The Role Of TLR2 In HBV Persistence And Memory NK Cell During HBV Infection

The liver, receiving blood from the intestine which contains abundant food antigens, is a unique lymphoid organ, as it usually induces immune tolerance rather than immunity to those antigens. Tolerance to food antigens can enable the body to reduce unnecessary immune response and protect hosts from liver inflammation caused by immune attack to foreign antigens. But the feature of liver is a double-sword, because hepatotrophic pathogens such as hepatitis B virus (HBV), hepatitis C virus (HCV) tend to establish chronic infections due to the liver immune tolerance, which are an initiator leading to liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). So it is impotant to further study the mechanism of liver immune tolerance and how to break liver immune tolerance during chronic virus infection.CD8+T cells are major players in HBV clearance, but the function of CD8+T cells in HBV-carrier humans and mice is seriously impaired, with decreased activating receptor, increased inhibitory receptor and lower production of the anti-viral cytokines IFN-γ and TNF-α, a status known as exhaustion. And the exhausted CD8+T cells can not exercise its antiviral effect, resulting in chronic virus infection. Previous studies have shown that regulatory T cells (Tregs), γδT cells and myeloid derived suppressor cells (MDSCs) inhibit HBV-specific CD8+T cell function and that deletion of these cells enhances the anti-viral function of CD8+T cells and HBV clearance. We study deeply the role of TLR2, an important compotent of innate immunity, in supporting HB V-mediated CD8+T cell exhaustion.In the process of pathogens infection and clearance, memory cells to these specific pathogens produced. And memory cells expanded robustly and exerted their function to wipe out pathogens when re-encountered the same pathogen. It is commonly believed that T lymphocytes and B lymphocytes have memory capacity. NK cells have traditionally been classified as cells of the innate immune system, kill pathogens during early infection and are not considered to have memory activity. But recent studies suggested that NK cells also showed adaptive immune feature and have memory-like functions. More importantly, these memory-like NK cells can protect the hosts from death caused by the same pathogen re-infection. So, we also concern whether there were memory NK cells in HBV-cleared hosts and whether memory NK cells could protect the hosts from HBV persistence induced by chronic HBV infection.I will describe the results obtained in this study from two aspects:I. TLR2 mediates HBV persistence-induced CD8+T cell exhaustion1. TLR2 deficiency improves HBV eliminationWe used an HBV-carrier mouse model in which the pAAV/HBV1.2 plasmid was hydrodynamically injected into the mouse to mimic chronic HBV infection. Using the mouse model, we found that TLR2 deficiency can improve HBV elimination and TLR2 activation by its agonist Pam3CSK4 injection resulted in more stable HBV persistence, as the concentration of HBsAg in serum was higher in mice treated with Pam3CSK4 than in mice treated with saline.2. Anti-HBV CD8+T cells are not exhausted in TLR2-/- miceCD8+T cells in HBV-carrier mice are in an exhausted state. Importantly, elevated numbers of CD8+T cells were found in the liver of HBV-carrier TLR2-/- mice compared with HBV-carrier WT mice. In addition, the percentage of HBV-specific cytotoxic T lymphocytes (CTLs) was elevated in HBV-carrier TLR2-/- mice and HBV-specific CTLs expressed less PD-1 in HBV-carrier TLR2-/- mice. Hepatic CD8+T cells showed increased CD44 and CD69 expression and decreased PD-1 and Tim-3 expression in HBV-carrier TLR2-/- mic. Furthermore, we found greatly increased IFN-y and TNF-a production in hepatic CD8+T cells from HBV-carrier TLR2-/- mice compared with HBV-carrier WT mice. More importantly, CD8-/- mice treated with CD8+T cells from HBV-carrier TLR2-/- mice had clearly lower HBsAg levels after HBV plasmid