| [BACKGROUND]More than350million people worldwide suffer from chronic Hepatitis B virus (HBV) infection, and93million of them are in China. Chronic HBV infection leads to a wide spectrum of clinical presentations, among which severe hepatitis B (liver failure) is the most severe disease manifestation. HBV induced acute-on-chronic liver failure (HBV-ACLF) is one of clinical manifestation of liver failure, the pathogenesis of which remains largely unclear. Although multiple factors have been demonstrated participating in disease development, it is generally accepted that immune responses play a critical role in the process. The virus-specific CTL play a critical role in the pathogenesis of HBV induced liver injury. It has been reported that there were large amount of CTLs infiltrating in the liver from HBV-ACLF patients. As the major type of effector cell, CTLs directing to various antigen epitopes of HBV are closely associated with virus clearance and cell injury. Releasing of a series of inflammatory cytokines induced by activation of super reacting CTL lead to a mass of hepatocytes necrosis, which is one of the mechanisms contributing to HBV-ACLF. At the moment, the pathogenesis leading to HBV-ACLF is considered to infect T cell responses directly or indirectly. On the other hand, our previous work first elaborated NK cells were accumulated in the liver of MHV-3infected mouse with acute liver injury, while the increased killing of hepatic NK cells contributed to hepatocyte necrosis through Fas/FasL and NKG2D/NKG2DL pathway.To further discuss the potential molecular and immunological mechanisms of HBV-ACLF, human whole genomic gene chip was adopted in our lab to analyze the disparate gene expression in PBMC from patients with HBV-ACLF or mild/moderate chronic hepatitis B. Among the8600cDNA probes,263genes were substantially up-regulated in HBV-ACLF group. KCNJ15and KCTD9, both ion channel associated genes, were most prominent identified, the expression of which were up-regulated7and6folds in PBMCs from HBV-ACLF patients comparing to mild/moderate CHB patients, respectively.KCNJ15is the member of inward-rectifier potassium channel family, also called Kir4.2. There are three transcript variants in human encoding the same protein for this gene. KCNJ15is an integral membrane protein which has a greater tendency to allow potassium to flow into a cell rather than out of a cell. Right now, the researches about the function of KCNJ15are most focused on its effect on electric potential of target cells. The study of KCNJ15in viral hepatitis has not been reported.[OBJECTIVE]The aim of this study is to illustrate the role and mechanism of KCNJ15in HBV induced liver failure.[CONTENTS]This study is classified into two parts, the concrete contents are as the followings:Part1The role and immune mechanism of potassium channel gene KCNJ15in HBV-ACLF.1. The expression of KCNJ15gene in patients with HBV-ACLF.(1) To detect the expression of KCNJ15at both gene and protein levels in PBMC from patients with HBV-ACLF or mild/moderate CHB;(2) To detect the expression of KCNJ15protein in liver tissues from patients with HBV-ACLF or mild/moderate CHB;(3) To locate the subpopulation of the peripheral lymphocytes with increased KCNJ15expression in patients with HBV-ACLF.2. Dissecting of the potential immunological function of KCNJ15in Jurkat cell line.3. In vivo dynamic investigation of KCNJ15gene expression and its contribution in a MHV-3induced fulminant hepatitis model in Balb/cJ mice. By the time-course of infection, the expression of KCNJ15in liver tissues as well as its expression on hepatic lymphocytes was determined by immunohistochemistry and flow cytometry, respectively.Based on the results of first part, we suppose that there was a correlation between KCNJ15and secretion of IL-17, which might participate in the mechanism of virus induced liver failure. Then we carried out the researches of the second part:Part2The role and immune mechanism of IL-17in MHV-3induced murine fulminant hepatitis model.1. To evaluate IL-17expression in liver and serum of fulminant hepatitis mice;2. To detect hepatic IL-17producing lymphocytes after infection;3. To analyze the correlation between IL-17level and severity of the disease;4. To investigate Th17cell associated cytokines in liver from MHV-3induced fulminant hepatitis mice.[METHODS]1. The mRNA level of KCNJ15in PBMC from patients with HBV-ACLF or mild/moderate CHB was studied by quantitative Realtime PCR, while its expression in PBMC and liver tissue was detected by Immunocytochemistry and Immunohistochemintry, respectively. The KCNJ15-producing peripheral lymphocytes were assayed by flowcytometry. By immunofluorescence, the KCNJ15expression in CD4+T cells was detected in liver tissue.2. The level of cytokines IL-2, IL-4, IL-6, IL-9, IL-17, TNF-a and IFN-y in supernatant were further determined in Jurkat cells in vitro by transfection of a human KCNJ15expression plasmid. The concentration of intracellular calcium after transfection was investigated by flowcytometry.3. The expression of KCNJ15in MHV-3induced fulminant hepatitis mice were studied by quantitative real-time PCR, immunohistochemistry and FACS, respectively.4. IL-17mRNA levels in liver tissue were quantified by quantitative real-time PCR, while cytokine IL-17levels in liver and serum were determined by ELISA in MHV-3induced murine fulminant hepatitis model. IL-17expressions in liver lymphocytes were determined by flow cytometry. The correlation between IL-17level and liver injury was studied. Th17associated cytokines were also investigated by intracellular staining.[RESULTS]1. KCNJ15was over-expressed in PBMCs and hepatic infiltrating lymphocytes from HBV-ACLF patients.2. KCNJ15had an abundant expression in peripheral and hepatic CD4+T cells in patients with HBV-ACLF.3. By the time-course of MHV-3infection, the expression of KCNJ15in liver tissue was elevated significantly. Moreover, the percentage of both KCNJ15-producing CD4+T cells and KCNJ15-producing CD8+T cells increased remarkably in the liver at48h post infection, while hepatic NK cells expressed KCNJ15increased significantly at24h post infection. 4. The IL-17expression was significantly elevated in liver and serum of BALB/cJ mice infected with MHV-3. Moreover, a time course study showed that the percentage of both IL-17-producing CD4+T cells and IL-17-producing CD8+T cells increased remarkably in the liver starting from48h and peaked at72h post infection. There was a close correlation between hepatic or serum IL-17concentration and the severity of liver injury determined by ALT level, respectively. Th17associated cytokines, IL-6, IL-21, IL-22, were also increased at72h post infection.5. In the supernatant of KCNJ15over-expressed Jurkat cells, increased secretion of IL-17was observed. Meanwhile, increased concentration of intracellular calcium was also found after transfection.[CONCLUSION]1. For the first time, this study explored prominently increased KCNJ15-producing CD4+T cells in peripheral blood and liver tissue from HBV-ACLF patients2. The increased expression of KCNJ15in liver tissue and heaptic T lymphocytes from MHV-3infected mice, which was similar to the phenomenon in human HBV infection, may correlate with immune regulatory function of hepatic T lymphocytes. This model contributes to illustrate the mechanism of KCNJ15involved with virus induced liver failure.3. There were significant increases of IL-17levels in liver and serum, as well as increased frequency and cytokine secretion of Thl7cells in MHV-3infected BALB/cJ mice, indicating IL-17may contribute to the pathogenesis of MHV-3induced acute liver failure.4. This study discovered increased IL-17and intracellular calcium concentration in KCNJ15over-expressed Jurkat cells, suggesting that KCNJ15promoted secretion of IL-17, in which process calcium influx might involve. However, the related mechanisms need further discuss and confimation.
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