Produzida por macrófagos e células dendríticas, a IL-27 é uma citocina heterodimérica capaz de induzir células Tr1 produtoras de IL-10 e consequentemente regular linfócitos Th1, Th2 e Th17, dependendo da doença envolvida. Partindo-se do pressuposto de que a infecção causada por Trypanosoma cruzi normalmente induz miocardite refletida pela migração intensa de linfócitos Th1 para o tecido cardíaco, nós analisamos o papel regulador da IL-27 nesta condição inflamatória. Nós inicialmente verificamos que a IL-27 foi prontamente induzida in vitro em células infectadas com T. cruzi. Para gerar miocardite intensa coordenada por linfócitos Th1, nós polarizamos linfócitos T naïves para o padrão Th1 na ausência de moléculas relacionadas ao perfil Th17 (camundongos IL-17R-/-, IL-23-/- e IL-6-/-). Como esperado, a inflamação cardíaca intensa e o dano tecidual foram observados na ausência das moléculas do padrão Th17, o que contribuiu para a morte prematura dos animais IL-17R-/-, IL-23-/- e IL-6-/-, precisa e notoriamente pela indução da migração excessiva de linfócitos Th1 para o tecido cardíaco via CXCL-9 e CXCL-10. Para explorar os mecanismos pelos quais a IL-27 controla a miocardite induzida pelo T. cruzi, nós encontramos um recrutamento substancial de macrófagos produtores de IL-27 para o tecido cardíaco, o qual foi mediado pelas quimiocinas CCL3 e CCL4 na ausência de moléculas do padrão Th17. Para determinar quais os receptores necessários para a produção de IL-27, nós observamos que macrófagos derivados da medula óssea de camundongos deficientes de TLR4-/-, TLR9-/- e NLRP3-/- aboliram completamente a produção desta citocina após a infecção in vitro com T. cruzi, enquanto o receptor TLR2 foi dispensável. Nós também verificamos que macrófagos produtores de IL-27 suprimiram linfócitos Th1 através da indução de células Tr1 produtoras de IL-10 após a infecção com T. cruzi. Em seguida, nós avaliamos se a IL-27 foi correlacionada com a proteção cardíaca durante a doença de Chagas. Nós observamos níveis séricos elevados de IL-27 tanto em pacientes com a forma clínica indeterminada ou cardíaca leve, enquanto pacientes com cardiomiopatia moderada ou grave produziram níveis reduzidos de IL-27. Neste estudo, nós descrevemos um novo mecanismo regulador desempenhado por macrófagos produtores de IL-27 no controle da miocardite induzida por T. cruzi. Macrófagos produtores de IL-27 podem suprimir processos inflamatórios desencadeados por linfócitos Th1, os principais vilões na doença de Chagas. / IL-27 is a heterodimeric cytokine produced by macrophages and dendritic cells known to induce IL-10-producing Tr1 cells and to regulate Th1, Th2, and Th17 lymphocytes, depending on the underlying disease. Because the infection caused by Trypanosoma cruzi normally induces myocarditis mirrored by an outstanding migration of Th1 cells to the heart tissue, we analyzed the regulatory role of IL-27 in this inflammatory condition. We firstly verified that IL-27 was promptly induced by in vitro T. cruzi-infected spleen cells. To generate a robust myocarditis coordinated by Th1 lymphocytes, we polarized lymphocytes to a Th1 pattern by infecting mice in the absence of Th17-related molecules (IL-17R-/-, IL-23-/-, and IL-6-/- mice). As expected, an impressive cardiac inflammation and damage was observed in the absence of Th17-related molecules, leading IL-17R-/-, IL-23-/-, and IL-6-/- mice to the premature death, precisely and notably by inducing an exuberant Th1 migration to the heart tissue via CXCL9 and CXCL10 chemokines. To explore the mechanisms by which IL-27 controls T. cruzi-induced myocarditis, we found a striking recruitment of IL-27-producing macrophages to the heart tissue mediated by increased levels of CCL3 and CCL4 chemokines in the absence of Th17-associated molecules. To gain further insights into the receptors required to IL-27 production, we observed that bone marrow-derived macrophages from TLR4-/-, TLR9-/-, and NLRP3-/- mice completely abolished IL-27 production after in vitro T. cruzi infection, while TLR2 was dispensable. We also verified that IL-27-producing macrophages supressed Th1 lymphocytes by inducing IL-10-producing Tr1 cells after T. cruzi infection. We next assessed whether IL-27 was correlated to cardiac protection during Chagas Disease. We observed augmented serum levels of IL-27 in either patients with indeterminate (asymptomatic) form or mild cardiac form, whereas patients with moderate or severe cardiomyopathy were poor producers of IL-27. Here, we described a novel regulatory mechanism developed by IL-27-producing macrophages in the control of T. cruzi-induced myocarditis. IL-27-producing macrophages can suppress inflammatory processes caused by Th1 lymphocytes, the bona fide culprits of Chagas Disease.

