The Role Of IL-17 And Adherent Invasive Escherichia Coli LF82 In The Pathogenesis Of Inflammatory Bowel Disease

【Background and aims】 Inflammatory bowel disease(IBD)represents an aberrant immune response to the gut microbiota companied with genetic and environmental disorders.Adherent-invasive Escherichia coli(AIEC)is one of the Escherichia coli strains which involved in the pathological processes in IBD through invading to the intestinal epithelial cells.In addition,Th17 cells were found to be involved in the development of inflammatory bowel disease(IBD)and many other autoimmune inflammatory disease in recent years.Especially,the IL-17 secreted by Th17 cells might play a key role among them.IL-17 are induced and accumulate in response to colonization with a subgroup of intestinal microbes and certain extracellular pathogens.In recent years,the precise role of IL-17 in IBD has been controversial,with dichotomous reports promoting both IL-17-dependent inflammation and protection.However,IL-17 seems to be both pathogenic and protected in the pathogenesis of IBD.However,the mechanism behind IL-17 and AIEC still unclear.In this study,we examined the involvements of IL-17 in AIEC mediated colonic disorders in colitis.The specific mechanism of IL-17 in IBD is still not clarified.This project is on the basis of our previous work,we plan to reveal the role and mechanism of differentiation and regulation of Th17 cell in IBD exacerbated by AIEC strain E.coli LF82 via animal models and some related experiments in vivo and in vitro.To carry out the project will help us to figure out the pathogenesis of IBD,which has important scientific significance.【Methods】1.To examine the impact of Escherichia coli LF82 on the colon of mice,wild-type(WT)mice were inoculated with 109 CFU Escherichia coli LF82 by oral gavage for 10 days.The colonic epithelial barrier was assessed after colonized with or without E.coli LF82.Viewed under transmission electron microscope(TEM)and scanning electron microscope(SEM),we detected the ultrastructure of the colonic epithelial cells and E.coli LF82 colonization.The cytokine expression in the colon was measured by ELISA.The expression level of IL-17 in the colon was measured by RT-PCR and immunohistochemistry.IL-17 KO mice were also inoculated with 109 CFU Escherichia coli LF82 by oral gavage for 10 days.Then the integrity of the epithelial barrier and cytokine expression were measure same as WT mice.2.WT mice were inoculated with 109 CFU AIEC reference strain LF82 by oral gavage at 4–6 weeks of age,and then placed in standard housing of specific-pathogen free(SPF).Then mice were fed 3% DSS dissolved in sterile and distilled water ad libitum for 5 days followed by 1 day of normal drinking water,resulting in a 6-day experimental period.The severity of colitis was assessed by body weight change,colon length change and colonic histological score.Cytokine expression was measured by ELISA and immunohistochemistry in colonic tissue.The expression level of IL-17 in the colon was measured by RT-PCR and immunohistochemistry.3.IL-17 KO mice were inoculated with 109 CFU Escherichia coli LF82 by oral gavage for 10 days.Then mice were fed 3% DSS dissolved in sterile and distilled water ad libitum for 5 days followed by 1 day of normal drinking water,resulting in a 6-day experimental period.Then the severity of colitis e expression were measure same as WT mice,included body weight change,colon length change and colonic histological score.Cytokine expression was measured by ELISA and immunohistochemistry in colonic tissue.【Results】1.Wild-type(WT)mice were inoculated with 109 CFU E.coli LF82 by oral gavage for 10 days.In contrast to no-colonization WT mice,colonization mice showed markedly disordered epithelium and more extensive infiltration of inflammatory cells.Immunohistochemical staining revealed elevated level of IL-17 in the colon colonized with E.coli LF82.In agreement,RT-PCR also showed the m RNA expression of IL-17 was remarkably increased in WT mice colonized E.coli LF82.To verify the role of IL-17 in colon infected with E.coli LF82,we then inoculated IL-17 knockout mice with E.coli LF82 for 10 days.Histopathological analysis revealed IL-17-knockout(IL-17-/-)mice exhibit more serious epithelial perturbations at baseline.Furthermore,transmission electron microscope and scanning electron microscopy indicated that E.coli LF82 infection was predominantly superficial in WT mice,whereas large numbers of bacteria penetrated deeply into the colonic epithelium in IL-17-/-mice.And we observed IL-22 in colon of IL-17 knockout mice colonized E.coli LF82 decreased significantly compared with WT mice colonized E.coli LF82.2.We found the severity of DSS-induced colitis have no markedly different between nocolonized and colonized E.coli LF82 in WT mice.PCR analysis revealed that IL-17 A was produced at significant levels in the WT colon colonized E.coli LF82.After daily administration with DSS,IL-17-/-mice colonized mice by E.coli LF82 showed more severe colitis than no-colonized IL-17-/-mice.Compared to WT mice colonized E.coli LF82,in the IL-17-/-mice colonized E.coli LF82 we observe the same phenomenon including weight loss,colon shortening,and colonic mucosa damage and severity.3.And in normal colon and DSS-induced colitis,we observed IL-22 in colon of IL-17-/-mice mice colonized E.coli LF82 decreased significantly compared with WT mice colonized E.coli LF82.【Conclusions】Our study indicated that in the normol colon over-expression of IL-17 protects colonic epithelial integrity in host defense against E.coli LF82.In the IL-17-/-mice,E.coli LF82 can exacerbate DSS-colitis.It suggest that E.coli LF82 could exacerbate the colitis in the absence of IL-17.Compared to WT mice colonized E.coli LF82,we observe the same phenomenon in the IL-17-/-mice colonized E.coli LF82.These results indicate that AIEC could not exacerbate colitis in the presence of IL-17.In brief,through over-expression and deletion of IL-17 in colon colitis models,we demonstrate IL-17 could protect the colon colonized with E.coli LF82 during DSS-induced colitis by evidence both pro and con.We also found IL-17 can promote the production of inflammatory protective cytokine IL-22.We hypothesis there is synergetic relationship between IL-17 and IL-22 in the protection of colitis.