The Effects Of Bacillus Amyloliquefaciens On Macrophage Polarization And Resistance To Salmonella Infection In Mice

Probiotics can regulate the intestinal flora balance and immune function of animals,thereby improving animal performance.Bacillus strains are widely used in feed industry and livestock because of their better stress resistance.We have obtained a Bacillus amyloliquefaciens strain SC06(BaSC06),which can significantly promote the growth of piglets and the intestinal mucosal immune function.The aim of this study was to investigate the effects of BaSC06 on macrophage polarization and the resistance to Salmonella enterica serovar Typhimurium(ST)infection in mice,and explore its mechanism.The main contents and results are as follows:(1)Effects of BaSC06 on ST infection and intestinal macrophage polarization in miceMale C57BL/6 mice were randomly divided into four groups:Control group,BaSC06 group,ST group and BaSC06 + ST group.Control group and ST group were fed with normal diet;BaSC06 group and BaSC06 + ST group were fed with diet contain BaSC06 for 8 weeks.Mice in ST and BaSC06 + ST groups were infected with ST and sacrificed 3 days post infection.Results showed that orally administration of BaSC06 significantly attenuated the weight loss,cecum atrophy,mucosal injury in ileum,cecum and colon of mice,which induced by ST infection.BaSC06 significantly reduced the translocation of ST in mesenteric lymph node(MLN)and spleen.Before and after ST infection,BaSC06 has no effect on the CD4:CD8 ratio in peyer's patches(PP),MLN and spleen,indicating that BaSC06 could not regulate T cell immune response.There was no significant effect of BaSC06 on serum IL-1?,IL-4,IL-12,IL-10 and IL-13 level in mice,but the significant increased expression of IFN-? was observed.BaSC06 significantly reduced the increased level of pro-inflammatory cytokines IL-1? and IL-12 induced by ST infection and promoted the expression of IL-10.It indicated that BaSC06 was effective against ST infection.Furthermore,BaSC06 significantly increased the number of M1 and M2 macrophages in the cecum and MLN.The above results implied that administration of BaSC06 effectively relieved intestinal and systemic inflammation induced by ST infection,and induced M1 and M2 polarization in intestine in mice.(2)The effects of BaSC06 on polarization of macrophageThe effects of BaSC06 on macrophage phenotype and function were determined in Raw264.7 macrophages and bone marrow derived macrophages.BaSC06 significantly enhanced phagocytosis and intracellular bactericidal function of Raw264.7 macrophages after co-culture for 6h.Gene expression and Western Blot results showed that BaSC06 induced Ml polarization of macrophages.Flow cytometry analysis showed that pre-treatment with BaSC06 inhibited M2 polarization of macrophages which induced by ST infection.Mice were treated with intraperitoneal injection of BaSC06(106 CFU/mouse)or(105 CFU/mice).12 hours later,the mice were sacrificed and the peritoneal macrophages were isolated for flow cytometry.The results showed that ST induced peritoneal macrophages M2 polarization,inhibited M1 polarization,whereas BaSC06 induced Ml type polarization,had no significant effect on M2 polarization.Tail vein injection of macrophages induced by BaSC06 significantly reduced the amount of ST in spleen.The above results indicated that BaSC06 enhanced the bactericidal ability of macrophage by inducing M1 polarization.The BaSC06-induced M1 polarization of macrophages was directly verified using an antibiotic-treated mouse model to exclude the interference of intestinal flora.Each mouse was fed with 20 mg streptomycin and then administered with ST and BaSC06.Compared with the antibiotic treatment group,ST infection could induce intestinal macrophages M2 polarization,and had no significant effect on Ml macrophages.BaSC06 promoted intestinal macrophages M1 polarization,inhibited M2 polarization induced by ST.The above results indicated that BaSC06 induced macrophage M1 polarization and reversed ST-induced M2 polarization,promoting the clearance of ST.(3)Effects of intestinal microbiota regulated by BaSC06 on ST infection and macrophage polarizationBaSC06 induced M1 and M2 macrophage in the intestine of mice,but only promoted M1 polarization in vitro and antibiotic-treated mouse.It is presumed that BaSC06 promoted M2 polarization by regulating intestinal flora.Sequencing analysis of intestinal flora in mice showed that adiministration of BaSC06 had no effect on the intestinal microflora diversity of healthy mice,significantly reduced the loss of microflora diversity caused by ST infection.ST infection reduced the proportion of Verrucomicrobia,increased the relative abundance of Bacterodetes.Adiministration of BaSC06 significantly increased the proportion of Verrucomicrobia and reduced Bacterodetes,indicating that BaSC06 could alleviate the imbalance of intestinal flora caused by ST.The intestinal flora of control group and BaSC06 group were transplanted into the antibiotic-treated mice to detect the inflammation and macrophage polarization in cecum.The results showed that the fecal microbiota from BaSC06 group relieved ST-induced cecal inflammation and promoted the M2polarization of intestinal macrophages.In summary,adiminstration of BaSC06 can directly promote macrophage M1 polarization,increased its bactericidal function on ST.While the intestinal microbiota regulated by BaSC06 promoted macrophage M2 polarization which maintained the intestinal microecological stability and inflammation balance.Both M1 and M2 polarization induced by feeding BaSC06 contribute to the resistance to ST infection.