The Novel KCTD9 Contributes To NK Cell Mediated Liver Injury In Experimental And Human Severe Viral Hepatitis

[BACKGROUND & OBJECTIVE]The prevalence of HBV infection is high in China, where HBV carriers account about 7.18%in the group of age 1-59 by 2010. HBV induced severe hepatitis or hepatic failure is still a common disease with acute and severe manifestation. According to the consensus from Chinese Association for the Study of Liver and Infectious Disease, the hepatic failure can be classified as Acute Liver Failure or Fulminant Hepatitis (ALF), Subacute Liver Failure (SALF), Acute-on-Chronic Liver Failure (ACLF), and Chronic Liver Failure (CLF). Virus induced hepatic failure, especially acute liver failure which can lead to a 70-90% death rate, usually presents a emergent clinical process and results in multiple complications. Due to lack of specific and effective treatment and interference targets, the clinical outcome of patients with liver failure is poor unless emergent liver transplantation is appliedThe pathogenesis of virus induced hepatic failure is complicated and not well defined. However, it is generally accepted as the result of complex interaction between the virus and the host. The virus directly or indirectly induces activation of immune system which results in primary liver injury, subsequently down stream liver injury is further led by endotoxin-cytokines axis. Patients with long term of chronic liver disease can easily undergo severe hepatitis upon the reactivation of virus or host immune system. As we know, hepatic failure is mainly caused by drug agents in Western, while mainly by HBV infection in China. Since HBV virus is a kind of non-cytopathic virus and does not cause damage of hepatocytes directly, the primary mechanism of hepatocytes injury after HBV infection lies on clearance of virus infected hepatocytes by active action of immune system. So, exploration of the immunological role and mechanism in virus infection may be specific in elaboration of pathogenesis of hepatitis B virus induced hepatic failure.In our previous work, human whole genomic gene chip was adopted to analyze the disparate gene expression in PBMC from patients with HBV-ACLF or mild chronic hepatitis B. Among the 8600 cDNA probes, widely-spread 263 genes were substantially up-regulated in HBV-ACLF group. KCTD9 and KCNJ15, two kinds of ion channel genes, were most prominent identified, the expression of which were up-regulated 6 and 7 times in PBMCs of HBV-ACLF patients respectively when compared to mild/moderate CHB patients. And the following studies from amplified cases of different clinical types with HBV infection confirmed the reliability. Furthermore, we detected KCTD9 expression in liver tissue or PBMC from 265 patients with mild/moderate CHB or HBV-ACLF and 36 healthy controls in gene and protein levels. The results showed the expression of KCTD9 was prominently increased only in liver tissue and PBMCs from HBV-ACLF patients, which suggested a close relationship between KCTD9 expression and severity of hepatitis B. To explore the role and mechanism of KCTD9 in pathogenesis of HBV induced hepatic failure, similar study was performed in the MHV-3 induced fulminant hepatitis model in Balb/cJ mice. The result showed that KCTD9 highly expressed in the liver tissue and hepatic lymphocytes after MHV-3 infection especially the hepatic NK cells. The results in the animal model study were similar with that in human disease, which suggested the applicability of this animal model in further research with regards to the identification of the function and mechanism of KCTD9 involved in virus induced hepatocytes injury.Our previous work first elaborated the increased killing of hepatic NK cells by Fas/FasL and NKG2D/NKG2DL contributed to hepatocyte necrosis in virus induced liver failure. The aim of this study wass to further elaborate the role and mechanism of NK cells involved with acute hepatocyte injury in virus induced liver failure, as well as to investigate the function and pathogenesis of increased expression of KCTD9 on NK cells in the disease. The concrete contents were as the followings:Part 