??T Cells Suppress Liver Fibrosis Via Strong Cytolysis And Enhanced NK Cell-mediated Cytotoxicity Against Hepatic Stellate Cells

BackgroundLiver fibrosis is a prevalent liver disease that can lead to cirrhosis and liver failure,accounting for about 45%of deaths in industrialized countries.Liver fibrosis is characterized by excessive accumulation of scar tissue resulting from ongoing inflammation and liver cell death during chronic liver diseases.The scar tissue,comprising collagen-rich extracellular matrix(ECM),replaces normal functional units of cells,leading to the progressive loss of organ function and eventual failure.Many studies have shown that liver fibrosis can be reversed when eliminating these pathogenic factors.Although elimination of pathogenic factors could inhibit inflammation and the progression of fibrosis,still lack of safe,effective,and specific treatment measures for liver fibrosis.Hepatic stellate cells(HSCs)are the main effector cells in liver fibrosis because of their capacity to transdifferentiate into collagen-producing myofibroblasts.Accordingly,several immune cell subtypes interact directly or via soluble mediators with HSC,possibly influencing the progress of liver fibrosis.Such interactions include NK cells,NKT cells,monocytes/macrophages,and so on on HSCs.??T cells are enriched in the liver.On activation,these cells can expand markedly and display various effector functions in immune responses.For example,chemokines and inflammatory cytokines release,potent cytolytic activity against tumor or microbial pathogens,and immunologic memory generation.Tese characteristics may contribute to the interection role of ??T cells cells and other immune cells.However,their role and underlying mechanisms are not fully understood.The present study aims to assess whether ??T cells contribute to liver fibrosis regression.Using a carbon tetrachloride(CCl4)-induced murine model of liver fibrosis in wild-type(WT)and y8T cell deficient(TCR?-/-)mice.Aims1.To investigate the role of y8T cells in CC14-induced liver fibrosis2.To determine the changes in the frequency and numbers of liver ??T cells,and the distribution of ??T cells in fibrotic liver;To determine the role of ??T cells in activated HSCs3.To investigate the cytotoxcity of ??T1 cells and ??T17cells against activated HSCs4.To investigate whether y8T cells prevent liver fibrosis by intercting with other immune cells5.To explore the mechanisms of ??T cells and their subsets by which inhibit liver fibrosisMethods1.The genotype identification of TCR8-/-mice2.Liver fibrosis mouse models were established by repeated administration of CCl43.To assay liver fibrosis by performing H&E staining,Sirus Red staining,IHC,ALT and ELISA for serum hydroxyproline4.To determine the changes in the frequency and numbers of liver ??T cells by FACs5.Bi-immunofluorescence staining in frozen liver sections6.Isolation of primary HSCs and establish culture-activated HSCs model7.To determine the cytotoxicity by lysis assay and co-culture experiments8.Hepatic ??T cells isolated from WT mice were transferred into TCR?-/-mice once per week during the process of treating with repetitive CCl4 injections9.To determin the expression of cytotoxicity-associated molecules on ??T cells and their subsets by FACs10.To examined whether IFN? or IL-17 is responsible for the development of liver fibrosis by comparing the degree of liver fibrosis upon in vivo blockade of IFNy and IL-17A in WT and TCR?-/-mice mice11.To determin the expression of cytotoxicity-associated molecules on NK cells and their subsets by FACs12.To determin the expression of CD137L on ??T cells and the expression of CD137 on cNK cells and lrNK cells13.To compare the lytic functions of ??T cells against activated HSCs in the presence of NCR1-Ig,FasL-specific and TRAIL-specific blocking antibodies,respectively.Results1.y8T cell deficiency exacerbates liver fibrosis upon chronic liver injuryThe TCR?