| The immune response of CD4+T cell to soluble antigen secreted by schistosome eggs,which may lead to cumulative liver granulomatous inflammation and subsequently fibrosis.CD4+helper T（Th,including Th1,Th2,Th17,Tregs）cells play critical roles in both host humoral immunity and cellular immunity against parasitic infection and immunopathology in schistosomiasis.Studies have shown that Th2,Th17 polarization was closely related to the development of immunopathology in host infected with S.japonicum.Tfh was reported to have an important role in type schistosomiasis and Japanese schistosomiasis.However,the mechanism of immunomodulatory still unknown.Research suggested that a direct correlation between the expression of ICOS and the differentiation and function of Tfh.ICOS-ICOSL signal play a key role in the formation of chronic inflammation and fibrosis.However,regulatory molecules related Tfh cells in the process of immune reponses and mechanism is not clear.In this study,we established ICOSL-/-,ICOSL+/-and wild-type controls mice as experimental animal models for schistosomiasis.To clarify the regulation role and key signal molecular of Tfh differention,polarization and GC reponses regulated by ICOS-ICOSL in the development of immunopathology in mice infected with S.japonicum.Schistosomiasis sexual disease it will provide a scientific basis for control the development of advanced fibrosis in schistosomiasis.一.The feature of immunomodulatory and molecular mechanism of Tfh in the development of immunopathology in mice infected S.japonicumObjective:to study the feature of immune response and immunomodulatory function of Tfh cells in the development of immunopathology in mice infected with S.japonicum.Methods:1.Kinetic analysis of costimulatory molecules ICOS,PD-1,OX40,CD40L,transcription factor Bcl-6,effector cytokine IL-21 of Tfh by flow cytometry on the day before infection（0 week）,the early stage of infection（4 weeks post-infection）,acute infected stage（7 weeks-9 weeks after infection）,chronic infected stage（12weeks post-infection）and late stage（16 weeks post-infection）.The correlation between dynamic changes of molecules associated with Tfh cells and the development of immunopathology was analyzed.2.Immunofluorescence technique was used to detect the dynamic changes of GC formation and numbers of Tfh cells have migrated to GC in the spleen in mice infected with S.japonicum.Isolated Tfh cells and B cells and coculture,dealing with IL-21R blocking antibody to observe its effect on B cells humoral immune response in the different period of infected mice.3.The liver granulomatous pathology in mice infected with S.japonicum was dynamically observed by hematoxylin and eosin（HE）staining.The hepatic fibrosis level in mice infected with S.japonicum was dynamically observed by Masson staining.Immunohistochemistry was used to detect the expression level of IL-21 andα-SMA of the mark molecules of HSCs activation in liver granuloma in the different infected period.Flow cytometry and Real-time PCR method to compare the expression level of IL-21R on HSCs in mice on the day before infection and acute infected stage（7 weeks postinfection）.Isolated HSCs in mice infected with S.japonicum and stimulated with IL-21 in vitro,discussed and analyzed the function of IL-21 on HSCs.4.IL-21 was injected into the mice infected with S.japonicum by abdominal cavity to observe the role of liver pathology and CD4+T cell immune response.Results:1.The results of flow cytometry showed that the expression level of Bcl-6were upregulated at 4 weeks post-infection and peaked at 7 weeks post-infection and keeping a comparative higher level subsequently.Tfh cells proliferation was positively correlated with the progression of liver fibrosis.costimulatory molecules and effector cytokines related with Tfh cells were elevated at 4 weeks after infection,further increased at 7 weeks post-infection（P<0.05 or P<0.01 or P<0.001）and down-regulated slowly at 9 weeks after infection and keeping a comparative higher level subsequently.2.Immunofluorescence data suggested that uninfected mice had rare normal GC structure in the spleen,while many normal GC were observed at 4 weeks post-infection（P<0.001）.A large number of GC still exist at 7 weeks after infection and then began reduced slowly（P<0.01）.Along with the progression of liver fibrosis,more Tfh cells migrate to GC.3.The expression level of IL-21 in liver granuloma was increased at 4 weeks after infection（P<0.01）,peaked 12 weeks after infection（P<0.001）and have a comparative higher level subsequently（P>0.05）.HSCs also express the IL-21R in normal mice and the mice infected with S.japonicum displayed markedly upregulated（P<0.05）.Stimulating with IL-21 cytokines could increase the secretion of HA produced from HSCs（P<0.05）.4.IL-21 stimulation increased the area of liver granuloma（P<0.001）and upregulated the expression of collagen in liver（P<0.001）.IL-21 stimulation had no role in CD69 expression of CD4+T cells and Tfh and Treg cells differentiation.IFN-γ/IL-4/IL-17A production of CD4+T cells were upregulated by IL-21 stimulation（P<0.05 or P<0.001）,but have no role in IL-21 expression of CD4+T cells.Conclusion:Tfh proliferation and polarization were positively correlated with hepatic fibrosis progression in mice infected with S.japonicum.Tfh cells migrate to GC promote B cells humoral immune response by secreted effectiveness factor IL-21.As well as IL-21 cytokine promote liver granulomatous inflammation and liver fibrosis.