1 The role and immune mechanism of hepatic NK cells in virus induced liver failure.1. To detect distribution of NK cells in liver tissue and expression of FasL, NKP30 and NKP46 in peripheral NK cells from HBV-ACLF patients, and to discuss possible mechanism of acute liver injury that NK cells involved in HBV induced hepatic failure.2. To investigate the effect by depletion of NK cells in MHV-3 induced fulminant hepatitis mice and discuss the role of NK cells in the model.Part 2 The role and immune mechanism of novel KCTD9 in virus induced liver failure.1. The cellular localization and role of KCTD9 expression in patients with HBV-ACLF.(1) To locate the subpopulation of the peripheral lymphocytes with increased KCTD9 expression in patients with HBV-ACLF.(2) To analyze the correlation between the percentage of peripheral NK cell that expressed KCTD9 and severity of liver injury in patients with HBV-ACLF.2. The functional study of KCTD9 in a NK cell line (NK92 cell line).3. Th role and functional study of KCTD9 in a MHV-3 induced fulminant hepatitis model in Balb/cJ mice.(1) Construction of specific shRNA interference plasmid targeting KCTD9, irrelevant plasmid, and full length mouse KCTD9 expression plasmid, and verification of their interference effects on KCTD9 expression in vitro and vivo, and mice suvival, hepatic biochemistry and histopathologic improvement in MHV-3 infected Balb/cJ mice.(2) Investigation of KCTD9 expression in NK lymphocytes and its contribution to NK cell functional change in MHV-3 infected Balb/cJ mice.[METHODS]1. The frequencies of NK cells in the liver tissue of patients with HBV-ACLF or mild/moderate CHB were analyzed by Immunohistochemistry. The expression levels of FasL, NKP30 and NKP46 in peripheral NK cells were detected by FACS. Survival rate after depletion of NK cells by injection of anti-AsGMl through tail vein was observed in the mice model of FHF in MHV-3 infected Balb/cJ mice. 2. The expression levels of KCTD9 in peripheral NK cells, CD4+T cells, and CD8+T cells from patients with HBV-ACLF or mild/moderate CHB were detected by FACS. The clinical information of these patients was documented and the correlation between the percentage of NK cells that expressed KCTD9 and severity of liver injury was analyzed in HBV-ACLF patients. Meanwhile, KCTD9 expression on hepatic NK cells in the consecutive section of liver tissue was observed by Immunohistochemistry.3. The human KCTD9 expression plasmid was transfected into NK92 cell line by electronic transfection in vitro, the expression level of KCTD9 protein and activation marker CD69 were detected by FACS at 24h after transfection. The transfected NK92 cells were co-cultured with HepG2.2.15 cells in gradients of effector/target ratio to determine cytotoxicity of NK92 cells by quantitative measurement of ALT release, whereas the secretion of IFN-γand TNF-αby NK92 cells was detected. The profiling of functional receptors of NK92 cells after transfection was also analyzed by FACS.4. The shRNA interference plasmid targeting mouse KCTD9 gene and KCTD9 full length expression plasmid were constructed and introduced into MHV-3 infected Balb/cJ mice respectively by hydrodynamic delivery through tail vein. The effect of KCTD9 on the process of the disease was elucidated by survival rate, serum ALT level, and change of liver histopathology. Meanwhile, the effects of shRNA interference on activation and KCTD9 expression of hepatic NK cells were also evaluated by FACS. Moreover, hepatic NK cells were isolated by microbeads after shRNA interference, and then the cytotoxicity to MHV-3 infected hepatocytes in vitro, production of IFN-γand TNF-αby intracellular staining were all analysed.