The Role Of NK Cells In Acute HBV Infection

Hepatitis B virus (HBV) is a human hepadnavirus that is the envelope protein containing3.2kb circular, double stranded DNA. Ahighly effective preventive vaccine has been available, however,there are still about350million people who are already infected by the virus,and these people are at high risk of inducing end-stage liver disease and hepatocellular carcinoma. Some researches have indicated the clearance of HBV depends on the age and immune status. The effective immune response can contribute to clear the virus, while the defective immune response may result in virus persistence. So the immune response play an important role in HBV clearance.The adaptive immune response is always thought to play critical roles in HBV elimination and pathology, however, as the main component of the innate immune system, the role of NK cells remains poorly understood during HBV infection. Although there are studies that depletion of NK cells lead to HBV persistence, and activated NK cells are accompanized with the HBV clearance in people with acute HBV infection, the mechanisms of how NK cells function is still unknown. Recently, there are hot researches about the role of NK cells in immune regulation. In contrast, the role of NK cells on immune regulation is still in its fancy during HBV infection. Therefore, are NK cells activated, and do they have immune regulation on adative immune system during HBV infection? These are the questions we want to find out.The aim of our studies is to investigate the precise mechanism in HBV clearance. We focus on the role of NK cells during acute HBV infection, and prove that NK cells contribute to orchestrate global antiviral defense. We hope to provide an important sight for the design of innovative therapeutic or vaccination strategies.In this study, we hydrodymically injected the mice with20-microgram pAAV-HBV1.2plasmids to develop acute murine HBV infection model. The role of CD8+T cells in acute HBV infection was assessed by the serum detection of HBV antigen in CD8-/-mice, and the secretion cytokines of liver MNCs after neutralizing CD8+T cells in vitro compared with control. To evaluate NK cell responses, we detected the phenotype of NK cells and the serum HBV antigens in NK-depleted mice.By selective depletion and adoptive transfer, the regulation on CD8+T cells were demonstrated. The role of CD4+T cells in NK-cell-viral control and regulating-CD8+T cells were assessed by serum HBV antigens detection and flow cytometry after selective depletion. The requirement of NK cells for CDllb+DCs were determined by flow cytometry. Additionally, to demonstrate the liver LN deliver anti-HBV immunity, the liver LN were surgically removed and the cells were asseseed by flow cytometry. The characteristics of HBV infection by determining serum levels of HBsAg, HBeAg, and anti-HBs using IRMA, and serum HBV DNA copies using real-time PCR were assessed. Serum transaminase ALT levels and the histopathology of liver by H&E staining were used to examine the liver injury. Cytokine levels after ex vivo activation including IFN-y, TNF, and IL-2were assessed by CBA. The proportion and phenotype of NK cells, CD8+T cells, CD4+T cells and DCs were determined by flow cytometry. Intracellular staining was used to examine the secretion of cytokines and other functional molecules. Our major results are shown as followed:1. CD8+T cells are important in acute HBV infectionpAAV-HBV1.2plasmid was injected hydrodynamically into the tail veins of male C57BL/6mice. After HBV plasmid injection, the infection mice were bled every week to monitor the serum levels of HBsAg, HBeAg, HBV DNA, and anti-HBs. The characteristics of replication and expression of HBV antigens detected in WT mice indicated that WT mice can eliminate the HBV within3-4weeks, but the ALT levels were within normal limits1-4-week after HBV plasmid injection. H&E staining of liver tissues revelsed that there were without lymphocyte infiltration. Immunohistochemical staining of liver tissues by rabbit anti-HBc, demonstrated kinetics of HBcAg expression in the liver. So the the characteristics of the HBV infection model was noncytopathic, which mimics the human acute HBV infection.Next, the cellular immune response was detected during HBV infection. The results indicated that serum HBsAg and HBeAg were higher in CD8-/-mice than WT mice. When liver CD8+T