| The mainly pathogenesis of schistosomiasis is the occurrence of egg granulomas in the liver and intestine, and can lead to more severe liver fibrosis. The immune response of CD4+T cell to antigen secreted by schistosome eggs, which lead to cumulative hepatic fibrosis. Studies have shown that Thl/Th2 immune deviation is closely related to the development of schistosome egg granuloma and immune regulation. Th17 and Tfh are the new T cell subsets in recent years. Studies have confirmed that ICOS L/ICOS signal play a key role in the formation of the chronic inflammation and fibrosis. New research have shown that a direct correlation between the expression of ICOS and the generation of Th17, and is also associated with the function of Tfh.In this study, we established ICOS transgenic (ICOS-Tg) mice and ICOSL knockout (ICOSL-KO) mice as experimental animal models for schistosomiasis. To clarify how ICOSL/ICOS signal pathway contributes to the development of immunopathology and polarizing Th17, Tfh immune responses in mice infected with schistosoma japonicum, as well as to analyze the molecular mechanism of Thl7, Tfh polarization mediated by ICOSL/ICOS signaling pathway correlated with hepatic egg granuloma and liver fibrosis formation. Furthermore, to explore the new effective way for controlling the hepatic egg granuloma formation and the development of hepatic fibrosis.1. Dynamic changes of molecules related with Th17 polarization mediated by ICOSL/ICOS signal in ICOSL-KO/ICOS-Tg mice infected with schistosoma japonicum.Objective:To investigate the enhance or down-regulation of ICOSL/ICOS signaling effection on the costimulatory molecules, transcription factors and cytokine expression levels associated with the polarization of Thl7 in ICOSL-KO/ICOS-Tg mice infected with Schistosoma japonicum.Methods:The expression of ICOS, CD154, IL-17A, RORyt on CD4+T lymphocytes and of ICOSL, CD40, IL-17R on CD19+B lymphocytes from ICOSL-KO/ICOS-Tg mice and their wild -type control mice were analyzed by Flow cytometry on the day before infection (0 week), and the early stages of infection (4 weeks postinfection), acute infected stage(7 weeks postinfection), chronic infected stage (12 weeks postinfection), advanced stage (16 weeks postinfection) Immunohistochemistry was used to detect the expression level of IL-17A in the liver egg granuloma in the same infected period of ICOSL-KO/ICOS-Tg mice and wild-type control mice.Results:The Flow cytometry analysis showed that the expression level of ICOS, CD 154, CD40 increased from 4 weeks post-infection, peaked at 12 weeks. The expression levels of ICOS, CD 154 on spleen lymphocytes from ICOSL-KO mice was significantly reduced at 4 weeks after infection compared to wild-type mice. CD 15412 weeks after infection significantly reduced. The expression level of ICOSL on CD19+B lymphocytes from ICOSL-KO mice are very low since ICOSL knockout after schistosome infection. Meanwhile, the expression level of Thl7-specific transcription factor RORyt factor as well as its important cytokine IL-17 in splenocytes were significantly reduced in ICOSL-KO mice; the results of ICOS-Tg mice were just opposite. The expression level of IL-17A in liver tissue tested by immunohistochemistry was consisted with the results of flow cytometry.Conclusion:Down-regulation of ICOSL/ICOS signaling pathway leading to inhibition of Th17 subsets and functions; Thl7 immune response were induced by enhancing the ICOSL/ICOS signal.2. Effection of ICOSL/ICOS signaling pathway on polarizing Thl7 immune response in ICOSL-KO/ICOS-Tg mice infected with Schistosoma japonicum.Objective:To investigate the down-regulation of ICOSL/ICOS signaling effection on Th17 polarization in ICOSL-KO/ICOS-Tg mice infected with Schistosoma japonicum.Methods:The spleen lymphocytes of mice were stimulated with SEA for 72 hours on the day before infection (Oweek) and at 4,7,12,16 and 20 weeks post-infection. The concentrations of Thl cytokines IFN-y, IL-2, IL-12; Th2 cytokines IL-13, IL-4, TGF-β1; Th17 cytokine IL-17, IL-23, IL-6 in the culture supernatants were measured by a sandwich ELISA according to the manufacture’s guideline.Results:The expression levels of Thl cytokines IFN-y, IL-2 from ICOSL-KO mice were significantly up-regulated compared to their wild-type control; Th2 cytokines IL-4, IL-13, TGF-β1 were significantly down-regulated compared to their wild-type control; Th17 cytokine IL-17, IL-6, IL-23 were significantly down-regulated compared to their wild-type control. However, compared to wild-type, the expression levels of Thl cytokine IFN-γ from ICOS-Tg mice was significantly down-regulated; Th2 cytokines IL-4, IL-13 were importantly up-regulated; Thl7 cytokine IL-17 was significantly increased.Conclusion:The ICOSL/ICOS signaling plays a critical role on the Th17 polarization in mice infected with Schistosoma japonicum.3. Effection of ICOSL/ICOS signaling pathway on formation of hapetic egg granuloma and fibrosis in ICOSL-KO/ICOS-Tg mice infected with schistosoma japonicum.Objective:To investigate the enhance or down-regulation of ICOSL/ICOS signal effection on formation of hapetic egg granuloma and fibrosis in ICOSL-KO/ICOS-Tg mice infected with Schistosoma japonicum.Methods:The dynamic changes of the liver and spleen weight were observed. Kaplan-Meier survival analysis was used to assess the survival rate in ICOSL-KO/ICOS-Tg mice infected with Schistosoma japonicum. The granulomatous pathology in liver of ICOSL-KO/ICOS-Tg