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Contudo, estudos clínicos e epidemiológicos demonstram uma associação entre as duas patologias, através da observação de que indivíduos obesos asmáticos representam um fenótipo clínico distinto da asma alérgica clássica, apresentando aumento na gravidade dos sintomas e resistência a terapias convencionais. Entretanto, os mecanismos imunológicos envolvidos na associação obesidade e asma não estão esclarecidos, devido à escassez de estudos e a uma heterogeneidade nos dados encontrados em modelos experimentais. Portanto, o objetivo do presente estudo foi avaliar a influência da obesidade sobre a inflamação alérgica pulmonar. Para isso, a obesidade foi induzida por dieta com alto teor de gordura durante dez semanas nos animais dos grupos OB e OB/AP, enquanto os animais dos grupos CN e AP foram alimentados com a dieta padrão. Da sexta a décima semana do protocolo de indução da obesidade, os animais dos grupos AP e OB/AP foram submetidos a subsequentes sensibilizações e desafios com a ovalbumina. As análises foram realizadas em 24 e 48 horas após o último desafio com a OVA. Os resultados demonstraram que após os desafios com o alérgeno, os animais do grupo AP apresentaram características marcantes da resposta imune alérgica, com elevado número de eosinófilos no LBA, no tecido pulmonar e na medula óssea, correlacionando com os níveis elevados de CCL11 e peroxidase eosinofílica, além de citocinas de eperfil Th2 como IL-4, IL-5, IL-9, IL-13, IL-25, IL-33 e TSLP e de IgE sérica anti-OVA. Contudo, foi observado em 48 horas um declíneo na resposta de perfil Th2 nos animais deste grupo. Já os animais do grupo OB/AP apresentaram em 24 horas, um menor número de eosinófilos no lavado broncoalveolar, no tecido pulmonar e na medula óssea, associado a menores níveis de CCL11, EPO e de IL-4, IL-5, TSLP e IL-25 assim como de IgE sérica anti-OVA. Em 48 horas, as análises de citocinas no grupo OB/AP demonstraram um aumento nos níveis de IL-1β, IL-4, IL-6, IL-9, IL-12, IL-13, IL-17A, TNF-α e IFN- associado ao maior influxo de macrófagos M1. Surpreendentemente, em 48 horas após o último desafio com a OVA, houve um aumento significativo de neutrófilos na medula óssea e de mieloperoxidase no tecido pulmonar. Paralelamente, os animais do grupo OB/AP, apresentaram um número maior de mastócitos e células caliciformes em ambos os tempos analisados, quando comparado aos animais do grupo AP. Conclusão: Somados estes resultados sugerem que a obesidade desenvolvida em camundongos BALB/c, foi capaz de influenciar a resposta imune no pulmão dos animais após as sensibilizações e os desafios com alérgeno, interferindo no desenvolvimento da resposta imune Th2 clássica e acarretando um atraso no desenvolvimento da resposta imune inflamatória. Adicionalmente, os animais obesos asmáticos apresentaram exacerbada resposta imune Th2, Th9 e altos níveis de IL-17A associada a um maior influxo de neutrófilos para o pulmão e a uma intensa produção de muco, sugerindo que estes animais apresentaram um perfil inflamatório mais grave de alergia pulmonar. / Asthma and obesity are chronic inflammatory diseases with opposite immune profiles. Although, clinical and epidemiological studies reveal the association between them, as obese asthmatic individuals represent a distinct phenotype from the classic allergic asthma. However, the immune mechanisms involved in this association are not established yet, due to the lack of studies and the heterogeneity of the data obtained in experimental models. Therefore, the present study aimed to evaluate the influence of obesity over the immune response pulmonary allergic. Female Balb/c mice were fed with high fat diet during ten weeks so as to induce obesity. From the sixth to the tenth week of the protocol, PA and PA/OB groups were sensitized and challenged with ovalbumin. The following analyses were performed 24 and 48 hours after the last OVA challenge. Striking features of the allergic immune response were observed in the PA group, as elevated eosinophil count in BAL, lung tissue bone marrow, in association with high IL-4, IL-5, IL-9, IL-13, IL-25, IL-33, TSLP and anti-OVA IgE