[RESULTS]1. NK cells play a significant role in virus induced acute liver injury. By immunohistochemistry the frequency of NK cells in liver tissue of patients with HBV-ACLF was much higher than that in patients with mild/moderate CHB. The expression level of FasL in liver tissue, percentage and MFI of NKP30, NKP46 and FasL on peripheral NK cells from patients with HBV-ACLF were prominently higher than those in patients with mild/moderate CHB.In MHV-3 induced fulminant hepatitis model, depletion of NK cells led to a increase of mice survival from 0.00%to 22.2%.2. There was increased expression of KCTD9 in peripheral lymphocytes, especially in NK cells from patients with HBV-ACLF. The percentage of peripheral NK and CD4+T cells but not CD8+T cells with elevated KCTD9 expression in patients(15 in total) with HBV-ACLF were evidently higher than that in patients (21 in total) with mild hepatitis B. The mean fluorescence index (MFI) of KCTD9 in peripheral NK, CD4+T, and CD8+T cells were all significantly increased in patients with HBV-ACLF. There was a positive correlation between the percentage of peripheral NK cells with increased KCTD9 expression and patients ALT or AST level, but not correlation with TBIL, DBIL, ALB, PTA, HBV DNA level and age, suggesting KCTD9 expression may contribute to NK cell mediated liver injury in virus induced hepatic failure.3. KCTD9 promoted activation of NK92 cells in vitro by down-regulating NKG2A expression. A human KCTD9 full-length expression recombinant plasmid (pcDNA3.1-hKCTD9) was constructed and introduced into NK92 cells, significantly elevated KCTD9 protein and activation marker CD69 were observed at 24h after transfection. Meanwhile, enhanced cytotoxicity of NK92 cells to HepG2.2.15 and increased secretion of IFN-r were also observed. After a series analysis of the functional receptors on transfected NK92 cells, a decrease of NKG2A expression was found indicating the mechanisms for KCTD9 to promot NK cells cytocoxicity may be mediated by downregulated expression of an inhibitory molecule NKG2D.4. The therapeutic shRNA interference plasmid targeting KCTD9 ameliorated the progress of MHV-3 induced fulminant hepatitis. A full length mouse KCTD9 expression plasmid, a specific KCTD9 shRNA and an irrelevant shRNA interference plasmid were successfully constructed. Introduction of a specific KCTD9 shRNA interference plasmids by hydrodynamic delivery into MHV-3 infected Balb/cJ mice, decreased KCTD9 expression in liver was observed and the survival rate elevated from 0 to 22.2%. Moreover, an extraordinary declined ALT level and significant improvement of liver histopathology was also evidenced when compared with the mice without treatment or with control plasmid. In contrast, hydrodynamic delivery of KCTD9 expression plasmid into MHV-3 infected Balb/cJ mice significantly deteriated the disease course. 5. The therapeutic shRNA interference plasmid targeting KCTD9 reduced activation and cytotoxicity of hepatic NK cells in MHV-3 incued fulminant hepatitis model. The KCTD9 and the activation marker CD69 in hepatic NK cells were notably down-regulated at 72 hours after delivery of a KCTD9 shRNA interference plasmid. Accordingly, the cytotoxicity of the hepatic NK cells from these mice to hepatocytes was decreased. These hepatic NK cells also displayed a decrease in IFN-y and TNF-a secreation in comparison with no treatment group or control plasmid treated mice. In contrast, an enhanced activation and cytotoxicity of hepatic NK cells was observed in mice when teil vein delivery of KCTD9 expression plasmid.[CONCLUSION]1. For the first time, this study explored prominently increased expression of NK cells in the liver tissue of HBV-ACLF patients and confirmed that activated hepatic NK cells contributed to hepatocytes necrosis by enhanced expression of FasL. This study also firstly elaborated the contribution of hepatic NK cells in virus induced FHF in a mice model.2. For the first time, this study discovered that the novel KCTD9 gene could promote the activation of NK92 cells in vitro via down-regulating the expression of its inhibotory molecule NKG2A.3. For the first time, this study discoved that KCTD9 gene plays an important role in the pathogenesis of acute hepatocyte injury and progress of virus induced hepatic failure by promoting activation of NK cells.4. For the first time, this study demonstrated a shRNA interference plasmid specific targeting KCTD9 gene inhibited NK cell activation and amelioated disease process in MHV-3 induced FHF, which in turn may shed light on the therapeutic stratitegies for disease control.