二.The immunomodulatory effect of Tfh mediated by ICOS-ICOSL signal in the development of immunopathology in mice infected S.japonicumObjective:to study the regulation function of Tfh differentiation,polarization and GC response mediated by ICOS-ICOSL signal in the development of immunopathology in mice infected with S.japonicum.Methods:1.The area of liver granuloma were observed by hematoxylin and eosin（HE）staining in ICOSL-/-,ICOSL+/-and wild-type control mice on the day before infection（0week）,the early stages of infection（4 weeks post-infection）,acute infected stage（7weeks post-infection）,acute infected stage（9 weeks post-infection）,chronic infected stage（12 weeks post-infection）and late stage（16 weeks pos-tinfection）.The expession of collagen in liver were dynamically observed by Masson staining in ICOSL-/-,ICOSL+/-and wild-type control mice infected with S.japonicum.2.The expression level of costimulatory molecules ICOS,PD-1,OX40,CD40L and transcription factor Bcl-6 of Tfh cells were dynamically analyzed by flow cytometry in ICOSL-/-,ICOSL+/-and wild-type control mice infected with S.japonicum.3.Kinetic analysis of the expression levels of effect factors（IFN-γ/IL-4/IL-17 A/IL-21）of Th1/Th2/Th17/Tfh cells by flow cytometry in ICOSL-/-,ICOSL+/-and wild-type control mice.4.Immunofluorescence technique was used to observe the changes of GC construction and numbers in spleen in ICOSL-/-,ICOSL+/-and wild-type control mice infected with S.japonicum on the day before infection（0 week）and acute infected stage（7 weeks post-infection）.5.The expression level of IL-21 and Bcl-6 in liver granuloma were observed by immunohistochemistry in ICOSL-/-,ICOSL+/-and wild-type control mice infected with S.japonicum.Results:1.The area of liver granuloma from ICOSL-/-mice was significantly down-regulated at acute infected stage（7 weeks post-infection and 9 weeks post-infection）compared to wild-type mice（P<0.001）,but have no difference at chronic infected stage（12 weeks post-infection）and late stage（16 weeks post-infection）.The level of collage in liver granuloma was significantly down-regulated at the same time points compared to wild-type mice（P<0.05 or P<0.01 or P<0.001）.2.The results of flow cytometry showed that transcription factor Bcl-6 and CXCR5expression level of CD4+T cells in spleen,lymph node,liver from ICOSL-/-mice were significantly down-regulated at the same time points compared to wild-type mice（P<0.05 or P<0.01 or P<0.001）.The expression level of costimulatory molecules related with Tfh cells in spleen,lymph node,liver from ICOSL-/-mice were also significantly down-regulated at the same time points compared to wild-type mice（P<0.05 or P<0.01 or P<0.001）and ICOSL-/-mice hold a lower level in the whole course of disease.The ICOSL+/-mice only have slightly down-regulated compared to wild-type mice,but no difference.3.The expression level of effect factors of IL-4/IL-17A/IL-21 in CD4+T lymphocytes from ICOSL-/-mice was significantly down-regulated at the same time points compared to wild-type mice（P<0.05 or P<0.01 or P<0.001）.The expression levels of IL-4/IL-17A/IL-21 from ICOSL+/-mice have no difference compared to wild-type mice.4.ICOSL-/-,ICOSL+/-and wild-type control mice had few germinal center before infection.ICOSL+/-and wild-type control mice exhibited a large number of germinal center in spleen after infection（7 weeks postinfection）,but germinal center in ICOSL-/-mice was showed reduction or deficiency.In other words,ICOSL knockout stronly impaired GC immune response.5.The results of immunohistochemistry showed that the expression level of IL-21and Bcl-6 in liver granuloma from ICOSL-/-mice were significantly down-regulated at the same time points compared to wild-type mice（P<0.05 or P<0.01 or P<0.001）.The expression levels of IL-21 and Bcl-6 from ICOSL+/-mice have no difference compared to wild-type mice.Conclusion:Blocking ICOS-ICOSL signal pathway might inhibite liver fibrosis progression.ICOSL deficiency strongly down-regulated Tfh differentiation,proliferation and the expression of effect factors of Th2/Th17/Tfh cells.In other words,regulating of Th2/Th17/Tfh immune response mediated by ICOS-ICOSL signaling pathway contribute to control hepatic fibrosis development of schistosomiasis.In summary,our findings indicate that Tfh and effect factors IL-21 play important role in the development of immunopathology in mice infected S.japonicum.Blocking ICOS-ICOSL signaling pathway could contribute to down-regulate or control the development of hepatic granulomatous inflammation and subsequent hepatic fibrosis via regulating Tfh differentiation and function. |
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