Effect And Mechanisms Of Constitutive TL1A On Epithelial-mesenchymal Transition During Chronic Colitis-related Intestinal Fibrosis

Intestinal stenosis and obstruction caused by intestinal fibrosis,are common complications of inflammatory bowel disease(IBD).Epithelial-mesenchymal transition(EMT)is one of the important mechanisms of intestinal fibrosis.During this process,epithelial cells lose cell polarity and intercellular connections,and their epithelial cell markers such as E-cadherin gradually disappear,while mesenchymal cell markers such as fibroblast-specific protein 1(FSP1),?-SMA gradually increase.A variety of cytokines and protein molecules can induce EMT.Many studies reported that EMT involved a large cellular signaling pathway and complex gene regulation processes,of which TGF-?/Smad is the most important EMT pathway.Tumor necrosis factor ligand-related molecule 1A(TL1A)is a member of the Tumor necrosis family(TNF),and its receptor Death receptor 3(DR3)is mainly expressed in CD4~+T lymphocytes,NK cells and CD8~+T lymphocytes.Recent studies has found that DR3 is also expressed in epithelial cells.The combination of the two can initiate a series of immune responses,such as the activation of T cells,innate lymphocytes,mononuclear macrophages and epithelial cells,and release inflammatory factors such as TNF-?,IL-17,IFN-?.Studies had shown that spontaneous colon inflammation in TL1A transgenic mice was related to increased IL-13 expression,and high level IL-13 could promote TGF-?1 secretion.Therefore,whether TL1A can activate the classical TGF-?/Smad pathway of EMT by directly binding to its receptor or indirectly secreting the fibrogenic factor IL-13 is still unknown.We studied the correlation between TL1A expression and markers of EMT in intestinal mucosa of patients with IBD-associated intestinal fibrosis to further validate the role of TL1A in EMT of intestinal fibrosis.Next,we used TL1A transgenic mice and HT-29 cells to investigate the effects and mechanisms of TL1A in EMT of intestinal fibrosis.We designed the experiment of the following three parts:Part one TL1A affected epithelial-mesenchymal transition in intestinal mucosal epithelial cells of patients with IBD-related intestinal fibrosis.Objective:To investigate the correlation between the expression of TL1A and EMT in intestinal mucosa of patients with IBD-related intestinal fibrosis.Methods:The study included 12 patients with UC-related intestinal fibrosis,10patients with CD-related intestinal fibrosis,and 8 individuals with control group.Firstly,we evaluated the degree of inflammation and fibrosis by H&E staining,Sirius red staining and Masson staining.Immunohistochemistry was used to determine the expression of TL1A and the indicators related with EMT,including E-cadherin,FSP1 and?-SMA.Then we further explored the correlation between TL1A and the indicators related to EMT.The expression of IL-13,TGF-?1,Smad3 and EMT related transcription molecules such as Snail1 and ZEB1 were also detected by immunohistochemistry.Results:Compared with the control group,mucosal inflammation and fibrosis were observed in UC and CD patients,and the expression of TL1A was significantly increased in the colonic mucosa in UC and CD patients.Further correlation analysis showed that the expression of TL1A was positively correlated with the degree of fibrosis,and with the interstitial markers FSP1,?-SMA,but negatively correlated with the epithelial marker E-cadherin.Compared with the control group,immunohistochemical showed that the expression of IL-13,TGF-?1,Smad3,Snail1 and ZEB1 in colonic mucosa was increased in UC group and CD group.Conclusions:TL1A is associated with intestinal fibrosis in patients with IBD and may be involved in the process of epithelial-mesenchymal transition in intestinal fibrosis via the TGF-?