Eenterobacter Ludwigii Protects Experimental Colitis Through CD103~+DC And Treg Cells

Objectives:Inflammatory bowel disease(IBD),represented by Crohn’s disease(CD)and Ulcerative colitis(UC),is a chronic inflammation of the gastrointestinal tract,which is related with genetics,environment,immune response and intestinal barrier factors.At present,the etiology and pathogenesis of IBD are not fully understood,and IBD is becoming a chronic disease,which is difficult to treat,easy to relapse,has long course,and seriously affects human health.The medical treatment of IBD involves pharmacological interventions,such as the use of anti-inflammatory drugs and/or antibiotics,but these therapies are usually associated with significant adverse side effects and drug tolerance,new therapies need to be developed.Nutritional therapy containing probiotics or prebiotics is increasingly recognized to have great potential application in the treatment of IBD.Recently,gut microbes have received widespread attention as a new therapeutic target for local and systemic inflammation.For example,fecal microbial transplantation(FMT)has been proposed as a potential treatment for Clostridium difficile infection(CDI).In addition,it has been proven that probiotics have a therapeutic effect on diseases related to gut microbiota dysbiosis.Further research on intestinal microbes will bring new treatment directions for refractory chronic inflammatory diseases.This paper intends to solve the following three problems:1.To compare the effects of multiple antibiotics on Dextran sulfate sodium(DSS)induced colitis sensitivity,and to study the effect of metronidazole on intestinal immune cells and intestinal flora;2.To isolate and identify bacterial strains from the mouse feces treated with metronidazole that can alleviate DSS colitis;3.To study the specific mechanism of the target strain to improve DSS colitis.Methods:1.To study antibiotics effect on mice susceptibility to DSS-induced colitis,male C57BL/6J mice were pretreated with antibiotics for seven days,and exposed with the same antibiotics in addition with 3%DSS in drinking water for another seven days.Body weight loss and DAI were scored every day during the DSS treatment.Colon tissues were collected on the seventh day when mice were euthanized,fixed in 4%paraformaldehyde and embedded into paraffin.Slides(5μm)were stained with hematoxylin and eosin,then images acquired with a microscope were used to assess histopathological scores.2.MLN and c LP of mice at the seventh day after DSS treatment in different groups were collected,and lymphocytes were isolated,stained and analyzed by flow cytometry,or frozen sections were made for immunofluorescence staining to detect the changes of dendritic cells and CD4~+T cells of different subtypes.We performed16S r RNA gene sequencing of bacterial DNA collected from mice feces or colon mucosal tissues to detect the diversities in microbial flora composition in different treatment groups.3.Fresh feces pellets collected from mice with metronidazole treatment were homogenized in 1 ml sterile phosphate buffered saline containing 2.5%L-cysteine and serially diluted with PBS,seeded onto multiple agar plates.After incubated under aerobic conditions or anaerobic conditions at 37℃for 48 h to 72 h,individual colonies with distinct morphologies were picked up.Bacterial genomic DNA was extracted from selective cultured bacteria,and 16S r RNA gene sequence was amplified by PCR to identify the bacterial species.4.The isolated strain was cultured and amplified in liquid medium,cultured bacteria were pelleted by centrifugation and resuspended in PBS to obtain a density of 5×10~9CFU/ml.Each male C57BL/6J mouse was gavaged with 200μl liquid bacterial strains(10~9CFU)once per day to test whether the bacterial gavage has a protective effect on DSS colitis.Diphtheria toxin was used to induce DC or Treg deficient mice,combined with antibiotics mix treatment,single bacteria colonization and DSS-induced acute colitis,to check whether the target strain has the effect of suppression colitis.5.We used quantitative real time PCR to detect the m RNA expression levels of tolerance-related protein in DC cells and Treg cells sorted from mouse mesenteric lymph nodes in different treatment groups.The DC cells sorted from the mesenteric lymph nodes of mouse with different treatments and the purified Naive T cells sorted from spleens of Foxp3-GFP-DTR mice were co-cultured in vitro for 72 h,and the percentage of CD25~+Foxp3~+Treg in CD4~+T cells after co-culture was assessed by flow cytometry and immunofluorescence.Results:1.Among ampicillin,chloramphenicol,ciprofloxacin,clindamycin,metronidazole,neomycin,polymycin,tetracycline and vancomycin,metronidazole was found to have the best effect on protecting mice against DSS-induced colitis.2.Metronidazole treatment increases CD103~+DCs and Foxp3~+Treg cells in mesenteric lymph nodes(MLN)and colonic lamina propria(c LP).3.Metronidazole treatment remodeled the composition of intestinal microbes,and increased the percent of facultative anaerobes.4.Enterobacter ludwigii,abundant in mouse feces after metronidazole treatment,was identified to decrease mice susceptibility to DSS-induced colitis.5.E.ludwigii gavage increased CD103~+DCs and Foxp3~+Treg cells in mesenteric lymph nodes(MLN)and colonic lamina propria(c LP),and effects of E.ludwigii on diminishing colitis were lost in DC or Treg depletion mouse.6.CD103~+DCs isolated from the mesenteric lymph nodes of E.ludwigii-treated mouse showed enhanced ability to promote the Treg differentiation from naive T cells.7.DCs,directly stimulated by live E.ludwigii strain or its culture supernatant,promoted the up-regulation of the transcription level of DC immune tolerance-related genes and promoted DC-mediated differentiation of Tregs.Conclusion:1.Metronidazole plays a role in protecting mice from DSS-induced colitis by reshaping the composition of intestinal microbes and increasing CD103~+DC and Foxp3~+Treg cells in the intestinal microenvironment.2.Our findings identify a facultative anaerobe bacterial strain E.ludwigii isolated from the feces of mouse in the metronidazole treatment group,enhances CD103~+DC and Treg-mediated immune tolerance by acting on DC cells directly,resulting in protecting mice against DSS-induced colitis.