levels. There was also elevated production of CCL11 and EPO correlated with the eosinophilia. In contrast, IL-4, IL-5, TSLP and IL-25 levels were diminished in PA/OB group. In association with the reduced eosinophil count, low levels of CCL11, EPO and Anti-OVA IgE were detected. However, 48 hours after the last challenge, IL-1β, IL-4, IL-6, IL-9, IL-12, IL-13, IL-17A, TNF-α and IFN- level were higher in the PA/OB, the was also an increased M1 macrophage influx. There was also more neutrophils in the bone marrow and MPO in the lung tissue, indicating their increased influx to the lung of PA/OB animals. Mast cells and goblet cells count was increased in this group, 24 and 48 hours after the last challenge. Taken together these results suggest that obesity developed in BALB/c mice was able to influence the immune response in the lungs of animals after sensitization and challenge with allergen, interfering with the immune response classical Th2 and causing a delay in the development inflammatory immune response. Additionally, asthmatic obese animals showed exaggerated Th2 immune response, Th9 and high IL-17A levels associated with an increased influx of neutrophils into the lung and an intense mucus production, suggesting that these animals showed an allergy more severe inflammatory profile lung.

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

L’asthme est une maladie multifactorielle hétérogène qui engendre une inflammation pulmonaire associée à une variété de manifestations cliniques, dont des difficultés respiratoires graves. Globalement, l’asthme touche environ une personne sur 6 et présente actuellement un sérieux problème de santé publique. Bien que de nombreux traitements soient disponibles pour soulager les symptômes de la maladie, aucun traitement curatif n’est actuellement disponible. La compréhension des mécanismes qui régissent l’état inflammatoire au cours de la maladie est primordiale à la découverte de nouvelles cibles thérapeutiques efficaces. Les cellules dendritiques captent les allergènes dans les poumons et migrent vers les ganglions drainants pour les présenter aux cellules T et engendrer la réponse inflammatoire pathogénique chez les asthmatiques. Nous avons contribué à l’avancement des connaissances mécanistiques de l’asthme en identifiant chez la souris la sous-population de cellules dendritiques responsable de l’initiation et du maintien de la réponse inflammatoire locale et systémique associée à l’asthme. En effet, nous avons démontré que le SIRPα, récepteur extracellulaire impliqué dans la régulation de la réponse immune, est sélectivement exprimé à la surface des cellules dendritiques immunogéniques. L’interruption de la liaison entre le SIRPα et son ligand, le CD47, interfère avec la migration des cellules dendritiques SIRPα+ et renverse la réponse inflammatoire allergique. Ce mécanisme constitue une avenue thérapeutique prometteuse. D’ailleurs, les molécules de fusion CD47-Fc et SIRPα-Fc se sont avérées efficaces pour inhiber l’asthme allergique dans le modèle murin. Nous avons également démontré l’implication des cellules dendritiques SIRPα dans un modèle d’inflammation pulmonaire sévère. L’administration répétée de ces cellules, localement par la voie intra-trachéale et systémiquement par la voie intra-veineuse, mène au développement d’une réponse inflammatoire mixte, de type Th2-Th17, similaire à celle observée chez les patients atteints d’asthme sévère. La présence de cellules T exprimant à la fois l’IL-17, l’IL-4, l’IL-13 et le GATA3 a été mise en évidence pour la première fois in vitro et in vivo dans les poumons et les ganglions médiastinaux grâce à ce modèle. Nos expériences suggèrent que ces cellules Th2-Th17 exploitent la plasticité des cellules T et sont générées à partir de la conversion de cellules Th17 qui acquièrent un phénotype Th2, et non l’inverse. Ces résultats approfondissent la compréhension des mécanismes impliqués dans l’initiation et le maintien de l’asthme allergique et non allergique, en plus d’ouvrir la voie à l’élaboration d’un traitement spécifique pour les patients asthmatiques, particulièrement ceux pour qui aucun traitement efficace n’est actuellement disponible. / Asthma