The Role Of TL1A In Experimental Intestinal Inflammation Associated Intestinal Fibrosis Model

The intestinal fibrosis, a common and serious complication of inflammatory bowel disease (IBD), may develop an intestinal stricture or obstruction. However, the pathogenesis of it remains elusive, and no ideal medical treatments for intestinal fibrosis have become available to date. So, studying the pathogenesis of IBD-associated intestinal fibrosis may offer a new research evidence and strategy for probing an effective therapeutic method.The intestinal fibrosis is a complex and dynamic process, which relates to a dysregulated mucosal immune response, leading to alterations in intestinal cytokines and the deposition of local collagens. These cytokines encompass transforming growth factor-β (TGF-β), insulin-like growth factor IGF), platelet-derived growth factor PDGF) and so on. The research has showed that the activated intestinal fibroblasts (IFBs) and intestinal myofibroblasts (IMFs) by the cytokines multiplied and synthetized resulted in an increased deposition of extracellular matrix (ECM), finally the lumen begins to narrow and the patient complains of obstructive symptoms.The imbalance of collagen catabolic enzymes resulted from chronic inflammation leads to a large number of ECM deposition, including matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) secreted by mesenchymal cells, and then the fibrosis will occur. Some studies indicated that synthesis and activity of MMPs and TIMPs increased in IBD. The mesenchymal cells activated by cytokines are regulated by signal transduction. TGF-β1/Smad3is a key signal transduction in process of fibrosis. The researches in vivo and vitro or in hunman have proved that TGF-(31/Smad3is upregulated in process of fibrosis, and meanwhile the fibrosis induced by mesenchymal cells becomes to strengthen. So, there are important significances to study the fibrotic factors and signal transduction for the pathogenesis of IBD-associated intestinal fibrosis.The tumor necrosis factor-like ligand1aberrance (TL1A) is a protein product of TNF superfamily member15(TNFSF15), which modulates the immune reponse as costimulatory factor of T lymphocyte activation. The latest researches have proved that TL1A was confirmed to be a susceptibility gene in IBD and played a proinflammatory role in process of IBD.The researches launched by Dr. Targan and his team have demonstrated that the higher expression of TL1A/DR3can induce the Th cells to polarize and aggravate inflammation, and dysfunctional immunoregulation leads to the emerging of IBD. Dr.Targan’s group found that TL1A over-expressed in lymphoid and myeloid cells would lead to intestinal inflammation and fibrosis. These results suggest that TL1A plays a crucial role in T cells and APCs of the intestinal mucosa in IBD, and upregulation of TL1A expression can promote inflammation and fibrosis of intestinal mucosa. Although the role of TL1A in IBD has improved, its influence in IBD-associated intestinal fibrosis is still unclear, and whether the anti-TL1A antibody may relieve fibrosis needs to be investigated. The purpose of the present study is to probe the role of TL1A in intestinal fibrosis and collagen metabolism with wild type (WT) mice and LCK-CD2-TL1A-GFP transgenic (L-Tg) mice. The experiment contains three parts as below:Part1:The role of TL1A in experimental colitis induced by TNBSObjective:To establish experimental colitis induced by TNBS for exploring the role of TL1A in experimental colitis.Methods:(1) LCK-CD2-TL1A-GFP Tg mice were identified by RT real-time PCR.(2) The WT and Tg mice were randomly divided as followed: a. Control/WT group:wild type mice were adminstered distilled water (n=6); b. EtOH/WT group:wild type mice were adminstered50%ethanol (n=6); c. TNBS+EtOH/WT:wild type mice were adminstered TNBS dissolved in a volume of150μL of50%ethanol (n=6); d. Control/Tg group:wild type mice were adminstered distilled water (n=6); e. EtOH/Tg group:wild type mice were adminstered50%ethanol (n=6); f. TNBS+EtOH/Tg group:wild type mice were adminstered TNBS dissolved in a volume of100μL of50%ethanol (n=6).The mice were deprived of feed for24h and were highly anesthetized with aether. A polyethylene catheter was inserted rectally until the splenic flexure about3-4cm to the anus, and adminstered100μL of TNBS solution contain2.0mg,3.0mg TNBS respectively into the colon lumen weekly. The mice were harvested3days after4weeks.(3) Severity of colitis was evaluated by changes of body weight (BW), disease activity index (DAI), colon histology and pathology score, changes of myeloperoxidase (MPO) in colon.(4) The levels of TNF-a, Interferon-y (IFN-y) and interleukin-17A (IL-17A) in serum were detected by ELISA assay.