L’organisme est exposé à divers agents infectieux et doit mettre en place une réponse immunitaire adéquate pour se protéger. Mes travaux de thèse m’ont permis d’étudier la réponse innée à l’infection par Listeria monocytogenes (L.m) et l’inflammation pulmonaire induite par la flagelline extraite de Salmonella typhimurium. Mes résultats ont mis en évidence l’association du co-récepteur CD14 avec TLR2 (Toll-like Receptor 2) dans la détection de L.m injectée par voie veineuse. En revanche, CD14 ne semble pas être associé au TLR5 dans la reconnaissance de la flagelline. Par ailleurs, l’activation des TLR par leurs ligands permet la synthèse de cytokines intervenant dans l’inflammation. J’ai ainsi pu étudier plus précisément le TNF (Tumor Necrosis Factor). Cette protéine pro-inflammatoire est un des médiateurs principaux de l’immunité et existe sous une forme membranaire qui a été peu étudiée (Mem-TNF) et sous une forme soluble bien connue (sTNF). Mes études ont montré que ce Mem-TNF active la production de cytokines et de médiateurs chimiques de l’inflammation conférant une protection partielle contre Listéria. L’étude de cette cytokine membranaire nous a permis de tester une nouvelle génération de traitements moins agressifs que les anti-TNF contre l’arthrite rhumatoïde ou la maladie de Crohn. / Toll-like receptors (TLRs) recognize a wide range of microbial pathogens and their products modulate the innate immune response that may lead to inflammation. In order to better understand the host-pathogen relationship, we have studied the implication of the co-receptor CD14 in the innate immune response to Listeria monocytogenes and to the bacterial flagellin from Salmonella typhimurium. Our results clearly show that TLR2 requires CD14 to control Listeria infection whereas TLR5 does not. Moreover, TLR activation leads to pro-inflammatory cytokines production such as Tumor Necrosis Factor (TNF). This pleiotropic protein is required for normal development and function of the immune system. TNF can be secreted (sTNF) or associated to the membrane (Mem-TNF). Our results suggest that Mem-TNF can activate the synthesis of cytokines and chemicals mediators of inflammation and partially protect mice from a moderate infection. These experiments open new avenues for the treatment of inflammatory disease like rheumatoid arthritis or Crohn disease.

The protein monomer of bacterial flagella, FliC, is known to stimulate human innate immunity through activation of Toll-like receptor five. Linking native Salmonella FliC with various antigens has demonstrated an increased immune response as compared to single antigen presentation. To drastically reduce production time and allow for a more cost effective recombinant vaccine adjuvant, a synthetic construct was created that enables genetic linkage of FliC to other known antigens. The construct contains the necessary components for immune system stimulation while the non-essential regions were replaced with commonly used restriction enzyme recognition sites to aid in ligation with other antigens and cloning into various expression vectors and hosts. After synthesis in the inducible expression vector pJ404, the construct was transformed into competent BL21 E. coli and expression was confirmed through SDS-PAGE, Western blot, and MALDI MS/MS. The cells were adapted to fermentation media and re-screened for expression, and upon confirmation a 20-liter fermentation was conducted. The resulting samples were analyzed for expression within the insoluble and soluble cellular fractions to further optimize fermentation conditions. Once purified, this synthetic FliC will serve as a platform technology for the standardized co-expression of the TRL5 activator with a variety of antigens in both prokaryotic and eukaryotic systems.