is a heterogeneous multifactorial disease resulting in airway inflammation associated with a variety of clinical manifestations, which include severe breathing difficulties. Asthma affects approximately one out of six people and is currently a serious public health problem. As of now, many treatments are available to relieve the symptoms of the disease, but no definitive cure is available. Understanding the mechanisms that regulate the inflammatory condition during the disease is essential to the discovery of effective new therapeutic targets. Dendritic cells capture allergens in the lungs, migrate to the draining lymph nodes where they activate cognate T cells, which cause the pathogenic inflammatory response. My work help defined and deepened the mechanistic understanding of asthma by identifying the subpopulation of dendritic cells responsible for the initiation and maintenance of local and systemic inflammatory response. We demonstrated that SIRPα is selectively expressed on the surface of immunogenic dendritic cells. Indeed, the interruption of the ligation between SIRPα and its ligand, CD47, interferes with the migration of SIRPα+ dendritic cells and reverses the allergic inflammatory response. This mechanism is a promising new therapeutic avenue. Moreover, we showed that the soluble fusion molecules CD47-Fc and SIRPα-Fc are potent inhibitors of the allergic asthma in a mouse model. In addition, we demonstrated the involvement of SIRPα+ dendritic cells in a model of severe airway inflammation induced upon local and systemic repeated administration of those cells. Either treatment led to the development of a mixed Th2-Th17 inflammation, a phenotype recently described in patients with severe asthma. This model allowed us to show the presence of T cells expressing at once IL-17, IL-4, IL-13 and GATA3 in vitro and in vivo in the lungs and in the mediastinal lymph nodes. Our results suggest that these Th2-Th17 cells are generated from the conversion of Th17 cells acquiring a Th2 phenotype, and not the other way around, a hallmark of Th17 cells plasticity. These results deepen the understanding of the mechanisms involved in the initiation and maintenance of allergic and non-allergic asthma. Besides, we open a way to the development of a specific treatment for asthmatic patients, particularly those for whom no effective treatment is currently available.

La maladie de Crohn (Crohn’s disease; CD) et la colite ulcéreuse (Ulcerative colitis ;CU) représentent deux formes distinctes de maladies inflammatoires chroniques de l’intestin (MICI), qui sont associées à une réponse immunitaire aberrante des tissus intestinaux à la flore intestinale. Les phagocytes mononucléés (MNPs) qui dialoguent, via les cytokines qu'ils produisent, avec les cellules immunitaires innées et adaptatives sont impliqués dans l’induction, la perpétuation et le maintien de la réponse inflammatoire des MICI. Chez la souris, les MNPs sont stratifiées en cellules dendritiques conventionnelles (cDCs), macrophages (M) et cellules dérivées de monocytes, une entité qui regroupe dans le tissu des cellules dendritiques dérivées de monocytes (Mo-DC), des M dérivés de monocytes et des « monocyte-like ». Toutefois, la diversité phénotypique, moléculaire et fonctionnelle des monocytes et des MNPs ainsi que la plasticité des monocytes restent à élucider dans les MICI. Les anticorps bloquant les cytokines IL12 et IL23 contrôlent la pathogénicité et la plasticité des cellules Th17, réduisant l'inflammation intestinale chez les patients atteints de MII. Cependant, il n’existe à ce jour aucun traitement curatif. L’étude approfondie de la plasticité des cellules T et du site tissulaire où elle pourrait se produire ne sont toujours pas clarifiés. Dans le premier chapitre, nous avons révélé l’existence de deux sous-populations distinctes de CD14+MNPs dans le colon de patients atteints de CU. Les cellules de type « inflammatory monocyte-like » CD14+CD163-CD64+ (P3) à l’opposé des CD14+CD163+CD64+ M (P4) s'accumulent dans le côlon inflammatoire. Nos résultats ont de plus établi un lien entre les P3 MNPs, l’IL12, l’IL1β et la détection de cellules Th17 mémoires produisant