La maladie de Berger est la glomérulonéphrite la plus fréquente avec une estimation selon laquelle 1% de la population mondiale serait touchée. L’agent causal est une immunoglobuline (Ig)A anormale (polymérique et hypogalactosylée) qui se dépose dans le mésangium et provoque un dysfonctionnement rénal (protéinurie, hématurie) et des lésions glomérulaires. Dans 25% des cas, la maladie évolue sur 20 ans vers l’insuffisance rénale terminale. Des évidences, de plus en plus nombreuses, montrent que l’environnement microbien, en particulier bactérien, commensal ou pathogène, a un impact important sur le développement de la maladie. Au cours de ma thèse, j’ai d’abord étudié l’effet d’une molécule de la paroi bactérienne, le lipopolysaccharide (LPS), sur la production des IgA dans les muqueuses chez la souris normale. Les résultats que j’ai obtenus et ceux publiés permettent de proposer que la stimulation chronique des muqueuses par l’environnement microbien conduit à une augmentation de la production d’IgA néphrotoxiques, qui, du fait d’un déficit de leur récepteur pIgR mucosal, sont anormalement dirigées vers la circulation plûtôt que dans la lumière es muqueuses. Dans une seconde partie de mon travail, j’ai étudié l’effet du LPS sur le développement de la maladie de Berger dans un modèle de souris 1KI. Ces souris génétiquement modifiées produisent de l’IgA humaine et développent spontanément des dépôts mésangiaux d’IgA mais n’ont pas de protéinurie, d’hématurie ou de lésions glomérulaires. Nos résultats montrent que le LPS provoque une forte hématurie dans les souris 1KI lorsque celles-ci expriment le récepteur des IgA humaines, CD89, à la surface des polymorphonucléaires neutrophiles. En conclusion, mon travail de thèse a permis de mettre en lumière un impact de l’environnement microbien sur pIgR et sur les polymorphonucléaires neutrophiles dont la déficience ou l’activation pourrait contribuer au développement de la maladie de Berger. / IgA nephropathy is the most frequent glomerulonephritis worldwide. It features mesangial immunoglobulin (Ig)A deposits and proteinuria, hematuria and glomerular histological lesions. In 25% patients, it evolves, within 20 years, towards the end stage renal disease. Microbial environment, through the interaction with mucosa, is believed to play a crucial role in the development of the disease. The objectives of my work were to evaluate the effect of the microbial compound lipopolysaccharide (LPS) on the production of the nephritogenic IgA in the mucosa of mice and on the development of IgA nephropathy in the murine model 1KI that spontaneously develops mesangial IgA deposits without the other signs of the disease. Our results and those previously published suggest that the chronic stimulation of mucosa by microbiota leads to the increased production of nephritogenic IgA in the mucosa. These IgA, thanks to a deficient mucosal receptor pIgR in patients with IgA nephropathy, may be abnormally routed in the blood. We also showed that LPS provokes hematuria in the 1KI mice, when they express a specific IgA receptor, CD89, on the surface of polymorphonuclear neutrophils. Altogether our findings highlight the impact of microbial environment on pIgR and on polymorphonuclear neutrophils and thus, potentially, on IgA nephropathy.

La Néphropathie à IgA est la glomérulonéphrite primitive la plus fréquente, responsable d’une évolution vers l’insuffisance rénale terminale dans 10 à 30% des cas après 20 ans d’évolution. Les déterminants de cette maladie sont nombreux, impliquant de multiples acteurs de l’inflammation, qu’ils soient cellulaires ou humoraux. La physiopathologie générale de la maladie est actuellement considérée comme se déroulant en quatre « coups », (i) la production d’IgA1 polymériques présentant un déficit de galactosylation de la région charnière, (ii) l’existence d’un élément circulant capable de complexer ces IgA1 anormales, pouvant être une IgG anti-glycane ou la portion soluble du récepteur de type I aux IgA (sCD89), (iii) la constitution de complexes immuns circulants et (iv) le dépôt glomérulaire de ces complexes, générant des lésions inflammatoires puis cicatricielles responsables de l’évolution vers la maladie rénale chronique. La médiation inflammatoire est impliquée à différents niveaux comprenant entre autres le rôle de l’infiltration de macrophages dans le tissu rénal, l’orchestration de cette réponse inflammatoire glomérulaire par les sous-populations de lymphocytes T et l’importance de l’activation du complément sur le déterminisme des lésions glomérulaires inflammatoires médiées par les complexes immuns déposés. Au cours de ce travail de thèse, l’implication de ces différents acteurs a été explorée. Les macrophages expriment le récepteur de type I aux IgA (CD89, issu du gène FCAR), dont une mutation de la portion intracytoplasmique modifie in vitro la transduction du signal. La première étude a visé à évaluer sur une grande cohorte rétrospective l’impact de cette mutation