cells were neutralized in vitro, it resulted in the decreased IFN-y secretion in the supernatant. Thus, CD8+T cells were important to eliminate HBV infection during acute HBV infection.2. NK cells are involved in HBV clearanceFurther, we also found that the frequency and the absolute number of NK cells (detected as NK1.1+CD3-cells) was higher on3day after HBV infection than control mice. And NK cells in HBV-injected mice homogeneously expressed CD69and, on in vitro stimulation, readily produced IFN-γ. Thus, these findings indicate that NK cells were activated and capable of producing IFN-γ. To investigate whether NK cells have a role in HBV clearance, selective depletion of NK cells by50μg of Anti-AsGM1monoclonal antibodies, HBV surface antigenemia persisted for8weeks, the same results for serum HBeAg between control mice and NK-depleted mice. Liver sections with anti-HBcAg staining also indicated that NK-depleted mice expressed more HBcAg. And Nfil3-/-mice (NK deficient mice) that cannot clear HBV even after13weeks of HBV plasmid injection.When Nfil3-/-mice were transferred with NK cells from WT mice and GKO mice selectively, transferred of NK cells from WT mice cleared HBV more quickly. In addition, when we injected mouse IFN-γ into the NK-depleted mice, the serum HBsAg also declined more quickly than without injection in NK-depleted mice. Taken together, these results demonstrated that NK cells derived IFN-γ was important for HBV clearance.3. NK cell-derived IFN-γ promotes generation and function of anti-HBV CD8+T cellsNext, to evaluate the role of NK cells on CD8+T cells in HBV infection, mice were treated with Anti-AsGMl to deplete NK cells. The total number of liver CD8+T cells declined significantly in NK-depleted mice. Liver and spleen CD44+CD62-CD8+T cells were lower in NK-depleted mice than control mice. After simulation, IFN-γ secretion of liver and spleen CD8+T cells declined more significantly in NK-depleted mice.We also found that NK-depleted mice secreted less IFN-γ than control mice by Core93or HBcAg stimulation. Collectively, these results suggest that NK cells affect the immune responses of CD8+T cells in acute HBV infection. Then we considered that NK cells might enhance CD8+T cell responses in vivo by providing IFN-γ. To test this hypothesis, two experimental system were constructed. Firstly, NK cell-depleted mice were reconstituted with highly purified NK cells isolated from the spleens of WT mice or IFNγ-/-mice. WT mice have higher proliferation and IFN-γ secretion than NK cell-depleted mice. Secondly, IFNγ-/-mice were transferred with CD8+T cells and hydrodynamically injected with HBV plasmid. Under these conditions, endogenous NK cells can not efficiently activate CD8+T cells. On the adoptive transfer of NK cells from IFN-γ-sufficient mice, however, CD8+T cell responses was reconstituted greatly. Therefore, these results demonstrate that NK cells participate in acquired CD8+T cell responses by providing an initial source IFN-γ which is necessary for the anti-HBV CD8+T cell response in HBV infection. 4. NK cells are involved in the recall responses of anti-HBV CD8+T cellsWe have shown here that NK cells were involved in the primary response of CD8+T cell response in acute HBV infection. To test whether NK cells participate in the recall response of CD8+T cells, NK cell-depleted recipient mice were transferred with CFSE labeled spleen HBV-activated CD8+T cells. We found that CFSE labeled CD8+T cells in NK-depleted mice exhibited poorer proliferation and IFN-y secretion. Similar with the transfer of HBV-activated spleen CD8+T cells, when transferred with CFSE labeled liver HBV-activated CD8+T cells, we also found that CFSE labeled liver CD8+T cells in NK-depleted recipient mice exhibited poorer proliferation and IFN-y secretion as well. Taken together, these results demonstrate that NK cells are not only involved in primary response of CD8+T cells, but also participate in recall response of CD8+T cell in acute HBV infection.5. Role of CD4+T cells in NK cell-midiated HBV control and antiviral CD8+T cells activationThe activities of CD4+T cells are critical for helping CD8+T cell function during virus infection. To evaluate whether CD4+T cells were participated in the NK-cell control of HBV and regulating anti-HBV CD8+T cell response, mice were treated with antibodies to deplete NK cells in Ragl-/-mce and CD4-/-mice. Whereas depletion