Antibiotic Treatment Protects Against Colitis By Increasing The Abundance Of Commensal Akkermansia Muciniphila In Gut

Background and aimUlcerative colitis(UC)is a chronic nonspecific inflammatory disease which mainly affects the large intestine(the colon and the rectum).The etiology of UC is still unclear and it could be attributed to many factors including environment,genetics,microorganism and immunity.Recently,increasing attention has been attached to the role of gut microecology in the pathogenesis of UC even though the specific pathogens that trigger the onset of UC remain to be determined.In fact,accumulating clinical evidence suggests that UC is probably caused by immune system dysfunction resulting from the imbalance of commensal bacteria rather than one single pathogen.Theoretically,UC could be owing to the condition that the abnormal interaction of micro-ecology and the host interferes with the balance of intestinal tract thereby destroying intestinal barrier function,increases bacterial adhesion and metastasis,affects innate immunity and adaptive immunity and eventually leads to the occurrence of inflammation in genetically predisposed individuals under the effect of environment.The use of antibiotics in patients with ulcerative colitis is somewhat controversial.On the one hand,UC patients could benefit from it due to the fact that antibiotics can kill pathogens;therefore,postoperative infection and/or the overgrowth of bacteria could be controlled.On the other hand,long-term use of broad-spectrum antibiotics leads to imbalance of intestinal flora and increases the risk of developing UC in gastroenteritis patients.A multicenter study by Ohkusa et al.showed that treatment with amoxicillin,tetracycline and metronidazole in moderate-severe UC patients increases the response rate to treatment.However,alteration of gut microbiota due to antibiotics use during infancy is shown to be correlated to the incidence of UC.Therefore,it is necessary to get a better understanding about the changes in gut microbiota under the context of antibiotics administration so as to facilitate the treatment for UC.As a broad-spectrum antibiotic,vancomycin is commonly used clinically in the treatment for drug-resistant bacterial infection,pseudomembranous colitis caused by dysbacteriosis and even for colitis.Study found that vancomycin treatment can reduce the incidence of type 1 diabetes in Non-obese diabetic(NOD)mice model.Also,the number of Akkermansia muciniphila(A.muciniphila/Akk)increases significantly in vancomycin treatment group.Therefore,it is hypothesized that the rise of A.muciniphila could be protective against type 1 diabetes.At present,a great many researches confirmed that A.muciniphila also plays an important role in the prevention and treatment of metabolic diseases such as high-fat diet-induced obesity and type II diabetes.Another research revealed that the relative abundance of A.muciniphila is negatively correlated with diseases such as inflammatory bowel disease(IBD),appendicitis and juvenile autism.As greater concerns have been attracted to the benefits of A.muciniphila in treating many other diseases,A.muciniphila is believed to be a new generation of probiotics.Notably,intestinal commensal Akkermansia can migrate to the site of injury and promote intestinal epithelial cell proliferation to help with wound repair.This specific mucosal repair is later found Akkermansia-dependent.As the damage of mucosa caused by chronic intestinal inflammation constitutes a constant scenario during UC progression,mucosal healing serves as a crucial therapy for it.Taken that antibiotics like vancomycin could modulate specific intestinal commensal bacteria(like A.muciniphila),which might subsequently affect the development of UC,our study aims to:1)firstly,investigate the effect of specific antibiotics treatment on UC development;2)then determine the specific alteration of gut microbiota caused by antibiotics administration;3)last,explore the underlying mechanism by which changes in gut microbiota affect UC progression.Methods and Results1.Pretreatment with vancomycin and metronidazole significantly alleviates DSS-induced experimental colitisC57 male mice aged 4-5 weeks were randomly divided into 4 groups.(1)Normal control;(2)DSS group;(3)Antibiotics-3d;(4)Antibiotics-2w.Mice received antibiotics or not at the first week and then were subjected 2.5%DSS water for the next week.We collected information including general condition,bodyweight changes and symptoms like diarrhea and bloody stool to conduct the Disease Activity Index(DAI)scoring.Mice were sacrificed on day 8(the first day after DSS challenge)and day 12(the fifth day after DSS challenge).Intestines were resected for length measure and HE staining was used for pathological analysis.Compared with the DSS group,the general condition and symptoms of diarrhea,rectal bleeding showed relieved in mice pretreated with antibiotic.Besides,survival rates were improved and the disease activity index(DAI),pathological score were reduced significantly in antibiotic groups,as compared to DSS group.2.Pretreatment with vancomycin and metronidazole exerts a protective effect on intestinal