Les cellules épithéliales jouent un rôle clé dans la mise en place et le maintien de l’homéostasie mais également dans les processus de défense contre les agents pathogènes, notamment via les récepteurs de l’immunité innée, comme le récepteur le Toll-like (TLR) 4. En effet, la stimulation de ce récepteur par son ligand, le lypopolysaccharide (LPS), le constituant majeur de la paroi des bactéries à Gram négatif, induit l’activation cellulaire et la production de médiateurs de l’inflammation, comme les cytokines et chimiokines pro-inflammatoires KC et MIP-2. Dans ce travail, j’ai étudié les mécanismes de régulation de la voie de signalisation du récepteur TLR4 au niveau des cellules épithéliales, notamment via, les microARN, de petits ARNs non codants qui peuvent réguler de manière extrêmement rapide et efficace l’expression de gènes cibles. Dans une première partie, en utilisant, une lignée de cellules du tubule collecteur rénal immortalisées, des cultures primaires de cellules de TC microdisséquées à partir de reins de souris transgéniques, ainsi qu’un modèle murin d’ITU ascendante, nous avons montré que la CsA induit l’expression du micro-ARN Let-7i, qui cible TLR4 et inhibe donc par conséquent l’activation cellulaire induite par le LPS. Dans une seconde partie, en utilisant une lignée de cellules épithéliales intestinales immortalisées ainsi qu’un modèle murin d’infection du tractus digestif par Citrobacter rodentium, nous avons pu montrer que l’IL-22 induit l’expression du miR-763 qui, en potentialisant la dégradation de la kinase IRAK1, une kinase clé de la voie de signalisation de TLR4, induit une inhibition de la voie de signalisation de TLR4 dans les cellules épithéliales intestinales. Mon travail de thèse a permis d’apporter un faisceau d’arguments montrant l’implication de microARN spécifiques dans la régulation de la voie de signalisation de TLR4 au niveau des épithéliums, suggérant que l’utilisation d’anti-miR synthétiques pourrait être efficace pour traiter certaines pathologies inflammatoires ou infectieuses. / Epithelial cells play a key role in the establishment and the maintenance of homeostasis, but also in the defense against pathogens, particulary via the receptors of innate immunity, such as the Toll-like receptor (TLR) 4. Indeed, the stimulation of this receptor by its ligand, the lypopolysaccharide (LPS), the major constituent of the wall of Gram-negative bacteria, induces cellular activation and production of inflammatory mediators, such as cytokines and pro-inflammatory chemokines KC and MIP-2. In this work, I studied the mechanisms of regulation of the signaling pathway of the TLR4 receptor in epithelial cells, specially via microRNAs, small non-coding RNAs which can regulate extremelys rapidly and efficiently the expression of genes. In a first part, using an immortalized renal collector tubule cell line, primary cultures of TC cells microdissected from transgenic mouse kidneys, as well as a mouse model of ascending UTI, CsA induces the expression of the Let-7i microRNA, which targets TLR4 and therefore inhibits the LPS induced cell activation. In a second part, using an immortalized intestinal epithelial cell line as well as a mouse model of Citrobacter rodentium digestive tract infection, we were able to show that IL-22 induces expression of miR-763 which, by potentiating the degradation of the kinase IRAK1, a key kinase of the TLR4 signaling pathway, induces an inhibition of the TLR4 signaling pathway in the intestinal epithelial cells. My thesis work has permitted to provide lines of evidence in the involvement of specific microRNAs in the regulation of the TLR4 signaling pathway in the epithelial cells, suggesting that the use of synthetic anti-miR could be effective to treat certain inflammatory or infectious diseases.

