifestations systémiques. Une des complications majeures est la survenue chez 5% des malades, d’un lymphome non-hodgkininen (NHL). La contribution majeure des lymphocytes B (LB) a récemment été démontrée. Dans ce travail, nous avons voulu aborder des sujets cliniques et fondamentaux concernant le rôle des LB dans le SGS. Dans un premier temps, nous avons démontré que des LB mémoires sont visibles dans des infiltrats des échantillons de la peau et sa présence peut aider au diagnostic. Dans un deuxième temps, nous avons démontré que la cytokine FLT3-L augmentée (une cytokine impliquée dans l’ontogenèse des LB et lympho-prolifération) est associée à une distribution anormale des LB dans les malades. En plus, le rôle prolifératif de FLT3-L sur les LB pourrait expliquer l’évolution vers le NHL. Dans un troisième temps, nous avons étudié une autre cytokine dérégulée dans le SGS (la cytokine BAFF) et nous avons confirmé le rôle d’un nouveau variant de BAFF produit par l’épissage alternatif de l’exon 4 (∆4BAFF) comme un facteur de transcription de son propre gène. Ce nouveau variant est beaucoup plus exprimé au cours des maladies autoimmunes, et son expression est contrôlée par l’interferon gamma et la protéine SC35. Tous ces données montrent pour la première fois, un nouveau concept à savoir la possibilité pour une cytokine d’être régulée par un variant provenant de l’épissage alternatif de son propre gène. Ensemble, ces résultats montrent le rôle des cytokines impliquées dans l’ontogenèse et la survie des LB, dans la physiopathologie du SGS. / Primary Sjögren’s Syndrome (pSS) is a systemic autoimmune disease characterized by sicca symptoms and a broad variety of systemic manifestations. The most severe complication of the disease is the development of non-Hodgkin’s lymphoma (NHL) in 5% of patients. Recent evidence indicates a major contribution of B cells. In this work, we developed clinical and basic research subjects, related to the role of B-cell in the pathogenesis of pSS. In the first section, we showed that memory B-cell infiltrates are present in pSS and may be used as an additional diagnostic and follow-up tool. In the second section, we showed that high serum levels of the cytokine called FLT3-L (a cytokine implicated in B-cell ontogenesis and lymphoproliferation) are associated with abnormal B-cell distribution, characteristic of pSS; and disease clinical activity. In addition, this cytokine may explain the development of lymphoma. In the third section, we demonstrated that ∆4BAFF (a new variant of BAFF, due to the alternative splicing of exon 4) is a transcription factor of its own gene. Interestingly, this new variant is mainly detected in autoimmune diseases and its expression is regulated by IFN-y and SC35 protein (one of the proteins implicated in the splicing machinery). This finding provides an expanded conceptual view of BAFF gene regulation in autoimmune diseases, and contributes to a better understanding of the mechanisms involved in BAFF up-regulation in autoimmunity. Collectively, these results are of clinical and fundamental basic interest in pSS, in the diagnostic, physiopathology and therapeutic contexts.

