| Schistosomiasis japonica,caused by Schistosoma japonicum,is an infectious disease and an enormous public health problem.The mainly pathogenesis of this disease is the occurrence of egg granulomatous inflammation in liver and intestine and subsequently fibrosis.The pathogenic mechanism is mainly Ⅳ type hypersensitivity reaction mediated by CD4+ T cells.A growing number of evidence suggests that Th2,Th17 and Tfh cells could up-regulate hepatic egg granuloma and fibrosis via secreting IL-4,IL-17 and IL-21,respectively,while Th1 and Treg cells play an opposite role by producing IFN-y and IL-10,respectively.Th9 has recently been described as a new subset of CD4+T cells.Recent studies have found that Th9 cells are involved in the development of hepatitis B hepatic fibrosis and contribute to the formation of hepatic fibrosis due to carbon tetrachloride(CC14)in an experimental mouse model.However,the roles of Th9 cells in the liver pathology induced by S.japonicum have never been explored.ICOSL/ICOS signaling pathway plays an important role in the polarization of Th cells.Our seminar in earlier research find that ICOSL/ICOS signal has a regulating role in the polarization of Thl,Th2,Th17 cells and Tfh cells,thereby affecting the process of egg granulomas and fibrosis in schistosomiasis.Nevertheless,it remains unclear whether polarization of Th9 is mediated by ICOSL/ICOS signal.In this study,we established ICOS transgenic mice(ICOS-Tg)as experimental animal models for schistosomiasis.To clarify whether ICOSL/ICOS signaling pathway is the key signal molecular of Th9 cells differentiation and polarization via specifically enhancing ICOSLACOS signal.To elucidate the molecular mechanism of Th9-mediated liver fibrosis formation in vivo and in vitro.In addition,to investigate the different immune response characteristics between Th9 and Th2 subsets.This project will be expected to provide a scientific basis for clarifying immunopathological mechanism of schistosomiasis and explore a new effective way for prevention and treatment of the hepatic egg granuloma and hepatic fibrosis.1.Dynamics of Th9/IL-9 in immunopathogenesis of murine schistosomiasisObjective:To explore the dynamic changes and potential roles of Th9/IL-9 in the pathogenesis of hepatic egg granulomatous inflammation and fibrosis in mice infected with Schistosoma japonicum.Methods:The average areas of egg granulomas were estimated by hematoxylin-eosin(H&E)staining.Hepatic IL-9 and transcription factor PU.1 levels were detected by immunohistochemistry.Flow cytometry techniques were used to analyze the proportions of Th9 cells.With the help of ELISA,serum levels of IL-9 were examined.Results:The egg granulomas began to form from four weeks after infection and continued to develop.In parallel with the development of egg granulomas,the hepatic levels of IL-9 and PU.1 increased very slowly during the first four weeks post-infection and increased rapidly thereafter.Moreover,the proportions of splenic Th9 cells and levels of serum IL-9 had similar developmental trends with the egg granulomas.Conclusion:The proliferation of Th9 cells and levels of IL-9 were significantly higher in S.japonicum-infected mice compared to non-infection.In addition,dynamic changes of Th9 and IL-9 were synchronous with the developmental trend of hepatic egg granulomatous inflammation,suggesting that Th9 cells might be a new subset in the pathogenesis of schistosomiasis.2.Effects of Th9/IL-9 in eggs granulomatous response and hepatic fibrogenesis of schistosomiasisObjective:In order to investigate effects of Th9/IL-9 in hepatic egg granulomatous inflammation,particularly in liver fibrosis in mice infected with Schistosoma japonicum.Methods:(1)Followed S.japonicum infection,eighteen mice were randomly divided into three groups(n=6 in each group):infected mice treated with anti-IL-9 antibody(anti-IL-9 group)and treated with IgG isotype control(IgG group).Six non-infected mice were used as control group.Nine weeks after infection,flow cytometry techniques were used to analyze the proportions of Th9 cells.With the help of ELISA,serum levels of IL-9 were examined.Histology was assessed by hematoxylineosin staining.Fibrillar collagen deposition was evaluated by Masson staining.