de l'IFN et de l'IL8, qui contribueraient collectivement à la pathogenèse de la CU. De plus, deux sous-populations CD14+ MNPs similaires sur le plan fonctionnel et moléculaire a ceux trouvés en CU, ont été détectées dans le côlon de patients atteints de CD. En revanche, dans le deuxième chapitre, nous fournissons des évidences que la sous-population monocytaire Slan+ pourrait contribuer à l’immunopathogenèse de la CD, mais pas à celle de la CU. La fréquence, le phénotype et la fonction des cellules Slan+ ont été examinés dans le sang, les ganglions mésentériques (MLN) et le côlon de patients atteints de MICI. Nous proposons que les cellules pro-inflammatoires CD14hiCD172α+Slan+ discriminent les tissus de CD et CU. En effet, elles ne s'accumulent que dans les MLNs et la muqueuse colique des patients atteints de CD. Dans le troisième chapitre, nous avons montré que les MLNs de CD et de CU, qui sont des tissus difficiles d'accès pour leur étude fonctionnelle en recherche, peuvent également être distingués par la distribution et le profil moléculaire des cellules T mémoire effectrices CXCR3-CCR6+ (Th17TEM). Nos données suggèrent également que la plasticité de Th17 se produit dans les MLNs avant leur migration vers l'intestin. Cette étude pourrait avoir des implications pour améliorer notre compréhension de la maladie. Enfin, il a été démontré qu’à l’homéostasie chez la souris, les monocytes sont continuellement recrutés dans la muqueuse intestinale où ils se différencient progressivement en M anti-inflammatoires. Ce processus de maturation est interrompu dans le contexte d'une inflammation. Les signaux environnementaux qui régulent la « cascade » de maturation d’un monocyte classique tissulaire demeurent inconnus chez l'homme. Dans le quatrième chapitre, nous avons récapitulé in vitro la cascade de différenciation des monocytes humains de «CD163- P3-like» en «CD163+P4-like» et avons montré leurs similitudes moléculaires avec les CD14+ MNP tissulaires. La manipulation de cette voie de différentiation pourrait ouvrir des pistes thérapeutiques pour restaurer l'homéostasie intestinale dans les MICI. En conclusion, une meilleure compréhension des sous-populations de MNPs, leurs fonction et plasticité dans la pathogenèse des MICI aidera à identifier des nouvelles cibles thérapeutiques et contribuera à augmenter les connaissances pour la mise au point de traitements personnalisés. / Crohn’s disease (CD) and ulcerative colitis (UC), the two forms of inflammatory bowel diseases (IBD), are associated with dysregulated immune response in the intestinal tissue. It is mediated by mononuclear phagocytes (MNPs) that dialogue via the cytokine they produce with innate and adaptive immune cells. In mice, MNPs are stratified into conventional dendritic cells (DCs), macrophages (M) and monocyte-derived cells that regroup tissue monocyte-derived DCs, monocyte-derived M and monocytes-like cells. However, the phenotypic, molecular and functional diversity of MNPs and their plasticity remain to be elucidated in IBD patients. Therapies in IBD employ antibodies that block IL12 and IL23, thus control Th17 pathogenicity and plasticity and decrease intestinal inflammation. However, no cure exist nowadays for the treatment of IBD. In-depth study of T cell plasticity and the tissue where it occurs remain to be investigated. In the first chapter, we revealed the existence of two distinct CD14+ MNP subsets in colon of UC patients. Only, CD163-CD64+ inflammatory monocyte-like cells (P3) but not anti-inflammatory CD163+CD64+ M (P4) accumulate in inflamed UC colon. Our findings further established a link between monocyte-like CD14hiCD172α+ CD163- MNPs, IL12, IL1β and the detection of colonic memory Th17 cells that produce IFN and IL8, which might all contribute to UC pathogenesis. Two CD14+ MNP subsets, resembling their counterparts in UC mucosa at the functional and molecular level, were also detected in CD colon. In contrast, in the second chapter, we provide evidence that Slan+ monocyte subset may contribute to CD but not UC immunopathogenesis. Frequency, phenotype, and function of Slan+ cells were examined in blood, colon, and mesenteric lymph nodes (MLN) of