<p>Autoimmune mechanisms and genetic susceptibility contribute to the pathogenesis of primary Sjögren’s syndrome and SLE. These chronic systemic autoimmune diseases have many serological and clinical features in common and have an impact on daily life. The studies in this thesis aim to elucidate their autoimmune mechanisms, define susceptibility genes and evaluate effects of androgen supplement on health-related quality of life.</p><p>Autoantibodies against α-fodrin, a widely distributed cytoskeletal protein, were detected at similar frequencies in sera from patients with primary and secondary Sjögren’s syndrome and SLE. Consequently, testing for antibodies against α-fodrin would not add diagnostic value compared to conventional serological analysis and does not discriminate between these diseases.</p><p>The type I interferon (IFN) system was found to be activated in primary Sjögren’s syndrome. IFN-α containing cells were detected in minor salivary gland biopsies, while sera from patients with primary Sjögren’s syndrome induced IFN-α production in the presence of apoptotic and necrotic cell material. This ability of sera correlated with the presence of antibodies against RNA-binding proteins and IFN-α production was dependent on RNA in immune complexes. The natural interferon producing cells/plasmacytoid dendritic cells (NIPC/PDC) were the IFN-α producers and blocking of FcγRIIa inhibited the production. Single nucleotide polymorphisms (SNPs) in two genes in the type I IFN signalling pathway, those for tyrosine kinase 2 and interferon regulatory factor 5, were strongly associated with SLE in a Swedish, Finnish and Icelandic population. The minor allele frequencies were lower in SLE patients than in healthy controls. These SNPs may decrease the function of the type I IFN system, thereby conferring protection against SLE. </p><p>Supplementation with dehydroepiandrosterone (DHEA) in glucocorticoid treated women with SLE led to mild improvements in health-related quality of life in respect of mental well-being and sexuality, whereas physical well-being was unaffected.</p>

Autoimmune mechanisms and genetic susceptibility contribute to the pathogenesis of primary Sjögren’s syndrome and SLE. These chronic systemic autoimmune diseases have many serological and clinical features in common and have an impact on daily life. The studies in this thesis aim to elucidate their autoimmune mechanisms, define susceptibility genes and evaluate effects of androgen supplement on health-related quality of life. Autoantibodies against α-fodrin, a widely distributed cytoskeletal protein, were detected at similar frequencies in sera from patients with primary and secondary Sjögren’s syndrome and SLE. Consequently, testing for antibodies against α-fodrin would not add diagnostic value compared to conventional serological analysis and does not discriminate between these diseases. The type I interferon (IFN) system was found to be activated in primary Sjögren’s syndrome. IFN-α containing cells were detected in minor salivary gland biopsies, while sera from patients with primary Sjögren’s syndrome induced IFN-α production in the presence of apoptotic and necrotic cell material. This ability of sera correlated with the presence of antibodies against RNA-binding proteins and IFN-α production was dependent on RNA in immune complexes. The natural interferon producing cells/plasmacytoid dendritic cells (NIPC/PDC) were the IFN-α producers and blocking of FcγRIIa inhibited the production. Single nucleotide polymorphisms (SNPs) in two genes in the type I IFN signalling pathway, those for tyrosine kinase 2 and interferon regulatory factor 5, were strongly associated with SLE in a Swedish, Finnish and Icelandic population. The minor allele frequencies were lower in SLE patients than in healthy controls. These SNPs may decrease the function of the type I IFN system, thereby conferring protection against SLE. Supplementation with dehydroepiandrosterone (DHEA) in glucocorticoid treated women with SLE led to mild improvements in health-related quality of life in respect of mental well-being and sexuality, whereas physical well-being was unaffected.