(5) The expressions of TNF-a, IFN-y and IL-17A were dected by western blot and real-time Q-PCR, respectively.Results:(1) The TL1A transgenic mice were identified by PCR. The TL1A DNA located at192bp in Tg mice and did not express at192bp in WT mice.(2) The proof of colitis showed that BW had greater loss in the TNBS+EtOH group compared with that in the Control group and EtOH group. DAI and pathology scores in the TNBS+EtOH group were higher than that of the Control and EtOH group, the length of colon was shorter. The degree of congestion and edema of colon, inflammation infiltration into mucosa superficial layers was much severer in the TNBS+EtOH group. Meanwhile, inflammation infiltrations were much more severer in TNBS+EtOH/Tg when compared to that in TNBS+EtOH/WT group, whereas no significant differences were found between the Control group and the EtOH group both in Tg or WT mice.(3) The data of MPO manifested that inflammatory reaction increased in the EtOH group compared to that in the Control group and the EtOH group. In TNBS+EtOH/Tg group, the activity of MPO showed much higher than that in TNBS+EtOH/WT group.(3) The levels of TNF-α, IFN-γ, IL-17A increased in the TNBS+EtOH group compared with that in the Control group and the EtOH group by ELISA assay, TNBS+EtOH/Tg group showed higher expressions of TNF-α, IFN-γ, IL-17A than that in TNBS+EtOH/WT group, and no obvious diffences were obversed between the Control group and the EtOH group.(4) The data dectetd by western blot and Real Time Q-PCR and showed that, the protein and mRNA expressions of IL-17, TNF-α, IFN-γ were increased in the TNBS+EtOH group compared with that in the Control group and the EtOH group, and the data indicated that the synthesis and secretion of inflammatory factors increased more obviously in the TNBS+EtOH/Tg group, and no significant differences were found between the Control group and the EtOH group both in Tg or WT mice.Conclusions:The inflammation emerged gradually as TNBS applied intracolonically. The inflammation was much severer in Tg-Model group. The evidence indicated that TL1A may promote the inflammation by upregulating the levels of TNF-α, IFN-γ and IL-17A.Part2:The role of TL1A in experimental colitis associated intestinal fibrosisObjective:To elucidate the role of TL1A in colitis associated intestinal fibrosis induced by intrarectal administration of TNBS.Methods:(1) The process of inducing colitis and experiment groups was the same as the Part1.(2) The deopsitons of ECM was by Masson’s trichrome staining and Sirius red staining.(3) The expressions of collagen I, collagen III, TIMP-1, Vimentin, α-SMA, and TGF-β1/Smad3were dected by immunohistochemistry, western blot and real-time Q-PCR, respectively.Results:(1) The thickness and collagen deposition of colon were increased in the TNBS+EtOH group, which were much severer in TNBS+EtOH/Tg group when compared to TNBS+EtOH/WT group, whereas no significant difference were found between the Control group and the EtOH group both in Tg or WT mice.(2) The data detected by immunohistochemistry showed that, the protein expressions of collagen Ⅰ, collagen Ⅲ, Vimentin, a-SMA were increased in the TNBS+EtOH group compared with that in the Control group and the EtOH group, and the data indicated that the synthesis and secretion of collagen increased more obviously with IFBs being activted in the TNBS+EtOH/Tg group. TGF-β1expressed slightly in colon from the Control group, but Smad3and TIMP-1either express a little or nothing. The level of all above proteins increased a bit in the EtOH group, however no statistical discrepancy was found compared to the Control groups. This may be caused by damages of intestinal mucous epithelium with50%ethanol and then normal wound-healing process started, and no statistical discrepancy was found between Tg mice and WT mice. In contrast, the level of all above proteins increased obviously both in TNBS+EtOH/Tg group and TNBS+EtOH/WT group, especially in TNBS+EtOH/Tg group, and their expressions located at cytoplasm and epicyte in each layers of colon.(3) The results dectetd by Real time Q-PCR and Western blot showed that, the levels of collagen I, collagen III, TIMP-1, Vimentin, a-SMA and TGF-β1\Smad3were increased in TNBS+EtOH compared with that in the Control group and the EtOH group both in Tg or WT mice, the level of expression in TNBS+EtOH/Tg group were higher than that in TNBS+EtOH/WT group, there were no statistical discrepancy compared to the Control group.Conclusions:The inflammation associated intetinal fibrosis model was established by TNBS applied intrarectally. The signal pathway of TGFβ1/Smad3could mediate IFBs to change the expression of collagen metabolism enzymes, and the disorder of collagen metabolism promoted the depostion of ECM and leaded to fibrosis. The TL1A may promote