The Effect Of Bacillus Amylobacillus SC06 On Macrophage Polarization

Bacillus amyloliquefaciens SC06（BaSC06）,a strain of probiotic screened by our research group,has been proved to have remarkable effects on growth and immunity function of animals.Regulation of macrophage polarization is one of the important reasons for BaSC06 to improve elimination of pathogenic bacteria and exerting probiotic effects in host.The aim of this research was to investigate the effects of BaSC06 on the polarization of macrophages and elucidate the potential mechanism of macrophage polarization.In this study,the effects of BaSC06 on the polarization of macrophages and signaling pathways were explored.Meanwhile,we further studied the effects of exosomes that treated by BaSC06（Ba-Exo）and without treatment BaSC06（Exo）produced by Raw264.7 cells on the polarized phenotype and their signaling pathway expression in Raw264.7 cells.The main results are as follow:1 Effects of BaSC06 on polarization in Raw264.7 cellsThe aim of study is to investigate the relative mechanism that exist on BaSC06 induced macrophages polarization.BaSC06 at 1×10^{^8} cfu/ml was used to treat Raw264.7 for 6 hours to assess phenotypic and function by western blotting and ELISA.The pathways of BaSC06 induced Raw264.7 were detected by western blotting.The research of BaSC06 induced IL-4 teratment of Raw264.7 cells was conducted by real-time PCR.The results showed that:BaSC06 treatment can enhance the expression of iNOS,IRF 5 protein and NO synthesis in macrophages after 6h,indicating that Raw264.7 treated with BaSC06 are polarized to MI polarization;significantly increase the phagocytosis of Ecoli K88 by macrophages and reduce the survival rate of macrophage intracellular bacteria after 12 hours,indicating that BaSC06 can enhance the phagocytosis and bactericidal ability of Raw264.7.The BaSC06 induce IL-4 treatment of macrophages result show the expression levels of IL-1β,IL-6,IL-12P40 and TNF-α in the IL-4+BaSC06 group were significantly increased,which significantly down-regulated Arg-1,Fizz],MR,and Ym1 genes,compared with the IL-4 group,indicating that BaSC06 can reverse M2 phenotypes.Moreover,we demonstrated that BaSC06 can activate the NF-κB signal pathway,p38 signal pathway and STAT3 signal pathway.It was found that the NF-κB signal pathway mediates the synthesis of NO,IFN-β,IL-1β,TNF-α,and iNOS.while the p38 signal pathway involved in the synthesis of NO and IL-1β,and inhibited Arg-1 and IL-10 expression through inhibitors.The result showed that both p38 and NF-κB signaling pathways were participated in the signal regulation of Raw264.7 cells towards M1 polarization.2 Effects of BaSC06-induced exosomes on Raw264.7 cell polarizationIn this study,Raw264.7 cells were treated with 1×10^{^8} cfu/ml heat-inactivated BaSC06 and Raw264.7 cells were treated with PBS for 24 h to induce the secretion of specific exosomes named Ba-Exo and normal exosomes named Exo.The study was focus on effect of exosomes to Raw264.7 cells about macrophages phenotypes,function and mechanism.Our result show that exosomes were analyzed by Western blotting,transmission electron microscopy and dynamic light scattering（DLS）.We found that their diameters ranged from 40 to 100 nm,with a typical cup-shaped or circular vesicle shape and the expression of CD9 and TSG101 on the surface of Ba-Exo and Exo were expressed positively.Ba-Exo treatment can enhance the expression of iNOS protein and NO synthesis in macrophages after 6 hours,and has no effect on IRF 5 protein.Exo did the same treatment and had no effect on the synthesis of iNOS,IRF 5 protein and NO.It shows that Ba-Exo-treated Raw264.7 macrophages are polarized toward M1 type,but Exo does not have this effect.Ba-Exo can significantly up-regulate the relative expression levels of M1 type marker genes iNOS,IFN-β,IL-1β,IL-6,IL-12P40,and TNF-α,down-regulate M2 type marker genes Arg-1,TGF-β,Fizzl,and Ym1,IL-10 and MR did not change significantly.Exo only increased the relative expression of IFN-β,down-regulated the relative expression of Arg-1,TGF-β,and Fizzl,and there was no significant change in the expression of Ym1,IL-10,and MR.The result indicated that Ba-Exo-treated Raw264.7 macrophages were polarized toward M1 type,but Exo did not have this effect.The phagocytosis experiments revealed Ba-Exo and Exo treated Raw264.7 cells had not the phagocytic ability against Ecoli K88 and survival rate of intracellular bacteria,indicating that neither Ba-Exo nor Exo could significantly affect the phagocytosis and sterilization ability of Raw264.7.We detected the mechanism of Ba-Exo and Exo effect on STAT signal,MAPK signal and NF-κB signal.The result showed that Ba-Exo can not activate ERK,STAT1 and STAT6 signal pathway in Raw264.7 cells,Exo can not activate MAPK signal,STAT1 and STAT3 and NF-κB signal,but activate STAT6 signal.Ba-Exo can activate the NF-κB,p38 and STAT3 signals pathway.It was found that p38 and NF-κB signaling pathways mediate IFN-β,IL-6,IL-12P40,and IL-1β synthesis in Ba-Exo-induced M1 macrophages through inhibitors.The p38 signaling pathway also inhibits the synthesis of IL-10 in M2-type macrophages induced by Ba-Exo.This indicates that p38 and NF-κB signaling pathways play a positive role in activating polarization of M1-type macrophages,and p38 signaling pathways play a negative role in activating polarization of M2-type macrophages.