Mechanism Of 5-HT/5-HT₇R Signaling In Regulating Regulatory B Cells In Patients With Ulcerative Colitis

Objective:The etiology and pathogenesis of ulcerative colitis（UC）is not clear,and accumulating evidence suggests that gastrointestinal neuroendocrine peptide/amine interaction with immune cells is one of the important pathogenic mechanisms.Regulatory B cells（Bregs）,as immunoregulatory cells,play an important role in maintaining intestinal immune homeostasis.The previous study of our group found that the number of Bregs decreased in UC patients at the active stage.Serotonin（5-hydroxytryptamine,5-HT）produced by intestinal Enterochromaffin cells（ECs）,affects the process of UC through immune regulation,but its immunoregulatory effect,especially its impact on Bregs,is still unclear.Thus,this study applied blood samples and DSS-induced colitis mice model to explore the molecular mechanism of 5-HT regulating Bregs via 5-HT receptor on the process of UC,which may be a new immunocell-based therapy for UC patients.Methods:（1）Detected the proportions of CD19⁺CD24^highCD38^highB cells in peripheral blood mononuclear cells（PBMCs）of UC patients and healthy controls,and analyzed their correlation with UC disease activity（Mayo clinic score）and relevant clinical indicators.（2）CD19⁺CD24^highCD38^highB cells in PBMCs of patients with UC and healthy controls were deleted by flow sorting and the remained cells were stained with CFSE and cultured in vitro for 5 days.The suppressive function of CD19⁺CD24^highCD38^highB cells in peripheral blood of patients with UC and healthy controls was detected by flow cytometry.（3）The serum levels of 5-HT and IL-10 were detected by ELISA and the correlation was conducted with CD19⁺CD24^highCD38^highB cells,Mayo clinic score and relevant clinical indicators in UC patients.（4）The expression of 5-HT receptors on different subsets of B cells in PBMC of UC patients were detected by flow cytometry.（5）B cells were isolated from PBMCs of healthy controls by magnetic beads.The isolated B cells were stimulated with 5-HT and cultured for 2 days in vitro.The number and proportion of CD19⁺CD24^highCD38^highB cells and CD19⁺CD24^highCD38^highIL-10⁺B cells were detected.Then,the expression of p-STAT3 and p-ERK1/2 on CD19⁺CD24^highCD38^highB cells were analyzed by flow cytometry.Meanwhile,5-HT₇R antagonist and STAT3 inhibitor were used to treated with B cells and then detected the number and proportion of CD19⁺CD24^highCD38^highB cells and CD19⁺CD24^highCD38^highIL-10⁺B cells.IL-10expression of B cells was also detected by q RT-PCR.（6）3.5%dextran sodium sulfate（DSS）was orally fed to C57BL/6 wild type mice for 7 days to obtain a colitis mouse model.Splenic B cells of wild mice were isolated by magnetic beads and cultured in vitro for 2 days with 5-HT stimulation.The 5-HT treated and untreated splenic B cells were adoptive transferred to wild type mice 3 days and 1 day before DSS drinking.The weight loss,stool consistency,and rectal bleeding were recorded every day after the start of colitis model to assess the disease activity index（DAI）.Colitis mice were sacrificed on the 8th day and the colon length was measured.Mice colonic tissues were taken for HE staining to determine the degree of mucosal injury and inflammatory infiltration.Mice colonic tissues were taken for immunofluorescence staining to determine the expression of IL-10 in intestinal tissues.The proportion of B220⁺IL-10⁺B cells in PBMCs,mesenteric lymph node（MLN）,spleen and intestinal mucosa LPMCs were measured by flow cytometry.Results:（1）The proportion of CD19⁺CD24^highCD38^highB cells in PBMCs of UC patients were decreased,and CD19⁺CD24^highCD38^highB cells were negatively correlated with Mayo clinic score and related clinical indicators.（2）CD19⁺CD24^highCD38^highB cells were deleted from PBMCs of UC patients and healthy controls.The suppressive rate of CD4⁺T cells was lower than that of UC patients,indicated that the suppressive function of CD19⁺CD24^highCD38^highB cells in PBMCs of UC patients was impaired.（3）The levels of 5-HT and IL-10 in the serum of UC patients were decreased,which were positively correlated with CD19⁺CD24^highCD38^highB cells in PBMC,Mayo clinic score and relevant clinical indicators.Serum 5-HT was positively correlated with IL-10 level.（4）The expression of 5-HT₇R on CD19⁺CD24^highCD38^highB cells in PBMCs of UC patients was higher than that of healthy controls.The expressions of 5-HT_1AR,5-HT_2AR,5-HT_3AR and 5-HT_3BR had no difference between UC patients and healthy controls.There was no difference in expression of 5-HT₇R on CD19⁺CD24^intCD38^intB cells and CD19⁺CD24^highCD38^-B cells in UC patients and healthy controls.（5）5-HTenhancedthenumberandproportionof CD19⁺CD24^highCD38^highIL-10⁺B cells,but had no effect on the number and proportion of CD19⁺CD24^highCD38^highB cells,and meanwhile increased the expression of IL-10 in B cells.The 5-HT promotion of Bregs production of IL-10could be abrogated by 5-HT₇R antagonist and STAT3 inhibitor.（6）The adoptive transfer of 5-HT-treated B cells could relieve the intestinal inflammation in DSS-induced colitis mice.The rectal bleeding was relieved and the weight loss decreased and the colon length was longer.HE staining indicated that the degree of intestinal inflammation was relatively mild.The adoptive transfer of5-HT-treated B cells could induce the proportion of B220⁺IL-10⁺B cells in PBMCs,MLN,spleen and LPMCs.Immunofluorescence staining indicated that the number of intestinal B-cells expressing IL-10 was increased.Conclusions:In this study,we found that Bregs were functionally and numerically impaired in UC patients,accompanied with reduced serum IL-10 and 5-HT.The serum IL-10 and5-HT were negatively correlated with disease activity.5-HT₇R was highly expressed in Bregs in UC patients,and 5-HT could activate STAT3 to promote the production of IL-10 in regulatory B cells through 5-HT₇R.In the DSS-induced colitis mice,5-HT-treated B cells were transferred to relieve intestinal inflammation via induced more B220⁺IL-10⁺B cells,suggesting that 5-HT-treated B cells may be a new potential immunocell-based therapy for UC patients.