injection, proving that the enhanced CD8+T cells were sufficient to promote HBV elimination. So our results suggested that TLR2 deficiency can reverse CD8+T cell exhaustion induced by HBV persistence.3. KCs induce CD8+T cells exhaustion in a TLR2-dependent mannerWe found that KCs from HBV-carrier mice expressed much more TLR2 than those from control mice. Similar to TLR2 deficiency, KC depletion resulted in significantly reduced serum HBsAg in HBV-carrier WT mice, and the difference between HBV- carrier WT mice and HBV-carrier TLR2-/- mice in serum HBsAg levels did not exist after KCs depletion, indicating that KC cells may exert their tolerance function via TLR2. Furthermore, consistent with HBV-carrier TLR2-/- mice, CD8+T cells had stronger anti-HBV ability in KC-depleted HBV-carrier mice. Taking these results together, the inhibitory function of KCs on CD8+ T cells depends on their TLR2 in HBV-carrier mice.4. IL-10 from HBcAg-responsive KCs drives CD8+T cell exhaustionKCs become more inhibitive by TLR2 up-regulation after HBV injection. Moreover, KCs from WT mice produced abundant inhibitory cytokine IL-10 after HBcAg stimulation in a TLR2-dependent manner. And elevated IL-10 directly inhibits CD8+T cell function, because IL-10-/- HBV carrier mice had more powerful CD8+T cells with stronger anti-viral function. In addition, the percent of HBV-specific CTLs was up-regulated in IL-10-/- HBV-carrier mice. Importantly, the secretion of IFN-y by CD8+T cells could be directly inhibited by HBV-carrier mice-derived KCs in an IL-10 dependent manner. Taken together, KCs-derived IL-10 upon interaction of TLR2 with HBcAg could lead to CD8+T cell dysfunction in HBV-carrier mice.Conclusion Ⅰ:TLR2 expression on KCs was up-regulated in HBV-carrier mice. And KCs produced more IL-10 upon TLR2 activation in response to direct HBcAg stimulation, and the elevated IL-10 inhibited CD8+T cells function in HBV-carrier mice, resulting in HBV persistence.Ⅱ. Study of memory NK cell in HBV-clearance mouse model1. NK cells are activated during HBVclearanceWe used an HBV-clearance mouse model in which high dose pAAV/HBV1.2 plasmid was hydrodynamically injected into the mouse to mimic HBV clearance. We found that after HBV plasmid injection, the percent of NK cells was similar with that in control mice but the percent of activated NK cells was up-regulated.2. High dose HBV plasmid injection promotes NK cell proliferation and enhances expression of memory cell-related molecule on NK cellsWe found that after HBV plasmid injection, the percent of proliferating NK cell increased quickly, and then reduced and returned to original level as time went by. In addition, we observed similar variation tendency of KLRG1 expression on NK cells, which was consistent with phenotype of memory NK cell in previous reports.3. Transfer of lymphocytes from high dose HBV plasmid sensitized mice can protect the recipients from HBV persistence induced by low dose HBV plasmid injectionHepatic lymphocytes from C57BL/6 mice hydrodynamically injected with 20μg pAAV/HBV1.2 or control plasmid were transferred into semi lethal irradiated C57BL/6 mice. The recipients were hydrodynamically injected with 6μg pAAV/HBV1.2 plasmid 1 day after cell transfer. We found that lymphocytes from HBV plasmid injected mice could help the recipients eliminate HBV plasmid.4. Transfer of NK cells from high dose HBV plasmid sensitized mice may not protect the recipients from HBV persistence induced by low dose HBV plasmid injectionHepatic NK cells from C57BL/6 mice hydrodynamically injected with 20μg pAAV/HBV1.2 or control plasmid were transferred into Rag-/- yc-/- mice. The recipients were hydrodynamically injected with 6μg pAAV/HBV1.2 plasmid 1 day after cell transfer. We found that NK cells from HBV plasmid injected mice could not help the recipients eliminate HBV plasmid.Conclusion Ⅱ:We observed memory-like NK cells in HBV-cleared mice but transfer of these NK cells could not help recipients eliminate low dose HBV plasmid.