Diarrheal disease caused by attaching and effacing pathogens, such as enteropathogenic E. coli (EPEC), is a worldwide health concern. As the second leading cause of diarrheal-related death in young children, new investigations into host defense against EPEC, as well as future therapeutics, is greatly needed. To elucidate the host immune responses to these enteric pathogens, the attaching and effacing (A/E) murine pathogen, Citrobacter rodentium, has been widely used. It is well understood that C. rodentium infection induces a robust Th1 response within the host. Yet how these pleiotropic IFNγ immune responses are initiated, propagated, and the accessory immune cell types involved remains poorly understood. In this thesis, I investigated how innate immune cell types such as natural killer cells, which are significant producers of IFNγ, mediate these Th1 directed responses. This work identified that both NK and NK-like innate lymphoid type 1 cells (ILC1s) are capable of producing IFNγ in response to C. rodentium, and NK cells rapidly increase in numbers within the colon during the early stages of infection. Depletion of these cell types causes a delayed Th1 CD4+ T cell response within the colon, resulting in increased bacterial load, and greater degree of colonic pathology at later time points. Additionally, depletion of these cells results in decreased CXCL9 chemokine expression in mice. I later determined that CXCL9 exhibited direct antimicrobial action against Citrobacter in vitro. Depletion of this chemokine in vivo, in the absence of adaptive immune responses, or its receptor CXCR3, results in increased mortality rates, elevated bacterial loads, greater degree of pathology, and deeper penetration of bacteria within the colonic crypts. These data indicate a potential direct antimicrobial role for this IFNγ-induced chemokine, independent of its known properties for the homing of T cells to the site of infection. These findings demonstrate the importance of accessory IFNγ-producing immune cells in not only mediating Th1 CD4+ T cells responses, but also other innate host defense mechanisms against A/E pathogens. / Thesis / Doctor of Philosophy (PhD)

Microbiota colonisation of the intestinal tract makes it difficult for pattern recognition receptors (PRR) to discriminate between beneficial microbes and harmful pathogens. We aim to define the roles of cytosolic Nod-like receptors (NLR) in intestinal immunity and homeostasis. Upon activation, some NLR form inflammasomes that mediate the release of inflammatory cytokines and pyroptosis, an inflammatory form of cell death. NLR activation in the non-hematopoietic compartment was shown to be protective during acute intestinal infection. To identify the cell type responsible for this protection, we generated transgenic mice in which the key inflammasome adaptor molecule Asc is selectively ablated in intestinal epithelial cells (IEC) (Asc^ΔVC) and observed that inflammasomes are important for controlling Citrobacter rodentium clearance in these mice. To further dissect the importance of pathogen clearance by IEC inflammasome, ex vivo cultures of primary IEC organoids were established. Thus far this system has revealed profound differences in inflammasome regulation between IEC organoids and bone marrow-derived macrophages (BMDM). This research will inform our understanding of cell type-specific regulation of inflammasomes.

No description available.

Enterohemorrhagic Escherichia coli (EHEC) colonizes the intestine and produces the phage-encoded Shiga toxin (Stx) which is absorbed systemically and can lead to hemolytic uremic syndrome (HUS) characterized by hemolytic anemia, thrombocytopenia, and renal failure. EHEC, and two related pathogens, Enteropathogenic E. coli (EPEC), and the murine pathogen, Citrobacter rodentium, are attaching and effacing (AE) pathogens that intimately adhere to enterocytes and form actin “pedestals” beneath bound bacteria. The actin pedestal, because it is a unique characteristic of AE pathogens, has been the subject of intense study for over 20 years. Investigations into the mechanism of pedestal formation have revealed that to generate AE lesions, EHEC injects the type III effector, Tir, into mammalian cells, which functions as a receptor for the bacterial adhesin intimin. Tir-intimin binding then triggers a signaling cascade leading to pedestal formation. In spite of these mechanistic insights, the role of intimin and pedestal formation in EHEC disease remains unclear, in part because of the paucity of murine models for EHEC infection. We found that the pathogenic significance of EHEC Stx, Tir, and intimin, as well as the actin assembly triggered by the interaction of the latter two factors, could be productively assessed during murine infection by recombinant C. rodentium expressing EHEC virulence factors. Here we show that EHEC intimin was able to promote colonization of C. rodentium in conventional mice. Additionally, previous in vitro data indicates that intimin may have also function in a Tir-independent manner, and we revealed this function using streptomycin pre-treated mice. Lastly, using a toxigenic C. rodentium strain, we assessed the function of pedestal formation mediated by Tir-intimin interaction and found that Tir-mediated actin polymerization promoted mucosal colonization and a systemic Stx-mediated disease that shares several key features with human HUS.