SIRP?-dependent Intestinal CD103~+CD11b~+DCs Maintain The Homeostasis Of Bile Acids Metabolism

Background and Purpose Dendritic cells(DCs),as the strongest antigen presenting cells,are considered to be a bridge connecting the specific immune response and non-specific immune response.DCs can determine the type of specific immune response.DCs originate from bone marrow precursor cells and could differentiate into a series of subets: mononuclear DCs,plasmacytoid DCs(p DCs)and conventional DCs(c DCs).Intestinal DCs mainly distribute in the intestinal lamina propria(Lamina propria,LP)and gut associated lymphoid tissues,including the Peyer's patches(PPs)and distal mesenteric lymph nodes(MLN).Intestinal DCs have many different specific subtypes and their formations are associated with of microenvironment and cytokines.CD103~+CD11b~+ DCs is the dominant subset in the small intestine lamina propria.Intestinal DCs monitor the commensal microbiota and pathogenic bacteria carefully in intestinal mucosal barrier and can carry them into the secondary lymphoid node.Then,intestinal DCs activate T and B lymphocytes in the lymph nodes.The distinct DCs subsets can influence the differentiation of T helper cells(Th cells,such as Th1,Th2 or Th1)and participate in different types of immune responses.The maintenance of the normal intestinal function depends on the precise balance between immunity and tolerance,abnormalities of microbiota immune regulation will lead to chronic intestinal inflammation,such as inflammatory bowel disease.Therefore,intestinal DCs play an important role in the intestinal mucosal immune homeostasis,but the chacateritics of distinct intestinal DC subets and its regulation are still unclear.Signal regulatory protein alpha(SIRP?),one of the glycoprotein molecules,is the members of immunoglobulin superfamily and selectively expressed on myeloid cell and nerve cell membrane surface.The cytoplasmic region is highly conserved and has immune receptor tyrosine inhibitory motifs(ITIMs).The extracellular domain of SIRP? is composed of three immunoglobulin super family domain(Ig SF).SIRP? mediates immune suppression through the interaction between its ligand CD47.Many studies have shown that SIRP?-CD47 pathway can participate in the development and maturation of DCs and promote the antigen presenting of DCs.The loss of SIRP? in the mouse intestinal lamina propria can lead to a decrease in the number of CD103~+CD11b~+ DCs subsets and Th17 cells in the intestinal mucosa,and Th17 cell responses caused by the infection of Citrobacter.rodentium were reduced.Studies have also shown that CD11c~+CD11b~+ DCs,highly expressed SIRP? in mouse intestinal lamina propria,could promote the production of flagellin-mediated IL-17 a,IFN-gamma and IL-6,thereby promoting the differentiation of naive CD4~+ T cells into Th17 and Th1.The results suggest that SIRP? participates in the regulation of intestinal immune homeostasis by regulating the number and function of DCs and Th17 cells.However,the exact mechanism of how SIRP? involved in DCs homeostasis and the impact of SIRP? on intestinal immune microenvironmentneed further researches.In this study,we constructed a SIRP? systemic knockout mouse by TALEN technology.We detected the number and proportion of DCs,M? and T cell subsets in spleen,intestine lamina propria and lymph nodes by flow cytometry,and explored the role of SIRP? in intestinal immune homeostasis.In order to further clarify the function of SIRP?,we constructed SIRP? cell specific knockout mouse by Cas9/RNA gene targeting strategies and the Loxp/Loxp-Cre system.We also detected the number and proportion of immune cells in intestinal lymphoid and non-lymphoid tissues by flow cytometry.In vitro,we tried to find the possible mechanism how SIRP? regulate intestinal DCs by using coculture research system and neutralizing antibody intervention.In order to further explore the regulation of SIRP? on intestinal homeostasis,we detected the composition and diversity of intestinal flora in SIRP?