Roles Of TIPE2in Hepatitis B Virus Infection By Regulating CD8~+T Cells Functions

There are millions of people who are infected with HBV all over the world. Virus can be eliminated by an acute immune response. But many of the immune systems are not that strong enough. Viruses proliferate in the liver all the time. Because of the weak immune system it becomes to chronic hepatitis. Chronic hepatitis is the main reason for cirrhosis and liver cancer. CD8+T cells have been considered as a kind of very important cells in eliminating HBV. Virus protein was processed and formed MHC-I-peptide complex with MHC-I molecules on liver cell surface. Once the CD8+T cells identified the complex they secret IFN, perforin, granzyme B, or increase the expression of Fas ligand aimed to induce the apoptosis of virus infected cells.TIPE2is a newly founded anti-inflammatory protein. It is mainly expressed in lymphocyte tissues and immune cells. TIPE2is very important in maintaining immune homeostasis through down-regulating TCR and TLR signal pathway. It has been demonstrated that TIPE2-defficient mouse shows a much more sever inflammation in HBV induced liver inflammation. CD8+T cell plays a very important role causing the liver inflammation in deleting virus. In this research we obtain a result that TIPE2plays an important role in regulating the function of CD8+T cell both in mouse model and peripheral blood from hepatitis B patients. Our results provide experimental and theoretical basis for further research.Materials and Methods1. Mouse model1.1Preparation of sensitized spleen lymphocytes TIPE2deficient mice and WT mice aged8weeks were immunized with HBV plasmid. Strengthen the immune after two weeks. All mice were killed by cutting neck a week after the twice immune. Take the immunized spleens out and grid them into single cell suspension.1.2Cy to toxic effect of HBV sensitized spleen lymphocytes in vitroHBV sensitized spleen lymphocytes were co-cultured with Hepal-6cell transfected with HBV plasmid with a ratio of50:1. Cell viability was determined by CCK-8assay after24h.1.3Cytotoxic effect of HBV sensitized spleen lymphocytes in vivoHBV sensitized spleen lymphocytes were injected into the tail vein of the HBV transgenic mice. Sacrifice the mice48h after the injection. Liver inflammation was detected by H&E staining.2. Hepatitis B peripheral blood samples2.1TIPE2expression in CD8+T cells of hepatitis B patientsPeripheral blood from hepatitis B patient was stained by fluorescent-labeled antibody and the expression levels of TIPE2protein in CD8+T cells were analyzed by FCM. Peripheral blood from healthy individuals was used as control. Correlation between TIPE2and levels of serum ALT, AST, HBV DNA load was analyzed. Analysis of CD8+T cells function was peformed based on different TIPE2levels.2.2Activation of CD8+T cells in Hepatitis B peripheral bloodSamples of peripheral blood from healthy individuals and hepatitis B patients were stained by fluorescent-labeled antibody. The number of CD8+T cells stained by T cell activation markers HLA-DR and CD38was analyzed by FCM.2.3CD8+T cell function assayPBMCs isolated from healthy individuals and hepatitis B patients were cultured in RPMI1640medium and incubated with HBC18-27peptide for6h. CD8+T cells were stained by fluorescent-labeled antibody. The number of CD8+T cells stained by perforin, granzyme B, and IFN-y was counted by FCM.2.4CD8+T cell apoptosis assayPMBCs were isolated from peripheral blood of healthy individuals and hepatitis B patients. CD8+T cells were stained by annexinV cells apoptosis assay kit. The number of CD8+T cells stained by annexinV was counted by FCM.2.5Differences of TIPE2expressed in PBMCs stimulated with HBC18-27PBMCs isolated from healthy individuals were adjusted the density to6×105/ml with RPMI1640contained10%human AB serum. PBMCs were stimulated with10μg/ml synthetic HBc18-27.hrIL-2was added at30U/ml every three days. Cells were cultured at37℃,5%CO2. The cells were collected after10days. Real-time quantitative PCR was used for detecting the mRNA levels of TIPE2.2.6Cytotoxicity of PBMCs stimulated by HBc18-27Density of PBMCs isolated from peripheral blood of hepatitis B patients were adjusted to6×105/ml in RPMI1640medium contained10%human AB