The Expression And Functional Role Of Tim-3 In Chronic Hepatitis B

Hepatitis B virus(HBV) primarily infects hepatocytes,which causes a series of necroinflammatory liver diseases worldwide.Persistent carriers of HBV,however, show a severe dysfunction in HBV-specific immune response.However,the mechanisms have not been well-defined.It has been shown that immune regulatory molecules play important roles in HBV-induced lymphocyte dysfunction.Tim-3 was identified to be specifically expressed on polarized Th1 cells in 2002,and might play great roles in immune regulation and tolerance induction.Engagement of Tim-3 by its ligand-galectin-9 negatively regulates IFN-γproduction and influences the ability to induce T cell tolerance.Recent studies have shown that Tim-3 is also expressed on Tcl cells, dendritic cells and natural killer cells,and may involve in the pathogenesis of a range of diseases by influencing these cell function.In our early studies,we demonstrated that Tim-3 mRNA is up-regulated on peripheral blood mononuclear cells(PBMCs) from patients with chronic hepatitis B(CHB),indicating Tim-3 might play an important role in the development of CHB.In this study,we investigated the expression and functional role of Tim-3 in CHB by clinical samples,in vitro HBV transfected cell lines and mouse model of HBV infection.Methods1.Tim-3 expression on lymphocytes from CHB patients.1.1 Tim-3 expression on lymphocytes from CHB patients.Fresh heparin-treated blood from CHB patients,healthy controls and patients with fatty liver disease(FLD) was collected to detect Tim-3 expression on PBMCs with flow cytometric analysis.Liver tissues from CHB patients and FLD patients were obtained to detect Tim-3 expression by immunohistochemical staining. 1.2 Correlation between Tim-3 expression on lymphocytes and levels of serum ALT activities and HBV DNA in CHB patientsSpearman correlation analysis was performed between Tim-3 expression on lymphocytes and serum alanine aminotransferase(ALT) activities or HBV DNA titers in CHB patients with Prism GraphPad software.2.Effects of HBV infection on Tim-3 expression2.1 Effects of HBV infection on Tim-3 expression in NK92 cells in vitroHuman NK cell line NK92 cells were electroporated with HBV expression plasmid,then grown in 200μg/ml of G418 for 1-2 weeks.Cells electroporated with pcDNA3 served as controls.Tim-3 expression was detected with RT-PCR and flow cytometric analysis.2.2 The effects of HBV infection on Tim-3 expression in HBV-transgenic (HBV-Tg) miceHepatic mononuclear cells(HMCs) from HBV transgenic mice were isolated by metrizamide gradient centrifugation in Percoll,and cells from normal Balb/c mice served as control.Tim-3 expression on HMCs was detected with flow cytometric analysis.3.The roles of Tim-3 in NK and CD8~+ T cell function3.1 The roles of Tim-3 in NK cell function3.1.1 The roles of Tim-3 in NK92 cytotoxicity and cytokine productionNK92 cells were treated with anti-Tim-3/Tim-3-Fc,isotype antibody or PBS respectively,then coincubated with HepG2 or HepG2.2.15 cells for 4h at different E/T ratio.Cytotoxicity was detected with Cell-counting kit-8 and IFN-γsecretion was assayed with ELISA kit3.1.2 The roles of Tim-3 in PBMCs from CHB patientsPBMCs were obtained from CHB patients as effector cells,treated with anti-Tim-3,isotype antibody or PBS,and coincubated with HepG2 or HepG2.2.15 cells.Cytotoxicity and IFN-γsecretion were detected as above.3.2 The roles of Tim-3 in hepatic CD8~+ T cell function in mouse model of HBV infection 3.2.1 The hydrodynamics-based mouse model of HBV infectionThe mouse model of HBV infection was induced by hydrodynamic injection of HBV expression plasmid pcDNA3-1.1HBV,and mice treated with pcDNA3 served as control.Mice were sacrificed at indicated time point,and levels of HBs antigen,HBs antibody and HBe antigen in the sera were detected with time-resolved immunofluorometric assay(IFMA) kit.HBc expression was detected in hepatocytes with immunohistochemical staining.Serum HBV DNA titers were detected with real-time PCR.Serum ALT activities were measured using the ALT Infinity Reagent.3.2.2 Tim-3 expression in mouse model of HBV infectionHMCs were obtained from model mice and control mice to detect Tim-3 expression with flow cytometric analysis.3.2.3 Construction of Tim-3 specific shRNA expression plasmidTim-3 specific short hairpin RNAs(shRNAs) or unrelated shRNA oligos were designed and chemical synthesized.After annealing,shRNA oligos were cloned into pmU6 vector and transformed into E.coli DH5α.Positive recombinant plasmids identified by restriction enzyme digestion and DNA sequencing,were named as Tim-3shRNA-1,Tim-3shRNA-2 and unshRNA.3.2.4 Inhibitory effects of Tim-3shRNA in vitroshRNA expression plasmids were transfected into murine macrophage line RAW264.7 