-KO mice by 16 S r DNA sequencing.We also built DSS-induced colitis to explore the role of SIRP? in the development of inflammatory bowel disease.We analyzed the bile acid composition and discussed the regulation of SIRP? for bile acid metabolism.These results suggested that SIRP? played an important role in regulation of intestinal immune and metabolic homeostasis.Our study provided new strategies and new ideas for the treatment of IBD and lipid metabolism related diseases.Method 1.We constructed the SIRP? systemic knockout mice by TALEN technology.We constructed SIRP? cell specific knockout mice by Cas9/RNA gene targeting technology and Loxp/Loxp-Cre system;2.We analyzed the number and proportion of DCs and M? in spleen,intestine lamina propria and mesenteric lymph nodes by flow cytometry.We detected the T cell subsets in tissues by intracellular staining;3.We induced and cultured the mouse bone marrow derived macrophages(BMDMs).The phagocytosis of M? on intestinal DCs,Th1 and Th17 was detected by transwell co-culture system;4.Antigen specific T cell proliferation test.In vitro experiments: Spleen purified DCs and the na?ve CD4~+ T cells from OT-II mice were co-cultured for 4 days.Then the concentration of IL-17 a in the supernatant was detected;In vivo experiments: Mice were injected OT-II~+ na?ve T cells by tail vein injection.1 day later,the mice were immunized.10 days later,detected the number and proportion of Th17/ Th1 cells in intestine;5.Bone marrow transplantation model: Recipient mice were irradiated by lethal gamma rays(Co60).Bone marrow cells were isolated from donor mice and then injected into irradiated recipient mice through tail vein;6.The colitis of mice were induced by fed with 2.5% dextran sodium sulfate(DSS)water solution;7.The mcie peritoneal M? were removed by intraperitoneal injection of chloronate liposomes;8.In vivo intestinal permeability assay.Fluorescein isothiocyanate(FITC)-conjugated detran dissolved freely in water.Mice were gavaged with FITC-detran for 4h.Then we measured the FITC-detran concentration and fluorescence intensity in serumby fluoresence spectrophotometer;9.We detected the composition and diversity of intestinal microflora by 16 S r DNA sequencing;10.The serum biochemical markers were detected by automatic biochemical analyzer.The concentration of IL-6,IL-1?,TNF-?,IFN-?,IL-17 a and FGF15 in the tissue and serum were measured by the enzyme linked immunosorbent assay;11.We detect the bile acids compositions in the feces,serum and tissues of mice by ultra high performance liquid chromatography(UPLC)-Mass spectrometry(MS)technology.Results 1.The M? in the spleen,the small intestine lamina propria and mesenteric lymph nodes were all highly expressed SIRP?,whereas DCs were heterogeneous for SIRP? expression;2.The number and proportion of CD4~+ c DCs in the spleen of SIRP?-KO mice were decreased.The number and proportion of CD103~+CD11b~+ DCs in the intestine lamina propria and mesenteric lymph nodes were decreased;3.SIRP? from hematopoietic cells was involved in the homeostasis of the intestinal DCs subsets;4.The specific deletion of SIRP? on DCs and M? were both resulted in the decreased in the number of CD4~+ c DCs of the spleen,CD103~+CD11b~+ DCs of the mesenteric lymph nodes and the small intestine lamina propria;5.SIRP?-dependent CD103~+CD11b~+ DCs affected the production and/or maintenance of intestinal Th17/Th1 cells;6.Blocked SIRP?-CD47 signaling pathway could enhance SLAMF7-mediated phagocytosis of M? on intestinal CD103~+CD11b~+ DCs and Th17/Th1;7.The number of intestinal epithelial bacteria was increased and the antibacterial functions of the intestinal mucosal epithelial were weakened in SIRP?-KO mice.Intestinal epithelial cells were abnormally activated and the bile acid negative feedback signaling pathway was inhibited in SIRP?-KO mice;8.The loss of SIRP? aggravated DSS-induced colitis;9.SIRP?-KO mice showed elevated systemic bile acids levelsConclusion In this study,we explored the role of SIRP? in intestinal immune homeostasis by SIRP? systemic knockout mice and SIRP? conditional knockout mice models.We found that the deletion of SIRP? can cause the reduced number of CD103~+CD11b~+ DCs and Th17/Th1 in intestine and lymph nodes.Blocked SIRP?-CD47 signaling pathway could enhance SLAMF7-mediated phagocytosis of M? on intestinal CD103~+CD11b~+ DCs and Th17/Th1.Meanwhile,we observed the loss of SIRP? weakened intestinal epithelial antibacterial function and led to abnormal NF-?B activation and FXR signaling in intestinal epithelial cells.Futhermore,SIRP? ablation led to the dysregulation of bile acids metabolism.Together,our results suggested a central role of SIRP?-dependent CD103~+CD11b~+ DCs in the immune and metabolism homeostasis.