Le syndrome de Gougerot-Sjögren (SGS) est une maladie autoimmune (MAI) systémique inflammatoire chronique caractérisée principalement par la diminution des secrétions salivaires et lacrymales, aboutissant à une sécheresse de la bouche et des yeux. Elle affecte principalement les femmes autour de la ménopause. Au niveau physiologique, le tissu épithélial des glandes salivaires (GS) est infiltré par des lymphocytes, ce qui provoque l’apoptose des cellules épithéliales (CE). L’épigénétique pouvant jouer un rôle important dans le développement des MAI comme le SGS, nous avons analysé l’expression d’éléments rétroviraux endogènes humains (HERV) et des microARN. Par ailleurs, afin de mieux comprendre les mécanismes cellulaires et moléculaires impliqués dans les interactions lymphocytes-CE au niveau de la GS pathologique, nous avons mis en place un modèle de co-culture in vitro entre des CE et des lymphocytes B ou T (LB, LT). Nous avons également étudié le rôle de BAFF sur les CE qui expriment l’un de ses récepteurs, BR3.Dans un premier temps, nous avons montré que dans les GS des patients, les HERV et les miARN ont un profil d’expression distinct de celui des personnes saines, montrant ainsi une participation de l’épigénétique dans la pathologie. Dans un deuxième temps, nous avons montré que les CE entrent en apoptose suite à l’interaction directe avec les lymphocytes B etT, en faisant intervenir la voie Fas pour les LT et la voie de la PKCδ pour les LB. En lien avec l’apoptose induite par les LB, nous avons démontré que BAFF est impliqué dans la survie des CE, et que le blocage de sa signalisation ou la sous-expression de son récepteur conduit à la mort des CE. Nous pouvons supposer que les LB entrent en compétition avec les CE pour le signal de survie apporté par BAFF, et que les CE qui s’en trouvent privées meurent. Finalement, nous avons montré que plusieurs formes de BAFF sont produites parles CE et reconnues de manière différente par les anticorps anti-BAFF. Il pourrait s’agir d’isoformes plus ou moins glycosylés ou de variants de BAFF. Cependant d’autres études sont nécessaires afin de les identifier. / Sjögren’s syndrome (SS) is an inflammatory systemic autoimmune disease (AID), mainly characterized by the decrease of salivary and lachrymal secretions. This leads to dry mouthand dry eyes. This chronic disease principally affects women around menopause. At the physiological level, salivary gland (SG) epithelial tissue is infiltrated by lymphocytes, leading to epithelial cells (EC) apoptosis. As epigenetics can play an important role in AID- such asSS development, we investigated human endogenous retroviral elements (HERV) and microRNA expression. Furthermore, in order to better understand the cellular and molecular mechanisms implied in lymphocyte-EC interactions in pathological SG, we set up an in vitroco-culture model between EC and B and T cells. We also studied BAFF role on EC which express one of its receptors, BR3. First, we demonstrated that in SS SG, HERVs and miRNAs show a distinct profile from healthy controls, indicating that epigenetics is implied in the pathology. Secondly, we showed that EC undergo apoptosis following direct interaction with T and B cells, via two distincts ignaling pathways: Fas pathway for T cells and PKCδ pathway for B cells. Linked with Bcell-induced apoptosis, we demonstrated that BAFF is implied in EC survival, and that BAFFsignaling blocking or BR3 down-regulation leads to EC death. We may suppose that B cells compete with EC for survival signaling from BAFF, and that EC die from lack of it. Finally, we showed that several forms of BAFF are expressed by EC and that they are differentially recognized by anti-BAFF antibodies. These may be more or less glycozylated BAFF isoformsor BAFF variants. Further studies are needed to identify them.

Les maladies auto-immunes et systémiques sont des maladies sur lequel le champ de la recherche pose son œil de façon appuyée depuis 15 ans, du fait de l’émergence de thérapies biologiques ciblées. Ces pathologies sont volontiers hétérogènes, au mieux de fréquence ou caractéristiques particulières dans les populations d’origine Africaine. La connaissance de l’épidémiologie, et des caractéristiques de ces maladies est un préalable essentiel à la mise en place de recherche plus fondamentale pour aider à décomposer leurs physiopathologies souvent extrêmement complexes. En effet, la comparaison de différences marquées entre deux expressions dans des populations différentes d’une même maladie peut permettre d’aider à en dénouer le fil. Nous proposons dans ce travail une estimation des caractéristiques du lupus cutané et du lupus systémique en Guyane Française qui retrouve une faible fréquence de la maladie, la plus faible jamais retrouvée dans une population subsaharienne. Nous décrivons en Martinique sur le plan épidémiologique comme clinique une forme rare de myosite appelée syndrome des anti-synthétases semblant très particulière, l’épidémiologie et la description de la maladie de Kikuchi-Fujimoto pour la première fois dans la littérature, l’épidémiologie et les caractéristiques à base de population de la maladie de Behcet, des principales vascularites (périartérite noueuse, micropolyangéite, granulomatose éosinophile avec polyangéite, granulomatose avec polyangéite), de l’hypertension pulmonaire des connectivites qui semblent plus fréquentes que chez