Fucose Improves Chronic Colitis Through AHR/IL-22 Pathway

Objective: The effect of fucose on inflammatory bowel disease(IBD)varies from one research to another,and it remains unclear how fucose ameliorates IBD.It is reported that IL-22 enhanced intestinal expression of fucosyltransferase 2(FUT2)and fucosylation in intestinal epithelium and mucus,and it remains to be proved whether exogenous fucose increases the level of IL-22 in gut.Whether fucose improves gut microbial dysbiosis in mice with chronic colitis also needs to be clarified.This study is designed to explore the impact of fucose on intestinal inflammation,colonic IL-22 and microbial dysbiosis in multiple sites of gastrointestinal tract in mice with chronic colitis,and figure out the underlying mechanism.Methods: C57BL/6 mice were divided into control(CON),treatment with fucose(FUC),chronic colitis(DSS)and chronic colitis treated with fucose(DF).We monitored disease activity index(DAI)and weight of all mice daily in the process of modeling,and colons were separated for recording their length and performing hematoxylin-eosin(HE)staining to assess colonic histology.Mucosal samples and luminal samples in colon and small intestine were collected from all mice for 16 S r DNA sequencing to learn the influence of fucose on gut microbiota.q PCR was used to assess transcription of IL-1,IL-6,TNF-α,CXCL1,IL-22,FUT2,NOTCH,Hes,STAT5 and CYP1A1,ELISA was performed to determine the expression of IL-22,and Western blot was used to detect IL-1,IL-6,TNF-α,IL-22,FUT2,CYP1A1,and nuclear AHR.Immunomagnetic beads were used to isolate CD4+ T cells from spleen of mice,and these cells were separated into three groups: Control,Fucose(intervention with Fucose)and Fucose+CH223191(intervention with Fucose and CH223191 which is AHR inhibitor).The level of IL-22,AHR and CYP1A1 were compared among these three groups.Lymphocytes were separated from spleen of mice and divided into three groups described above,and flow cytometry was performed to evaluate ratio of CD4+ IL-22+ cells in splenic lymphocytes.Colonic lamina propria mononuclear cells(LPMC)separated from mice were also divided into Control,Fucose and Fucose+CH223191.IL-22,AHR,CYP1A1 and ratio of CD4-RORγt+ IL-22+ cells and CD4-RORγt+ AHR+ cells were compared among these three groups.Results: ⑴ In comparison with CON,DSS mice showed significantly lower body weight change rate,decreased length of colon,increased DAI and histological score,more severe inflammatory infiltration of colon mucosa and more crypt destruction,which were all improved significantly by fucose.⑵ Mice in DSS exhibited increased level of proinflammatory cytokines and chemokine compared with CON,involving IL-1,IL-6,TNF-α and CXCL1,which were downregulated by fucose.Mice in DSS group showed lower level of IL-22 and FUT2 than CON,and fucose upregulated transcription and expression of IL-22 in DSS,as well as FUT2.⑶ Transcription factors for regulating IL-22,involving NOTCH,Hes and STAT5,were not affected significantly by fucose in DSS group,but fucose could enhance CYP1A1(a target gene of AHR)and expression of nuclear AHR.⑷ In vitro,fucose increased level of IL-22,AHR and CYP1A1 in CD4+ T cells,and promoted the proportion of CD4+ IL-22+ cells in splenic lymphocytes.CH223191 inhibited IL-22,AHR and CYP1A1 in CD4+ T cells treated with fucose,and decreased the ratio of CD4+ IL-22+ cells in splenic lymphocytes stimulated with fucose.⑸ Moreover,fucose increased the level of IL-22,enhanced transcription of AHR and CYP1A1,and elevated proportion of CD4-RORγt+ IL-22+ cells and CD4-RORγt+ AHR+ cells in LPMC.IL-22,AHR and CYP1A1 of LPMC were suppressed,and ratio of CD4-RORγt+ IL-22+ cells and CD4-RORγt+ AHR+ cells were reduced in Fucose+CH223191 compared with Fucose group.⑹ As far as luminal and mucosal microbiota in colon and small intestine were concerned,fucose increased microbial diversity,restored microbial structure to CON,adjusted the distribution of microbial phyla and genera,and improved relative abundance of some genera and microbial functions in DSS.Conclusion: Fucose ameliorated inflammatory phenotype,inhibited colonic chemokine and inflammatory cytokines,and increased the expression of IL-22(a critical factor for intestinal homeostasis)and FUT2(a protective factor for gut barrier)in chronic colitis.Experiment in vitro demonstrated fucose promoted the production of IL-22 via AHR in CD4+ T cells and innate lymphoid cells(ILC).Furthermore,fucose improved microbial dysbiosis in lumen and mucosa of colon and small intestine moderately in chronic colitis.