The Effect And Mechanism Of AIF-1 In Type Ⅰ/Type Ⅱ Immune Response In Mice Infected With Schistosoma Japonicum

Objective:Egg granuloma and liver fibrosis are the main pathological damage in Schistosomiasis.During 2⁴ weeks of schistosoma infection,a type I immune response is observed.Egg deposition,a prounced type II immune response comes up.Allograft inflammatory factor 1（AIF-1）,an important factor involved in many inflammatory diseases and autoimmune diseases,can regulate the balance of type I/II immune response.An enhanced expression of AIF-1 was observed in mice infected with Schistosoma in our previous study.Given the AIF-1 peptide can reduce the fibrosis in schistosomiasis.In order to confirm that AIF-1 can participate in the development of inflammatory reaction in schistosomiasis by regulating the balance of type I/II reactions.The research was administered to demonstrate the role of AIF-1 in immune regulation by observing the pathologic manifestations in liver and polarization of type I/II response.Method:AIF-1 transgenic mice(AIF-1^Tg mice)was constructed.All group mice were challenged through percutaneous exposure to 25 cercariae of Schistsoma.At eight weeks post infection,the worm burden,calculated amount of a single female fecundity,survival rate,and the levels of ALT and hydroxyproline in serum were detected.Liver granuloma and fibrosis were detected by H&E and Masson staining at8 weeks and 14 weeks post infection.The spleen was isolated from mice at 4 weeks,8weeks and 14 weeks post infection,and the mononuclear cells were labeled with fluorescent antibody CD3,CD8,IFN-γand IL-4.The levels of Th1/Th2 cell surface markers（CD3⁺CD8^-IFN-γ⁺and CD3⁺CD8^-IL-4⁺）were detected by Flow cytometry.Real-time PCR was used to detect the expression of transcription factors T-bet and GATA-3 from spleen cells.The spleen lymphocytes were cultured and the supernatant was separated after 72 hours.The expression of cytokines IFN-γand IL-4 was detected by ELISA kit.In order to further study the effect of AIF-1 on macrophage differentiation.Peritoneal macrophages and liver macrophages were separated at 8weeks post infection.Cells were labeled with fluorescent antibody F4/80,CD16/32,CD206.The surface markers of M1/M2 in the peritoneal macrophage and liver macrophage were detected by Flow cytometry.RAW264.7 cells were cultured and stimulated with AIF-1 peptide,LPS and SEA in vitro.After 48 hours of stimulation,the supernatant and the cells were isolated.The expression levels of TNF-αand TGF-βin culture supernatant were detected by ELISA.Cells were used to extract RNA and protein,Real-time PCR was used to detect the expression of iNOS and Arg-1.Then the expression of NF-κB and p-P38 was detected by Western blot.Result:There is no difference on the worm burden and liver eggs quantity between AIF-1^Tg mice and WT mice at 8 weeks post infection,but the area of the egg granulomas in liver,and hepatic fibrosis and liver damage of the AIF-1^Tg mice were reduced.The result from flow cytometry showed that the number of CD3⁺CD8^-IFN-γ⁺cells in the spleen in AIF-1^Tgg mice increased comparing with the control group at 4 weeks post infection.The number of CD3⁺CD8^-IL-4⁺cells in the spleen in AIF-1^Tg mice was decreased after 8 weeks infection.The ratio of CD3⁺CD8^-IFN-γ⁺/CD3⁺CD8^-IL-4⁺showed that the relative proportions of Th1/Th2 cells in AIF-1^Tg mice were significantly higher at 4 weeks and 8 weeks post infection and there was no difference at 14 weeks post infection.AIF-1 suppressed the expression of T-bet at 4 weeks while enhanced the expression of GATA-3 at 4 weeks and 8 weeks post infection in spleen cells by Real-time PCR.the expression of IFN-γin AIF-1^Tg group was increased at 4weeks post infection.At 8 weeks post infection,the proporation of M1 macrophage in AIF-1^Tg mice was higher than that in WT mice.The result from ELISA showed that the expression of TNF-αand TGF-βwas increased in RAW264.7 under the stimulation of AIF-1.But the expression of TGF-βin RAW264.7 stimulated by AIF-1 was less than that stimulated by SEA.The expression of p-P38,NF-κB was increased in RAW264.7 under the stimulation of AIF-1 comaparing to that stimulated by SEA by western blot.Conclusions:AIF-1 can promote the Th cell response to Th1 at the early and acute stages during Schistosoma infection,and promote the macrophage to M1 polarization by activating the P38 MAPK/NF-κB pathway.Thus,AIF-1 can affect the balance of type I/II responses resulting in pathologic changes of schistosomiasis by promoting type I immune response.