mucosal barrier in mice with DSS-induced colitisThe intestinal tissue of mice was fixed,dehydrated,embedded and cut into sections.Alcian blue staining was used to detect the goblet cells.Immunofluorescence was used to detect MUC2 expression.Immunohistochemistry was used to detect the expression of Reg??.Compared with DSS group,the number of goblet cells and the expressions of MUC2 and Reg?? were increased significantly in the antibiotic groups.3.Pretreatment with vancomycin and metronidazole enhances intestinal epithelial proliferation and mucosal repair in mice with DSS-induced colitisIntestinal epithelial cells were collected from small intestines and colons of mice for flow cytometry cell cycle assay.Western blot(WB)assay was used to detect cell cycle proteins.Immunohistochemistry was used to detect the expression of Ki67and p-ERK.As compared to DSS group,the expression of Cyclin A2,B1,E1 and CDK2,CDK4 were increased significantly in antibiotic groups.In line with the above changes,epithelial cell proportion in S and G2/M phase were also increased in antibiotic groups.Furthermore,pretreatment with antibiotic led to dramatically increased expression of mucosal Ki67 and p-ERK in mice.4.Pretreatment with vancomycin and metronidazole promotes the intestinal colonization of Akkermansia in mice with DSS-induced colitisMice fecal microbial DNA was extracted from stool samples and 16s rRNA sequencing was performed for bacteria analysis.Probes for specific bacteria were constructed and used to detect bacterial localization by FISH.As compared to DSS group,increased proportion of Akkermansia were detected in mice pretreated with antibiotic by 16s rRNA sequencing.Besides,the images of FISH showed that Akkermansia was mainly colonized in the mucus layer of mice pretreated with antibiotic.5.Dysbiosis of the intestinal microbiota is a characteristic of UCWe included 65 UC patients and 30 healthy volunteers and 16s rRNA sequencing was performed for bacteria analysis.Our findings showed that the beneficial microorganisms,Akkermansia,Clostridium ?,Faecalibacterium and Ruminococcus were decreased significantly in UC patients,as compared to healthy volunteers.while the genera Dorea?Proteus?Fusobacterium,all of which demonstrate pathogenic characteristics,were enriched in UC patients.6.A.muciniphila treatment significantly alleviates DSS-induced colitis in miceC57 male mice aged 4-5 weeks were randomly divided into 5 groups.(1)Normal control;(2)DSS group;(3)B.fragilis treatment group;(4)A.muciniphila treatment group;(5)Antibiotics-2w group.Mice received normal water or B.fragilis or A.muciniphila or antibiotics at the first week and then were subjected 2.5%DSS water for the next week.The monitored items were the same as above.Compared with the DSS group,the general condition and symptoms of diarrhea,rectal bleeding showed relieved in A.muciniphila treatment group.Besides,survival rates were improved and the disease activity index(DAI))pathological score were reduced significantly in A.muciniphila group,as compared to DSS group.The protective effect of A.muciniphila on experimental colitis was similar to that of B.fragilis.7.A.muciniphila treatment exerts a protective effect on intestinal mucosal barrier in mice with DSS-induced colitisIntestinal permeability assay was performed by detecting FITC intensity in serum.Alcian blue staining was used to detect the goblet cells and qPCR was used to measured the expression of MUC2.A.muciniphila treatment increased goblet cells and expressions of MUC2 as well as decreased intestinal permeability,as compared to DSS group.8.A.muciniphila exerts the protective effect through various pathwaysProteomic analysis reveals that A.muciniphila activates expression of proteins involved in MAPK signaling pathway,cell cycle progression,cell proliferation,cell differentiation,antigen presentation and differentiation of immune cells,whereas B.fragilis mainly affects expression of proteins associated with cell cycle progression,cell proliferation and differentiation.9.A.muciniphila enhances intestinal epithelial proliferation and repair via activation of IL-6/STAT3 or ERK signaling pathwayTotal proteins of cells or intestines were extracted and the protein expressions of several pathways were analyzed by WB,Immunofluorescence or ELISA.Both A.muciniphila and B.fragilis enhanced the expression of proteins associated with cell cycle progression via activation of IL-6/STAT3 axis or ERK signaling pathway,though the activation of these pathways by Akk and Bf were at different time points.Conclusion1.Pretreatment with vancomycin and metronidazole protects against DSS-induced experimental colitis.2.Pretreatment with vancomycin and metronidazole promotes the mocosal colonization of A.muciniphila in mice with DSS-induced colitis.3.The protective role of A.muciniphila in DSS-induced colitis is probably dependent on promotion of epithelial proliferation and mucosal repair via the activation of IL-6/STAT3 axis or ERK signaling pathway.4.The protective effect of A.muciniphila on experimental colitis was similar to that of B.fragilis,though the activation of these pathways by Akk and Bf were at different time points.