-/-mice displayed more severe liver fibrosis than the WT mice by detecting liver morphology,H&E and Sirius Red(SR)staining,serum ALT and hydroxyproline.These data strongly suggested that ??T cells have a protective role during CCl4-induced l iver fibrosis.2.Increased ??T cells infiltration and their co-localization with activated HSCs in fibrotic liversThe proportion and numbers of ??T cells increased significantly after repeated CCl4 administration compared with the controls.We analyzed the distribution of ??T cells and activated HSCs in fibrotic liver using bi-color immunofluorescence staining for ??T cells(??TCR)and activated HSCs(a-SMA).We observed larger numbers of??TCR+ T cells(green)accumulated in the fibrotic livers,and these ??T cells were located close to activated HSCs(?-SMA+,Red)in the periportal region and fibrotic septa during liver fibrosis,which suggested that these infiltrating y8T cells might directly interact with activated HSCs.3.??T cells isolated from fibrotic livers could induce apoptosis of activated HSCsImmunofluorescence double staining for a-SMA and TUNEL was performed to assess HSC apoptosis.The activated HSCs were shown undergo apoptosis after co-culturing with ??T cells.Significant cytolytic activity of ??T cells against activated HSCs were detected by LDH.Severe fibrosis and liver damage were markedly alleviated after transferring hepatic ??T cells from WT mice,as displayed by reduced extracellular collagen deposition and serum ALT levels.These data confirmed the protective effects of ??T cells by directly killing activated HSCs or inducing inactivation of activated HSCs in CCl4-induced liver fibrosis.4.Chronic inflammation promotes hepatic ??T cells expression of NKp46To investigate the mechanism by with ??T cells kill activated HSCs,we detected the expression of cytotoxicity-associated molecules on ??T cells in fibrotic livers.CCl4-induced chronic inflammation significantly increased the expression of NKp46,TRAIL,FasL and cytotoxic molecules.We compared the lytic functions of ??T cells against activated HSCs in the presence of NCR1-Ig,FasL-specific and TRAIL-specific blocking antibodies,respectively.Our results revealed that hepatic??T cells exert their lytic functions against activated HSCs in NKp46-,TRAIL-and FasL-dependent manner.5.??T1 cells exhibit more significant protection than ??T17 cells upon chronic liver injury??T1 cells exhibited higher expression levels of NKp46,CD 107a,GmB,perforin,TRAIL and FasL than ??T17 cells upon chronic liver injury.in vitro killing assay showed that ??T1 cells exhibited higher cytotoxicity against activated HSCs than??T17 cells.6.??T cells increase the cytotoxicity of cNK cells as well as IrNK cells against activated HSCsThrough in vitro co-culture experiments,we observed that both cNK and IrNK cells could induce the apoptosis of HSCs and efficiently kill activated HSCs.o further investigate whether ??T cells affect the anti-fibrotic effects of cNK and IrNK cells in fibrotic livers,we isolated hepatic cNK and lrNK cells from CCl4-treated WT mice or TCR?-/-mice,and then detected their cytotoxicity against activated HSCs.Both hepatic cNK and lrNK cells from CCl4-treated TCR8-/-mice exhibited markedly lower lysis ability against activated HSCs,compared to those from CCl4-treated WT mice,suggesting that loss of ??T cells impaired the cytolytic ability of both cNK and lrNK cells.We observed that ??T cells might promote the functions of lrNK and cNK cells by enhancing NK cell activation and augmenting the expression of cytotoxic effector molecules(perforin,GmB,FasL and IFNy).7.??T cells increase the cytotoxicity of cNK cells as well as lrNK cells against activated HSCs by CD137L/CD137 signalThe expression of CD137L on hepatic ??T cells increased after liver fibrosis.Correspondingly,the levels of CD137 on lrNK and cNK cells also increased in the liver fibrosis model.Then,we compared the cytotoxicity of hepatic cNK or lrNK cells co-cultured with y8T cells from fibrotic livers against activated HSCs in the presence or absence of a CD137L-specific blocking antibody.We found that the increased lytic functions of hepatic cNK and lrNK cells against activated HSCs promoted by y8T cells were significantly attenuated in the presence of the CD137L-specific blocking antibody,which suggested that the CD137-CD137L interaction plays a major role in the process of y8T cells' contribution to the cytotoxicity of cNK and lrNK cells against activated HSCs.