Machanism Of Clostridium Tyrobutyricum On Regulating Intestinal Immune Response In Mice Based On Interactions Between Intestinal Epithelial Cell And Immune Cell

Young animals are susceptible to Enterotoxigenic Escherichia coli(ETEC),leading to diarrhea,immune disruption,and death.At present,probiotic is one of the therapeutic or prophylactic method for diarrhea and intestinal inflammation.Clostridium tyrobutyricum(C.tyrobutyricum/Ct)is one of the anaerobic Clostridium strains producing high butyric acid.Our previous study has revealed that C.tyrobutyricum efficiently alleviates LPS-induced intestinal epithelial barrier dysfunction,indicating that C.tyrobutyricum could be a potential probiotic in regulating intestinal health.Herein,LPS,derived from Escherichia coli,is used to induce diarrhea and gut dysfunction.The effects of C.tyrobutyricum on regulating intestinal immune response and barrier function are studied via evaluating the growth,diarrhea,intestinal immune response,and barrier function.RNA-sequencing,flow cytometry,and in vivo interference with the adenoassociated virus are used to investigate the mechanism of C.tyrobutyricum in regulating intestinal immune response based on the interactions between epithelial cell and immune cell.Moreover,the co-culture cell model with epithelial cells and immune cells is applied to further verify the mechanism of C.tyrobutyricum in epithelial cell-immune cell interactions.The main results are described in the following sections:1 Establishment of intestinal bowel disease and dose screening of C.tyrobutyricum in mice 1.1 Establishment of intestinal bowel disease in miceMice were intraperitoneally injected with 10 mg/kg body weight of lipopolysaccharide(LPS).The mice presented serious watery diarrhea and weight losses after injection with LPS.The expression of inflammatory cytokines was significantly increased in the duodenum,jejunum,and ileum after treatment with LPS.These phenomenons were more serious after in jecting with LPS for 24 h.These results indicated that a single intraperitoneal injection with LPS(10 mg/kg Body weight)with a duration of 24 h successfully induced intestinal bowel disease in mice.1.2 Dose screening of C.tyrobutyricum in miceMice were treated with different concentrations of C.tyrobutyricum by gavage followed by injection with LPS to investigate the effects of C.tyrobutyricum in vivo.Although,107 CF/mL C.tyrobutyricum affected the growth of mice,generally,C.tyrobutyricum itself had no harmful effects on the intestinal immune function.In response to LPS,C.tyrobutyricum efficiently maintained the body weight gain,107 CFU/mL C.tyrobutyricum decreased the mRNA expression of inflammatory cytokines in the duodenum,and 108 CFU/mL C.tyrobutyricum decreased the mRNA expression of inflammatory cytokines in both duodenum and ileum.Altogether,107 CFU/mL C.tyrobutyricum efficiently regulated immune response in the duodenum,and 108 CFU/mL C.tyrobutyricum could regulate the immune response in the ileum.108 CFU/mL C.tyrobutyricum was used to investigate ileal immune response in the follow-up experiment.2 Mechanism of C.tyrobutyricum in regulating the intestinal immune response in mice2.1 Effects of C.tyrobutyricum on intestinal barrier functionC.tyrobutyricum was labeled with bacteria-specific fluorescence and the mice were treated with 108 CFU/mL C.tyrobutyricum once by gavage.After 4 h,fluorescence C.tyrobutyricum was observed on the epithelium of duodenum,jejunum,and ileum,while no fluorescence could be observed in the colon,indicating that C.tyrobutyricum could colonize or adhere on the surface of the epithelium.Mice were then treated with C.tyrobutyricum by gavage,followed by injection with LPS.In response to LPS,C.tyrobutyricum effectively maintained the body weight gain,prevented diarrhea,decreased the levels of inflammatory cytokines in the serum,maintained the intestinal permeability,protected epithelial morphology,and tight junction structures.These results further verified that C.tyrobutyricum efficiently alleviated LPS-induced ileal dysfunction.2.2 Mechanism of C.tyrobutyricum in regulating the intestinal immune response in miceThe underlying mechanisms of C.tyrobutyricum in regulating intestinal immune response were studied using RNA-sequencing.Three potential mechanisms were identified according to the transcriptomic analysis:1)Nerve-intestinal immune regulation,2)Metabolism-intestinal immune regulation,3)Cytokines-intestinal immune regulation.Herein,we focused on the cytokines-intestinal immune regulation.We used RT-qPCR to verify the expression of differentially expressed genes in the immune responses.In response to LPS,C.tyrobutyricum enhanced the mRNA expression of IL-18,IL-17rc,and IL-22.Moreover,C.tyrobutyricum enhanced the mRNA expression of genes in the MHC-II process.In this study,we focused on IL-22 and further investigated whether C.tyrobutyricum regulated intestinal immune response depending on IL-22.2.3 Investigation of C.tyrobutyricum in regulating intestinal immune response based on immune cellsAccording to the transcriptomic analysis,some differentially expressed genes enriched in the biomarkers of immune cells were identified.The immune cells in different sections of small intestines were isolated and analyzed using flow cytometry.C.tyrobutyricum decreased the proportions of mast cells in both duodenum and ileum,indicating that C.tyrobutyricum inhibited the overactivation of mast cells under the inflammatory condition.In response to LPS,C.tyrobutyricum enhanced the proportions of DCs,Tregs,Th17,and ILC3s in the duodenum,while in the ileum,C.tyrobutyricum dramatically decreased the proportions of DCs,Tregs,and ILC3s,and significantly enhanced the percentage of Th17 cells.As for the