/Smad3 pathway.Part two TL1A induced epithelial-mesenchymal transition in chronic colitis-associated intestinal fibrosisObjective:To investigate the effect of TL1A on EMT during the formation of intestinal fibrosis in experimental colon mice and in epithelial cell line HT-29.Methods:We divided TL1A Transgenic mice and C57BL/6 WT mice into four groups:Con/WT,DSS/WT,Con/Tg,and DSS/Tg.Chronic colitis was induced by oral administration of 2%DSS for 7 days followed by a recovery period of2 weeks with normal drinking water.This was repeated three times to establish a fibrotic model.At the end of modeling,we evaluated the colonic inflam-mation and fibrosis in mice.Furthermore,we used immunofluorescence,immunohistochemical staining,RT-PCR and Western Blot to detected the expression of E-cadherin,FSP1 and?-SMA.HT-29 cells were treated with different concentrations of TL1A at different time points,and the proliferation rate of HT-29 cells was detected by CCK-8,in order to obtain the optimal stimulation concentration and time of TL1A.The mean density of E-cadherin~+FSP1~+was analyzed by immunofluorescence.To assess the concentration and time dependence of TL1A induced epithelial-mesenchymal transition in HT-29 cells,the E-cadherin,FSP1 and?-SMA proteins were analyzed by Western Blot.Results:Compared with the control group,the DSS group showed more severe inflammation,more weight loss,higher DAI score and MPO score.Moreover,mice in the DSS/Tg group had more colonic inflammation compared with the DSS/WT group.Compared with the control group,the length of colon in the DSS group was shortened,the colonic fibrosis was more severe in the DSS/Tg group than in the DSS/WT group.Immunofluorescence showed that the expression of E-cadherin~+FSP1~+in the DSS group increased compared with the Control group,and that in DSS/Tg group was more than that in the DSS/WT group.We further found that the expression of E-cadherin protein and mRNA levels in the DSS group decreased than the control group by Immunohistochemical,Western Blot and RT-PCR,and the expression of E-cadherin in DSS/Tg group decreased more.While the expression of FSP1and?-SMA protein and mRNA were increased in the DSS/Tg group.Immunofluorescence staining and Western Blot showed that TL1A significantly increased FSP1 and?-SMA expression and reduced E-cadherin expression.Conclusions:In DSS-induced chronic colitis mice,the intestinal collagen deposition was more obvious and the degree of fibrosis was more severe,while the expression of epithelial cell markers were decreased.The DSS/Tg group changed more significantly,suggesting that TL1A may promote intestinal fibrosis in mice with chronic colitis through EMT.TL1A induced EMT in the epithelial HT-29 cell line in vitro.Part three Mechanisms of TL1A promoting epithelial-mesenchymal transition in chronic colitis-associated intestinal fibrosisObjective:To explore the mechanisms of TL1A promoting epithelial-mesenchymal transition in intestinal fibrosis.Methods:Mice were grouped as before.For experimental mice,the expression levels of IL-13 and TGF-?1 in serum were detected by Elisa,The protein and mRNA expression of TGF-?/Smad3 pathway and EMT-related transcription molecules such as Snail1 and ZEB1 were detected by Western Blot and RT-PCR.Next,HT-29 cells were divided into five groups:Control,TL1A,TL1A+TL1A Ab,TL1A+BMP-7,and TL1A+TL1A Ab+BMP-7.Immuno-fluorescence was applied for the colocalization E-cadherin~+FSP1~+,and the expression of EMT-related markers and transcription factors were further detected by Western Blot to investigate the effect of TL1A on epithelial-mesenchymal transition in epithelial cells.Results:In mice,IL-13,TGF-?/Smad3 pathway protein,Snail1 and ZEB1 protein levels and mRNA levels were elevated,and serum IL-13 and TGF-?1expression were elevated,especially in the DSS/Tg group.Epithelial-mesenchymal transition can occur after induction of TL1A.Expression of E-cadherin were increased in TL1A Ab and/or BMP-7 group,while the expression of interstitial markers FSP1 and?-SMA were decreased,and TL1A+TL1AAb+BMP-7 group changed most obviously.Conclusions:TL1A may induce epithelial-mesenchymal transition in intestinal epithelial cells by interfering with the TGF-?/Smad3 pathway,which through affected the EMT-associated transcription factors Snail1 and ZEB1.