| Hepatitis B virus (HBV) infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma and is an important public-health threat worldwide. As a hepatotropic, non-cytopathic virus, hepatitis B virus primarily infects hepatocytes and causes liver injury mostly results from the abnormal anti-HBV immune response of the host. It is well recognized that CD4+T and CD8+ T played an important role in the defense against HBV infection. However, as the new definition was put forward that liver is a lymphoid organ with an overwhelming innate immune system, the effect of innate immune cells, especially natural killer (NK) cells, on the pathogenesis of HBV infection need to be further investigated. Studies suggest that NK cells involved in the immune response against chronic HBV infection and HBV persistent infection correlated with dysfunction of NK cells. Meantime, patients suffered from chronic HBV infection are more sensitive to liver injury induced by a variety of environmental factors such as chemical toxins, alcohol and pathogen infection, which influence the capacity of liver regeneration.In this study, we assorted the patients into 4 groups according to the viral load of HBV DNA to explore the effect of NK cells on the pathogensis of chronic hepatitis B (CHB) patients. We compared the frequency, function and NK cell receptors of NK cells in the peripheral blood by flow cytometry and found that inhibitory receptor NKG2A has an obvious effect on the function of NK cells, which is likely the cause of HBV escape from the attack of NK cells. At the same time, we also investigated in the chemical toxin carbon tetrachloride (CC14)-induced liver injury mouse model, the effect of liver NK cells on the regeneration after liver injury so that to get some clues that whether NK cells influence the capacity of liver repair in the HBV infected patients contacted with chemical toxins. In these studies, our major findings were divided into two parts:â… . Up-regulated expression of inhibitory receptor NKG2A facilitate HBV persistent infection1. The percentage of NK cells was higher in the patients with low-level viremiaIn the CHB patients with low-level viremia (HBV DNA< 105copies/ml), the frequency of NK cells in the peripheral blood was significantly higher than healthy control, patients with high-level viremia and patients with liver cirrhosis, and the frequency of NK cells was negatively correlated with the copies of HBV DNA and serum ALT levels, which indicate that decreased NK cells will not helpful to clearance of HBV and remission of diseases. Analysis function of NK cells showed that cytokine IFN-y production by NK cells after stimulation with IL-12 was significantly higher in the CHB patients with low-level viremia than in the patients with high-level viremia and healthy controls. And the cytotoxicity assay showed that the capacity of NK cells to kill target cells K562 and expression of CD 107a on NK cells were elevated in the patients with low-level viremia.2. The percentage of NKG2A4 NK cells was increased in patients with high-level viremiaWe investigated expression of activating and inhibitory NK cell receptors on NK cells by flow cytometry. And the results showed that there were no difference in the expression of these activating receptors including NKG2D, NKG2C, NKp30, NKp44 and NKp46 and inhibitory receptors including KIR2DL3, KIR3DL1, CD 158a and CD 158b on NK cells, except for inhibitory receptor NKG2A. The frequency of NKG2A+NK cells was lower in the patients with low-level viremia, while in the patients with high-level viremia and liver cirrhosis, expression of NKG2A on NK cells was significantly increased. Furthermore, the frequency of NKG2A+NK cells was positively correlated with the copies of HBV DNA and serum ALT levels, suggest that upregulated NKG2A expression on NK cells may temper activity of NK cells.3. Blockade of NKG2A recognition reversed the HBV-related inhibition of NK cell activitiesBlockade of NKG2A signaling by mAb enhanced the NK cell-mediated cytotoxicity against K562 cells only in patients with high-level viremia. There were no changes in the results for patients with low-level viremia and healthy controls after using anti-NKG2A mAb in in vitro culture. These results were confirmed by using purified NK cells against three liver cell lines, that is human hepatic cell line LO2, hepatoma cell line HepG2 and HBV-expressing HepG2 cell line HepG2.2.15. An immunocompetent mouse model for human CHB virus infection was utilized to test the role of NKG2A signaling in the control of HBV replication in vivo. We used anti-NKG2A or anti-Qa-1 (ligand of NKG2A) to block the NKG2A/Qa-1 signaling. The results showed that the serum HBsAg and viral load were markedly reduced in mice treated with anti-NKG2A or anti-Qa-1, and these effects were dependent on the presence of NK cells, cause depletion on NK cells lead to slight change in the levels serum HBsAg and viral load. These results indicate that blockade of NKG2A recognition may be used to treat persistent infection of HBV.â…¡. NK cells inhibits liver regeneration via TNF-a dependent mechanismPrevious results showed that NK cells played important roles in the HBV clearance and liver injury during HBV infection, and the liver have the capacity to repair after liver injury. Therefore, this study is to investigate the role of NK cells in the liver regeneration. CC14-induced liver injury was used as the regeneration model to examine the effect of activation of NK cells on the liver regeneration. The results showed that administration of poly I:C suppressed liver regeneration in CC14-treated mice. Depletion of NK cells or NK1.1+cells (including NK and NKT cells) by anti-AsGM1 or anti-NK1.1 antibodies restored the liver regeneration in poly I:C/CC14-treated mice, indicate that NK cells involved in the inhibition of liver regeneration. Poly I:C and CC14 co-treatment synergistically induced accumulation and activation of NK cells in the liver and NK cell production of IFN-y and TNF-a. Finally, blockage of TNF-a but not IFN-y restored liver regeneration in poly I:C/CC14-treated mice. These findings suggest that poly I:C treatment inhibits liver regeneration in the CC14-induced liver injury model via induction of NK cell production of TNF-a.Above all, activated NK cells could control HBV replication, however, upregulation of inhibitory receptor NKG2A may temper anti-virus activity of NK cells; at the same time, activated NK cells exert negative regulation in the liver repair of damaged hepatocytes. These findings will be helpful to understand the mechanisms of persistent HBV infection and provide some theoretical basis on the treatment of hepatitis.
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