The Effects Of Gasdermin D In Salmonella Typhimurium Mucosal Infection

Gasdermin D?GSDMD?,a member of the Gasdermin family,is widely expressed in the gastrointestinal tract and other tissues or cells.According to the study in 2015,GSDMD is a key downstream molecule of the natural pattern recognition signal of the inflammasome.GSDMD is cleaved by caspases to produce GSDMD-N with the property of binding to biomembrane and mediating biomembrane perforation,causing pyroptosis and promoting the release of IL-1?and IL-18.At present,reports on the biological function of GSDMD mainly focus on cell death.However,it is unclear whether GSDMD is involved in regulating intestinal mucosal barrier function.Salmonella Typhimurium?This article is referred to as Salmonella?an important zoonotic pathogen with high pathogenic mortality and widespread epidemics,which poses a serious threat to human health and hinders the development of animal husbandry.Innate immunity is the first line of defense against pathogen invasion.The molecular mechanism of regulating intestinal mucosal immune response is studied to find intervention targets and facilate to the prevention and control of Salmonella infectious enteritis.In this paper,the role and mechanism of GSDMD in host defense against Salmonella mucosal infection were studied.This study first analyzed the role of GSDMD in Salmonella infection by wild-type?WT?and GSDMD gene-deficient(GSDMD^-/-)mice Salmonella enteritis models.GSDMD^-/-mice were more susceptible than WT mice,mainly charcterizied by the increased weight loss and the decreased survival rate.Moreover,at 48 h after infection,the characteristics of intestinal pathological damage such as cecal weight loss,intestinal epithelial integrity destruction,submucosal edema,and goblet cell reduction were more significant.After entering the host through the digestive tract,Salmonella colonizes the intestinal tract and then breaks through the intestinal mucosal barrier and spreads to the systemic sites,causing gastroenteritis and systemic infection of the host.The study found that the colonization of cecal tissue,feces,mesenteric lymph nodes,liver and spleen in GSDMD^-/-mice was significantly higher than that in WT mice after infection with Salmonella?48 h?.These data indicated that GSDMD contributes to the protection against Salmonella mucosal infection.The integrity of the intestinal mucosal barrier function is beneficial to prevent invasion and spread of intestinal pathogens.To reveal the phenotype and mechanism of GSDMD limiting Salmonella mucosal infection,this study further analyzed the effects of GSDMD on the intestinal mucosal physical barrier,chemical barrier,and inflammatory response.At 48 h after infection,inflammatory cell infiltration and various inflammatory mediators in GSDMD^-/-mice were significantly higher than WT mice,but IFN-?was lower than WT mice?P<0.05?.In order to analyze whether GSDMD affects intestinal mucosal physical barrier and chemical barrier,the proliferation and apoptosis of intestinal epithelial cells in cecal tissues were detected by immunofluorescence and TUNEL staining.The expression of tight junction protein claudin-3 in cecal tissues was detected by immunofluorescence.And detection of mucin expression in cecal tissues by immunohistochemistry and AB-PAS staining.Compared with WT mice,the proliferation of epithelial cells in GSDMD^-/-mice was significantly reduced,apoptosis was significantly increased,the expression of claudin-3 was significantly decreased,and the expression of mucin and mucin 2 was significantly decreased at 48 h after infection.However,there was no significant difference in epithelial cell proliferation and apoptosis,claudin-3,mucin and mucin 2expression between the two genotype mice before infection.The above results indicate that the integrity of intestinal mucosa is more severe ly damaged after infection of Salmonella in GSDMD^-/-mice.However,these results do not account for the causal relationship between increased inflammation,impaired intestinal mucosal barrier,and colonization and spread of Salmonella.Therefore,the acute infection of Salmonella in two genotype mice was designed,and the bacterial colonization,inflammatory reaction and physical and chemical barrier function were