Aryl Hydrocarbon Receptor/IL-22/Stat3 Signaling Pathway Is Involved In The Modulation Of Intestinal Mucosa Antimicrobial Molecules By Commensal Microbiota In Mice

The human gastrointestinal tract harbors a great number of microorganisms,including bacteria,viruses,fungi,protozoa,and helminths,which are collectively referred to as the commensal microbiota.In recent years,the commensal microbiota has been established as an indispensable factor in host physiology processes such as food digestion,vitamin synthesis,and fat metabolism.Moreover,an important aspect underlining the indispensability of gut mierobiota is their contribution to the development,maturation,and regulation of the immune system,including systemic immunity of the host and local immunity of the gut.Studies in isolator-raised germ-free(GF)mice revealed fundamental impairments in mucosal immunity,including decreased IgA levels,atrophic intestinal epithelia,and reduced lamina propria lymphocytes.The intestinal epithelium is regarded as the physical and immune barrier of the gut mucosa.Intestinal enterocytes consists of columnar cells,goblet cells,Paneth cells,and enteroendocrine cells,which can produce and secrete a series of antimicrobial molecules for epithelial protection,including lysozyme,regenerating islet-derived protein 3 gamma(Reg?g),cryptdins,and secretory phospholipase A2.The creation and release of these products from enterocytes are promoted by IL-22 via the IL-22Ral/IL-10Rb/Stat3 pathway.These products limit excessive growth of the gut microbiota by disrupting the integrity of the bacterial cell membrane or wall to regulate the balance of intestinal homeostasis.Aryl hydrocarbon receptor(AhR),a basic helix-loop-helix protein,is a member of the Per-AhR-nuclear translocator-Sim superfamily of proteins.Recently,AhR has gained attention because it represents an important link between the environment and immunity.Recent studies showed that activation of AhR by the microbiota within the gastrointestinal tract is essential for promoting local IL-22 production.Specifically,tryptophan catabolites generated via metabolism by the microbiota are involved in mucosal immune responses via AhR modulation.However,whether the expression of AhR depends on the commensal microbiota and its role in maintaining antimicrobial molecules is unknown.In this study,we hypothesized that the gut microbiota can modulate the production and secretion of antimicrobial molecules and the AhR/IL-22/Stat3 signaling pathway might be involved in this process.Part1 Intestinal microbiota affects the intestinal barrier and expression of antimicrobial peptidesObjective:Observing the effects of intestinal symbiotic bacteria on intestinal barrier function and antimicrobial peptidesMethods:Eighteen 6-week-old male mice were randomly divided into three groups,antibiotic treatment group(Abx),and the distilled water treatment group(Normal)the fecal microbiota transplantation group(FMT).Here,the effect of antibiotics on the mice's intestinal commensal bacteria,the changes of intestinal mucosa morphology and secretion of antimicrobial peptides in different groups were observedResults:More than 95%of the intestinal microbiota was depleted by broad-spectrum antibiotics and was reconstituted by FMT.Intestinal mucosa of Abx group was obviously atrophied,intestinal villi shortened,and intestinal mucosa became shallow.The morphology of intestinal mucosa was significantly improved in the FMT group compared with the Abx group,and there was no difference with Normal.The secretion of antimicrobial peptides in the Abx group was significantly lower than that in the other two groups.Conclusion:Depletion of commensal microbiota leads to intestinal mucosa atrophy and reduction of antimicrobial molecules,including lysozyme,regenerating islet-derived protein 3 gamma(ReglllY),and cryptdin 5 mRNA,whereas subsequent reconstitution of intestinal microbiota by fecal microbiota transplantation(FMT)rescues mucosa morphology and antimicrobials.Part2 AhR/IL-22/Stat3 signal:ing pathway is involved in the reduction of antimicrobial peptide secretion in AbxObjective:Clarifying the role of AhR/IL-22/Stat3 signaling pathway between the symbiotic bacteria and intestinal mucosal barrier functionMethods:Eighteen 6-week-old male mice were randomly divided into three groups,antibiotic treatment group(Abx),and the distilled water treatment group(Normal)the fecal microbiota transplantation group(FMT).Here,The expression of AhR and the expression levels of IL-22 and Stat3 were detected.Results:AhR was down-regulated significantly following antibiotic treatment and restored partially by subsequent FMT.The protein levels of AhR as measured by Western blot analysis showed the same changes.IL-22 levels in the Abx group were significantly lower than those in the Normal group and subsequent FMT rescued this change to normal levels.Conclusion:AhR/IL-22/Stat3 signaling pathway is associated with the modulation of intestinal mucosa antimicrobial molecules by commensal microbiotaDown-regulation of aryl hydrocarbon receptor(AhR),interleukin-22(IL-22),and phosphorylated Stat3(p-Stat3)is associated with decreased antimicrobials,which might mediate the antibiotic-associated intestinal mucosa injury.Part3 Activation of AhR/IL-22/Stat3 signaling pathway can repair intestinal mucosal damage in AbxObjective:Exogenous injection of AhR agonists to activates AhR,then the activation of IL-22/Stat3 pathway,morphological of intestinal mucosa and expression of antimicrobial peptides were observed.To further clarified the role of this pathway between the flora and the intestinal barrier.Methods:Twenty-four 6-week-old male mice were randomly divided into four groups,antibiotic treatment group(Abx),the distilled water treatment group(Normal),the fecal microbiota transplantation group(FMT),and the antibiotic plus agonist group treatment group(Abx+Ficz).Here,the effect of antibiotics on the mice's intestinal commensal bacteria,the changes of intestinal mucosa morphology and secretion of antimicrobial peptides in different groups were observed;The expression of AhR and the expression levels of IL-22 and Stat3 were detected.Results:After the addition of the AhR agonist,the morphology of the intestinal mucosa in the Abx+Ficz group was close to the FMT group.The level of antimicrobial peptide secreted by intestinal mueosa was significantly increased in Abx+Ficz group compared with Abx group,but still lower than the normal group.Detection of its IL-22/Stat3 pathway showed that after the addition of the AhR agonist,the transcription level of this pathway and the level of Stat3 phosphorylation was significantly increased.Conclusion:Exogenous activation of the AhR/IL-22/Stat3 signaling pathway with the AhR agonist 6-formylindolo(3,2-b)carbazole(Ficz)rescued antimicrobial molecule levels markedly after antibiotic treatment to levels similar to those following reconstitution of intestinal microbiota by FMT.