Clostridium difficile (CD) est l’entéropathogène le plus fréquemment responsable d'infections nosocomiales intestinales post-antibiotiques. L'apparition de cas graves liés à l'émergence de souches hypervirulentes ces dernières années a contribué à accroître la morbidité et la mortalité. Les toxines TcdA et TcdB contribuent directement aux lésions intestinales associées aux infections à CD (ICD), mais d'autres facteurs bactériens sont nécessaires à l’adhésion et la colonisation intestinale. Les flagelles de CD, qui confèrent la mobilité et la chimiotaxie, pourraient jouer un rôle dans la pathogenèse en contribuant à la réponse inflammatoire de l’hôte et aux lésions de la muqueuse. En effet, en activant le récepteur TLR5, les flagelles peuvent provoquer l'activation des cascades de signalisation cellulaire des MAPK et de NF-κB conduisant à la sécrétion de cytokines pro-inflammatoires. Notre objectif était d’étudier ce rôle potentiel des flagelles de CD in vitro et in vivo. Nous avons montré que l'interaction de la flagelline de CD avec le TLR5 active principalement la voie de NF-κB, et, dans une moindre mesure, la voie des MAPK, conduisant ainsi à la régulation de l'expression de gènes pro-inflammatoires et à la synthèse de médiateurs pro-inflammatoires. De plus, en utilisant un modèle murin d’ICD, nous avons démontré un effet synergique des flagelles et des toxines dans l’induction d’une réponse inflammatoire de la muqueuse caecale. Dans ce modèle, l'absence de flagelles diminue considérablement le degré d'inflammation de la muqueuse caecale et la seule présence de toxines, sans flagelles, ne suffit pas à provoquer d’importantes lésions épithéliales. Ces résultats mettent en évidence le rôle des flagelles de CD dans l’induction d’une réponse inflammatoire intestinale en synergie avec l’action des toxines bactériennes sur l'épithélium. / Clostridium difficile (CD) is the most common enteropathogen responsible for intestinal nosocomial post-antibiotic infections. The appearance of severe cases related to the emergence of hypervirulent strains these last years has contributed to increased mortality and morbidity. The CD TcdA and TcdB toxins contribute directly to CD infection (CDI)-associated lesions of the gut, but other bacterial factors are needed for the bacteria to adhere and colonize the gut. The CD flagella, which confer motility and chemotaxis for successful intestinal colonization, could play an additional role in bacterial pathogenesis by contributing to the inflammatory response of the host and mucosal injury. Indeed, by activating the TLR5, flagella can elicit activation of the MAPK and NF-κB cascades of cell signaling, leading to the secretion of pro-inflammatory cytokines. Our objective was to study the potential role of CD flagella in vitro and in vivo. We reported that the interaction of CD flagellin-TLR5 predominantly activates the NF-κB, and, in a lesser degree, the MAPKs pathways, thus leading to up-regulation of pro-inflammatory gene expression and subsequent synthesis of pro-inflammatory mediators. Moreover, by using a mouse model of CDI, we demonstrated a synergic effect of flagella and toxins in eliciting an inflammatory mucosal response. In this model, the absence of flagella dramatically decreased the degree of mucosal inflammation in mice and the sole presence of toxins without flagella was not enough to elicit epithelial lesions. These results highlight the important role of CD flagella in eliciting mucosal lesions as long as the toxins exert their action on the epithelium.

Clostridium difficile (CD) représente la première cause d'infections digestives nosocomiales dans les pays développés. Les infections à CD (ICD) induisent une inflammation intestinale importante qui se manifeste principalement par des colites pseudomembraneuses ainsi que par un taux de mortalité élevé. Les facteurs majeurs de virulence sont les toxines TcdA et TcdB. Dans la première partie des travaux de cette thèse, nous avons validé le rôle in vivo d'un autre facteur bactérien, les flagelles, dans un modèle murin d'ICD, en montrant que les flagelles induisent une réponse inflammatoire au niveau de la muqueuse caecale en synergie avec les toxines. Nous avons ensuite montré une régulation de de cette réponse par un microARN (miARN) exerçant un rôle anti-inflammatoire en modulant l'activation de la voie de signalisation de NF-KB. Le traitement des souris infectées par ce miARN réduit l'inflammation intestinale apportant la preuve du concept pour une nouvelle approche thérapeutique. / Clostridium difficile (CD) is the leading cause of nosocomial digestive infections in developed countries. CD infections (CDI) induce significant intestinal inflammation that is manifested primarily by pseudomembranous colitis and a high mortality rate. The major virulence factors are TcdA and TcdB toxins. In the first part of the work of this thesis, we validated the in vivo role of another bacterial factor, the flagella, in a mouse model of CDI, showing that the flagella induce an inflammatory response in ceacal mucosa in synergy with toxins. We then showed a regulation of this response by a microRNA (miRNA) exerting an anti-inflammatory role by modulating the activation of the NF-KB signaling pathway. The treatment of mice infected with this miRNA reduces intestinal inflammation providing proof of concept for a new therapeutic approach.

L'organisme est exposé à divers agents infectieux et doit mettre en place une réponse immunitaire adéquate pour se protéger. Mes travaux de thèse m'ont permis d'étudier la réponse innée à l'infection par Listeria monocytogenes (L.m) et l'inflammation pulmonaire induite par la flagelline extraite de Salmonella typhimurium. Mes résultats ont mis en évidence l'association du co-récepteur CD14 avec TLR2 (Toll-like Receptor 2) dans la détection de L.m injectée par voie veineuse. En revanche, CD14 ne semble pas être associé au TLR5 dans la reconnaissance de la flagelline. Par ailleurs, l'activation des TLR par leurs ligands permet la synthèse de cytokines intervenant dans l'inflammation. J'ai ainsi pu étudier plus précisément le TNF (Tumor Necrosis Factor). Cette protéine pro-inflammatoire est un des médiateurs principaux de l'immunité et existe sous une forme membranaire qui a été peu étudiée (Mem-TNF) et sous une forme soluble bien connue (sTNF). Mes études ont montré que ce Mem-TNF active la production de cytokines et de médiateurs chimiques de l'inflammation conférant une protection partielle contre Listéria. L'étude de cette cytokine membranaire nous a permis de tester une nouvelle génération de traitements moins agressifs que les anti-TNF contre l'arthrite rhumatoïde ou la maladie de Crohn.