patients with IBD. We showed that pro-inflammatory CD14hiCD172α+Slan+ cells are a distinguishing feature between CD and UC, as they only accumulate in MLNs and colonic mucosa of CD patients. In the third chapter, we showed that MLNs of CD and UC, tissues that were hard to access for research use, can also be distinguished by frequencies of CXCR3−CCR6+ Th17 effector memory T cells (TEM) and their molecular profile. Our data further suggested that Th17 plasticity is taking place in MLN, before T cell homing to gut tissues. This investigation has clear implications in furthering our understanding of the disease. Finally, it has been demonstrated that monocytes are continuously recruited into murine gut mucosa and progressively differentiate into macrophages under homeostatic conditions, a maturation process interrupted in the context of inflammation. However, the environmental cues that regulate tissue inflammatory monocyte “waterfall” remain to be investigated in humans. In the fourth chapter we recapitulated in vitro human monocyte differentiation cascade, from CD163- inflammatory monocyte-like cells (P3) towards anti-inflammatory CD163+ macrophages (P4) and showed their molecular similarities to tissue CD14+ MNPs. Manipulating this pathway might open therapeutic avenues to restore tissue homeostasis. In conclusion, a better understanding of MNP subsets, function and plasticity in IBD pathogenesis would help identify novel therapeutic targets and shed light for the development of personalized treatments.

Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome cansignificantly vary among patients within the same stage. Data collected from largecohorts of human cancers has demonstrated the impact of immune-classification, which has a prognostic value that may add largely to the significance of the AJCC/UICC TNM-classification. In our study we examined the immune cells that infiltrated the tumor tissues of colorectal and ovarian cancer patients. In a cohort of newly diagnosed colorectal cancer patients we examined the correlations between the KRAS mutational status, patterns of tumor-infiltrating immune cells and the presence of tumor recurrence. Our data suggest that colorectal cancer patients with low levels of tumor-infiltrating lymphocytes, a high CD1a/DC-LAMP tumor-infiltrating dendritic cells ratio, and a KRAS mutation in codon 13 are at a high risk of disease recurrence. In ovarian cancer patients we focused on the dynamics of the tumor-infiltrating...

L'épilepsie est un trouble chronique du système nerveux central qui touche 70 millions de personnes dans le monde. Un tiers des patients souffrent d'épilepsie réfractaire (ER), caractérisée par des crises récurrentes malgré l’utilisation de médicaments antiépileptiques (MAE) appropriés. Plusieurs données soutiennent un lien biologique entre la neuroinflammation et l'épilepsie chronique. De plus, des études suggèrent que certains MAE de nouvelle génération comme le brivaracétam présentent des propriétés immunomodulatrices qui pourraient contribuer à leur bénéfice dans l’ER et d’autres pathologies neurologiques. Les objectifs de cette thèse étaient tout d’abord de caractériser ex vivo le profil des cellules immunitaires périphériques, les niveaux de cytokines pro- et anti-inflammatoires et les biomarqueurs neurodégénératifs dans le sang périphérique de sujets souffrant d’épilepsie (ER vs. bien contrôlée) en comparaison à celui de donneurs sains. Par la suite, nous avons évalué l’impact du brivaracétam sur l’activation des cellules immunitaires humaines in vitro et son potentiel immunomodulateur et neuroprotecteur in vivo dans le modèle de l'encéphalomyélite auto-immune expérimentale (EAE) active, un modèle animal de la sclérose en plaques, la pathologie inflammatoire du SNC la plus fréquente. Méthodologie : Après isolation des cellules mononucléées et du sérum à partir du sang périphérique d'adultes souffrant d'épilepsie focale et de donneurs sains, nous avons effectué des analyses par cytométrie en flux ex vivo, ELISA multiplex et technique ultrasensible de détection de biomarqueurs par technologie single molecule array (SIMOA). Nous avons comparé l'influence du