Study On Adhesion Of Lactobacillus To Peyer's Patches And Its Immune Modulation

Peyer's patches (PP) are the main site of intestinal mucosa immunity system through which Lactobacillus come into contact with intestinal mucosal immunity system to modulate their functions.This study was focused on relationship with adhesion of Lactobacillus to immobilized mucus from rat small intestine or mice PP in vitro and Lactobacillus surface properties, Lactobacillus with different adhesive properties or treatment (three states: live,ultraviolet (UV)-inactivated and heat-killed(HK)) in the role of immune regulation, anti-infection and regulation of gene expression in PP with strong adhesive strain.The aim was to give some explanation on mechanism that Lactobacillus modulates mucosal immunity and to help developing immunomodulatory agents related to Lactobacillus.Adhesive properties of twenty-three bacteria were studied by using immobilized mucus from rat or PP under spectroscopic methodology or radioactivity.The results indicted that Lactobacillus plantarum Fn008, Lactobacillus acidophilus Fn037, Lactobacillus rhamnose GG (LGG) and Salmonella Typhimurium were the strong adhesive strains. Bacterial surface hydrophobicity was studied by hexadecane (BATH) and water contact angle method. Bacterial surface electric charge was determined by Zeta potential. Surface free energy was calculated by surface tension, electric charge and water contact angle. Expression of lectin was studied by hemagglutination of rabbit red cells.The results indicated that surface free energy of Lactobacillus had a negative correlation with adhesion ability to PP. Expression of lectin on bacterial surface was studied by hemagglutination of rabbit red cells.The results indicated that there was no correlation with hemagglutination of Lactobacillus and adhesion ability.In order to determine the relationship between the immune modulation and adhesion ability, LGG, Fn008, Fn022 and Fn036 were oral administrated to mice with consecutive 3 days.Spleen lymphocyte proliferation and IL-12 gene expression, phagocytosis of peritoneal and PP macrophage, cytokine (IL-4, IL-10, IFN-γ, TGF-β, IL-17), mucosal immunity indicators sIgA, immune-related genes (CD80,Mfge8,CXCL13 and CD79a)and bacterial translocation were analysised respectively.The results indicated that adhesion ability with PP of Lactobacillus(LGG,Fn008,Fn022 and Fn036) was correlated with macrophage phagocytic activity in PP and ability to production of sIgA. Lactobacillus (LGG, Fn008, Fn022 and Fn036) could induce antigen presenting cell specific genes(CD80 and Mfge8)and B-cell chemokines (CXCL13)expression up-regulated,but inhibit B cell specific gene CD79a expression.This function was dependent on adhesion ability to PP.The immune response induced by Lactobacillus was strong than Salmonella; they all induced Th1 immune response. Lactobacillus (Fn008, Fn022, LGG and Fn036) could not destroy mucosa barrier, but Salmonella could cause bacterial translocation significantly through destroying mucosa barrier.In order to determine the relationship between the immune modulation and adhesion ability of different-treated Fn008, V-Fn008,UV-Fn008, HK-Fn008,Fn008+Salmonella and Salmonella were oral administrated to mice with consecutive 3 days. Spleen lymphocyte proliferation and IL-12 gene expression, phagocytosis of peritoneal and PP macrophage, cytokine (IL-4, IL-10, IFN-γ, TGF-β, IL-17), mucosal immunity indicator sIgA, immune-related genes (CD80, Mfge8, CXCL13 and CD79a) and bacterial translocation were analysised respectively.The results indicated that adhesion ability with PP of V-Fn008, UV-Fn008 and HK-Fn008 were correlated with macrophage phagocytic activity in PP and ability to production of sIgA.V-Fn008,UV-Fn008 and HK-Fn008 could induce antigen presenting cell specific genes(CD80 and Mfge8)and B-cell chemokines (CXCL13)expression up-regulated,but inhibit B cell specific gene CD79a expression.This function was dependent on the adhesion ability to PP.The immune response induced byUV-Fn008 and HK-Fn008 was weaker than V-Fn008; they all induced Th1 immune response. Lactobacillus (Fn008, Fn022, LGG and Fn036) could not destroy mucosa barrier, but Salmonella could cause bacterial translocation significantly through destroying mucosa barrier. V-Fn008 can inhibit bacterial translocation induced by Salmonella.Onto-Tools were applied to explore significant genes in jejunal Peyer's patch induced by oral administration Fn008 with consecutive 14 days. The results indicated that responsive genes in jejunal Peyer's patch mainly related to immunity, mucosal barrier and other pathways (regulation of actin cytoskeleton, natural killer cell-mediated cytotoxicity and etc.). The results of quantitative PCR indicated that Lactobacillus (LGG,Fn008,Fn022,Fn036, UV-Fn008 and HK-Fn008) could induce focal adhesion gene(Itgb1,lamaα3 and Thbs2) expression upregulated.This function was dependent on the adhesion ability to PP.V-Fn008 can induce key gene FAK down-regulated and genes ( Itgb1,lamaα3,Itgb4,Thbs2) up-regulated in focal adhesion.This caused inhibition of bacterial translocation induced by Salmonella.In conclusion, the results of present study had revealed that adhesive ability of Lactobacillus to PP mainly determined by surface free energy.Its adhesive ability to PP had correlation with macrophage phagocytic activity in PP and ability to production sIgA. Lactobacillus can induce focal adhesion gene(Itgb1,lamaα3 and Thbs2),antigen presenting cell specific genes(CD80 and Mfge8)and B-cell chemokines (CXCL13)expression up-regulated,but inhibit B cell specific gene CD79a expression.This function was dependent on the adhesion ability to PP. V-Fn008 can induce key gene FAK down-regulated and genes (Itgb1,lamaα3,Itgb4,Thbs2) up-regulated in focal adhesion.This caused inhibition of bacterial translocation induced by Salmonella.