the intestinal fibrostenosis.Part3:The regulation effects of anti-TL1A antibody on experimental colitis associated intestinal fibrosisObjective:To explore regulation effects of anti-TL1A antibody on intestinal fibrosis in adoptive T-cell transfer colitis.Methods:(1) The adoptive transfer colitis model was induced by intraperitoneal injection of CD4+CD45RBhigh naive T cells isolated from either C57BL/6wild type (WT) mice or LCK-CD2-TL1A-GFP transgenic (L-Tg) mice to Rag-/-mice. The colitis model mice were treated prophylactically with a dose of500μg anti-TL1A antibody twice per week after T-cell transfer for6weeks; The colitis model mice were treated therapeutically with a dose of500 μg anti-TL1A antibody twice per week in the forth week after T-cell transfer or when weight loss is more than10%of their original body weight for4weeks; The mice were treated with Isotype prophylactically and therapeutically. The Rag-/-mice injected were randomly divided as followed: a.WT-Prevention Isotype group:CD4+CD45RBhigh naive T cells isolated from WT mice treated with Isotype prophylactically; b.WT-Prevention Ab group: naive T cells isolated from WT mice treated with anti-TL1A antibody prophylactically; c.Tg-Prevention Isotype group:naive T cells isolated from Tg mice treated with Isotype prophylactically; d.Tg-Prevention Ab group: naive T cells isolated from Tg mice treated with anti-TL1A antibody prophylactically; e.WT-Treatment Isotype group:naive T cells isolated from WT mice treated with Isotype therapeutically; f.WT-Treatment Ab group: naive T cells isolated from WT mice treated with anti-TL1A antibody therapeutically; g.Tg-Treatment Isotype group:naive T cells isolated from Tg mice treated with Isotype therapeutically; h. Tg-Treatment Ab group:naive T cells isolated from Tg mice treated with anti-TL1A antibody therapeutically;(2) Haematoxylin and eosin staining (H&E staining), Masson’s trichrome staining (MT staining) and sirius red staining were used to detect colon histopathological changes;(3) The expressions of collagen Ⅰ, collagen Ⅲ, TIMP1, Vimentin, α-SMA and TGF-β1/Smad3were detected by immunohistochemical staining.Results:(1) H&E, MT staining and sirius red staining confirmed that:the colitis-associated intestinal fibrosis was established by adoptive T-cell transfer. In the Tg-Prevention Isotype group and Tg-Treatment Isotype group, the structures of colon were destroyed, granulocytes infiltration and collagen deposition into the mucosa superficial layers were increased and the pathology score of inflammation and fibrosis were more severer than that in the WT-Prevention Isotype group and WT-Treatment Isotype group. After administration of anti-TL1A antibody, the pathology score of inflammation and fibrosis decreased in the WT-Prevention Ab group, Tg-Prevention Ab group, WT-Treatmeant Ab group, Tg-Treatment Ab group, WT-Prevention Ab group compared with that in WT-Prevention Isotype group, Tg-Prevention Isotype group, WT-Treatment Isotype group, Tg-Treatment Isotype group; There were no statistical discrepancy between WT-Prevention Isotype group and WT-Treatment Isotype or Tg-Prevention Isotype and Tg-Treatment Isotype.(2) The expressions of Vimentin and a-SMA in colon by immunohistochemistry assay:the overexpressions of Vimentin and a-SMA in colon demonstrated that the IFBs were activated and multiplied. In the Tg-prevention Isotype group and Tg-Treatment Isotype group, the expressions of collagen Ⅰ, collagen Ⅲ, TIMP-1, TGF-β1/Smad3in colon were increased and the rates of postive expressions were more higher than that in the WT-Prevention Isotype group and WT-Treatment Isotype group. After administration of anti-TL1A antibody, rates of postive expressions decreased in the WT-Prevention Ab group, Tg-Prevention Ab group, WT-Treatment Ab group, Tg-Treatment Ab group, WT-Prevention Ab group compared with that in WT-Prevention Isotype, Tg-Prevention Isotype group, WT-Treatment Isotype group, Tg-Treatment Isotype group; There were no statistical discrepancy between WT-Prevention Isotype and WT-Treatment Isotype or Tg-Prevention Isotype group and Tg-Treatment Isotype. There were no statistical discrepancy between WT-Prevention Isotype group and WT-Treatment Isotype group or Tg-Prevention Isotype and Tg-Treatment Isotype group.Conclusions:The adoptive T cells from either WT mice or Tg mice transfer can induce colitis associated intestinal in Rag1-/-mice, and the obvious changes were noted in Tg mice. TL1A may promote the immunoreaction which activate IFBs to synthesis ECM, anti-TL1A antibody alleviate the changes of inflammation and fibrosis in Prevention groups and Treatment groups, especially in the Prevention group, the decreasing level of fibrosis maybe achieve due to blocking the development of infammation mediated by TL1A.