Role Of Toll-like Receptor Pathway In Th17Cell Activation And The Pathogenesis Of Hepatitis B Virus (HBV)-related Acute-on-chronic Liver Failure

BackgroundImmune-mediated inflammatory injury is an important feature of hepatitis B virus (HBV)-related acute-on-chronic liver failure. IL-17, the signature cytokine, produced by T helper17(Th17) cells involved in liver damage, and the activation of Th17cells is considered to play an significant role in HBV-related acute-on-chronic liver failure, but the precipitating cause of Th17cell activation is still not very clear. Research of Autoimmune diseases have shown that Toll-like receptor pathway is involved in promoting the differentiation of Th17cells and development of the disease. Currently, Role of Toll-like receptor pathway in pathogenesis of hepatitis B virus-related acute-on-chronic liver failure and its effect on Th17cell differentiation research remains blank. In this study, hepatitis B virus-related acute-on-chronic liver failure paitents as research objects, the system analyzes the correlation between Toll-like receptor expression on T cells in HBV infected patients and its clinical indicators of disease, and to explore that the Toll-like receptor2expression affect activation and function of Th17cell. This study try to elucidate the pathogenesis of hepatitis B virus-related acute-on-chronic liver failure and provide important clues and theories to looking for a new immunotherapy programs Objective1. To investigate the expression of Toll-like receptor family (TLR1-10) on PBMC and TLR2/4on CD4+/CD8+T cells of HBV infection of acute-on-chronic liver failure and CHB, and analyze their correlation with clinical biochemical indexes and serum viral markers.2. Through the transverse observed in vitro by TLR2ligand (Pam3Csk4) stimulation of T cells on PBMCs in patients with HBV-related acute-on-chronic liver failure and CHB, to detection the change before and after stimulation of Th17cells and cytokine expression, in order to determination the correlation between Th17cells expression and clinical biochemical indexes.3. Through the longitudinal observation in vitro by TLR2ligand (Pam3Csk4) stimulation of T cells on PBMCs in patients with HBV-related acute-on-chronic liver failure and CHB, to observe the Thl7cells and their secreted cytokines, and to understand the correlation between TLR2expression and the diseases development, so as to explore the role of TLR2in Th17response and pathogenic, and provide experimental and theoretical basis in pathogenesis for elucidating Th17cells in HBV-related acute-on-chronic liver failure.Methods1. Forty-four patients with chronic HBV infection (including22patients with HBV-related acute-on-chronic liver failure, and22patients with CHB) and20healthy subjects were enrolled in this study. All the patients were recruted from the Department of Infectious Disease. Union Hospital. The study was approved by the local medical ethics committee of WuHan Union Hospital, and each individual signed the informed consent and have been excluded other virus infection.2. heparin anticoagulant tube collected venous blood and peripheral blood was separated by centrifugation of Ficoll density gradient. RNA was extracted by TRIZOL method, and used for the amplification of TLR1-10gene expression.3. Part of the separated PBMC added into96-well plate, cultured for4hours, according to the need to added the phorbol-12-myristate13-acetate (PMA), ionomycin (Ionomycin), or phorbol ester, ionomycin and TLR2ligand (Pam3Csk4, P3C) repectively, while a medium well setting as negative control, the expression of TLR2on CD4+T cells in PBMCs were evaluated by Flow cytometry, and the Thl7cell related cytokines IL-21, IL-22, IL-17, IFN-y and IFN-a were detected by intracellular cytokine staining.4. The expression of TLR2and TLR4on CD4+and CD8+T cells in PBMCs were measured by Flow cytometry.5. The serological viral markers were detected by ELISA Kit:HBsAg, anti-HBsAg, anti-ABcAg, anti-HIV, anti-HCV and anti-HDV.6. The clinical biochemistry indexes come from the Department of Laboratory.7. All statistical analyses were performed by using SPSS17.0software. Multiple comparisons were made between the different groups using Mann-Whitney U-test, whereas comparisons between the same individual were made using the Wilcoxon matched