The Anti-inflammatory Role And Mechanism Of Estrogen Receptor β In Inflammatory Bowel Disease

Background:Ulcerative colitis(UC)and Crohn’s disease(CD)are the two major forms of inflammatory bowel disease(IBD).Although the etiology of IBD remain elusive,it is thought to be multifactorial and primarily involves an aberrant gut immune response to intestinal microbiota.As the crucial mediators in regulating immune responses,CD4~+T cells have been implicated in the pathogenesis of IBD,mainly resulting from the imbalance of effector T(Teff)cells and regulatory T(Treg)cells.In all,modulation of the imbalance will be useful in developing new IBD strategies.Estrogen receptor(ER)βactivation has anti-inflammatory activity,and beneficial effects of ERβagonists in inflammatory disease models have been reported.There is an ample expression of ERβin healthy intestinal mucosa in both men and women,but its expression is markedly down-regulated in active UC and CD.In addition,treatment with ERβ-specific agonists inhibits inflammation in rodent colitis model.However,little is known about how the ERβactivation inhibits the development of colitis,and whether ERβhas a role in regulating intestinal immune responses during the process of colitis has not been clarified.Especially,the expression of ERβin CD4~+T cells as well as its association with IBD severity and whether the ERβactivation can modulate pro-inflammatory and Treg responses are unclear.This study aimed to assess the clinical value of ERβ~+CD4~+T cells in IBD patients and to examine the anti-inflammatory role of ERβactivation in Teff and Treg responses on a Dextran sulfate sodium(DSS)-induced chronic colitis.Methods:1.30 UC patients,31 CD patients and 29 healthy controls(HCs)were recruited.The frequency of circulating ERβ~+CD4~+T cells was analyzed by flow cytometry.We analyzed the ERβexpression in the intestinal tissues by immunofluorescent assays and RT-PCR.2.Male C57BL/6 mice were randomly divided into three groups,control group,colitis group and colitis+ERB041 group.2%DSS was used to induce the experimental chronic colitis.The mice were injected s.c with vehicle(the colitis group)or with a specific agonist of ERβ,ERB041(5 mg/kg body weight)daily.The severity of colitis in individual mice was evaluated by monitoring body weight,stool consistency,rectal bleeding daily in a blinded manner.At the end of the experiment,all mice were sacrificed and their colon tissues were collected to evaluate the colon length and pathological changes by H&E stain.3.We detected the relative levels of mice colonic ERβ,inflammatory cytokines and pro-inflammatory cytokines m RNA transcripts by quantitative RT-PCR analyses.4.We investigated the protective effects of ERB041 on ERβ~+CD4~+T cells,pro-inflammatory T cells(CD4~+CD25~-Teffs,CD3~+CD4~+and CD3~+CD8~+T cells)and Treg responses of colitis mice in vivo by flow cytometric analysis.5.We tested the role of ERβactivation on Treg differentiation and its activity to suppress T-cell proliferation in vitro.Results:1.We found that impaired frequency of circulating ERβ~+CD4~+T cells in IBD patients was negatively correlated with inflammation and disease severity.ERβexpression were downregulated in the intestinal tissues of IBD patients.2.Treatment with ERB041 significantly mitigated the colitis-reduced body weights and colon length,intestinal inflammation and disease severity,and inhibit the release of inflammatory cytokines.3.ERB041 treatment restored the reduced frequency of ERβ~+CD4~+T cells in the spleen and the colonic lamina propria of mice.4.ERB041 treatment inhibited Teff,CD4~+and CD8~+T cells infiltrate and restores Tregs and activated TIGIT~+Tregs quantity in the colon lamina propria.5.ERβactivation enhanced Treg differentiation,immunosuppressive activity and the phosphorylated Smad2/3 signaling in CD4~+T cells in vitro.Conclusions:1.Impaired ERβ~+CD4~+T cells has the potential to be a biomarker for evaluating disease severity of IBD.2.ERB041 has anti-inflammatory effect on mice with chronic colitis,which can significantly restore the colitis-impaired ERβ~+CD4~+T cell population,inhibit the release of inflammatory cytokines in the intestinal tract,reduce excessive pathogenic T cells infiltration and enhance Treg responses in the lamina propria.ERβactivation may be valuable for intervention of IBD by regulating the Teff/Treg balance.3.The activation of ERβmay initiate downstream signaling crosstalk with the TGF-β1-related Smad signaling pathway to promote Fox P3 expression and functional Treg development,leading to the inhibition of autoimmune inflammation.