Control Of Mucosal Immune Responses By DNA And Lipoprotein In Classical CD103+ Dendritic Cells

Inflammatory bowel disease (including Crohn’s disease and ulcer colitis) is a non-specific, chronic and inflammatory disease with increasing prevalence in recent decades. The pathogenesis of IBD is still elusive. Genetic susceptibility, immunological imbalance and dysregulated intestinal microbiota contribute to the IBD occurrence. Among them, immunological dysregulation is the most critical element that significantly determines the development of IBD.Mucosal immune system is the primary defense for the host against intestinal pathogens. Delicate mechanism of mucosal immune system in response to commensal and pathogenic stimuli has been suggested in recent studies, which contributes to the immune homeostasis in intestine. Dysregulated mucosal immune responses are associated with multiple autoimmune diseases, including IBD.Dendritic cell is one of antigen-presenting innate immune cells that is responsible for the surveillance, recognition and capture of intestinal antigens. DCs are capable to produce various inflammatory mediators and express various receptors to initiate and maintain immune responses in intestine. DCs are divided into two major types-classical DCs (cDC) and plasmacytoid DCs (pDC). Subsets of cDCs are further defined by major surface markers (such as CD11b and CD103) that determine distinct phenotypes and functions. Nevertheless, detailed mechanism that how DCs are involved in the pathogenesis of IBD and corresponding molecular signaling pathways is still elusive.In current study, we firstly identified the morphology, distribution and surface marker expression of classical DCs in small intestine lamina propria. Afterwards, we analyzed the effect and mechanism of the recognition of microbial DNA by STING in cDCs shapes adaptive immune responses (especially TH17 responses) in intestine. Furthermore, we investigated the antagonistical effect and underlying molecular mechanism of microbial lipoprotein recognition by TLR2 complex on DNA-STING-mediated TH17 activation. Finally, we established multiple murine infection models, and discovered that IL-17RA on CD 103+cDCs is the target shared by TLR2 and STING signaling that determines adaptive immune responses and tolerance in intestine. The whole thesis can be divided into three parts.PART I. Identification of classical dendritic cells in murine small intestine lamina propria. We performed bone marrow transplantation (BMT) on zbtb46-GFP knock-in mice, and observed the morphology and distribution of cDCs in small intestine lamina propria. We isolated and sorted zbtb46+ cDCs from zbtb46-GFP mice, and analyzed the subset of cDCs according to the expression of surface marker (CD11b and CD 103) on cDCs derived from small intestine lamina propria.PART Ⅱ. STING-mediated microbial DNA recognition shapes adaptive immune responses in intestine. We investigated the effect of DNA stimulation on TH17 development in intestine. Afterwards, we injected DT into zbtb46-DTR knock-in mice to deplete all zbtb46-expressing DCs, leading to cDC-deficient mice in which we were able to analyze the role of cDCs in the activation of TH17 responses by DNA-STING. Furthermore, we adopted STING-deficient mice to reveal that STING is the exclusive signaling that is capable to recognize microbial DNA in mice. Finally, we used IL-17RA-/- and IRF5-/-mice to demonstrate that STING requires IRF5 and IL-17RA to activate TH17 responses after DNA stimulation.PART Ⅲ. TLR2-mediated microbial lipoprotein recognition competes with DNA-STING on TH17 responses in intestine. We initially stimulated primary CD103+ cDCs freshly derived from murine small intestine lamina propria with various ligands of TLRs and RLRs. We identified that TLR2 signaling complex is the only suppressor for TH17 development and stimulator for immune tolerance. Subsequently, we used TLR2-deficient mice to analyze the effect of lipoprotein recognition mediated by TLR2 on TH17 development in the presence of DNA stimulation. To elucidate the antagonistic mechanism between TLR2 and STING signaling, we established Listeria monocytogenes and Salmonalla enterica serovar Typhimurium infection models in STING-/-, TLR2-/- and IL-17RA-/-mice, and discovered that IL-17RA on CD103+ DCs is the primary target shared by TLR2 and STING that determines the outcome of immune responses or tolerance. When pathogens release both DNA and lipoprotein into cytosols, they achieve immune evasion through the regulation of IL-17RA on CD 103+ classical dendritic cells.Our study explains the role of CD 103+ cDCs in the initiation of immune responses in intestine. We elucidate the mechanism of TH17 activation in response to DNA recognition by STING signaling pathway. We also reveal the molecular basis that TLR2 competes with STING signaling to determine adaptive immune responses while both lipoprotein and DNA are released by microbiome, which uncovers the mechanism of "immune evasion" of intestinal pathogens.