Effection Of The Th2Polarization And HSCs Activation Mediated By ICOSL/ICOS Signaling Pathway On The Formation Of Fibrosis In ICOS-Tg Mice Infected With Schistosoma Japonicum

The main pathological liver injury schistosomiasis is caused by the deposition ofschistosome eggs, which may lead to cumulative hepatic fibrosis. Studies have shownthat SEA of Schistosoma japonicum infection in the pathogenesis of chronic host Th2polarization induced the liver egg granuloma formation and lead to hepatic fibrosisplays a key role. Hepatic stellate cells activation is the central part of development,which leads to deposition of the extracellular matrix (ECM) and liver fibrosis.This study established ICOS transgenic(ICOS-Tg) which mice has FVB/NJ geneticbackground as experimental schistosomiasis model for Schistosoma japonicum.Tofurther investigate how ICOSL/ICOS signaling pathway contributes to the developmentof Th2polarization and activation of HSCs in mice infected with Schistosomajaponicum. Also, to establish the Th2polarization and HSCs activation mediated byICOSL/ICOS signaling pathway correlated with hepatic fibrosis formation.Futhermore,to explore the new effective way for controlling the hepatic fibrosis.1. Expression of costimulatory molecules related with polarizing Th2immuneresponses in ICOS-Tg mice infected with Schistosoma japonicumObjective: To investigate the enhance of ICOSL/ICOS effection on ICOS, CD28and CD154in ICOS-Tg mice infected with Schistosoma japonicum.Methods: The expression of costimulatory molecules related with polarizing Th2immune responses ICOS, CD28, CD154in ICOS-Tg mice infected with Schistosomajaponicum were analyzed by flow cytometry on the day before infection (0week), andin early stage (4weeks postinfection), in acute infected stage (7weeks postinfection), in chronic infectd stage (12weeks postinfection).The expression of costimulatorymolecules on inflammatory cells around granulomatous infiltration in liver fromICOS-Tg mice was assessed by immunohistochemical stainning in the development ofschistosomiasis.Results: Flow cytometry analysis showed that the expression of CD28on CD4+Tsplenocytes from infected mice increased in4weeks postinfection and peaked at7weeks postinfection, then decreased at12weeks postinfection. The expression of ICOSand CD154on CD4+T splenocytes from infected mice increased in4weekspostinfection and peaked at12weeks postinfection. Immunohistochemical resultsshowed that the liver egg granuloma costimulatory molecules surrounding inflammatorycells from ICOS-Tg mice presented at a high level in7weeks postinfection, and stillrised slightly at12weeks postinfectin. ICOS-Tg mice splenic CD4+T lymphocytesICOS, CD28, CD154expression levels were higher than the same period of thewild-type mice. Inflammation of the liver egg granuloma surrounding inflammatorycells, the expression levels of ICOS, CD28, CD154in seven weeks postinfection,12weeks was also higher than the same period of the wild-type mice.Conclusion: The ICOSL/ICOS signaling pathway has a regulatory effect on Th2polarization associated with the costimulatory molecules ICOS, CD28, CD154.2. Effection of ICOSL/ICOS signaling on polarizing Th2immune response inICOS-Tg mice infected with Schistosoma japonicum.Objective: To explore the enhance of the ICOSL/ICOS costimulatory moleculessignaling effection on Th2polarization in ICOS-Tg mice infected with Schistosomajaponicum.Methods: The spleen lymphocytes of ICOS-Tg mice were stimulated with SEA for72hours on the day before infection and at4,7,12weeks postinfection.Theconcentrations of Th1cytokines IFN-γ,IL-12and Th2cytokines IL-4,IL-13in theculture supernatants were measured by a sandwich ELISA according to themanufacturer’s guideline.Results: The Th1-type cytokines IFN-γ, IL-12begin increased at4weekspostinfection, the production mounted on peak at7weeks postinfection,then,downregulated