mediated by lipofectamine 2000.Cells were harvested at 24h,48h and 72h post the transfection to detect Tim-3 andβ-actin expression with RT-PCR and western blot.Expression of Tim-1,another member of Tim family,was detected for the specificity of Tim-3 shRNA.3.2.5 Effects of Tim-3 shRNA expression plasmid on hepatic CD8~+ T cell functionHMCs were prepared from mice treated with hydrodynamic injection of pcDNA3-1.1HBV with Tim-3shRNA or unshRNA.Tim-3 expression and IFN-γproduction were analyzed by flow cytometry.4.Statistical analysesAll data were analyzed using the GraphPad Prism 4.The Kruskal-Wallis nonparametric H test and Mann-Whitney nonparametric U test were used for comparison between groups.Spearman correlation analysis was performed between Tim-3 expression on CD8~+ T cells and serum ALT levels or HBV DNA.Value of p＜0.05 or p＜0.01 is considered as significant difference.Results1.Tim-3 expression was increased on PBMCs and HMCs from CHB patients1.1 Tim-3 expression was augmented on PBMCs,especially on NK cells and CD8~+ T cells from CHB patientsFlow cytometric analysis showed that Tim-3 expression was significantly increased on PBMCs from CHB patients compared to healthy controls and FLD patients(p＜0.05).Further analysis showed that Tim-3 expression was abundant on NK cells,and was significantly increased on NK cells from CHB patients compared to healthy controls and FLD patients(p＜0.05).Besides,Tim-3 was significantly up-regulated on CD8~+ T cells from CHB patients compared to controls(p＜0.0001).1.2 Tim-3 expression was increased in liver tissue from CHB patientsImmunohistochemical staining showed that CHB patients exhibited an increased number of Tim-3~+ cells compared to FLD controls.Morphologically,these cells appeared to be infiltrating lymphocytes.1.3 Tim-3 expression on T cells or CD8~+ T cells was correlated with ALT levels in sera from CHB patientsSpearman correlation analysis showed that Tim-3 expression on T cells or CD8~+ T cells was positively correlated with serum ALT levels in CHB patients(p＜0.01). There was no correlation between Tim-3 expression and HBV DNA titers.2.Up-regulation of Tim-3 by HBV infection2.1 Up-regulation of Tim-3 on NK92 cells transfected with HBV expression plasmidSemi-quantitative RT-PCR revealed that HBx and HBc mRNA could be detected in NK92 cells transfected with pcDNA3-1.1HBV(NK92-HBV) which showed higher Tim-3 expression than cells transfected with control plasmid.Consistent with RT-PCR, flow cytometric analysis showed that Tim-3 protein was significantly increased on NK92-HBV cells.2.2 Up-regulation of Tim-3 on HMCs and NK cells from HBV transgenic mice.Flow cytometric analysis showed that Tim-3 expression was significantly up-regulated on HMCs and NK cells from HBV transgenic mice compared to control mice(p＜0.05).3.Regulatory roles of Tim-3 in NK and CD8~+ T cell function3.1 Tim-3 blockade up-regulates cytotoxicity of NK cells3.1.1 Tim-3 blockade up-regulates cytotoxicity of NK92 cells1) Cytotoxicity:CCK-8 assay showed an increase in cytotoxicity in anti-Tim-3/Tim-3-Fc-treated NK92 cells against HepG2 or HepG2.2.15 cells at different E/T ratio,compared to isotype-and PBS-treated control(p＜0.05 or p＜0.01).However,Tim-3 blockade could not up-regulate the cytotoxicity of NK92 cells against K562 cells.Semi-quantitative RT-PCR was used to detect galectin-9 expression in HepG2,HepG2.2.15 and K562 cells.Our data showed that galectin-9 was abundant in HepG2 and HepG2.2.15 cells,and in low levels in K562 cells.2) IFN-γproduction:After coincubation of NK92 cells and HepG2.2.15 cells for 4h,cell culture supernatant was collected to detect IFN-γproduction with ELISA kit.Our data showed that compared to isotype control and PBS control,Tim-3 blockade could significantly up-regulate IFN-γproduction from NK92 cells against HepG2.2.15 cells(p＜0.05).3.1.2 Tim-3 blockade up-regulates cytotoxicity of PBMCs from CHB patientsPBMCs from CHB patients were obtained and incubated with HepG2 or HepG2.2.15 cells for 4h at different E/T ratio.CCK-8 reagent was used to detect cytotoxicity.Our data showed that compared to isotype control and PBS control, Tim-3 blocking antibody could up-regulate cytotoxicity of PBMCs from CHB patients(p＜0.05).3.2 Tim-3 knockdown increased IFN-γproduction of hepatic CD8~+ T cells from mouse model of HBV infection3.2.1 The mouse model of HBV-induced hepatitis After hydrodynamic injection of pCDNA3-HBV1.1,HBV antigens(HBsAg, HBeAg) and antibody(anti-HBs) were detected in sera with IFMA kit.The production of neutralizing antibody against HBsAg(anti-HBs) became detectable from day 7,accompanying with the decline of HBsAg.Real-time PCR showed that HBV DNA appeared after day 1,peaked at day 7,and became undetectable at day 18 (＜10~3 copies/ml),indicating HBV replication in model