serum. PBMCs were stimulated with10μg/ml synthetic HBc18-27.30U/ml hrIL-2was added for every three days. Cells were cultured at37℃,5%CO2. After10days PBMCs were cocultrued with HepG2.2.15cells for24h with a ratio of50:1. CCK-8assay was used to determine the cell viability. Results1. Increased kill rate of the HBV sensitized TIPE2-/-splenocytesHepal-6cells viability cocultured with TIPE2-/-splenocytes sensitized by HBV was much lower than the controls （WT vs. TIPE2-/-,21.33±3.18%vs.60.33±3.53%, P=0.0038）. There was more severe inflammation in the livers of HBV transgenic mice adoptive transferred HBV sensitized TIPE2-/-splenocytes than the controls.2. Decreased TIPE2levels in CD8+T cells and enhanced cell function2.1Decreased TIPE2levels in hepatitis B CD8+T cellsFCM showed that TIPE2expression levels in CD8+T cells of the hepatitis B patients were much lower than that of the healthy individuals（31.32±3.57vs.19.19±1.18, P=0.0323）. And TIPE2levels in CD8+T cells negatively correlated with ALT/AST/HBV DNA （ALT<47U/L vs. ALT>47U/L,22.33±1.26vs.15.06±1.44, P=0.0126. AST<47U/L vs. AST>47U/L,22.83±0.97vs.16.69±1.44, P=0.0064. HBV-DNA<1000vs. HBV-DNA>1000,21.24±0.89vs.15.45±1.89, P=0.0273）. 2.2Increased activated CD8+T cell number in hepatitis B peripheral bloodThere are more active CD8+T cells in the hepatitis B patients’peripheral blood. According to the FCM CD8+T cells express the activation markers HLA-DR and CD38on cell surface in hepatitis B patients’ peripheral blood are much more than that of the controls （1.56±0.38%vs.19.18±3.43%,P<0.001）.2.3Negative correlation between CD8T cell function and TIPE2levelsThere are more CD8+T cells in the hepatitis B patients that express perform, granzyme B, and IFN-y after stimulating by the synthetic HBc18-27for6h than that of the controls （perforin:healthy control vs. hepatitis B,10.96±3.29%vs.47.78±9.93%, P=0.0244. granzymB:healthy control vs. hepatitis B,12.00±5.73%vs.54.50±11.37, P=0.0206. IFN-y:healthy control vs. hepatitis B,0.60±0.09%vs.4.65±0.40%, P=0.0022）. But there are not significant differences of the total number of CD8+T cells between the two. And the number of CD8+T cells of low TIPE2level that secreting perforin, granzyme B, IFN-y was much more than that of high TIPE2level （perforin:TIPE2high vs. TIPE2low,33.18±3.65%vs.55.90±7.85%, P=0.039. granzymeB:TIPE2high vs. TIPE2low,31.90±2.89%vs.61.23±7.21%,P=0.0092. IFN-y:TIPE2high vs.TIPE2low,2.70±0.52%vs.5.25±0.31%, P=0.0245）.2.4Increased cell apoptosisThe number of CD8+T cells that can be stained by annexinV in peripheral blood of hepatitis B patients was much more than that in the healthy controls （24.13±4.13%vs.50.61±6.35%, P=0.0081）.3. Decreased TIPE2mRNA and enhanced cell fouction induced by HBc18-27The levels of TIPE2mRNA in PBMCs were obviously decreased （control vs. stimulate with HBc18-27,1±0.04vs.0.667±0.01, P=0.0018） after induced by HBc18₂7synthetic peptide according to real-time PCR. Cell viability of HepaG2.2.15after cocutured with HBc18-27stimulated PBMCs from hepatitis B patients detected by CCK-8kit decreased obviously （control vs. stimulate with HBc18-27,1.00±0.08vs.2.28±0.05, P<0.001）. Conclusions1. Cytotoxicity of the splenocytes sensitized by HBV from the TIPE2deficient mice is stronger than that of the control both in vitro and in vivo.2. Down-regulation of TIPE2increased the function of CD8+T cells in hepatitis B.3. The increased apoptosis of CD8+T cells may be one of the way in which our bodies maintain the immune homeostasis. And on the other hand, the increased CD8+T cells apoptosis may be the reason for chronic hepatitis B.4. Our study performed both from mouse model and peripheral blood samples showed that down-regulation of TIPE2in CD8+T cells is beneficial to the removal of the virus. Our results provide experimental and theoretical basis for further clinical and basic research.Innovations and significancesIt is the first time to show that HBV sensitized TIPE2deficiency spleen cells acquire increased killing function both in vitro and in vivo. According to FCM and induction of synthetic HBc18-27, it is the first time to show that the down-regulation of TIPE2enhance the function of CD8+T cells. Our results may provide theoretical and experimental guidance for further research.