Bone marrow (BM) transfer from flagellin-treated mice has been reported to improve the survival of lethally-irradiated mice. Although the mechanism for flagellin’s antiviral and antibacterial effects have been elucidated, there remains a gap in knowledge regarding its radioprotective effects. Here, we report that flagellin treatment results in a 5-fold increase in the proliferation of Lin-Sca-1+C-Kit+(LSK) cells, a heterogeneous stem and multipotent cell population in BM, with the most striking increase within the ST-HSC, MPP2 and MPP3 subpopulations. Furthermore, the presence of TLR5 but not NLRC4 on radiosensitive, non-LSK cells in BM was both sufficient and necessary for the observed phenomenon. Finally, adoptive transfer of MPP3 cells along with an insufficient amount of whole bone marrow cells (WBM) to lethally-irradiated mice significantly improved their survival, recapitulating the effects of Whole bone marrow from flagellin-treated mice.

Systemic Lupus Erythematosus (SLE) stands as a multifaceted autoimmune disorder, characterized by a spectrum of clinical manifestations and the generation of autoantibodies against self-antigens. Our focus was on the pivotal role of B cells in the development of SLE. The study also underscored the significant contribution of regulatory B (Breg) cells in the context of SLE, suggesting their potential as key regulators of the disease process. Our results provided a deeper understanding of the intricate interplay between B cells and SLE, offering insights that were valuable for both scientific research and future designs of therapeutic approaches. Cutting-edge single-cell RNA sequencing was employed to analyze the differences in splenic Breg subsets and their molecular profiles across different stages of lupus development in mice. Transcriptome-based changes in Bregs during active disease were confirmed through phenotypic analysis. These findings provided crucial insights into the dynamic role of B cells in the pathogenesis of SLE. In addition, we delved into the intricate connection between SLE and the gut microbiota. A literature review offered a comprehensive analysis of current research, with a particular emphasis on potential interactions between bacterial flagellin and Toll-Like Receptor 5 (TLR5) on immune cells. These interactions garnered substantial attention due to their potential implications in the pathogenesis of SLE. We synthesized existing research, providing valuable insights into the complex interplay between SLE and the microbiota and suggesting promising avenues for further investigation and potential therapeutic interventions. In the final study, we explored lupus-like disease in mice with global Tlr5 deletion, initially expecting disease attenuation. Surprisingly, the results revealed an exacerbation of lupus-like symptoms, particularly in female mice lacking Tlr5. Future research will seek to uncover the mechanisms by which Tlr5 deletion modulates interactions between the host and the gut microbiota, ultimately contributing to the exacerbation of lupus-like disease. / Doctor of Philosophy / Systemic Lupus Erythematosus (SLE) is characterized by a range of health issues and the body attacking itself. In this exploration, we journey through the intricate landscape of SLE, uncovering key players and unexpected twists. In this dissertation, we journeyed through the intricate landscape of SLE, uncovering key players and unexpected twists. In this dissertation, we closely examined these immune cells, revealing how different types of B cells contributed to SLE's development. We also introduced the enigmatic regulatory B (Breg) cells, which acted as potential peacekeepers in this autoimmune reaction. Our results illuminated the complex relationship between B cells and SLE, offering insights that benefited both researchers and those seeking new treatments. We employed cutting-edge technology, single-cell RNA sequencing, to scrutinize the genetic fingerprints of B cells in mice with SLE. The results unveiled changes in Breg cells during active disease, providing critical clues about how B cells impacted SLE progression. In addition, this dissertation took us into the microscopic world of our gut inhabitants, the microbiota. We dived into a treasure trove of research, focusing on how interactions between bacterial flagellin and various microbiota elements affected immune cells through a special receptor called Toll-Like Receptor 5 (TLR5). These interactions, like hidden clues, had piqued scientists' interest for their potential role in SLE development. We synthesized existing research, offering valuable insights into the complex interplay between SLE and our microbiota. The discussion also suggested promising paths for future research and potential therapies. In the final study, we encountered a plot twist. We anticipated that deleting the Tlr5 gene would improve lupus-like disease in mice. To our surprise, the opposite happened. Lupus-like symptoms worsened, especially in female mice lacking Tlr5. Clinical signs included enlarged spleens and lymph nodes, increased immune cell activity, and kidney inflammation. But Tlr5 deletion didn't change the mice's metabolism or the leaky gut. Instead, it reshaped their gut's microbial residents. Future research aimed to uncover how Tlr5 deletion altered the interactions between the host and gut microbes, ultimately making lupus-like disease more severe. In a nutshell, this journey through SLE's complex world provided a deeper understanding of its intricacies. We met the B cells, explored the microbiota, and encountered surprises along the way. These discoveries were vital pieces of the puzzle, bringing us closer to unlocking the secrets of SLE and, perhaps, finding new ways to manage and treat this challenging autoimmune disorder.