sur le risque d’occurrence de la maladie ainsi que son impact pronostique. Le rôle des sous-populations T a été abordé au cours d’une seconde étude, suivant l’hypothèse que l’orientation pro inflammatoire au cours de la NIgA pourrait être liée à un déficit de régulation par une sous-population de lymphocytes T, les Tregs. Cette étude prospective a évalué la représentation de la sous-population CD4+CD25+CD127low et le profil d’expression génique de gènes prototypiques des sous-populations Th1, Tregs et Th17. Le rôle du complément comme médiateur inflammatoire à l’interface entre les complexes immuns à IgA1 et les cellules mésangiales a été exploré par une étude in vitro. La mutation 844 A->G de FCAR n’a pas été associée à un risque supplémentaire de développer la maladie et n’impactait pas le pronostic des patients. L’étude des lymphocytes T n’a pas montré de différence de quantité de cellulesCD4+CD25+CD127low entre patients et volontaires sains et ne suggérait qu’une tendance en faveur d’un déficit fonctionnel de régulation (plus faible expression des gènes FoxP3, IL10, TGFβ chez les patients). Enfin, des fragments issus de l’activation et de la protéolyse par le facteur I de C3 ont été mis en évidence par immunoblot et spectrométrie de masse au sein des complexes immuns générés artificiellement en présence de serum. Ces études suggèrent qu’une modification du rôle fonctionnel du CD89 n’impacte pas le devenir de la NIgA, ce qui est en défaveur d’un rôle critique de ce récepteur dans la pathogénie de la maladie. La tendance au déficit fonctionnel Tregs nécessite d’être confirmée au sein d’un effectif plus conséquent et présentant une forme plus active de la maladie, mais elle corrobore deux autres études comparables dans leur méthodologie. Enfin, le rôle du complément se situe à l’interface entre les complexes immuns et les effecteurs inflammatoires glomérulaires tels que les cellules mésangiales / IgA Nephropathy (IgAN) is the most common primary glomerulonephritis, leading to end stage renal failure in 10 to 30% of cases after 20 years. This disease is determined by numerous inflammatory players, including cells and molecules. The pathogeny of the disease is likely to be driven by a 4 « hits » model, (i) increased systemic production of aberrantly O galactosylated polymeric IgA1, (ii) the existence of circulating abnormal IgA1 binding element, which could be either an anti-glycan IgG or the soluble fragment of the main IgA receptor (sCD89), (iii) the formation of circulating immune complexes, and (iv) the glomerular deposition of these complexes, that accounts for a variable local inflammation leading to scarring processes and finally to the chronic kidney disease. Inflammatory mechanisms operate at several levels, including the macrophage cells infiltration in the kidney tissue, the orchestration of the immune response by T-cells subsets, including regulatory T-cells, and the role of complement activation to induce the glomerular inflammatory response from the immune complexes deposition. In the present work, we aimed to explore the implication of these inflammation response players. Macrophages express the type I IgA receptor (CD89, downstream from its gene FCAR), whose function can be affected in vitro by a common mutation of its intracytoplasmic portion. A first study evaluated the impact of this mutation on the risk to develop the disease as well as on the global prognosis. A second study evaluated the role of T-cell subsets during IgAN, following the hypothesis that the pro-inflammatory balance of the disease could be a consequence of a defect in the immune regulation by the Tregs. This prospective study aimed to assessing the frequency of CD4+CD25+CD127low cells in peripheral blood and the characteristic gene expression profile from Th1, Th17 and Tregs subsets. The role of complement as an inflammatory player at the interface between IgA1 containing immune complexes and mesangial cells was explored by an in vitro study. The single nucleotide polymorphism 844 A->G of FCAR had no impact neither on disease risk of occurrence neither on the renal survival. The T-cell subsets study failed to demonstrate any difference in the proportion of CD4+CD25+CD127low cells and only suggested a defect in functional activity of Tregs, according to a lower expression of FoxP3, IL10, TGFβ genes. The third in vitro study demonstrated by immunoblot and mass spectrometry the presence of C3 breakdown products accompanying IgA1 based engineered immune complexes formed in presence of normal immunoglobulin depleted serum. The lack of effect of the mutation of FCAR on the IgAN prognosis is not in favour to a critical role of this receptor on the pathogeny of the disease. The trend in the functional defect of Tregs subset needs to be confirmed in a larger study, including patients with a more severe form of their disease. This result is however consistent with two other studies displaying a similar methodology. The role of complement is confirmed to be a key player, as it is likely to act at the interface between the IgAN particular immune complexes and mesangial cells