les Européens. Les néphropathies du lupus systémiques sont décrites dans la population Guadeloupéenne montrant une grande fréquence des néphropathies prolifératives. Le protocole EUROLUPUS qui permet le traitement de ces néphropathies prolifératives du lupus systémique avec de faibles doses de cyclophosphamide et de corticoïdes, est évalué en Martinique sur 30 patients alors qu’il ne l’a jamais été dans une population d’origine Africaine. Il semble y être aussi efficace que chez les patients d’origine Européenne, alors que les néphropathies y ont un pronostic meilleur. La maladie de Sjögren primaire est décrite en Martinique très proche de ce qu’on trouve en Europe sur le plan du tableau clinique et évolutif alors que cela n’est l’objet d’aucune étude dans une population d’origine noire Africaine. Nous avons par ailleurs montré en Martinique l’amélioration de la prise en charge du lupus systémique en Martinique au travers de la régression au fil du temps d’une des complications de la corticothérapie les plus pénibles pour les patients, l’ostéonécrose aseptique. La sclérodermie systémique est décrite à base de population avec épidémiologie dans les deux départements de Guadeloupe et Martinique, montrant des caractéristiques proches de celles retrouvées chez les AfroAméricains. Nous avons aussi montré la fréquence et la gravité des atteintes ORL des myopathies inflammatoires sur ces 2 départements avec une fréquence inhabituelle de certaines maladies auto-immunes dont le lupus systémique et les myosites inflammatoires associées aux anticorps anti-SRP, et l’absence de myosite à inclusion. Au total, nous apportons une somme de connaissance descriptive de ces maladies auto-immunes et systémiques permettant la mise en place de recherches plus fondamentales avec des bases solides par rapport aux profils hétérogènes de ces maladies. / Auto-immunes and systemic diseases are priorities for researchers since 15 years. This is related to the emergence of biological therapies, associated to great efficacy. Although, these diseases are heterogeneous, depending of different parameters such as ethnicity or geography. In the African descent population, we encounter unusual or particular manifestations of these diseases. Also, the knowledge of epidemiology and population based descriptions are crucial to properly initiate works on these populations, but also to understand a particularly complex physiopathology by using differences between populations. We describe in this work the population based characteristics of pure cutaneous lupus and systemic lupus, including an epidemiology of the incidence of the lowest incidence ever found in a population of African heritage. We also describe a population based series of anti-synthetase syndrome, confirming that the presentation is totally different compared to caucasians, and allows in Martinique the incidence, never explored before. We also provide the first evaluation of Kikuchi-Fujimoto disease in a population of African origin, and the first incidence ever realized. We do the same evaluation of the epidemiology of Behcet’s disease in a black origin population that shows that this disease was at a similar frequency in Martinique and in Europe. Micropolyangeitis, polyarteritis, eosinophilic granulomatosis with polyangeitis and Granulomatosis with polyangeitis were evaluated in an epidemiologic study in Martinique, with addition of some cases from other French American region for a more powerful characteristics description. These diseases seem less frequent than in Europe, associated with less severity except for micropolyangeitis. EUROLUPUS, a protocol with low dose IV cyclophosphamide and low dose steroids, used to treat proliferative nephritis of systemic lupus is shown to have the same efficacy in Martinique than in patients of European origin. Primary Sjögren syndrome, evaluated in Martinique, is very similar in expression than what is found in Europe. The decrease overtime of aseptic osteonecrosis, a steroid side effect, is a witness of better control of systemic lupus activity with less usage permitted by protocols and new immunosuppressive drugs such as mycophenolate. Systemic sclerosis is described as very close to African American in a population based study in Martinique and Guadeloupe. We finally show that the rare ENT involvement of idiopathic inflammatory myositis is frequent in our population, associated with poor outcome, and surprisingly frequently related to systemic lupus and necrotizing myositis associated to SRP antibody but not to inclusion body myositis. To conclude, we allow an amount of description of these diseases in our region, including pioneer studies. This works tends to be the basis for studies to be continued in a more fundamental way in our countries.

