Innate Immunity In Over-Sensitive Liver Injury Of Murine Chronic HBsAg Carriers

Hepatitis B virus (HBV) as a hepatotropic, noncytopathic virus, primarily infects hepatocytes and causes a series of liver diseases such as acute and chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, which are among the most important human health problems worldwide. It has been reported that the immune responses are fundamental for viral clearance and pathogenesis during HBV infection; however, the precise pathogenesis has not been well known until now. With the new recognition that liver is a lymphoid organ with an overwhelming innate immune system, the roles of innate immune cells during HBV infection, especially the hepatic NK cells, deserve further investigating. NK cells are abundant in the normal liver, accounting for about one third of intrahepatic lymphocytes. Available evidences have indicated that hepatic NK cells play an important anti-viral role during HBV infection, but the role in the development of HBV-associated liver injury remains obscure. Noticeably, human healthy HBsAg carriers are susceptible to hepatocyte injury induced by a variety of elements such as stresses, infections, pregnancy and use or withdrawal of immunosuppressive therapies, which is a serious risk for the health of chronic HBsAg carriers. The basis for the susceptibility has not been understood yet. It has been argued that NK cells were involved in the over-sensitive liver injury in HBV transgenic mice triggered byα-GalCer, but the actual mechanisms of NK cells in hepatocyte injury remain elusive.In this study, we used HBV transgenic mice C57BL/6J-TgN (AlblHBV) 44Bri, which mimic human healthy chronic HBsAg carriers, to determine the functions of hepatic NK cells and the precise roles of innate immune responses in HBV-associated liver injury. The lymphocytes of central and peripheral immune organs were compared between HBV transgenic mice and wild C57BL/6 mice, especially hepatic NK cells. Poly I:C, Con A and CCl₄ were used to induce liver injury of HBV transgenic mice, and then immune responses of hepatic NK cells and precise roles of NK cells in the liver injury were investigated further. We examined the liver injury by determining serum transaminase ALT/AST level and liver pathologic changes. By cell depletion and cell transfer experiments, effects of the specific lymphocyte population and cellular interactions in the process of liver injury were investigated. The FACS analysis provided us with the precise information about lymphocyte phenotype, activation and cytokine production. Western blotting was applied to determine signals in heptocytes. Moreover, specific neutralization with antibody was applied to explore the potential mechanisms involved in cell interaction. Blocking with antibody and 4-h AST release experiments were applied to explore the molecular interaction between lymphocytes and heptocytes. Our major findings are shown as followed:1. Impaired function of hepatic NK cells from murine chronic HBsAg carriersCompared with wild C57BL/6 mice, in HBV transgenic mice the number of hepatic NK cells was decreased; natural activation (CD69 expression) of hepatic NK cells was declined; and cytotoxicity of hepatic NK cells was attenuated, which might relate to the down-regulated expression of TRAIL on hepatic NK cells. Additionally, although in the liver of HBV transgenic mice NK cells could be accumulated and activated by Poly I:C injection, the increase in anti-tumor cytotoxic activity of intrahepatic activated NK cells was markedly impaired in HBV transgenic mice compared with that in wild C57BL/6 mice.2. Murine chronic HBsAg carriers were over-sensitive to liver injuryA much low dose of Poly I:C (3μg/g body weight) injection induced much higher elevations of serum ALT and AST, severe hepatocyte necrosis and inflammation in HBV transgenic mice, which was similar to a high dose of Poly I:C (30μg/g body weight) injection. However, in wild C57BL/6 mice, there were only slight elevations of serum ALT and AST after 30μg/g body weight Poly I:C injection. These results demonstrated that HBV transgenic mice were over-sensitive to Poly I:C-induced liver injury.The nonhepatotoxic low dose of Con A (3μg/g body weight) for wild C57BL/6 mice induced severe liver injury in HBV transgenic mice, demonstrated by high level of serum ALT/AST and liver massive necrosis and inflammation. High dose of Con A (15μg/g body weight) injection induced the peak level of serum ALT/AST hardly with death in wild C57BL/6 mice, but caused death of most HBV transgenic mice.CCl₄ injection also induced much more severe hepatocyte injury in HBV transgenic mice than that of wild C57BL/6 mice shown by the level of serum ALT after 24 h of injection.3. Murine chronic HBsAg carriers were over-sensitive to Poly I:C-induced liver injury in NK cell- and IFN-γ-dependent mannerPoly I:C-induced over-sensitive liver injury was absolutely dependent on the presence of NK cells in HBV transgenic mice. After Poly I:C injection, intrahepatic NK cells were activated and produced IFN-γ. Neutralization of endogenous IFN-γsignificantly inhibited the over-sensitive liver injury. HBV transgenic mice were hypersensitive to IFN-γin liver injury, indicated by the results of serum ALT/AST after recombinant mIFN-γadministration. Further, much stronger IFN-γreceptor expression was observed on hepatocytes of HBV transgenic mice, which was significantly enhanced by Poly I:C injection. Treatment with IFN-γin vitro triggered much higher activation of downstream signals (pSTAT1-IRF-1) in hepatocytes of HBV transgenic mice. Depletion of Kupffer cells and neutralization of endogenous IL-12 did not affect Poly I:C-induced over-sensitive liver injury in HBV transgenic mice.4. Involvement of NKG2D activation in Con A-induced over-sensitive liver injury of murine chronic HBsAg carriersAfter low dose of Con A stimulation, hepatic MNCs became more cytotoxic and hepatocytes more sensitive to cytotoxicity from HBV transgenic mice compared with wild C57BL/6 mice. Low dose of Con A-induced over-sensitive liver injury was dependent on the more accumulated intraheptic NK cells in HBV transgenic mice, demonstrated by cell depletion. Adoptive transfer of purified hepatic NK cells from low dose of Con A-treated HBV transgenic mice further confirmed activated intrahepatic NK cells were cytotoxic to hepatocytes as effectors. Upon stimulation of low dose of Con A, expressions of NKG2D ligands (Rae-1 and Mult-1) in hepatocytes of HBV transgenic mice were markedly enhanced, but not in wild C57BL/6 mice. NKG2D activation of NK cells via recognition with Rae-1 or Mult-1 on hepatocytes accounted for the over-sensitive hepatocyte injury in HBV transgenic mice. Interestingly, NKT cells triggered by low dose of Con A served as regulator necessary forNK cell activation via secreting IFN-γand IL-4. 5. HBV down-regulated expressions of MHC class I molecules on hepatoplastoma cell lineIt was found that the expressions of HLA-ABC protein, HLA-E mRNA, membrane MICA protein and MICA mRNA were much lower in HepG2.2.15 cells compared with HepG2 cells. The expressing HBV in human hepatoplastoma cell line significantly down-regulated the expressions of MHC class 1 molecules.Here, we for the first time demonstrated the characterization of hepatic NK cells and revealed the precise roles of NK cells in the over-sensitive liver injury of HBV transgenic mice. Additionally, the possible effect of HBV on MHC class I molecules in human hepatocytes was investigated. These findings would be helpful to interpret the immune responses of hepatic NK cells and the immunological mechanisms of hepatocyte susceptibility to injury in human chronic HBsAg carriers.