of NK cells before hydrodynamically injection of HBV plasmid, serum HBsAg were identical with the control mice. In contrast, IFN-y produced by HBV-specific CD8+T cells from both CD4-/-mice and CD4-/-mice depleted with NK cells decreased much more significantly than wild-type mice. However, depletion of NK cells in CD4-/-mice did not induce a decrease in IFNy+HBV-specific CD8+T cells than CD4-/-mice. Together, these results demonstrate that NK cells did not directly control HBV infection, and did not directly regulate the activation of CD8+T cells as well. Thus, CD4+T cells are needed for NK-cell modulation of antiviral CD8+T-cell responses associated with viral control.6. CD4+T cells provide help for CD8+T cell activationBecause CD4+T cells mediate NK-cell modulation of antiviral CD8+T cell-response and viral control, we next examined that whether CD4+T cells are involved in HBV clearance. In CD4-/-mice, serum HBsAg and HBeAg were higher than wild-type mice. Thus, CD4+T cells is critical in HBV clearance as well. Adoptive transfer of5×106purified spleen CD8+T cells from C57BL/6mice into Rag1-/-mice. However, CD8+T cells can not help Rag1-/-mice to clear the viral antigen until8weeks after injection. Neutralizing intraepatic CD4+T cells also lead decreased supernatant IFN-γ secretion. Together, CD4+T cells are required for anti-HBV CD8+T cells activation during HBV infection.7. NK cells promote CD4+T cells activationWe next examined wheather CD4+T cells were required for NK cell activation. C57BL/6mice and CD4-/-mice were injected hydrodynamically with20μg of HBV plasmid.5days after injection,we found that NK cell activation was not affected in CD4-/-mice. In addition, adoptive transfer of CFSE labled CD4+T cells into CD4-/-mice, the IFN-y secretion and proliferation of CD4+T cells declined in NK-depleted CD4-/-mice. By PMA and ionomycin stimulation, liver CD8+T cells secreted less IFN-y in NK-depleted mice. We also found that CD4+T cells from NK-depleted mice secreted less IFN-y than control mice by Core129stimulation. Collectively, these results suggest that NK cells promote CD4+T cells activation.8. The effects of NK cells on the migration and maturity of CD11b+DCsSpleen CDllchigh DCs increased significantly on5day after HBV plasmid injection compared to control mice. However, spleen CDllchlgh DCs,especially CDllb+DCs,but not CD8α+DCs, declined significantly in NK-depleted mice.Advanced experiments demonstrated that expression of CD80and CD86on spleen CD11b+DCs declined significantly in NK-depleted mice, and the mRNA levels of IL-18,,IL-15and IFN-α in NK-depleted splenocytes decreased as well. Selective netralizing IFN-γ in vivo, we found that the frequency of spleen CD11b+DCs, and the mRNA levels of IL-15and IFN-α in IFN-γ-neutralizing splenocytes decreased as well. Thus, IFN-γ mediated the influenced the migration and maturity of CD11b+DCs by NK cells. Given the full expression of CD4on CD11b+DCs, we hypothesized that CD11b+DCs might mediate the regulation of NK cells on CD4+T cells.9. The sinificance of liver LN in delivering anti-HBV immunityAdditionally, through intrahepatic injection and hydrodynamic injection of Ad-EGFP, we found that liver LN can drain the EGFP+cells from liver. Further exprements demonstrated that liver LN cells delivered anti-HBV immunity. To verify the effects, we surgically removed liver LN, and found that such experiments resulted in about30%HBV persistant mice, while surgically removed spleen did not influence the HBV clearance. And we also found that the frequency of CD8+T cells and DCs increased significantly in liver LN after HBV infection. Thus, in this part, our research first provides the evidence that liver LN is important in HBV clearance in vivo.Taken together, the present work described the immune mechanisms involved in acute HBV infection.We demonstrated that NK cells played a pivotal role by secreting IFN-γ in HBV clearance, and found that the indirect role of NK cells in viral control and activation of CD8+T cells. Further experiments indicated that CD4+T cells mediated the function of NK cells on viral control and regulating of CD8+T cells. Aditionally, we found that liver LN is critical in delivering anti-HBV immunity. Thus, based on these results, the contribution of NK cells and liver LN for anti-HBV specific CD8+T cells provide a new sight for understanding the mechanisms involved in HBV clearance.