Study On Polarization Of M1 Macrophages Induced By Lactobacillus Plantarum Against Salmonella Enterica Serovar Typhimurium Infection

Probiotics are usually colonized in the intestinal tract with their good adhesion ability,regulating the balance of intestinal flora,activating the innate immune cells in the bottom of the mucosa,and playing a variety of beneficial effects on the host.Macrophages are important bridge cells mediating innate and acquired immunity.They can differentiate into classical activated macrophages(M1)and alternative activated macrophages(M2)under different stimuli.Among them,M1 has a strong antigen presenting effect and the ability to remove pathogens,and promotes the inflammatory response of the body.M2 is mainly immune regulation and immune tolerance,inhibiting excessive inflammatory response and promoting tissue repair.Studies have shown that the regulation of probiotics on inflammation may be achieved by affecting macrophage polarization.However,the effect and mechanism of probiotics on macrophage polarization have not been clarified,especially in the case of exogenous pathogen infection.Therefore,in this study,Lactobacillus plantarum(LP)isolated from the intestinal tract of piglets in the early stage was used as the research object.Through in vivo and in vitro experiments,the regulatory effects of Lactobacillus plantarum on macrophage polarization and key signaling pathway molecules were studied to clarify the mechanism of Lactobacillus affecting macrophage polarization.(1)Inhibition of Salmonella typhimurium infection by LP-induced macrophage polarizationIn the in vivo experiment,C57BL/6 mice were intragastrically administrated with LP in advance.Seven days later,Salmonella enterica serovar Typhimurium(S.Typhimurium)was infected.The surface markers(CD80~+,CD86~+and CD206~+)of mesenteric lymph nodes(MLNs)and spleen macrophages(F4/80~+)were detected by flow cytometry;By monitoring the weight loss rate and survival rate of mice,the number of S.Typhimurium bacteria in organs(liver and spleen)was detected to determine the effect of LP-induced macrophage polarization on S.Typhimurium infection.The results showed that LP could up-regulate the expressions of CD80~+and CD86~+in mouse spleen and MLNs;Slow down the weight loss caused by S.Typhimurium and improve the survival rate of mice;Inhibition of S.Typhimurium migration and colonization to liver and spleen.This indicated that LP could increase the polarization of M1 macrophages in the spleen and MLNs of mice infected with Salmonella,protect mice from the invasion of S.Typhimurium,and enhance the resistance of mice.In vitro,LP(1×10~7 CFU)and RAW264.7(1×10~6)were co-cultured for 1 h and12 h,respectively.Western blotting was used to detect the expression of M1polarization marker protein(i NOS)and M2 polarization marker protein(Arg-1).The secretion of TNF-?,IL-6 and IL-10 in the culture supernatant of macrophages was detected by ELISA to reveal the effect of LP on macrophage polarization.The effect of LP on the phagocytosis of Salmonella by macrophages was detected by immunofluorescence,and the number of viable Salmonella in macrophages was detected by plate counting method to further verify the effect of LP on the function of polarized macrophages.The results showed that LP promoted the expression of M1polarization marker protein i NOS,increased the secretion levels of TNF-?and IL-6,promoted the expression of M2 polarization marker Arg-1,and down-regulated the secretion of IL-10;Moreover,LP significantly increased the phagocytosis and killing ability of macrophages to S.Typhimurium.In conclusion,LP can enhance the phagocytosis and killing ability of S.Typhimurium by promoting the polarization of RAW 264.7 to M1.(2)LP synergistically promoted M1 macrophage differentiationIn order to further clarify whether LP can synergistically enhance the polarization and function of M1 macrophages,we used known M1 inducer LPS and IFN-?,M2 inducer IL-4 to induce murine bone marrow-derived macrophages(BMDM)in non-polarized state(M0)to differentiate into M1 and M2,respectively.LP(1×10~7 CFU)was co-cultured with M0,M1 and M2 cells for 6 h.The expression of M1 marker CD86~+,TNF-?and IL-6,M2 marker CD206~+and IL-10 were detected by flow cytometry and ELISA.The effects of LP on the phagocytosis and killing of S.Typhimurium by polarized M1 and M2 were detected by immunofluorescence and plate counting.The results showed that LP could synergistically up-regulate the expression of CD86~+molecules on the surface of M1 macrophages with LPS and IFN-?,and synergistically increase the secretion of TNF-?and IL-6.LP down-regulated the expression of CD206~+molecules on the surface of M2 cells induced by IL-4,and reduced the secretion of IL-10.In addition,the results of phagocytosis and killing effect of macrophages showed that LP did not significantly affect the phagocytosis of M1 macrophages induced by LPS and IFN-?,but its ability to kill S.Typhimurium was improved.LP enhanced the phagocytic function of IL-4-induced M2 cells,but did not enhance the cytotoxicity of cells.In summary,LP can enhance the polarization of M1 macrophages and improve their ability to kill Salmonella in coordination with LPS and IFN-?,but there is no synergistic effect on M2 macrophages.Since NF-?B is a key signal molecule mediating M1 polarization,western blot was used to further detect the activation of NF-?B.The results showed that after LP intervention,the expression of p-I?B?in macrophages was up-regulated,indicating that LP activated the classical pathway and activated the NF-?B signaling pathway by promoting the expression of p-I?B?to mediate the polarization of M1 macrophages.(3)Preliminary study on the polarization of LP to porcine macrophagesIn order to compare the polarization effects of LP on macrophages from different species,porcine macrophages(3D4/21)were used as the research objects in this experiment.LP(1×10~7 CFU,1×10~8 CFU)were used to stimulate cells(1×10~6 cells)for 12 h.The effects of LP on the expression levels of i NOS and Arg-1 in porcine macrophages and the levels of cytokines(including TNF-?,IL-6 and IL-10)were detected,and the effects of LP on the phagocytosis and killing ability of macrophages were further detected.The results showed that LP could still promote the M1polarization of porcine macrophages and improve their ability to phagocytize and kill Salmonella,which was basically consistent with that of mouse-derived macrophages.In conclusion,Lactobacillus plantarum can promote the polarization of M1macrophages and resist Salmonella typhimurium infection;Moreover,Lactobacillus plantarum synergistically enhanced the polarization of M1 macrophages by activating the classical NF-?B pathway and enhanced its phagocytosis and killing ability against Salmonella typhimurium.The polarization effect of porcine macrophages was basically consistent with that of mouse macrophages.