Toll-like receptors (TLRs) expressed by cells of the immune system play a central role in the generation of immune responses against pathogens. Following TLR ligation, both pro-inflammatory and anti-inflammatory mediators are produced in order to elicit an immune response that controls the microbial infection while limiting tissue damage. Among these mediators, the proinflammatory cytokine IL-12 and the anti-inflammatory cytokine IL-10 are known to play major roles. Here, we show that in vitro or in vivo stimulation with flagellin, the TLR5 ligand, does not result in IL-10 production. Furthermore flagellin inhibits IL-10 production by other specific TLR ligands at the protein and mRNA levels while increasing IL-12p70 production. Several studies have linked the activation of extracellular signal regulated kinases (ERKs) with IL-10 induction by TLRs. Our findings that LPS-induced ERK activation is significantly decreased in flagellin-treated macrophages suggest that this pathway might play a role in the inhibition of IL-10 production by flagellin. Flagellin-mediated IL-10 inhibition was not observed in cells that do not express TLR5 supporting that this effect is TLR5-dependent.Flagellin used as an adjuvant is capable of priming antigen specific T cell responses in an in vivo model of tolerance using high dose peptide. Furthermore, DCs differentiated in tolerogenic conditions (tolerogenic-DCs) express higher levels of TLR5 mRNA than standard BM-DCs and respond more vigorously to flagellin stimulation. Antigen presentation by LPS-matured tolerogenic-DCs results in the differentiation of IL-10 producing T cells with a Tr1-like phenotype. On the contrary, antigen presentation by tolerogenic-DCs that have been stimulated with flagellin results in the differentiation of a typical Th1 responseThis study provides a new insight of the role of flagellin recognition by TLR5 in shaping the immune response elicited by flagellated microorganisms.

No description available.

River buffalo are economically important to many countries and only recently has their genome been explored for the purpose of mapping genetic variation in traits of economic and biologic interest. The purpose of this research is to characterize the genetic and evolutionary profile of Toll-like receptor 5 (TLR5), which mediates the mammalian innate immune response to bacterial flagellin. This study is comprised of three parts: 1) generating a radiation hybrid (RH) map of river buffalo chromosome 5 (BBU5) where the TLR5 gene is located and building a comparative map with homologous cattle chromosomes; 2) conducting a single-nucleotide polymorphism (SNP) survey of the TLR5 gene to reveal variation within river buffalo and other species; and 3) performing an evolutionary study by inferring phylogenetic trees of TLR5 across multiple taxa and determining the possible evolutionary constraints within the TLR5 coding region. River buffalo chromosome 5 is a bi-armed chromosome with arms corresponding to cattle chromosomes 16 and 29. A BBU5 RH map was developed using the previously published river buffalo RH mapping panel and cattle-derived markers. The RH map developed in this study became an integral part of the first river buffalo whole genome RH map. Genetic variation of the TLR5 gene was evaluated in a small domestic herd of river buffalo. Sequencing of the TLR5 coding region and partial associated 5'- and 3'-untranslated regions yielded 16 novel SNPs. Six SNPs were identified as non-synonymous with one predicted to potentially code for a functionally altered product. For the evolutionary study of the TLR5 coding region, phylogenetic trees were inferred based on TLR5 variation across multiple orders and another for artiodactyla. Species that are closely related to river buffalo appear to have undergone negative selection in TLR5 while those that diverged from river buffalo earlier may be retaining alleles that river buffalo are removing from the population. In conclusion, putative chromosomal rearrangements were identified between river buffalo and cattle, the variation that was uncovered in the TLR5 coding region could potentially lead to differential immunity across species, and there appears be some evolutionary flexibility in the DNA sequence of the TLR5 coding region.