| Schistosomiasis is a zoonotic disease that could lead cirrhosis in patients.Liver fibrosis is usually regard irreversible,to date,there is no specific clinical anti-fibrotic treatment.Thus,it is urgent for us to find new therapeutic targets for liver fibrosis.The pathological feature of schistosomiasis liver fibrosis is the massive accumulation of soluble egg antigen(SEA)that were secreted by schistosomiasis eggs,and the dominant cells around the eggs include neutrophils,macrophages,hepatic stellate cells,B cells and T cells.IL-33 is a novel member of the IL-1 family,many studies have found that IL-33 is involved in the pathological process of CCL4-induced fibrosis model.However,the mechanism of IL-33 in liver granulomas and fibrosis in Schistosoma japonicum(S.japonicum)has not been elucidated.Inducible co-stimulator ligand(ICOSL)is expressed on macrophages.When specifically blocked the ICOSL/ICOS signaling,it could affect polarization of macrophage phenotype and cause immunopathological changes in S.japonicum egg granulomas and liver fibrosis.Whether ICOSL/ICOS signaling pathway could influence Mcp polarization regulating of IL-33 in schistosomiasis liver fibrosis has not been reported.In addition,the roles of miR-130a-3p on liver macrophages and HSCs and its biological functions in schistosomiasis liver fibrosis are still needed to be further verified by in vitro and in vivo experiments.In this study,we used S.japonicum-infected mice as a model to investigate the effect of ICOSL/ICOS pathway on macrophage phenotypes through specific down-regulation of ICOSL/ICOS signaling;we observe the relationships of IL-33 on the effects of ICOSL/ICOS signaling and IL-33 aggravate liver fibrosis in vivo experiments;in addition,in vitro experiments we investigated the effects of IL-33 on macrophage phenotypes,and the activation,autophagy and apoptosis of HSCs;furthermore,we examined the effects of miR-130a-3p on macrophages and HSCs in vitro and in vivo experiments.Our study focused on the key cells Mφ and HSCs,which play the important role in Immunopathological reaction of schistosomiasis,from different perspectives of ICOSL/ICOS signals and miR-130a-3p,to provide a richer basis for further uncovering the immunopathological process of schistosomiasis liver fibrosis and providing new ways for the prognostic identification and therapeutic application of liver fibrosis.Part One The molecular mechanism of ICOSL/ICOS signal regulating Mφ to M1 polarization to induce HSCs activation in liver fibrosis of schistosomiasis japonica1.Effects of IL-33 regulating HSCs on liver immunopathology of Schistosoma japonicum(S.japonicum)-infected miceObjective:To investigate the changes of IL-33 and its immunomodulatory effects in mice at different stages of S.japonicum infection.Methods:The H&E and Masson staining were applied to detect the pathological trends of liver egg granulomas and liver fibrosis in non-infected mice,4,8 and 12 weeks after infection.The qRT-PCR was used to explore the changes of fibrosis-related factors and the expression of IL-33 during the different stages of infection.In addition,HSCs were isolated,and then used for measuring the expression of ST2 by immunofluorescent staining.FCM,TUNEL staining and qRT-PCR were determined to analyze the effect of IL-33 on apoptosis and activation of HSCs.Results:H&E staining and Masson staining showed that there were liver egg granulomas at 4 weeks post-infection,there were the largest granuloma and the most severe liver fibrosis at 8 weeks post-infection.The results of qRT-PCR indicated that there were increased levels of α-SMA,Col I,TGF-β,IL-4,IL-13 and IL-33 after 4 weeks of S.japonicum infection,and reached to the peak at 8 weeks,then showed the decreasing trends with the prolonging of infection.The finding of immunofluorescence showed that ST2 was expressed on activated HSCs,and FCM and TUNEL staining results indicated that IL-33 inhibited the apoptosis of HSCs.In addition,we also found that IL-33 enabled to promote the expression of α-SMA and Col I in HSCs,suggesting that IL-33 could induce the activation of HSCs.Conclusions:IL-33 levels were significantly increased in S.japonicum-infected mice,and the dynamic changes of IL-33 were consistent with the trend of liver granuloma area and the degree of liver fibrosis,suggesting that IL-33 is involved in the immune response process of S.japonicum-infected liver fibrosis,which could promote the activation of HSCs,and inhibite the apoptosis of HSCs.2.ICOSL-KO mice mitigating liver fibrosis by polarizing Mφ to M1 typeObjective:To investigate the effect of ICOSL/ICOS signaling on the development of granulomas and fibrosis in mice infected with S.japonicum by affecting macrophage phenotype.Methods:The H&E and Masson staining were applied to detect the pathological trends of liver egg granulomas and liver fibrosis in non-infected mice,4,8 and 12 weeks Sjaponicum-infected mice.The IHC was applied to measure the levels of liverα-SMA,IL-33 and ST2 in WT and ICOSL-KO mice.The expression of α-SMA,IL-33 and ST2 was measured by IHC.The expression levels of HYP and serum HA were measured by alkaline lysis methods and ELISA.The fibrosis-related factors,iNOS and Arg 1 were detected by qRT-PCR.FCM was used to investigate the expression of ICOSL,CD86 and CD206 on macrophages.The expression of IL-33 and CD206 on macrophages was tested by immunofluorescence and FCM.Results:The results of H&E,Masson staining and qRT-PCR showed that ICOSL-KO mice showed a significant decrease in granuloma area,the degree of fibrosis and the levels of fibrosis-related factor compared to those WT mice at 4,8 and 12 weeks of S.japonicum