Tumor Necrosis Factor-like Cytokine 1A Plays A Novel Role In Inflammatory Bowel Disease Pathogenesis

Objective:Inflammatory bowel disease(IBD),including Crohn's disease(CD)and ulcerative colitis(UC),is characterized by chronic and spontaneously relapsing inflammation of the intestine.Although the precise etiology of IBD is not well understood,genetic susceptibility factors,dysbacteriosis,environmental triggers,and mucosal immune imbalance are considered as the key causes for IBD pathogenesis.TL1A(Tumor necrosis factor-like cytokine 1A,TNFSF15)is a member of the TNF superfamily of proteins(TNFSF)that binds to death domain receptor 3(DR3),expressed on a variety of cell types including myeloid lineage cells,endothelial cells and activated T cells,whereas DR3 is predominantly expressed on the surface of T-cells.TL1A/DR3interaction is important for maintaining homeostasis in the intestinal mucosal immune system.Previous studies on transgenic mice and the application of neutralizing antibody or agonistic antibodies demonstrated that TL1A/DR3 signaling may play an important role in normal immune response and have altered expression in multiple immune-mediated disorders in IBD.However,the function of TL1A in the development of IBD remains unknown.So we utilized Tl1a^-/-mice to construct different types of experimental colitis models to explore the direct impact of TL1A deletion on the occurrence and development of colitis,and investigate the immune regulation mechanism of TL1A in mouse IBD models.Meanwhile,the regulation effect of TL1A/DR3 signal transduction on T cell differentiation.Methods:(1)Using WT mice to establish animal models of IBD:taking mice inflamed colon tissue,extracting m RNA and use q PCR technology to detect the gene expression changes of Tl1a in colon.Using western blot technology to detect TL1A protein changes in inflamed colon tissue.Using Flow Cytometry technology to detect the expression of TL1A and DR3 on the surface of several subsets of immune cells in inflamed colon Lamina propria lymphocytes.(2)Establish IBD animal model with WT and Tl1a^-/-mice:dynamically compare and observe the changes of weight,survival rate,stools,colon length and disease activity index(DAI).At the same time,the intestinal changes of WT and Tl1a^-/-mice before and after inducing colitis were detected by mice endoscope,and the histopathological changes of mice intestinal tract were detected by HE staining technology.(3)Establish IBD animal models with WT and Tl1a^-/-mice,and explore the effect of TL1A depletion in process of immune regulation.The numbers of each immune cell of lamina propria lymphocyte(LPL)and mesenteric lymph node(MLN)subset were calculated,and the changes of each immune cell subset proportion by means of the ratio of flow cytometry.The regulatory effects of TL1A on accumulation and infiltration of gut associated lymphoid tissue(GALT)were analyzed.(4)Establish IBD animal models with WT and Tl1a^-/-mice to explore the mechanism of TL1A in regulating the differentiation of CD4⁺T cells in GALT.ELISA was used to detect the cytokines(IL-6,IFN-?,IL-17A,IL-17F,IL-21,IL-22,IL-4,1L-5,IL-10,IL-13)secreted by T cells under the stimulation condition of CD3/CD28 and cytokines(IL-1?,IL-12,IL-23)known to contribute to the innate immune response in LPL and MLN.Flow cytometry was used to analyze Th1,Th2,Th17,and Treg cells related cytokines and nuclear transcription factors(IFN-?,IL-17A,IL-4,IL-10,IL-17F,IL-22,Foxp3,ROR-?t,T-bet,GATA3).(5)Using Tl1a^-/-and WT naive CD4⁺T cells to successfully induce Th1,Th17 and Treg differentiation under T cell differentiation conditions,and analyze Th cell differentiation related cytokines and nuclear transcription factor(IFN-?,IL-17A and Foxp3)to analyze the regulation mechanism of TL1A on Th cell differentiation in vitro.(6)Establish T cell transfer animal model with Rag1^-/-and Tl1a^-/-Rag1^-/-mice to explore the Th cell differentiation in vivo.Dynamically compare and observe the changes of weight,stools,colon length,disease activity index.The histopathological changes of mice intestinal tract were detected by HE staining technology.Flow cytometry was used to analyze Th1 and Th17 cells related cytokines(IFN-?,IL-17A).To investigate TL1A/DR3 signaling play a vital role in regulating the differentiation of various T cell subsets(T-T or T-APC),as well as effector function in vivo.(7)Using Tl1a^-/-and WT mice to explore the functional changes of bone marrow-derived dendritic cell(BMDC).Detect the expression of MHC-II and co-stimulatory molecules CD80 and CD86,and ELISA was used to detect the cytokines(IL-1?,IL-12,IL-23)in supernatant after LPS stimulation.Phagocytotic ability of BMDC was detected by FITC-dextran uptake assay.