jejunum,in response to LPS,although the proportions of macrophages,DCs,Th17,and Th2 cells were decreased,while no differences could be observed in other cell types.Interestingly,we found that the role of C.tyrobutyricum in regulating immune cells varied dramatically in the intestinal sections.These significant differences may rely on the anatomical and physiological distinctions among the duodenum,j ejunum,and ileum.The number of Tregs in the duodenum was highest among these three small intestinal sections,and Th17 cells were highest in the ileum after treatment with C.tyrobutyricum.These results suggested that C.tyrobutyricum regulated the immune response via activating Tregs in the duodenum and stimulating Th17 cells in the ileum.3 Mechanism of C.tyrobutyricum in epithelial cells-immune cell interactions mediated by IL-223.1 The role of IL-22 in C.tyrobutyricum regulating the intestinal immune response in the miceIn vivo interference IL-22 with the adeno-associated virus was conducted.After knocking down IL-22,no differences were observed in body weight gain,intestinal morphology,mRNA expression of inflammatory cytokines,and tight junction proteins.In mice with IL-22,C.tyrobutyricum enhanced the mRNA expression of IL-22RA1 and had no effects on IL-22BP expression.Besides,C.tyrobutyricum had no effects on the levels of SCFAs,and C.tyrobutyricum decreased the levels of SCFAs in response to LPS.These results indicated that C.tyrobutyricum regulated intestinal immune response depending on IL-22.C.tyrobutyricum protected epithelial barrier function via IL-22-IL-22RA1 signaling and its role in regulating ileal function was independent of SCFAs.3.2 The role of C.tyrobutyricum in intestinal epithelial cells-immune cell interactions mediated by IL-22The numbers of ileal immune cells were analyzed after knocking down with IL-22 in mice.After knocking down IL-22,no differences could be observed in the numbers of macrophages and Th17 cells between the LPS group and Ct+LPS group.The ileal sections were then labeled with EpCAM(epithelial cells),CD45(immune cells),IL-22,and IL-22RA1.In general,IL-22 and EpCAM were colonized in the epithelial cells and crypt,little could be observed in the lamina propria.In response to LPS or C.tyrobutyricum,colocalization of IL-22,EpCAM,and CD45 were observed in the epithelial cells and crypt.More importantly,C.tyrobutyricum treatments enhanced the colocalization of IL-22 and CD45 in the lamina propria.Altogether,these results indicated that IL-22 in the IL-22+EpCAM+CD45+cells was from the intraepithelial lymphocyte,and C.tyrobutyricum stimulated lymphocytes in the lamina propria,thereby inducing the production of IL-22.Generally,IL-22RA1 was located in the epithelial and crypt and little colocalization of IL-22 and IL-22RA1 could be observed.C.tyrobutyricum enhanced the colocalization of IL-22 and IL-22RA1 in the epithelial cells,while LPS decreased the IL-22 and IL-22RA1 colocalization.These results indicated that C.tyrobutyricum stimulated lymphocytes in the lamina propria to produce IL-22,which could bind with IL-22RA1 in the epithelial cells.The ileal sections were then labeled with CD3e,RORyt,EpCAM,and IL-22.Compared with the control,the numbers of CD3e+RORyt+IL-22+cells were enhanced after treatment with C.tyrobutyricum.The numbers of CD3e+RORyt+IL-22+cells were significantly higher in the Ct+LPS group than that in the LPS group.These results suggested that C.tyrobutyricum stimulated Th17 cells,thereby inducing IL-22 production.Altogether,C.tyrobutyricum stimulated Th17 cells to produce IL-22,which combined with IL-22RA1 in the epithelial cells,thereby protecting the intestinal barrier function and immune system.3.3 Mechanism of C.tyrobutyricum in epithelial cells-immune cell interactions mediated by IL-22 in vitroIn the single cultured Caco-2 cells,C.tyrobutyricum had no effects on the mRNA expression of inflammatory cytokines and ZO-1 expression in response to LPS,indicating that the regulation of C.tyrobutyricum in Caco-2 cells relied on immune cells.To fully understand the mechanism of C.tyrobutyricum in the epithelial cells-immune cell interactions,the co-culture cell model with Caco-2 cells and Naive T cells was established,and shRNA,overexpression,and rescue experiments were conducted in the coculture cell model.The results further indicated that the regulation of C.tyrobutyricum in Caco-2 cells relied on immune cells and C.tyrobutyricum enhanced IL-22 expression depending on Th17 cells,which were consistent with the results in vivo.3.4 Effects and mechanism of C.tyrobutyricum in regulating the colonic immune response in miceC.tyrobutyricum enhanced the expression of MUC2 and alleviated inflammation in the colon and this process was dependent on IL-22.After knocking down IL-22,no significant differences could be observed in the number of Th17 cells between the LPS group and Ct+LPS group,indicating that C.tyrobutyricum might regulate immune response via Th17,while the mechanism needed further investigation.This work investigated the role of C.tyrobutyricum in regulating intestinal barrier function and immune response.The underlying mechanism of C.tyrobutyricum in regulating intestinal immune response based on the interaction between epithelial cells and immune cell was studied.The results showed that C.tyrobutyricum could trigger Th17 cells in the lamina propria to induce IL-22 production,which combined with IL-22RA1 expressed in the epithelial cells,thereby protecting the intestinal barrier function and immune system.This study suggested that C.tyrobutyricum could be a potential probiotic in regulating intestinal health and provided a scientific basis for its application in preventing diarrhea and inflammatory bowel disease in young animals.