mice.Immunohistochemical staining clearly showed HBcAg~+ hepatocytes in HBV model mice,illustrating HBV expression in hepatocytes.ALT activities were slightly increased in both HBV model mice and control mice at day 1 and 3,then declined to normal levels.3.2.2 Increased Tim-3 expression on hepatic T cells,especially on CD8~+ T cells from HBV model miceFlow cytometric analysis showed that Tim-3 was slightly induced on hepatic T cells from both HBV model mice and control mice at day 1 and 3.However,no difference in Tim-3 expression was found in two groups.In contrast,Tim-3 expression was dramatically increased in HBV model mice from day 7 to day 18(p＜0.05 at day 7 and 18,p＜0.01 at day 10 and 14).The increase in Tim-3 expression was prominent on CD8~+ T cells(p＜0.05 at day 10),especially on IFN-γ-producing CD8~+ T cells.However,we did not detect any changes in Tim-3 expression in the spleen.3.2.3 Relationship between Tim-3 expression on CD8~+ T cells and serum neutralizing antibody in HBV model miceAt day 10 post the hydradynamic injection,certain amount of neutralizing antibody against HBsAg could be detected in HBV model mice although antibody titer differed in various mice.HBV model mice were divided into two groups based on the serum anti-HBs levels.A total of 50 mIU/ml was taken as the cutoff value of anti-HBs production since it closes to the average anti-HBs production(58 mIU/ml) of three independent experiments in the study.It was showed that mice with serum anti-HBs production＞50 mIU/ml(HBV model-anti-HBs~H) had a higher expression of Tim-3 on hepatic CD8~+ T cells than mice with serum anti-HBs production＜50 mIU/ml(HBV model-anti-HBs~L)(p＜0.05) 3.3.4 Tim-3shRNA expression plasmid effectively inhibits Tim-3 expression in vitroTim-3shRNA expression plasmids and unrelated shRNA control plasmid were successfully constructed and named as Tim-3shRNA-1,Tim-3shRNA-2 and unshRNA.Transfection of Tim-3shRNA-1 suppressed Tim-3 mRNA and protein expression in murine macrophage cell line RAW264.7(compared to control shRNA (unshRNA),p＜0.05).This suppression was specific because the transfection of Tim-3shRNA-1 had no inhibitory effect on Tim-1,another Tim family member.3.3.5 Tim-3 knockdown up-regulates IFN-γproduction from hepatic CD8~+ T cells in vivoFlow cytometric analysis showed that Tim-3 expression on hepatic CD8~+ T cells from HBV model mice treated with Tim-3shRNA-1 was significantly decreased than that from unshRNA-treated HBV model mice(p＜0.05).Further analysis showed that IFN-γproduction was also significantly increased in hepatic CD8~+ T cells from Tim-3shRNA-1-treated HBV model mice(p＜0.05).3.3.6 Tim-3 knockdown could down-regulate the titer of serum neutralizing antibodyOur data showed that production of serum anti-HBs decreased in Tim-3shRNA-1-treated HBV model mice compared to that in unshRNA-treated HBV model mice(p＜0.05).Conclusions1.Compared to controls,Tim-3 expression on PBMCs(especially on NK cells and CD8~+ T cells) and HMCs was significantly increased in CHB patients.2.Tim-3 was up-regulated on NK cells and CD8~+ T cells from HBV-Tg mice and hydrodynamics-based HBV model mice.Consistently,HBV transfection in NK92 cells leads to Tim-3 up-regulation.All indicates that HBV infection could induce increased Tim-3 expression.3.Tim-3 blockade could up-regulate cytotoxicity and IFN-γsecretion from NK92 cells and PBMCs in CHB patients. 4.Tim-3 knockdown with shRNA expression plasmid could up-regulate IFN-γproduction from hepatic CD8~+ T cells,accompanied with decreased levels of serum neutralizing antibody.Innovations and significance1.In this study,we firstly reported the increased expression of Tim-3 on PBMCs (especially on NK cells and CD8~+ T cells) and HMCs from CHB patients,which might be a basic for further mechanism study and act as a new target for clinical therapy.2.In this study three different models were involved:NK92 cells transfected with HBV expression plasmid(NK92-HBV),HBV-Tg mice and mouse model of HBV-induced hepatitis,to investigate the possibility that HBV infection could up-regulate Tim-3 expression,and to supply new data for the involvement of Tim-3 in HBV infection..3.NK92 cells and PBMCs from hepatitis B patients were used to investigate the possibility that Tim-3 blockade might help to up-regulate the cytotoxicity of NK cells, indicating Tim-3 might be a new therapeutic target for chronic HBV infection.4.With Tim-3 specific shRNA expression plasmid,we found that Tim-3 knockdown could up-regulate IFN-γfrom CD8~+ T cells in the mouse model of HBV-induced hepatitis.We also detected decreased levels of neutralizing antibody,indicating that in HBV infection Tim-3 may act as a potent regulator of hepatic CD8~+ T cells and indirectly affect neutralizing antibody production by regulating CD8~+ T cell function.