Regulation Of TL1A On Intestinal Dendritic Cells In Chronic Experimental Colitis

Inflammatory bowel disease(IBD)is a group of inflammatory conditions of the colon and small intestine.Crohn's disease(CD)and ulcerative colitis(UC)are the principal types of IBD.The aberrant mucosal immunology that consists of innate immune and adaptive immune plays a key role in IBD.Intestinal dendritic cells(DCs)are one of the most important group of innate immune cells.Their main functions are processing antigen material and presenting it on the cell surface to the T cells of the immune system.Firstly,intestinal DCs are active and expressed with high T-cell activation stimulation molecules,such as CD80 and CD86.Multiple clinical studies proved more active DCs in inflammatory site of intestine.Secondly,DCs immigrate to Payer's node and Mesenteric lymph nodes(MLN)via chemokine receptor 2(CCR2),CCR5 or CCR7 pathway.Finally,dendritic cells present antigenic peptides in association with major histocompatibility complex class II(MHCII)to na?ve CD4~+T lymphocytes,which promote na?ve T cells differentiate into different T helper cells and thus activates adaptive immune.Recently,Tumor necrosis factor-like ligand 1 aberrance(TL1A)has been proved to connect innate immune and adaptive immune.Previous studies mainly focused its role in adaptive.Shih et al showed that mice with constitutive TL1A expression in myeloid cells developed spontaneous enteritis.However,the regulation of TL1A in DCs activation,migration and antigen presentation hasn't yet to be addressed.Nuclear factor-kappa B(NF-?B)pathway has been shown to participate in DCs maturation.Human vascular endothelial growth inhibitor(VEGI,TNF superfamily 15)-stimulated bone immature marrow-derived DCs(BMDC)displayed early activation of maturation signaling molecules NF-?B p50(65)and p-I?B?.As a VEGI isoform,TL1A does not induce similar activities under otherwise identical experimental conditions.To further investigated its role in regulation of DCs activation,we generated FMS-TL1A-GFP-transgenic(over 70%DCs express GFP)and wild-type mice and intestine DCs and BMDCs were isolated.Dextran sulfate sodium(DSS)-induced chronic colitis model was established to determine how TL1A regulate activation,migration and T cell stimulation function of DCs in vivo and in vitro.In addition,this work will determine the regulation role of TL1A in NF-?B signaling pathway and provide important therapeutic revenue for treatment in IBD.Part one The regulation of TL1A on colon inflammation in chronic experimental colitis Objective:To investigate the effects of TL1A on colon inflammation and T cell activation in chronic experimental colitis.Methods:1.Both Tg mice and WT mice were administrated by 2.5%DSS drinking water discontinuously to induce chronic colitis.2.Severity of colitis was evaluated by body weight(BW)change,the disease activity index(DAI),colon morphology,histopathology change and myeloperoxidase(MPO)activity.3.Expressions of CD44 and CD69 in MLN and LPMC were detected by FACS.Interferon-?(IFN-?)and Interleukin-17A(IL-17A)in serum were measured by ELISA.MLN cells and Lamina propria mononuclear cells(LPMC)were measured by FACS.4.Expressions of TL1A and DR3 in colon were detected by Real-time qPCR and Western blot.Results:1.Significantly increased DAI,gross inflammation score,histological score and MPO activity in Tg group were observed in DSS-induced chronic experimental colitis models as compared to WT group mice(P<0.05).The colon length in Tg group was shorter than in WT group,but there was no difference between two groups(P>0.05).2.The expressions of CD44 and CD69 in MLN and LPMC were significantly higher in Tg group as compared to WT group(P<0.05).The level of IFN-?and IL-17A in serum and supernatant of MLN cells and LPMC were significantly increased in Tg group(P<0.05).The percentage of CD4~+IFN-?