The Role Of LL-37/CRAMP Expression Regulated By IL-27in Protecing Intestinal Clostridium Difficile Infection

Background:Clostridium difficile is a leading cause of nosocomial gastrointestinal infections and antibiotic-associated diarrhea.The immunity mediated by intestinal epithelial cells plays an important role in combating Clostridium difficile infection.However,its molecular and immuno-regulatory mechanisms remain to be elucidated.Recent studies have found that IL-27 is an important immune regulatory factor that plays a protective role in clostridium difficile infection.LL-37 is the only cathelicidin-like antimicrobial peptide currently found in humans.CRAMP is mouse derived LL-37,which is mainly expressed in intestinal epithelial cells and plays an anti-infection immune function.Thus,we hypothesized that IL-27 might play a protective role against clostridium difficile infection by inducing the expression of LL-37/CRAMP in intestinal epithelial cells.Objective:This study focused on the clostridium difficile infection of the public health problems,from the angle of interaction between pathogen and host,with IL-27 regulation of intestinal epithelial cell immune response as the breakthrough point,to explore the IL-27 mediated immune protection mechanism of clostridium difficile infection.Methods:1.The expression of IL-27 in serum and feces of clinical CDI patients was detected by ELISA to understand the correlation between the expression of IL-27 and the incidence of CDI;In addition,serum CXCL10levels were detected in CDI patients to assess whether it could be used as a biomarker to monitor the severity of CDI disease and the efficacy of treatment.2.CDI animal model was constructed by WT mice and WSX-1^-/-gene knockout mice,and serum and intestinal CRAMP expression levels of CDI mice were determined by ELISA and quantitative PCR.Recombinant antimicrobial peptides were used to treat WSX-1^-/-CDI mice,to assess the differences in intestinal inflammation and tissue damage,inflammatory molecule expression,bacterial load and survival rate in mice,and to identify the immune protection of IL-27 in promoting intestinal epithelial cell expression of CRAM in CDI.3.In vitro,human primary colonic epithelial cells were used as models.Expression of LL-37 gene and protein levels were detected after co-stimulation of intestinal epithelial cells with IL-27 and Clostridium difficile.Then,intestinal epithelial cells were treated with specific small-molecule inhibitors,and the expression of LL-37 was detected by Western blot.The expression of LL-37 intracellular signaling molecules in intestinal epithelial cells induced by IL-27 was preliminarily explored.Results:1.The expression of serum and fecal IL-27 in CDI patients was significantly higher than those of patients with diarrhea,which was positively correlated with WBC,PCT and TNF-αand negatively correlated with serum albumin;CXCL10 is highly expressed in CDI,significantly correlated with disease severity,and decreased after effective treatment,which can be used as a novel serum marker to evaluate CDI severity and therapeutic effect.2.The expression level of CRAMP was significantly decreased in WSX-1^-/-CDI mice.In WSX-1^-/-mice,the intestinal damage was alleviated,bacterial clearance and the survival rate of mice were significantly improved,which may be related to the production of anti-inflammatory cytokines（IL-10,IFN-γ）after treatment.3.The co-stimulation of IL-27 and clostridium difficile induced the expression of LL-37 in intestinal epithelial cells in a time-dependent and dose-dependent manner.After pretreatment with LY294002,AG490 and SB203580 inhibitors,the production of LL-37 in intestinal epithelial cells was significantly reduced,suggesting that IL-27 may regulate the expression of ll-37 in intestinal epithelial cells through the three pathways of AKT,JAK and MAPK.Conclusion:IL-27 can regulate the expression of CRAMP/LL-37 in intestinal epithelial cells to resist clostridium difficile infection,reduce intestinal inflammation,and play its immune protection role,providing a new perspective on the mechanism of CDI interaction with the host.In addition,CXCL10 can be served as a new target for the diagnosis and prognostic assessment of CDI.