compared and analyzed.At 6 h after Salmonella infection,the two genotype mice had no obvious symptoms of enteritis,and there were no significant difference in proliferation and apoptosis,mucin,and claudin-3.However,compared with WT mice,the number of bacterial colonization increased significantly of feces in GSDMD^-/-mice?P<0.05?.This shows that GSDMD mainly inhibits the intestinal infection of Salmonella by restricting the early colonization of Salmonella in the intestinal lumen.How does GSDMD limit the early colonization of Salmonella in the intestinal lumen?This study further analyzed this.As mentioned earlier,IFN-?in the intestinal mucosa of GSDMD^-/-mice was significantly lower than that in WT mice at 48 h after infection.IFN-?has the function of inhibiting the proliferation of pathogenic bacteria,it was mainly produced by natural killer?NK?cells,intrinsic lymphocytes and T cells,and IL-18 is an important factor in inducing its secretion;and,under the action of inflammasome activation signals,GSDMD can mediate the release of IL-18.For these reasons,this study analyzed the dynamic expression of IL-18 and IFN-?during infection.The results showed that there was no difference in the expression of IL-18and IFN-?between WT and GSDMD^-/-mice under normal conditions;IL-18 and IFN-?were significantly higher in WT than GSDMD^-/-mice after streptomycin treatment and Salmonella infection.It is speculated that the reduction in the production of IL-18 and IFN-?in GSDMD^-/-mice may be responsible for the increased spread of Salmonella and increased susceptibility.Therefore,we replenished IL-18 and IFN-?recombinant proteins,and collected tissues at 48 h after infection.It was found that the pathological damage and Salmonella colonization of GSDMD^-/-mice were significantly improved after replenishing the recombinant protein,especially in the effect of limiting Salmonella colonization in the intestinal lumen was significant?P<0.001?.Therefore,IL-18 and IFN-?are important effector molecules of GSDMD inhibiting the colonization of Salmonella in the intestinal lumen.Since the induction of IL-18 and IFN-?was mainly caused by streptomycin treatment,and there was no obvious induction effect at 6 h of Salmonella infection,it is speculated that the IL-18 and IFN-?may be produced by the stimulation of intestinal bacteria,and the difference in IL-18 and IFN-?between the two genotype mice after streptomycin treatment may also be due to the difference in intestinal bacteria between the two genotype mice.To verify this hypothesis,the intestinal microbiota composition of the two genotype mice was examined by quantitative PCR and 16S rRNA sequencing.The results showed that the intestinal microbiota of GSDMD^-/-mice was significantly different from that of WT mice after streptomycin treatment.Two genotype mice were co-housed,and the gut microbiota of the two genotype mice tended to be similar,and GSDMD^-/-was consistent with WT mice for Salmonella susceptibility after co-housing,especially.The difference of IL-18 and IFN?in the two genotypes mice disappeared after co-housing.The intestinal bacteria of the two genotype mice were cleared by compound antibiotics,and the colonization of Salmonella in WT and GSDMD^-/-mice was no longer different;furthermore,after streptomycin treatment the bacteria of WT and GSDMD^-/-mice were transplanted into WT mice,and the colonization of feces and cecal Salmonella in WT mice(GSDMD^-/--strep?WT)was significantly higher than that of the WT mice?WT-strep?WT?.Similar to the co-housing conditions,the difference in IL-18 and IFN-?expression between the two genotype mice also disappeared under the condition of colony clearance before the Salmonella infection;and in the GSDMD^-/--strep?WT mice the colonization of Salmonella was higher,and the IL-18and IFN-?levels were lower than those of the WT-strep?WT mice.The above results indicate that GSDMD forms a specific intestinal microbiota that induces the production of IL-18 and IFN-?,thereby limiting the colonization of Salmonella in the intestinal lumen.In summary,this study reveals that GSDMD may induce the production of IL18and IFN-?by shaping specific intestinal microbiota,and restrict the colonization of Salmonella,thereby achieving its anti-infective protection.