Células produtoras de IFN-? e IL-17A são envolvidas na proteção contra infecção por Leishmania infantum (L. infantum). Ainda não está claro como o sistema imune coordena, ou o parasito manipula, o balanço entre Th1 e Th17 durante a leishmaniose visceral (LV). Utilizando RNAseq, PCR array e citometria de fluxo, nós demonstramos que, enquanto o padrão Th1 é altamente induzido, o perfil Th17 é inibido durante a infecção por L. infantum, e que as células B compõem uma fonte importante de IL-17A nesse modelo. Usando animais Nod2-/- e Rip2-/- nós caracterizamos essa via como um regulador negativo de células Th17 na LV. Por outro lado, a potente indução de Th1 foi dependente da sinalização via NOD2-RIP2 em células dendríticas CD8??+XCR1+, o que foi crucial para produção de IL-12 através da fosforilação de p38 e JNK. Como consequência, camundongos Nod2-/- e Rip2-/- tiveram defeito na resposta Th1, aumento de Th17, e maiores cargas parasitárias comparado com camundongos WT. Juntos, os dados mostram que a via de NOD2-RIP2 desempenha um papel importante na modulação da resposta imune adaptativa e promove proteção contra LV causada por L. infantum / IFN-? and IL-17A-producing cells are described to be related to protection against Leishmania infantum (L. infantum) infection. How the immune system coordinates, or the parasite manipulates, the balance between Th1 and Th17 during visceral leishmaniasis (VL) is still unknown. We showed here that Th17 is suppressed during L. infantum infection and B cells are an important source of IL-17A in this model. By using Nod2-/- and Rip2-/- mice we characterized this pathway as a negative regulator for Th17 cells in VL. On the other hand, the high level of Th1 induction was dependent on the NOD2-RIP2 signaling in CD8?+XCR1+ dendritic cells (DCs), which was crucial for IL-12 production through the phosphorylation of p38 and JNK. As a consequence, Nod2-/- and Rip2-/- mice showed a Th1 defective response, more Th17, and higher parasite loads compared to WT mice. Together, the data demonstrate that NOD2-RIP2 signaling pathway plays a role in shaping adaptive immunity and promotes protection against VL caused by L. infantum

Células produtoras de IFN-? e IL-17A são envolvidas na proteção contra infecção por Leishmania infantum (L. infantum). Ainda não está claro como o sistema imune coordena, ou o parasito manipula, o balanço entre Th1 e Th17 durante a leishmaniose visceral (LV). Utilizando RNAseq, PCR array e citometria de fluxo, nós demonstramos que, enquanto o padrão Th1 é altamente induzido, o perfil Th17 é inibido durante a infecção por L. infantum, e que as células B compõem uma fonte importante de IL-17A nesse modelo. Usando animais Nod2-/- e Rip2-/- nós caracterizamos essa via como um regulador negativo de células Th17 na LV. Por outro lado, a potente indução de Th1 foi dependente da sinalização via NOD2-RIP2 em células dendríticas CD8??+XCR1+, o que foi crucial para produção de IL-12 através da fosforilação de p38 e JNK. Como consequência, camundongos Nod2-/- e Rip2-/- tiveram defeito na resposta Th1, aumento de Th17, e maiores cargas parasitárias comparado com camundongos WT. Juntos, os dados mostram que a via de NOD2-RIP2 desempenha um papel importante na modulação da resposta imune adaptativa e promove proteção contra LV causada por L. infantum / IFN-? and IL-17A-producing cells are described to be related to protection against Leishmania infantum (L. infantum) infection. How the immune system coordinates, or the parasite manipulates, the balance between Th1 and Th17 during visceral leishmaniasis (VL) is still unknown. We showed here that Th17 is suppressed during L. infantum infection and B cells are an important source of IL-17A in this model. By using Nod2-/- and Rip2-/- mice we characterized this pathway as a negative regulator for Th17 cells in VL. On the other hand, the high level of Th1 induction was dependent on the NOD2-RIP2 signaling in CD8?+XCR1+ dendritic cells (DCs), which was crucial for IL-12 production through the phosphorylation of p38 and JNK. As a consequence, Nod2-/- and Rip2-/- mice showed a Th1 defective response, more Th17, and higher parasite loads compared to WT mice. Together, the data demonstrate that NOD2-RIP2 signaling pathway plays a role in shaping adaptive immunity and promotes protection against VL caused by L. infantum

Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIP2) is a kinase which modulates signaling downstream of the bacterial peptidoglycan sensors NOD1 and NOD2. It is known that activation of RIP2 by engaging NOD receptors increases the production of pro-inflammatory cytokine and lipid mediators. We have some data indicating that RIP2 may also be involved in specialized pro-resolution lipid mediator (SPM) production. However, the molecular mechanisms by which RIP2 is involved in lipid mediator biosynthesis, are currently unknown. Understanding this process may have significant implications for RIP2-targeted therapies, which may not only inhibit pro-inflammatory cytokine and lipid mediator production but may also disrupt SPM production and resolution programs. This thesis aims to demonstrate that RIP2 is involved in promoting the activation of ALOX5 in a transient overexpression setting but also in an endogenous setting using relevant bacterial stimuli. These aims were accomplished through the optimization of a fluorescent assay to assess ALOX5 enzymatic activity, by optimization of ALOX5 enzyme purification and through molecular cloning of ALOX5 into a retroviral vector followed by viral transduction of the THP-1 human monocytic cell line. We find that co-expression of RIP2 with ALOX5 significantly enhances the enzymatic activity of ALOX5. We have successfully cloned NTAP-tagged ALOX5 into the pBABE retroviral vector and are currently selecting transduced cells so that we might test if this effect also occurs endogenously. Understanding the mechanisms underlying the production and regulation of SPMs would provide greater insight into potential new therapeutic approaches to promote resolution in chronic inflammatory diseases.

NOD1 est une protéine de surveillance intracellulaire jouant un rôle crucial dans la défense immunitaire innée. Suite à la reconnaissance d'un ligand bactérien spécifique, NOD1 recrute RICK au moyen d'une interaction CARD-CARD. RICK module ensuite l'activation du facteur de transcription NF-kB et initie une cascade de signalisation pro-inflammatoire. Le but de cette thèse était de déterminer la base structurale de ce processus. Le domaine CARD de NOD1 a été cloné, exprimé et purifié à la station EMBL de Grenoble. La structure de ce domaine a été résolue par résonance magnétique nucléaire à l'IBS de Grenoble. Cette structure est généralement similaire à celle des autres domaines CARD connus, étant constituée d'un paquet compact de 6 hélices a. Cependant elle présente des caractéristiques inhabituelles concernant la conformation de certaines boucles inter-hélices ainsi que la longueur et l'orientation de la sixième hélice. Des mutations dans les domaines CARDs des protéines NOD1 et RICK entières ont été réalisées pour identifier les résidus importants pour l'interaction ou la signalisation. Des expériences de co-immunoprécipitations et d'activation de NF-kB ont été effectuées à l'Université du Michigan. Trois résidus d'un patch acide de NOD1 (E53, D54 et E56) et trois résidus d'un patch basique de RICK (R444, R483 et R488) sont indispensables à l'interaction. Celle-ci serait donc de nature électrostatique. Deux autres résidus du patch acide du CARD de NOD1, L44 et I57, ont été identifiés comme importants pour la signalisation. Ces résidus peuvent potentiellement contribuer à la formation d'une interaction CARD-CARD fonctionnelle, ou bien également interagir avec d'autres régions de RICK.