mice was dynamically observed by hematoxylin and eosin (HE) staining. The fibrosis level in liver of ICOSL-KO/ICOS-Tg mice was dynamically observed by Masson staining. The expression of IL-13, TGF-β1, MMP-9, TIMP-1 in liver from ICOSL-KO/ICOS-Tg mice was assessed by immunohistochemical staining in the develpoment of schistosomiasis.Results:Pathology of hapetic tissue sections were observed and found that the volume of hepatic egg granuloma in mice was biggest at 7 weeks postinfection and gradually shrinked with the development of schistosomiasis. Throughout the course, the volume of liver egg granulomas of ICOSL-KO mice was notablely smaller than that of wild-type mice, however, the volume of liver egg granulomas of ICOS-Tg mice was strikingly greater than that of wild-type mice. With the develpoment of schistosomiasis, the level of hapetic fibrosis and the expression of TGF-β1、IL-13、MMP-9、TIMP-1in liver gradually increased in infected mice. The levels of TGF-β1、IL-13 of ICOSL-KO mice were significantly lower than that of wildtype C57BL/6J mice. Also, the expression of TIMP-1, MMP-9 in liver of ICOSL-KO mice were decreased compared with wildtype C57BL/6J mice. Furthermore, the level of hapetic fibrosis in ICOSL-KO mice was significantly lower than that of wildtype C57BL/6J mice; ICOS-Tg mice were just opposite compared with ICOSL-KO mice.In addition, ICOSL-KO mice showed improved survival compared with wildtype C57BL/6J mice; whereas deteriorated survival showed in ICOS-Tg mice compared with wildtype FVB/NJ mice.Conclusion:ICOSL/1COS signaling pathway involved in the process of hapetic egg granuloma and fibrosis formation in mice infected with Schistosoma japonicum.4. Effection on blocking ICOSL/ICOS signaling pathway in ICOS-Tg mice infected with Schistosoma japonicum in vitro.Objective:To investigate the up-regulation of ICOSL/ICOS signaling effection on Th17 polarization in ICOS-Tg mice infected with Schistosoma japonicum; and observe the effect of blocking ICOSL/ICOS signaling pathway on the Th17 polarization in vitro.Methods:The spleen lymphocytes of mice were stimulated with SEA and anti-ICOSL/ICOSmAb were used for 72 hours on the day before infection (Oweek) and at 4,7,12,16 and 20 weeks post-infection. The concentrations of Thl/Th2/Thl7 cytokines in the culture supernatants were measured by CBA (cytometric bead array system).Results:Compared with wild-type, ICOS-Tg mice produced higher levels of IL-17, IL-4 and lower levels of IFN-γ. After blocking ICOSL, IL-4, IL-10, IL-13 were down-regulated in the supernatan of spleen lymphocyte from ICOS-Tg mice; IL-17 was also decreased. Meanwhile, the production of Thl cytokine IFN-y was up-regulated. Blocking ICOS, this phenomenon also occurred.Conclusion:Blocking ICOSL/ICOS signal can regulate the balance of immune responses and Th2/Th17 polarization.5. Dynamic changes of molecules related with Tfh polarization mediated by ICOSL/ICOS signal in ICOS-Tg mice infected with schistosoma japonicum.Objective:To explore the enhance of ICOSL/ICOS signaling effection on the costimulatory molecules, transcription factors and cytokine expression levels associated with the polarization of Tfh in ICOS-Tg mice infected with Schistosoma japonicum.Methods:The expression of CXCR5, BCL-6, CXCR5+IL-21+cell on CD4+T lymphocytes and of ICOS, CD40L on CXCR5+CD4+T lymphocytes from ICOS-Tg mice and their wild-type control mice were analyzed by flow cytometry on the day before infection (0 week), and the early stages of infection (4 weeks postinfection), acute infected stage(7 weeks postinfection), chronic infected stage (12 weeks postinfection), advanced stage (16 weeks postinfection). Immunohistochemistry was used to detect the expression of BCL-6, CXCR5 in the liver egg granuloma in the same infected period of ICOSL-KO/ICOS-Tg mice and wild-type control mice. Tfh cytokine IL-21 in the culture supernatants was measured by cytometric bead array system (CBA).Results:The Flow cytometry analysis showed that the expression level of CXCR5, BCL-6, CXCR5+IL-21+cell on CD4+T lymphocytes and of ICOS, CD40L on CXCR5+CD4+T lymphocytes increased from 4 weeks after infection, peaked 12 weeks post-infection; The expression level of CXCR5,BCL-6,CXCR5+IL-21+cell on CD4+T lymphocytes and of ICOS,CD40L on CXCR5+CD4+T lymphocytes from ICOS-Tg mice was significantly up-regulated after 4 weeks postinfection compared to wild-type mice, the peak 12 weeks after infection, and then decreased slowly. Meanwhile, the expression level of CXCR5 as well as Tfh-specific transcription factor BCL-6 in liver tissue tested by Immunohistochemistry were significantly up-regulated in ICOS-Tg mice. Tfh cytokine IL-21 was down regulated compared to wild type control after using Anti-ICOSL/ICOSmAb..Conclusion:Enhancing the ICOSL/ICOS signaling pathway leading to up-regulation of Tfh subsets and functions; IL-21 may be involved in the immune response of the host following Schistosoma japonicum infection. The CD4+T cells and their surface molecules CD40L may also play an important role.In summary, our findings indicate that ICOSL/ICOS signaling mediates the Th17/Tfh responses and could contribute to severe hepatic granulomatous inflammation and subsequent fibrosis via Thl7/Tfh polarizing. This study further clarifies the immune regulatory mechanism of fibrosis and provides a new approach to understand the immunopathogenesis of Schistosoma-induced fibrosis. |
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