IL-33 Alleviates Murine Ulcerative Colitis By Regulating Intestinal Neurotrophic Factor Expression

Inflammatory bowel disease（IBD）,which mainly includes ulcerative colitis（UC）and Crohn’s disease（CD）,is a complex chronic intestinal disease with an unclear pathogenesis.IBD has been short of cure methods,which can only induce remission and reduce relapse through surgery,drugs and so on.Therefore,it is very important to clarify the pathogenesis of IBD and look for new drugs to treat this disease.Interleukin 33（IL-33）is a pluripotent cytokine of the IL-1 family,which regulates gene expression as a nuclear transcription factor.During infection or cell injury,IL-33 is released into tissue as an"alarm pheromone’’,activating a variety of immune cells via ST2 receptor and playing an immunomodulatory role in inflammatory diseases.There have been many studies on the role of IL-33 in IBD,but the exact mechanism is still controversial.Glial cell line-derived neurotrophic factor（GDNF）is a distant relative of the TGF-βsuperfamily and widely exists in a variety of tissues,such as brain,intestine,kidney,etc.GDNF is considered to play an important role in maintaining the integrity of the intestinal epithelium and alleviating intestinal inflammation.Our recent studies found that IL-33binds to ST2 receptor,and promotes the secretion of multiple trophic factors by astrocytes to reduce ischemic brain damage.Therefore,we further studied the potential effect of IL-33 in ulcerative colitis in mice.Objectives:To investigate the effect of IL-33 on the expression of neurotrophic factor from intestinal epithelial cells in murine ulcerative colitis and its in the progression of IBD.Methods:The acute colitis model was induced by dextran sulfate sodium（DSS）.the difference between wild type（WT）and IL-33 receptor deficiency(ST2^-/-)mice was investigated.After treatment with IL-33,the changes of body weight,disease activity index（DAI）and histopathology were measured to evaluate the macroscopic and microscopic pathological damage of the experimental mice.The integrity of intestinal mucus layer was determined by AB-PAS staining.The expression and distribution of IL-33,ST2,tight junction protein,RET,GDNF family ligands and MLCK in colonic tissue were detected by RT-PCR,Western blot and immunohistochemistry.The cell experiments were mainly based on 3D organoid to establish a DSS-induced intestinal epithelial barrier injury model.The effects of IL-33 on the permeability,tight junction protein,neurotrophic factors and receptor expression of intestinal organs were detected.Results:1.The role of IL-33 in DSS-induced ulcerative colitis in miceThe murine acute colitis model was induced by drinking 2%DSS for7 days.IL-33 treatment increased the body weight and the number of goblet cells,and relieved the pathological damage of colon tissue.Western blot and immunofluorescence results showed that IL-33significantly up-regulated the expression of ZO-1,Claudin-1 and Occludin.Compared with the WT mice,ST2^-/-mice received DSS showed significant weight loss,severe colonic atrophy,increased DAI score and histopathological score,severe intestinal barrier function impairmen.IL-33 treatment improved the WT mice pathological injury,but no effect on ST2^-/-mice.2.IL-33 alleviates ulcerative colitis via GDNF/RET signal pathwayRT-PCR and immunohistochemical staining results showed that IL-33 up-regulated the level of GDNF family ligand（GDNF,ARTN,NRTN,PSPN）and receptor RET in mice with ulcerative colitis.Treatment with the RET kinase receptor inhibitor GSK3179106（GSK31）impeded the effect of IL-33 on ulcerative colitis,and resulted in weight loss,severe colonic atrophy,significantly increased DAI score and histopathological score,decreased tight junction protein expression,and increased MLCK expression.While GDNF can alleviate ulcerative colitis and repair intestinal epithelial barrier.3.The mechanism of IL-33 in repair intestinal epithelial barrier injuryEnteroids induced by DSS showed significantly reduced viability and increased enteroids permeability.IL-33 increased the viability of enteroids,decreased the enteroid permeability,up-regulated the expression of GDNF,receptor RET and tight junction protein m RNA and decreased the level of MLCK m RNA.Correspondingly the effect of IL-33 on ST2^-/-enteroids induced by DSS was incapable.The effect of IL-33 on repairing WT enteroids barrier was inhibited after blocking RET kinase.GDNF similarly decreased enteroid permeability,up-regulated tight junction protein m RNA expression,and inhibited MLCK m RNA expression after treatment with MLCK inhibitor,ML-7 hydrochloride（ML-7）.Conclusion:By binding to its specific receptor ST2,IL-33 promotes the expression of GDNF and its receptor RET,down-regulates the expression of permeability regulator protein MLCK,up-regulates the production of tight junction proteins,and improves the permeability of intestinal epithelial barrier,thus promoting the repair and functional remodeling of intestinal epithelial barrier in murine ulcerative colitis.