brivaracétam et du lacosamide sur l'activation des cellules immunitaires périphériques humaines in vitro et in vivo dans l'EAE active induite par immunisation avec le MOG, le modèle animal le plus commun de la sclérose en plaques. Résultats : Nous avons rapporté une augmentation de la proportion des lymphocytes T CD4 dans le sang périphérique des adultes souffrant d’ER, avec une fréquence plus élevée de lymphocytes pro-inflammatoires Th17/Th1 en comparaison avec les contrôles. Nous avons également rapporté des niveaux significativement plus élevés du marqueur de lésion neuronale sNfL chez les sujets plus âgés avec l’ER par rapport aux témoins appariés pour l’âge. De plus, nous avons montré que l'administration prophylactique du brivaracétam ou du lacosamide ne retardait pas l'apparition de l'EAE mais était associée à une évolution clinique significativement moins sévère dans la phase chronique de l'EAE active chez les souris femelles C57BL/6 par rapport au contrôle (véhicule). Conclusion : Dans l'ensemble, nos données soutiennent d'une part que l’ER est associée à un profil immunitaire pro-inflammatoire des lymphocytes Th17/Th1 dans le sang périphérique et à des niveaux pathologiques de sNfL, soutenant la présence de composantes inflammatoire et neurodégénérative potentielles dans l’ER. D'autre part, les MAE de nouvelle génération (brivaracétam et lacosamide) n'altèrent pas de façon importante la réponse à l'immunisation avec le peptide MOG, mais améliorent l'évolution de l'EAE par le biais principalement de mécanismes neuroprotecteurs. / Epilepsy is a chronic central nervous system condition affecting 70 million people around the world. One third of patients suffer from drug-resistant epilepsy (DRE), characterized by recurrent seizures despite appropriate trials of anti-epileptic drugs (AEDs). Accumulating data support a biological link between neuroinflammation and chronic epilepsy. Recent studies suggest that some of the new generation AEDs, like brivaracetam, can display immunomodulatory properties that could contribute to their beneficial impact in DRE and other neurological disease. We aimed to investigate differences in immune cell populations, cytokines, and neurodegenerative biomarkers in the peripheral blood of subjects with epilepsy (DRE vs. well-controlled) compared to healthy controls and to assess the impact of brivaracétam on activation of human immune cells in vitro and its immunomodulatory and neuroprotective potential in vivo in the active experimental autoimmune encephalomyelitis (EAE), a common model of the prototypical CNS neuroinflammatory disease multiple sclerosis. Methodology: Using peripheral blood mononuclear cells and serum isolated from the peripheral blood of adults suffering from focal onset epilepsy and healthy donors, we performed flow cytometry analysis ex vivo, multiplex immunoassays, and ultrasensitive single molecule array. We compared the influence of brivaracetam and lacosamide on activation of human peripheral immune cells in vitro and in vivo in MOG-induced active EAE, the most common animal model of multiple sclerosis. Results: We observed an increased proportion of CD4 T cells in the peripheral blood compartment of adults suffering from DRE, with a higher frequency of proinflammatory Th17/Th1 cells compared to controls. We also reported significantly higher levels of the marker of neuronal injury sNfL in aging subjects with DRE compared to age-matched controls. Furthermore, we showed that prophylactic administration of brivaracetam or lacosamide did not delay EAE onset but was associated with a significantly less severe clinical course in the chronic phase of active EAE in C57BL/6 female mice compared to control (vehicle).Conclusions: Taken together, our data support that DRE is associated with a proinflammatory Th17/Th1 CD4 T cell immune profile in peripheral blood and pathological levels of sNfL, supporting both potential inflammatory and neurodegenerative components in DRE. On the other hand, the novel generation AEDs (brivaracetam and lacosamide) do not impair the response to immunization with MOG peptide but improve the course of EAE through mostly neuroprotective mechanisms.