pairs t-test. Spearman’s correlation test was used to assess the correlation of immune factors and clinical characters. P<0.05was set for statistical significance. Results1. Compared to the healthy controls, the expression of TLR1/2/4/5/6/10mRNA in patients with ACLF was significantly increased, the TLR1/2/3/4/5/6/8/9/10mRNA expression in CHB patients was markedly increased, and the TLR2/4expression was significantly higher than that of other TLRs. Compared with the CHB, the expression of TLR2/6mRNA in patients with ACLF was significantly increased, while the TLR8/9mRNA expression was lower than CHB.2. Compared with the healthy controls, the expression of TLR2/4/5/6/8/9/10mRNA in patients with HBeAg+/HBeAg-was significantly increased, and the TLR2/4mRNA expression was highest. Although the TLR2/4/5/6/8/9/10mRNA expression in patients with HBeAg+was lower than in HBeAg" patients, the difference was not statistically significant.3. The TLR2/4expression on CD4+T cells in ACLF group obviously higher than that of CHB group and healthy control group; the TLR2expression on CD4+T cells in CHB group was higher than the healthy control group. The expression of TLR2/4on CD8+T cells in ACLF group were significantly increased than that of CHB patients and healthy subjects; and the expression of TLR2on CD8+T cells in CHB were higher than the healthy controls.4. The TLR3mRNA expression in patients with CHB was positively correlated with the expression of HBV DNA, and the expression of TLR7mRNA in patients with CHB was positively correlated with serum ALT levels.5. In the HBV patients, the TLR2/4expression on CD4+and CD8+T cells was positively correlated with TBIL, DBIL, ALP and INR, and was negatively correlated with ALB. In addition, the expression of TLR2on CD4+and CD8+T cells were positively correlated with AST, PT and WBC, and were negatively correlated with TP and PLT. The expression of TLR4on CD4+T cells were positively correlated with PT and WBC.6. IL-17mRNA expression in PBMCs of ACHBLF Patients was obviously higher than that in CHB patients and healthy subjects.7. IL-17mRNA expression in PBMCs of ACHBLF Patients was positively correlated with TBIL and DBIL.8. The frequency of peripheral Th17cells in ACHBLF patients was significantly higher than that in CHB patients and healthy subjects.9. After administration with TLR2ligand in PBMCs of ACHBLF, CHB and Healthy controls:1) The expression of IL17on CD4+T cells in patients with ACHBLF and CHB was significantly increased, but the healthy control group had no statistical differences.2) The secreted cytokines (IL-22, IL-21) and pro-inflammatory factor (IFN-y, IFN-a) of Thl7cells was significantly increased, but no statistical differences in healthy controls.3) The expression of cytokines (IL-22, IL-21) and pro-inflammatory factor (IFN-y, IFN-a) in patients with ACHBLF were higher than CHB and healthy controls, but the IFN-y expression no statistical differences.4) The expression of Th17cells in patients with ACHBLF was positively correlated with TBIL and DBIL, and was negatively correlated with albumin.5) The expression of Thl7cells in patients with ACHBLF was positively correlated with the expression of TLR2.10. The expression of IL-17and TLR2in patients with ACHBLF was increased when the disease aggravating, but the IL-17and TLR2expression decreased obviously along with the improvement of the disease.Conclusions1. The expression of TLR2/4mRNA and protein levels on PBMCs in patients with ACHBLF was significantly higher than that of CHB and healthy controls, showed that the patients of ACHBLF exists innate immune activation and presence of infection.2. The Th17cells expression and secreted cytokines on PBMCs in patients with ACHBLF were obviously higher than those of CHB and healthy controls, suggesting that Th17was involved in liver injury in patients with ACHBLF. 3. Under the stimulation of TLR2ligand, the expression frequency of Th17cells and secreted cytokines in patients of ACHBLF was significantly increased. Th17expression related to clinical severity index, and the expression of Th17cells was positively associated with TLR2expression, suggested that the activation mechanism of Th17cells in patients with ACHBLF may be involved in TLR2initiate the response of Th17cells, and aggravate the liver injury in patients with ACHBLF.