~+and CD4~+IL-17A~+cells in MLN and LPMC from Tg group were significantly higher than in WT group(P<0.05).3.Significantly increased TL1A and DR3 were observed in Tg group as compared to WT group(P<0.05).Conclusions:1.Myeloid cells with constitutive expression of TL1A and wild type mice were used to build DSS induced chronic experimental colitis.2.Myeloid cells with constitutive expression of TL1A promoted the expression of CD44 and CD69 which are T cells activation markers.3.Myeloid cells with constitutive expression of TL1A enhanced differentiation of Th1 and Th17 cells,upregulated the level of IFN-?and IL-17A,and thus aggravated colonic inflammation.Part two The regulation of TL1A on dendritic cells activation in chronic experimental colitis Objective:To investigate the effects of TL1A on dendritic cells activation in chronic experimental colitis.Methods:1.The model of chronic colitis was established the same as the Part One..2.Percentages of intestinal DCs were detected by both FACS and IF.Expression of CD44 and CD69 in MLN and LPMC were detected by FACS.3.Level of IL-1?,TNF-?and IL-12/23 p40 in serum were measured by ELISA.The number of MLN cells and LPMC were measured by FACS.4.Expressions of NF-?B pathway of colon were detected by Western blot.5.Expressions of chemokine receptors of colon were detected by Real-time qPCR and Western blot in chronic experimental colitis.Results:1.The expressions of CD80 and CD86 in MLN and LPMC were significantly higher in Tg group as compared to WT group(P<0.05).2.The levels of IL-1?,TNF-?and IL-12/23 p40 in serum and supernatant of MLN cells and LPMC were significantly increased in Tg group(P<0.05).The percentage of IL-1?,TNF-?and IL-12/23 p40 positive DCs in MLN and LPMC from Tg group were significantly higher than in WT group(P<0.05).3.Significantly increased phosop-NF-?B p65 and phosop-I?B?protein were observed in Tg group as compared to WT group,whereas total I?B?protein was significantly decreased in Tg group(P<0.05).4.Significantly increased CCR2,CCR5,CCR7 and CX3CR1 mRNA were observed in Tg group as compared to WT group(P<0.05).Increased CCR2,CCR5,CCR7 and CX3CR1 protein were observed fin Tg group as compared to WT group(P<0.05),however there's no significance in CCR2protein expression(P>0.05).Conclusions:1.Myeloid cells with constitutive expression of TL1A were found to increase the number of intestinal DCs.2.Myeloid cells with constitutive expression of TL1A promoted the expression of CD80 and CD86 which are DCs activation markers via activation of NF-?B pathway.3.Myeloid cells with constitutive expression of TL1A increased the number of DCs in MLN and promoted the expression of CCR2?CCR5?CCR7and CX3CR1,which enhanced migration of intestinal DCs.Part three The Regulatory effect of TL1A on bone marrow derived dendritic cellsObjective:To investigate the regulation of TL1A on bone marrow derived dendritic cells function.Methods:1.Expressions of TL1A and DR3 in BMDC were detected by Real-time qPCR and Western blot.2.Phagocytotic ability of BMDC was detected by FITC-dextran uptake assay.Mixed lymphocyte reaction(MLR)assay was used to detect the ability of BMDC on stimulation of CD4~+T cells.3.Level of IL-1?,TNF-?and IL-12/23 p40 in supernatant of BMDC were detected by ELISA.4.Expression of NF-?B pathway of BMDC was detected by Western blot.5.Expression of chemokine receptors of BMDC were detected by Real-time qPCR and Western blot.Results:1.Significantly increased TL1A and DR3 were observed in Tg group as compared to WT group(P<0.05).2.The percentage of BMDC uptake of FITC-dextran and median fluorescence intensity from Tg group was significantly higher than WT group(P<0.05).The stimulation index of BMDC from Tg group co-cultured with T cells was significantly higher than WT group(P<0.05).Tg group increased the expression of CD44 and CD69 significantly as compared to WT group(P<0.05).3.The level of IL-1?,TNF-?and IL-12/23 p40 in supernatant of BMDC from Tg group were significantly higher than in WT group(P<0.05).4.Significantly increased phosop-NF-?B p65 and phosop-I?B?were observed from Tg group as compared to WT group,whereas total I?B?protein was significantly decreased from Tg group as compared to WT group(P<0.05).5.Significantly increased CCR2,CCR5,CCR7 and CX3CR1 mRNA were observed from Tg group as compared to WT group(P<0.05).Increased CCR2,CCR5,CCR7 and CX3CR1 protein were observed from Tg group as compared to WT group,however there's no significance in CX3CR1 protein expression(P>0.05).Conclusions:1.TL1A could enhance the phagocytotic ability of BMDC and promote T cell-dependent stimulation.2.TL1A promoted the production of IL-1?,TNF-?and IL-12/23 p40 in BMDC.3.TL1A could promote activation and migration of DCs via activating NF-?B pathway and upregulating the expression of chemokines receptors.