infection.FCM revealed that ICOSL was expressed on activated macrophages.Compared with WT mice,the expression of CD86 showed an increase,but the expression of CD206 showed a decrease from 4 weeks of of S.japonicum infection.Furthermore,immunofluorescence and FCM results observed that IL-33 was expressed by macrophages,and IL-33 showed the ability to skew macrophage toward M2 type.Conclusion:Activated macrophages expressed ICOSL,ICOSL-KO attenuated the immunopathological process of S.japonicum-infected mouse liver by skewing macrophage to M1 type and reducing the expression of IL-33.3.Effects of intervening IL-33/ST2 signaling on liver fibrosis of S japonicum-infected ICOSL-KO miceObjectives:To investigate the immunomodulatory effect of IL-33/ST2 signal on liver fibrosis in acute stage of S japonicum-infected ICOSL-KO mice.Methods:ICOSL-KO mice were randomly divided into 2 groups,group I:ICOSL-KO mice,and group Ⅱ:IL-33 intervente ICOSL-KO mice.Group II was defined as the injection of 1μg recombinant IL-33 into ICOSL-KO mice after infected with S.japonicum once a week,totally for 4 weeks.Specimens were collected at 6,8 and 10 weeks of infection for testing.The liver egg granulomas and the degree of liver fibrosis in group Ⅰ and group Ⅱ mice were detected by H&E and Masson staining.The levels of HYP and serum HA were measured by alkaline lysis methods and ELISA.FCM was applied to measure the number of different types of Ly6C and the expression of IL-4 and TNF-α in splenic lymphocytes.The expression of chemokines CCL2,CCL5 and CXCL2 were tested by qRT-PCR,and the levels of ERK,Smad2/3 and TGF-β in the liver were detected by IHC.After stimulated with IL-33,the apoptosis rate of JS1 cells was evaluated by FCM,and the activation and autophagy of JS1 cells were detected by WB.Results:Compared to group I,H&E and Masson staining results showed a significant increase in granuloma area and the severe degree of liver fibrosis at 6,8 and 10 weeks in group II;FCM revealed significant infiltrations of KCs and monocytes,and an elevated level of in Ly6Chi type macrophages at 6 weeks of infection,and the expression of IL-4 in splenic lymphocytes showed an increase in group Ⅱ from 8 to 10 weeks of infection,but the expression of TNF-α was decreased from 6 to 10 weeks of infection in group II.In addition,the expressions of CCL2,CCL5 and CXCL2 were consistent with the levels of Ly6Chi.The results of IHC indicated that,there were increased levels of ERK,Smad2/3 and TGF-β in ICOSL-KO+IL-33 group at 6,8 and 10 weeks of S.japonicum infection.After stimulated with rIL-33,there was decreased apoptosis rate of JS1 cells,however,the degrees of activation and autophagy of JS1 cells showed increased.Conclusions:IL-33 showed the ability to skew the macrophages towards to Ly6Chi,and promote the autophagy and activation of HSCs through TGF-β/Smad and ERK signaling,thus inhibited the apoptosis of HSCs and aggravated the liver pathological process of schistosomiasis.Parter Two A novel immune check-point of miR-130a-3p in liver fibrosis by regulating Mφ polarization and inhibiting HSCs activation in schistosomiasis miceObjectives:Emerging evidence highlights the important role of microRNAs(miRNAs)in liver cirrhosis,but the relationships between miR-130a-3p and liver cirrhosis are still unclear.The current study aimed to investigate the biological function of miR-130a-3p in schistosomiasis liver fibrosis through in vitro and in vivo experiments.Methods:The serum from cirrhosis patients and liver tissues of S.japonicum-infected mice for 8 weeks were collected and extracted,then qRT-PCR was applied to detect the levels of miR-130a-3p.After mice infected with S.japonicum cercaria,the lentivirus LV-miR-130a-3p or LV-NC were injected by tail vein,and the distribution of LV-miR-130a-3p or LV-NC were detected by In-vivo imaging systems.At 8 weeks of S.japonicum infection,mice were euthanatized,and liver granulomas and the degree of fibrosis were evaluated by H&E and MASSON staining.Immunohistochemistry and qRT-PCR were applied to detect the indexes for liver fibrosis,such as SMA and Col I.Flow cytometry was used to investigate the expression of CD206 and Ly6C in different groups of macrophages.Moreover,the expression of MMP2,TIMP1,CCL2,CXCL2,CCL3 and,CCL4 were also measured by qRT-PCR.JS1 cells were transfected with AgomiR-130a-3p/AgomiR NC or AntagomiR-130a-3p/AntagomiR-NC,and the effects of miR-130a-3p on the activation,proliferation and apoptosis of JS1 cells were detected by flow cytometry and CCK8.Venn diagram,WB,qRT-PCR and dual-luciferase reporter gene were applied to predict and validate the target genes of miR-130a-3p.Results:Our results showed that there were decreased levels of miR-130a-3p in the serum of cirrhotic patients and in the liver of S.japonicum-infected mice.LV-miR-130a-3p was mainly distributed in the liver tissue,and attenuated liver granulomatous inflammation and collagen deposition.Meanwhile,LV-miR-130a-3p skew macrophages toward to Ly6Clo phenotype.There was also a decreased level of TIMP-1,but an elevated MMP-2 level,which would be helpful to collagen deposition.In addition,the overexpression of miR-130a-3p not only inhibited the activation and proliferation of HSCs,but also induced apoptosis of HSCs.Furthermore,we demonstrated that miR-130a-3p was able to bind to MAPK1,TGFBR1 and TGFBR2 genes and inhibit the expression of these genes.Conclusions:Our results indicated that miR-130a-3p could inhibit liver fibrosis by skewing macrophages toward to Ly6Clo phenotype,promoting apoptosis of HSCs,and inhibiting the activation and proliferation of HSCs. |
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