at12weeks postinfection, indicating that the duration of the acutephase to chronic phase, the expression levels of Th1-type cytokines decreased; whereasTh2cytokines IL-4, IL-13expression levels from4weeks postinfection began to increase,7weeks,12weeks after the infection is still in its gradual upward trend.ICOS-Tg mice Th2cytokines IL-4, IL-13expression levels were higher than the sameperiod of the wild-type mice.Conclusions: The ICOSL/ICOS costimulatory molecules signaling plays a keyrole on the Th2polarization process in mice after the Schistosoma japonicum infection.3. Effection of ICOSL/ICOS signaling on formation of liver fibrosis in ICOS-Tgmice infected with Schistosoma japonicum.Objective: To investigate the enhance of the ICOSL/ICOS signaling effection onthe formation of liver fibrosis in mice infected with Schistosoma japonicum.Methods: The sura of ICOS-Tg mice were collected on the day before infection0week,and at4,7and12weeks postinfection. The concentrations of HYP and HA in surameasured by ELISA.The liver fibrosis of ICOS-Tg mice was dynamically observed byMasson staining. The expression levels of Collagen-Ⅰ, α-SMA, TGF-β1in liver fromICOS-Tg mice was detected by Immunohistochemical staining in the development ofschistosomiasisResults: In the course of migration, the ICOS-Tg mice serum HYP, HA expressionlevels gradually increase, gradually increased liver fibrosis, liver Collagen-Ⅰ, α-SMA,TGF-β1expression levels also gradually increase. ICOS-Tg mouse serum HYP, HAexpression levels were higher than the same period of the wild-type mice; ICOS-Tgmouse liver Collagen-Ⅰ, α-SMA, TGF-β1expression levels in infected after sevenweeks,12weeks was also higher than in the same period of the wild-type mice.Conclusion: The ICOSL/ICOS signaling pathway has an important impact on thedevelopment of hapetic fibrosis formation in mouse infected with Schistosomajaponicum.4. Effection of ICOSL/ICOS signaling on activation of HSCs effects and liverfibrosis in ICOS-Tg mice infection with Schistosoma japonicum.Methods: The liver of ICOS-Tg and wild-type mice was perfused in situsuccessively under agitation. HSCs were then separated by density gradientcentrifugation through16%and12%OptiPrep on the day before infection0week,andat4,6and9weeks postinfection. The cells viability were tested by erythrosin exclusion.The purity of the HSCs were detected by fluorescence of retinoid-containing vacuolesunder ultraviolet excitation and immunocytochemisy of GFAP,which is a specificmarker of the HSCs and not expressed in other hepatic cell types.Total RNA of cultuered7days primary HSCs was extracted by using Trizol Reagent. QRT-PCR wasperformed using SYBR Premix Ex Taq to detect the expression levels in the primaryHSCs Collagen-Ⅰ, Collagen-Ⅲ, α-SMA, TGF-β1and other genes in the developmentof schistosomiasis.Results: The purity of HSCs isolated were higher than90%, and the cell viabilityexceeded95%. Cells in good candition during the primary culture. With the develop ofschistosomiasis, the level of activation of HSCs was increasing, and the expression ofCollagen-Ⅰ, Collagen-Ⅲ, α-SMA and TGF-β1in HSCs were enhancing too. Thedegree of HSCs activation in ICOS-Tg mice were significantly higher than wildtypemouse. Also, the expresson on Collagen-Ⅰ, Collagen-Ⅲ, α-SMA and TGF-β1in HSCsof ICOS-Tg mice were higher than that in wildtype mice.Conclusion: The ICOSL/ICOS signaling pathway has a very important impact onthe development of HSCs activation and fibrosis formation in the mouse infected withSchistosoma japonicum.In summary, the ICOSL/ICOS costimulatory molecules signaling has a keyregulatory in HSCs activation and Th2polarization in host infected with Schistosomajaponicum. The results of this experiment has improtant potential applications to controlhepatic fibrosis, and to find the development of therapeutic targets, to provide veryimportant theoretical remains.