Le syndrome de Sjögren primitif (SSp) est une maladie auto-immune (MAI) systémique ayant des caractéristiques communes avec le lupus érythémateux. Ces caractéristiques incluent des mécanismes physiopathologiques et des facteurs de predispositions génétiques. Notre équipe et d’autres groupes ont pu mettre en evidence une signature interféron (IFN) dans les glandes salivaires et les PBMCs de patients ayant un SSp. Cette découverte a permis de mettre en évidence de nouvelles voies à explorer dans la pathogénie du SLE et SSp en permettant la focalisation des recherches sur le rôle de l’immunité innée et de la voie IFN.Nous avons confirmé le rôle de 2 gènes importants dans le SSp, impliqués dans les voies des IFN. Le premier est IRF5 sur la voie IFN de type I et STAT4 sur la voie IFN de type II. Nous avons pu mettre en évidence une fonctionnalité de l’allèle à risque d’IRF5 (Polymorphisme Indel situé dans le promoteur). Concernant STAT4, son expression n’était pas altérée par le SNP associé à la maladie. Toutefois, l’ARNm de STAT4 était corrélé à l’expression des gènes IFN de type I. Les dérégulations épigénétique pourraient jouer un rôle important dans la pathogénie de nombreuses MAI, en particulier la méthylation de l’ADN qui est hautement liée à l’extinction de l’expression des gènes. Nous avons étudié la méthylation du promoteur d’IRF5 et nous n’avons pas trouvé de régulation de ce promoteur par le méthylation. Une analyse de la méthylation avec une approche globale du méthylome est en cours dans notre équipe et permettra d’identifier de gènes cibles d’une dérégulation épigénétique pouvant être impliqués dans les MAI.Nous avons essayé de comprendre la relation entre STAT4 et gènes IFN de type I. Ainsi, nous rapportons que l’IL-12 induit spécifiquement l’IFN de type I par intéraction entre deux partenaires cellulaires, les lymphocytes T CD4+ et les cellules dendritiques plasmacytoïdes. Ces résultats pourraient expliquer l’implication des polymorphismes de STAT4 dans les MAI dépendantes de l’IFN de type I. Ces résultats suggèrent également que les MAI dépendantes des IFN de type I et II ne s’opposent pas. Elles seraient seulement le Yin et le Yang d’un facteur d’activation commun, STAT4, capable d’induire les IFNs de type I et II. / Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease (AID) that presents similar characteristics to systemic lupus erythematosus. These characteristics include pathophysiology and genetic factors. Our team and other groups have highlighted an interferon (IFN) signature in salivary glands and PBMCs from patients with Sjögren syndrome. This signature demonstrates new pathways in pSS and lupus, focusing research on innate immunity and in the IFN pathway.We have confirmed the implication of 2 genes in the pSS, and these genes are involved in the IFN pathway. The first gene is IRF5 which is in the type I IFN pathway and the second is STAT4 which is in the type II IFN pathway. We have shown a functional consequence of IRF5 at-risk allele. Regarding STAT4, the associated SNP did not altered STAT4 mRNA expression but was highly correlated with type I IFN genes expression.The epigenetic deregulation could play a triggering role in autoimmune diseases, particularly through DNA methylation which is highly implicated in the suppression of gene expression. We studied the methylation of IRF5 promoter and found no methylation. Our team is currently undertaking a global approach with methylome analysis. This methylome study will assess specific gene methylation patterns and will allow a better understanding of the role of these genes in autoimmune diseases.We further demonstrated that IL-12 specifically induces a type I IFN signature through a CD4+ T cells and pDCs crosstalk. These results could explain the implication of STAT4 polymorphism not only in type II IFN-dependent AIDs but also in type I IFN-dependent AIDs. Our data confirm that type I IFN- and type II IFN-mediated AIDs do not have to be opposed. They are only the yin and the yang of a common STAT4 activation which may induce secretion of both cytokines.