<p>CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.</p><p>microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases. </p><p>To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis. </p><p>A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.</p><p>Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.</p> / Dissertation

Une série d’études récentes chez l’homme et dans les modèles murins a conduit à la mise en évidence d’une réponse immune, qui met en jeu des cellules de l’immunité innée et de l’immunité adaptative dans le tissu adipeux (TA) obèse. Nous avons mis en évidence un dialogue pro-inflammatoire entre les macrophages et les LT CD4+ dans le TA humain obèse mettant en jeu l’IL-1β, l’IL-17 et l’IL-22. Le pourcentage de Th17 est positivement corrélé au % d’HbA1c. De plus, la sécrétion des cytokines impliquées dans cette boucle pro-inflammatoire diminuent après une perte de poids induite par la chirurgie bariatrique. Par la suite, nous avons montré que l’IL-1β et l’IL-17 induisent des programmes transcriptionnels pro-inflammatoires concordant dans trois types de cellules non-immunitaires du TA, les pré-adipocytes, les cellules endothéliales et les adipocytes, et diminuent les gènes du métabolisme dans les adipocytes. Les effets d’IL-1β sont nettement plus prononcés que ceux de l’IL-17. Le milieu conditionné de cellules immunitaires CD45+ reproduit les réponses pro-inflammatoire et catabolique induites par les cytokines recombinantes dans les adipocytes, et ces réponses sont inhibées après la neutralisation de l’IL-17 et l’IL-1β. Ces résultats démontrent une implication pathologique de l'IL-1β et de l'IL-17 dans les altérations du TA induites par l'obésité. / A series of recent studies in humans and mouse models has led to the detection of an immune response, which involves cells of the innate immunity and adaptive immunity in obese adipose tissue (AT).We highlighted a proinflammatory crosstalk between macrophages and CD4+ T cells in obese human AT involving IL-1β, IL-17 and IL-22. Percentage of Th17 is positively correlated with HbA1c %. Moreover, the secretion of the cytokines implicated in this proinflammatory loop decreased after weight loss induced by bariatric surgery.Subsequently, we have shown that IL-1β and IL-17 induce proinflammatory transcriptional programs in three types of non-immune cells of the TA, preadipocytes, endothelial cells and adipocytes , and decrease metabolism genes in adipocytes. IL-1β effects are much more pronounced than those of IL-17. The conditioned medium of CD45+ immune cells reproduces the pro-inflammatory and catabolic responses induced by the recombinant cytokines in adipocytes, and these responses are inhibited after the neutralization of IL-17 and IL-1β.These results demonstrate a pathological involvement of IL-1β and IL-17 in the AT dysfunction induced by obesity.

Pathogenesis of autoimmune and auto-inflammatory diseases is induced by auto-aggressive helper T (Th) cells (i.e. Th1 and Th17 cells), and can be controlled by regulatory T cells (Tregs) characterized by expression of the transcription factor Foxp3. Thus, development of autoimmunity is regulated by the balance of Tregs and Th1/Th17 cells. Prostaglandin E₂ (PGE₂) is a bioactive lipid mediator with immune-modulatory potential that acts through 4 receptors (EP1-4). It has been shown that PGE₂ facilitates Th1 and Th17 cell development and expansion, therefore promoting autoimmune inflammation. However, the role of PGE₂ in Treg development and function is largely unclear. The aim of this PhD was to test the hypothesis that PGE₂ regulates Treg development, function and subsequent immune response. I observed that in vivo inhibition of endogenous PGE₂ biosynthesis using a COX inhibitor resulted in increased Foxp3+ Tregs in various lymphoid organs. This response was prevented by addition of an EP4 agonist. PGE₂-EP4 signalling particularly inhibits RORγt+ Tregs in the intestine. This was not observed in either antibiotic-treated mice or MyD88/TRIF double-knockout mice, suggesting gut commensal microbiota involvement. In addition, PGE₂ has a role in microbiota-dependent regulation of intestinal CD11c+MHCII+CD11b+CD103- mononuclear phagocytes (MNPs) which drive intestinal Treg expansion through production of type 1 interferons. Consistent with these in vivo observations, gut microbial metabolites from indomethacin treated mice enhanced in vitro RORγt+ Treg differentiation in the dendritic cell- T cell co-culture system. Adoptive transfer of caecal microbiota from COX inhibitor- treated mice into naïve mice also provided protective benefits in a chemical (DSS)-induced colitis disease model. In summary, this work has demonstrated that PGE₂ affects intestinal Tregs, indicating a novel mechanism for interaction of PGE₂, the adaptive immune system and the gut microbiota in homeostasis within this environment. These findings increase our understanding of the role of PGE₂ in development of inflammatory bowel disease and offer potential therapeutic strategies for treating this disease.