Introdução: Os receptores Fc são proteínas de importância crucial no processo saúde-doença. São responsáveis pela ativação de mecanismos efetores e modulam a resposta imune e inflamatória. Têm papel central na patogênese de doenças autoimunes, sepse e doenças neoplásicas. O lúpus, protótipo das doenças autoimunes e a sepse, infecção grave que causa disfunção orgânica, são doenças inflamatórias nas quais o papel dos receptores Fc vêm sendo desvendados. Essas patologias têm alta morbidade e mortalidade, impondo enormes custos para sociedade. A descoberta que a E. coli se liga ao receptor CD16 (FcGamaRIII) para evadir-se do sistema imune, através da ligação com a proteína wzxe presente em sua membrana, torna esse receptor um alvo terapêutico interessante. O CD16 é um FcGamaR com ITAM que classicamente tem função ativadora e gera respostas inflamatórias ao se ligar a imunocomplexos. Porém, na sepse, a ligação direta com a E. coli induz uma ativação ITAMi, que bloqueia a produção de ROS e inibe a fagocitose e a morte desta bactéria. A manipulação dessa ativação inibitória (ITAMi), aparentemente anti-inflamatória, pode ser uma estratégia efetiva para o tratamento de doenças inflamatórias como a sepse e o lúpus. Inicialmente visamos avaliar a importância fisiológica e o papel terapêutico do peptídeo ligante do CD16, em modelo de sepse e em modelo de lúpus induzido por pristane, respectivamente. De maneira similar ao CD16, o CD89 (FcAlfaRI) é capaz de mediar uma sinalização dual, ativatória ou inibitória, que depende da forma como se dá sua ligação às imunoglobulinas e imunocomplexos. Por isso, num segundo momento, averiguamos se o FcAlfaRI (CD89) poderia se ligar à bactérias de maneira direta, na ausência de ligantes cognatos e mediar respostas pro ou anti-inflamatórias, protegendo ou não o hospedeiro. Metodologia: Camundongos C57Bl/6, WT e CD16KO com lúpus induzido por pristane, foram tratados com o peptídeo CYWGGTEGAC(IRG Bioscience,USA). A expressão gênica e protéica de diversas citocinas, assim como genes associados a assinaturas de interferon foram avaliados nos pulmões desses animais. Utilizamos também um modelo de sepse através da injeção intra-peritoneal de E. coli WT e E. coli mutante wzxe -/-, no qual avaliamos mortalidade e produção de citocinas. Realizamos experimentos in vitro com BMM e BMDC murinos, fagócitos humanos e bactérias. A expressão de CD89 e de receptores cognatos foi avaliada através de citometria de fluxo. Empregamos a citometria de fluxo com imagem para análise da fagocitose. Foram realizados também, experimentos in vivo com camundongos WT e transgênicos: CD89tg, CD89R209Ltg CD89tgCD16KO, CD16KO e PCRKO. Comparamos mortalidade, produção de citocinas, quantidade de bactérias e lesão tecidual em modelos de CLP e de pneumonia por administração nasal de S. pneumoniae. Produção de ROS pelos BMM foi avaliada por microscopia confocal e, nos PMN, por quimioluminescência. Imunoprecipitação e immunoblotting foram utilizados para avaliar recrutamento de syk e SHP-1. Utilizamos ELISA para ensaios de ligação de bactérias com CD89 e para quantificar TNF-Alfa, IL-1 e IL-6. Resultados: Camundongos injetados com a bactéria mutante wzxe-/- sobreviveram mais e produziram quantidade menor de citocinas reforçando o papel chave da proteína wzxe, no mecanismo de evasão imune da E. coli. Camundongos WT e CD16KO com lupus induzido por pristane, tratados ou não com peptídeo CYWGGTEGAC, não apresentaram diferenças na expressão gênica nem protéica de citocinas nem em genes associados a assinaturas de interferon em seus pulmões. O CD89 interage diretamente com bactérias gram-positivas e gram-negativas. A interação bactéria-CD89 em macrófagos murinos induz ativação celular, fagocitose e morte bacteriana, que são dependentes da cadeia FcRGama. Essa mesma interação protege contra a mortalidade em dois modelos de sepse (CLP e pneumonia) e é dependente da cadeia FcRGama e indepedente de PCR e IgA anti-bactéria. Conclusões: O CD16 e o CD89 são FcRs com ITAM que apresentam uma dualidade na forma de ativação através do ITAM, que em algumas situações pode ser inibitória (ITAMi). Nessa tese reforçamos o papel chave da proteína wzxe, ligante do CD16, como responsável pela evasão bacteriana da E. coli através de sinalização ITAMi. Por outro lado, falhamos em demostrar diferenças após o tratamento de camundongos com lúpus induzido por pristane usando o peptídeo CYWGGTEGAC. Acreditamos que isso tenha ocorrido devido a dose inadequada do peptídeo ou proteólise por enzimas endógenas do camundongo, assim que o peptídeo é injetado. Novas doses ou a manipulação da estrutura do peptídeo são perspectivas futuras para este projeto. Quanto ao CD89, provamos seu papel extremamente importante na imunidade inata. Esse receptor, à semelhança do CD16, foi capaz de ligar-se diretamente a bactérias, na ausência de opsoninas