Effects Of Bacillus Subtilis Expressing Different Lipopeptides On Congenital Immunity Of Chicken Mucosa

Bacillus subtilis(B.subtilis)can improve the intestinal environment and promote the absorption of nutrients.Previous studies have shown that Bacillus subtilis as mucosal adjuvants can improve the local immune response,but the mechanism has been unclear.Bacillus subtilis can express antimicrobial lipopeptides such as surfactin?fengycin?Iturin etc.These lipopeptides have a good effect in the anti-virus,anti-bacterial,anti-parasite and so on.We hypothesize that antimicrobial lipopeptide may be associated with the mucosal immune response.In order to explore this mechanism,we conducts this test.First of all,we use Bacillus subtilis,Surfactin B.subtilis,Fengycin B.subtilis,Iturin B.subtilis to explore its function in the local cellular immunity and humoral immunity.And then we observe its effect on DCs phenotype and function in vitro experiments.By studying the effect of Bacillus subtilis expressing different lipopeptides on congenital immunity of chicken mucosa,we can further understand the mechanism of Bacillus subtilis and its lipopeptide in improving local mucosal immunity,and provide reference for its future application.1.Effects of Bacillus subtilis expressing different lipopeptides on local-cellular immunity in chickens.Cytokines are a key regulator of innate immunity and adaptive immune systems.Many cytokines play an important role in the local cellular immunity,such as IL-1?,IL-6,IL-12,IFN-y.By detecting its expression,we can know the localized cellular immunity to a certain extent.In this study,36 SPF chickens were randomly divided into 6 groups.The first immunization was performed at 3 days old and the second time after one week.Respectively,PBS,Bacillus subtilis,Surfectin B.subtilis,Fengycin B.subtilis,Iturin B.subtilis,Poly(I:C).The third day after the second immunization,we slaughtered animal and take the nasal cavity,trachea and jejunum.The cytokines in the tissue were tested using an enzyme-linked immunosorbent assay(ELISA)kit.The results showed that different Bacillus subtilis strains could increase the expression of IL-1?,IL-6,IL-12 and IFN-y in the measured tissues.Surfactin B.subtilis could significantly increase the expression of cytokines in the tissues compared with other Bacillus subtilis strains,followed by Fengycin B.subtilis;Bacillus subtilis and Iturin B.subtilis did not improve the effect of IL-6 in the nasal cavity.Bacillus subtilis expressing different lipopeptides has different effects on improving the immunity of local mucosal cells in chickens.This suggest that lipopeptide could enhance the innate immune effect.And surfactant can better stimulate the local mucosa to produce cytokines.2.Effects of Bacillus subtilis expressing different lipopeptides on local humoral immunity in chickens.SIgA is the main antibody of humoral immunity in the mucosal mucosa,and plays an important role in the humoral immunity of various mucosal tissues.Non-specific SIgA can be used as a measure of local mucosal humoral immunity level.In this study,chickens were immunized with different Bacillus subtilis and PBS,Poly(I;C).And then take 13,24.38 days of age serum,nasal swabs,cloacal swabs,nasal rinse liquid,trachea rinse lotion,jejunum rinse and cecal feces.The expression level of Non-specific SIgA and IgA in serum was detected by ELISA kit.The results showed that the expression of Non-specific SIgA and IgA increased first and then decreased in all the detection sites.Compared with the control group,different kinds of Bacillus subtilis in the measured age improved the body mucosal antibody level.The expression levels of SIgA and IgA in Surfactin B.subtilis treated group were significantly higher than those in control group and other Bacillus subtilis group.Compared with B.subtilis and Iturin B.subtilis,Fengycin B.subtilis group could improve the expression level of SIgA more effectively.This suggests that Bacillus subtilis,which expresses different lipopeptides,can improve the level of congenital humoral immunity in chickens.And the effect of Surfactin B.subtilis is the best,followed by the Fengycin B.subtilis.This indicate that lipopeptide can improve mucosal congenital humoral immunity,and surfactant can effectively enhance the immune effect.3.Effects of Bacillus subtilis expressing different lipopeptides on the function of chicken dendritic cells in vitroDCs are a powerful antigen presenting cell(APC).DCs can promote the body's immune response by stimulating T,B lymphocytes to produce cytokines and antibodies.The expression of MHCII can reflect the ability of DCs to deliver.Mature chicken DCs overexpress MHCII,and the ability to deliver increases.In order to investigate the effect of Bacillus subtilis expressing different lipopeptides on the maturation and function of dendritic cells,chicken bone marrow-derived DCs were isolated in vitro and their morphology and phenotype were identified.Flow cytometry was used to detect the expression of MHCII and the proliferation of T cells after treatment with different Bacillus subtilis.The results showed that Surfactin B.subtilis and Fengycin B.subtilis could significantly up-regulate the expression of MHCII,and the effect of Surfactin B.subtilis was significantly higher than that of Fengycin B.subtilis.The effect of Bacillus subtilis and Iturin B.subtilis treatment group is not obvious.In addition,the proliferation of DCs stimulated by Surfactin B.subtilis and Fengycin B.subtilis treated group was significantly increased.This suggests that Surfactin B.subtilis can more effectively improve the ability of DCs to elicit and activate T cells.This Indicates that lipopeptides can promote dendritic cell maturation,and Surfactants are more effective in promoting dendritic cell maturation than other lipopeptides.