Investigation On The Mechanism Of IL-4 In The Protective Effect Of DnaJ-ΔA146Ply Vaccine

Objective:Streptococcus pneumoniae can normally colonize in the respiratory tract.It can cross the mucosal barrier and cause various diseases.Vaccination is an effective method to prevent S.pn infection.Our previous studies have proved that the fusion protein Dna J-ΔA146Ply had protective effect in immunized mice.Further study showed that the survival rate of IL-4^-/-mice was significantly lower than that of WT mice,and the bacterial loads were increased.These results suggest that IL-4 plays an important role in the immune effect induced by Dna J-ΔA146Ply,but the mechanism is not clear.This study aims to explore the specific mechanism of the role of IL-4 in the protective effect of Dna J-ΔA146Ply vaccine.Methods:To determine whether IL-4 affects the antibody production or function,WT and IL-4^-/-mice were subcutaneously immunized with Dna J-ΔA146Ply,and the antibody titers of specific antibody and Ig G subtypes were compared.Additionally,WT and IL-4^-/-mice were intraperitoneal injected WT-Dna J-ΔA146Ply and IL-4^-/--Dna J-ΔA146Ply antiserum,and the bacterial loads in nasopharynx and lung were compared.The expression of inflammatory factors and the pathology of lung tissue were analyzed.To determine whether IL-4 affects the cellular immune induced by Dna J-ΔA146Ply,spleen cells of immunized WT and IL-4^-/-mice were extracted and stimulated with Dna J-ΔA146Ply.The expression of cytokines was detected by ELISA.Immunized IL-4^-/-mice were injected with anti-IFN-γantibody to neutralize Th1 cytokine IFN-γ,and the bacterial load,expression of inflammatory factors and lung pathological damage were compared to identify whether Th1/Th2 imbalance affects the protective effect of Dna J-ΔA146Ply.Macrophages and neutrophils of WT and IL-4^-/-mice were isolated,and the results of opsono-phagocytosis tests were compared.Western blotting was used to analyze the expression of Syk and p-Syk to explore the specific molecules activated by IL-4.Furthermore,we used piceatannol to inhibit the expression of Syk,and analyzed the opsono-phagocytosis mediated by Dna J-ΔA146Ply and the expression of Syk and p-Syk used by real-time PCR and western blotting to determine whether IL-4 can participate in the protective effect of Dna J-ΔA146Ply by activating Syk.Results:There was no significant difference in antibody titers between WT and IL-4^-/-mice immunized with Dna J-ΔA146Ply（p>0.05）.The antibody titers of Ig G2b were decreased in immunized IL-4^-/-mice（p<0.05）.WT and IL-4^-/-mice were passively immunized with WT-Dna J-ΔA146Ply and IL-4^-/--Dna J-ΔA146Ply antiserum,separately.IL-4^-/-mice showed significantly higher bacterial load than did WT mice（p<0.05）.There was no significant difference between two groups in WT mice（p>0.05）.These results suggest that IL-4 does not affect the antibody production and function.The expression of inflammatory factors of immunized IL-4^-/-mice were increased,and the lung pathological damage aggravated.The expression of IFN-γand IL-17A of immunized IL-4^-/-mice were significantly higher than that of WT mice.These results suggest that IL-4 deficiency promotes Th1and Th17 cellular immune responses induced by Dna J-ΔA146Ply,leading to Th1/Th2 imbalance.After neutralizing IFN-γ,there was no significant difference in the bacterial load,the expression of inflammatory factors and lung pathological damage.These results indicate that IL-4 deficiency can enhance the inflammatory response after bacterial infection,but the imbalance of Th1/Th2 is not the key factor leading to the disappearance of the protective effect of antibody in IL-4^-/-mice.The results showed that the opsono-phagocytosis of IL-4^-/-macrophages was enhanced compared with WT mice（p<0.05）,while that of IL-4^-/-neutrophils was decreased compared with WT mice（p<0.05）.The expression of Syk and p-Syk in IL-4^-/-neutrophils were down regulated.Inhibition of Syk expression by piceatannol attenuated the opsono-phagocytosis of neutrophils in WT mice（p<0.05）,and down regulated the expression of Syk and p-Syk.Additionally,there was no significant difference in the opsono-phagocytosis and the expression of p-Syk in IL-4^-/-mice.These results suggested that IL-4 could enhance the opsono-phagocytosis of neutrophils by up regulating the expression of Syk and p-Syk.Conclusion:IL-4 deficiency does not affect the antibody production and function of Dna J-ΔA146Ply,but can change the cellular immune response.Additionally,IL-4 can enhance the opsono-phagocytosis of neutrophils by activating Syk,and participate in the protective effect of Dna J-ΔA146Ply.This study provides fundamental basis for the mechanism research of IL-4in vaccine immunity.