e ligantes cognatos. O CD89 foi protetor tanto para infecção por gram-positivo quanto por gram-negativo enquanto o CD16 foi protetor apenas em modelo de pneumonia por gram-positivo / Introduction: Fc receptors are proteins of crucial importance in the health-disease process. They are responsible for the activation of effector mechanisms and modulate the immune and inflammatory responses. They play a central role in the pathogenesis of autoimmune diseases, sepsis and neoplastic diseases. Lupus, a prototype of autoimmune diseases and sepsis, a serious infection that causes organ dysfunction, are inflammatory diseases in which the role of Fc receptors has been unraveled. These pathologies have high morbidity and mortality, imposing enormous costs for society. E. coli has been found to bind directly to the FcGammaRIII(CD16) receptor to evade the immune system. This is due to the binding to the wzxe protein present in its membrane, making this receptor an interesting therapeutic target. CD16 is an FcGammaR with ITAM that classically has an activating function and generates inflammatory responses when binding to immunocomplexes. However, in sepsis, direct binding with E. coli induces an ITAMi activation, which blocks ROS production and inhibits phagocytosis and death of this bacterium. The manipulation of this apparently anti-inflammatory inhibitory signaling (ITAMi) may be an effective strategy for the treatment of inflammatory diseases such as sepsis and lupus. Initially we aimed to evaluate the physiological importance and therapeutic role of the CD16 binding peptide, in a sepsis model and pristane-induced lupus model, respectively. Similarly to CD16, CD89 (FcAlphaRI) is capable of mediating dual, activating or inhibitory signaling, which depends on how it binds to immunoglobulins and immunocomplexes. Therefore, we assessed whether FcAlphaRI (CD89) could bind to bacteria directly, in the absence of cognate ligands and mediate pro or anti-inflammatory responses, protecting or not the host. Methods: C57Bl/6 mice, WT and CD16KO with pristane-induced lupus were treated with the peptide CYWGGTEGAC (IRG Bioscience, USA). Gene and protein expression of cytokines, as well as genes associated to interferon signatures were evaluated in the lungs of these animals. We also used a sepsis model through the intra-peritoneal injection of E. coli WT and E. coli mutant wzxe-/-, in which we evaluated mortality and production of cytokines. We performed in vitro experiments with murine BMM and BMDC, human phagocytes and bacteria. Expression of CD89 and cognate receptors was assessed by flow cytometry. Flow cytometry with imaging was employed for phagocytosis analysis. In vivo experiments were also performed on WT and transgenic mice: CD89tg, CD89R209Ltg CD89tgCD16KO, CD16KO and CRPKO. We compared the mortality, cytokine production, amount of bacteria and tissue injury in CLP and pneumonia by nasal administration of S. pneumoniae. ROS production by BMM was evaluated with confocal microscopy and, in PMN, by chemiluminescence. Immunoprecipitation and immunoblotting were used to evaluate recruitment of syk and SHP-1. We used ELISA for binding assays with CD89 and bacteria and quantification of TNF-Aphla, IL-1 and IL-6. Results: Mice injected with wzxe-/- mutant E. coli survived more and produced smaller amounts of cytokines, reinforcing the key role of the wzxe protein in the mechanism of immune evasion of E. coli. WT and CD16KO pristane induced lupus mice, treated or not with the peptide CYWGGTEGAC didn\'t show differences in gene or protein expression of cytokines nor in interferon signature genes in their lungs. The bacterial-CD89 interaction in murine macrophages induces cellular activation, phagocytosis and bacterial death, which are dependent on the FcRGamma chain. This same interaction protects against mortality in two models of sepsis (CLP and pneumonia) and is dependent on the FcRGamma chain and independent of PCR and IgA anti-bacterium. Conclusions: CD16 and CD89 are ITAM-bearing FcRs that present a duality in the form of activation through ITAM, which in some situations may be inhibitory (ITAMi). In this thesis we reinforce the key role of wzxe protein, a CD16 ligand, as responsible for the bacterial evasion of E. coli through ITAMi signaling. On the other hand, we failed to demonstrate differences after treatment of pristane-induced lupus mice using the CYWGGTEGAC peptide. We believe that this was due to inadequate dose of the peptide or proteolysis by endogenous mouse enzymes, so the peptide is injected. New doses or manipulation of the peptide structure are future prospects for this project. As to CD89, we proved the extremely important role of CD89 in innate immunity. That receptor, similarly to CD16, was able to bind directly to bacteria, in the absence of opsonins and cognate ligands. CD89 was protective for both gram-positive and gram-negative infection while the CD16 was protective only in a model of gram-positive pneumonia