(2)By taking situ liver perfusion and density gradient centrifugation,HSCs were separated from mice primary of before infection(0 Week)and after infection(7 Weeks).HSCs were randomly assigned to control group and stimulation group after cultured 24 hours.Control group continued to culture in DMEM complete medium.Stimulation group continued to culture with IL-9 cytokines(20 ng/ml)in DMEM complete medium.After 48 hours and 72 hours,primary HSCs were harvested and using Western blotting detect a-SMA,Collagen-I,Collagen-III protein expression levels in HSCs.Results:Nine weeks post-infection,there were obvious hepatic egg granulomatous inflammation and fibrosis in both anti-IL-9 group and IgG group.However,neutralization of IL-9 reduced frequencies of Th9 in spleen,suppressed expression of IL-9 in serum,more importantly,attenuated egg granulomas and fibrosis in liver compare to injection of IgG.In vitro,HSCs were remarkably activated and secreted more Collagen-I and Collagen-III after 48 hours and 72 hours stimulation of IL-9.Conclusion:Th9/IL-9 could promote liver granulomatous inflammation and fibrosis in schistosomiasis by inducing HSCs activation and driving HSCs to secrete Collagens.3.Th9 immune response in front Th2 immune response in immunopat hogenesis of murine schistosomiasisObjective:To observe the immune network of Th9 cells and Th2 cells in the pathogenesis of schistosomiasis.Methods:Twenty-five mice were euthanized at 0,4,7,9 and 12weeks after infection.The dynamics of Th9 cells and Th2 cells in spleen were analyzed by flow cytometry techniques after infection.The serum levels of IL-9 and IL-4 were measured by ELISA,and the expressions of IL-9 and IL-4 in liver were detected by immunohistochemistry.Results:Flow cytometry results showed that the proportion of Th9 was increased very rapidly at 4 weeks and peaked at 7 weeks.Subsequently,the number of Th9 cells declined gradually.While The proportion of Th2 was increased very slowly and peaked at 12 weeks.More importantly,the results of ELISA and immunohistochemistry were synchronous with the flow cytometry results.The peak of IL-9 in serum and liver appeared at 7 weeks post-infection,while the peak of IL-4 occurred at 12 weeks post-infection.Conclusion:Th9/IL-9 might exert functions in the regulation of immuno pathogenesis of schistosomiasis in front of Th2/IL-4.4.Effection of ICOSL/ICOS signaling pathway on Th9 and Th2 immune respone in the development of immunopathogenesis in mice infected S.japonicumObjective:To study the regulative effects of Th9 cells and Th2 cells differentiation and polarization mediated by ICOSL/ICOS signal in the development of immunopathogenesis in mice infected S.japonicum.Methods:Using the ICOS-Tg mice and wild-type(WT)mice as experimental animal models for schistosomiasis,the dynamics of Th9 cells and Th2 cells in spleen were analyzed by flow cytometry techniques after infection.The serum levels of IL-9 and IL-4 were measured by ELISA,and the expressions of IL-9 and IL-4 in liver were detected by immunohistochemistry.Results:Flow cytometry results showed that the proportions of Th9 from ICOS-Tg mice were significantly increased at 4 weeks,7 weeks and 9 weeks compared to their WT control;The proportions of Th2 from ICOS-Tg mice were distinctly higher at 7 weeks and 9 weeks than their WT control.ELISA results showed that the levels of IL-9 in serum from ICOS-Tg mice were obviously up-regulated at 4 weeks,7 weeks,9 weeks and 12 weeks compared to their WT control;The levels of IL-4 in serum from ICOS-Tg mice were evidently increased at 7 weeks compared to their WT control.Immunohistochemistry results showed that the expressions of IL-9 in liver from ICOS-Tg mice were statistically up-regulated at 4 weeks,7 weeks,9 weeks and 12 weeks compared to their WT control;The expressions of IL-4 in liver from ICOS-Tg mice were dramatically higher at 7 weeks,9 weeks and 12 weeks compared to their WT control.Conclusion:Enhanced ICOSL/ICOS signal could strongly up-regulate Th9 and Th2 differentiation and polarization in the immune response of murine schistosomiasis.In summary,Th9 cells not only promoted liver granulomatous inflammation and fibrosis but also exerted functions in front of Th2.ICOSL/ICOS signaling pathway could mediate the Th9 and Th2 responses,and thereby affect immunopathogenesis of schistosomiasis.This study might provide a new approach to control the development of hepatic granulomatous inflammation and subsequent fibrosis in schistosomiasis. |