(8)Tl1a^-/-and WT bone marrow-derived dendritic cells were used to co-culture with WT and Tl1a^-/--derived naive CD4⁺T cells in vitro to induce Th1,Th17 and Treg differentiation.Detect the expression of Th cell differentiation-related cytokines and nuclear transcription factors(IFN-?,IL-17A and Foxp3),and analyze the regulatory mechanism of TL1A in DC on Th cell differentiation in vitro.(9)Tl1a^-/-and WT BMDC were used to explore the molecular mechanism of TL1A in regulating the phagocytosis.Results:(1)Using WT mice to establish DSS induced acute colitis model successfully.Compared with WT control mice,Tl1a m RNA expression in inflamed mucosal tissues was increased,and TL1A protein expression was also upregulated.TL1A were expressed mainly on myeloid dendritic cells(m DC),macrophages,neutrophils and DR3 were expressed mainly on CD4⁺T cells from freshly isolated LPL(p<0.05).(2)Tl1a^-/-mice exhibited alleviated symptoms of DSS-induced acute and chronic colitis compared with WT mice,characterized by reductions in body weight loss,low disease activity index(DAI)and longer survival time.H&E staining of colon sections revealed reduced infiltrating inflamed cells in the lamina propria and the colon length was longer in Tl1a^-/-mice than that in WT mice(p<0.05).(3)Populations of LPL and MLN from Tl1a^-/-and WT mice after DSS administration were analyzed by flow cytometry.The proportions and the absolute numbers of neutrophils and plasmacytoid dendritic cells(p DC)in Tl1a^-/-mice were significantly higher than WT mice;Whereas the proportions and absolute numbers of macrophage and myeloid dendritic cells(m DC)markedly decreased in DSS-treated Tl1a^-/-mice compared with WT mice.In addition,the proportions and the absolute numbers of CD4⁺and CD44⁺CD4⁺T cells had distinctly decreased in DSS-treated Tl1a^-/-mice as compared with WT mice(p<0.05).(4)After establishing acute and chronic DSS-induced colitis,the spontaneous release levels of IFN-?,IL-17A,IL-17F,IL-21,IL-6,TNF?were significantly lower in Tl1a^-/-mice than WT mice,but the levels of IL-10,IL-4,IL-22 significantly higher in Tl1a^-/-mice.A significant reduction in Th1 and Th17 cytokines and Th2 and Treg activity were enhanced in Tl1a^-/-mice(p<0.05).(5)Using naive CD4⁺T cells from WT and Tl1a^-/-mice to establish T cell differentiation system in vitro.Compared with the WT group,Th1/Th17 cells were significantly down-regulated in the Tl1a^-/-mice,while Treg cells were significantly up-regulated(p<0.05).(6)Rag1^-/-and Tl1a^-/-Rag1^-/-mice were used to establish a Th cell differentiation system in vivo.WT and Tl1a^-/-naive CD4⁺T cells transferred into Rag1^-/-hosts respectively,the susceptibility of Tl1a^-/-naive CD4⁺T cells transferred into Rag1^-/-hosts group was decreased,which was manifested in the fact that the weight change rate,disease activity score(DAI),histopathology scores of Tl1a^-/-group were significantly decreased,Th1/Th17 immune response and related cytokines were significantly weakened.In addition,colitis was significantly exacerbated when WT CD4⁺T cells were transferred into Rag1^-/-recipients compared Tl1a^-/-Rag1^-/-recipients respectively.The attenuated intestinal pathology observed in recipients of Tl1a^-/-Rag1^-/-mice correlated with a significantly decreased frequency and number of IFN-?⁺CD4⁺and IL-17A⁺CD4⁺cells(p<0.05).(7)Tl1a^-/-BMDC were stimulated in vitro with LPS for 24 hours,the MFI of costimulatory molecules CD80,CD86 and MHCII;IL-12 and IL-23 concentrations in supernatant were decreased compared with WT BMDC.In addition,a significant decrease in phagocytosis of Tl1a^-/-BMDC(p<0.05).(8)WT or Tl1a^-/-BMDC were co-cultured with Tl1a^-/-or WT naive CD4⁺T cells,Tl1a^-/-BMDC impaired the ability of naive CD4⁺T cells differentiation to Th1/Th17,while enhance Treg response(p<0.05).(9)TL1A is located in the cytoplasm and nucleus,and TL1A promotes TLR4signaling-mediated DC activation thro?gh the DC-SIGN/RAF1/NF?B pathway,releasing a large amount of IL-12 and IL-23,thereby enhancing Th1 and Th17differentiation(p<0.05).Conclusion:(1)TL1A via its effects on colonic Th1 and Th17 cells,plays a novel regulatory role in IBD pathogenesis.(2)TL1A binding to death receptor 3(DR3)promotes Th1 and Th17 cell differentiation in a T-T and DC-T cell interaction manner.(3)TL1A is located in the cytoplasm and nucleus,and TL1A promotes TLR4signaling-mediated DC activation thro?gh the DC-SIGN/RAF1/NF?B pathway.