Therapeutic Recovery Of HBV-induced Hepatocyte-intrinsic Immune Defect Reverses Systemic Adaptive Immune Tolerance

Object:Hepatitis B virus (HBV) infection, with400million carriers worldwide, is a major risk factor for hepatocellular carcinoma (HCC). Although both innate and adaptive immunity are capable of controlling HBV replication in acute infection, HBV persistence impairs innate and adaptive immune responses in chronic HBV infection, leading to cell-intrinsic immunotolerance and systemic immunotolerance, especially CD8+T cell exhaustion.CD8+T cells play a critical role in HBV clearance, especially intrahepatic HBV-specific CD8+T cells. Although HBV-specific CD8+T cell numbers remain low during infection, their cytokines, including IFN-y and TNF-α, are essential for suppressing HBV gene expression and replication. Unfortunately, in CHB patients, CD8+T cells lose their ability to proliferate and mediate antiviral function; this dysfunctional state is characterized by co-inhibitory molecule overexpression (e.g. PD-1, Tim-3, CTLA-4), low cytokine production, and T cell exhaustion.PD-1, as an inhibitory receptor, not only mediate the negative signals by forming a coinhibitory microclusters and directly inhibiting TCR signaling via recruiting phosphatase SHP2, but also promotes T cell apoptosis. PD-L1, the ligand of PD-1, is expressed on broadly tissues and a lot of different cell types, and provides inhibitory signals for T cells. In CHB patients, PD-L1expression is reported to be higher on hepatocytes than that in health donors, which contributes to HBV induced immune tolerance by interaction with PD-1. But how PD-L1was induced under HBV infection state was still not clear.HBV induced immunotolerance is becoming the major obstacle for chronic HBV therapy. So, to achieve effective HBV therapy, there is a pressing need to develop strategies to break cell-intrinsic tolerance and reconstitute adaptive immunity against HBV. One promising strategy to treat CHB infection is simultaneous use of immune stimulation and HBV gene-expression silencing to reduce virus load; recently, bifunctional5’-triphosphate-siRNAs (3p-siRNAs) silenced HBV expression and simultaneously activated the host retinoic acid inducible gene Ⅰ (RIG-I) signaling pathway to successfully reverse hepatocyte-intrinsic immunotolerance.In this study, to achieve effective HBV therapy, we constructed a dual-function shRNA vector, exerting both immunostimulatory and HBx-silencing effects. To explore its effect, an HBV-persistent mouse was established by hydrodynamic injection of an HBV-genome-containing plasmid. This mouse model exhibited not only hepatocyte-intrinsic but also systemic immunotolerance to HBV rechallenge. HBV-specific CD8+T cell and anti-HBs antibody generation were systemically impaired by HBV persistence in hepatocytes. Interestingly, HBV-induced hepatocyte-intrinsic immune tolerance was reversed when the therapeutic dual vector was administered, and the systemic anti-HBV adaptive immune responses, including CD8+T cell and anti-HBs Ab responses, were efficiently recovered. During this process, CD8+T cells and IFN-γ secreted play a critical role in clearance of HBV. However, when IFN-α/β receptor was blocked or TLR7signaling pathway was inhibited, the activation of CD8+T cells and clearance of HBV was significantly impaired.In this HBV-persistent mice model, we also found that HBV could induce PD-L1expression on hepatocytes. Through transfection of predicted miRNA mimics or inhibitor, we found miR-200c mimics could decrease but miR-200c inhibitor could increase PD-L1expression. And the dual luciferase reporter assay showed that miR-200c could directly target3’-UTR of PD-L1. Moreover, miR-200c was found down-regulated in primary hepatocytes from the HBV-persistent mice. And over-expression of miR-200c could reverse the up-regulation of PD-L1and CD8+T cell exhaustion in HBV persistent mice.Methods:On the one hand, we compared the IFN-I and ISGs expression in HepG2.2.15and HepG2; HBV+HCC and HBV-HCC; HBV+and HBV-primary murine hepatocytes, using qPCR and Western blot. Then the percentage and activation of CD8+T cells was detected by FACS. For HBV therapy, a dual-function shRNA vector, exerting both immunostimulatory and HBx-silencing effects, was constructed. And after treatments with the dual-function vector, we detected the IFN-I expression in HepG2.2.15and HepG2; HBV+HCC and HBV-HCC; HBV+and HBV-primary murine hepatocytes, using qPCR. The activation of CD8+T cells and IFN-y producing CD8+T cells were detected by FACS. The serum HBV DNA level was detected by qPCR. And the serum HBsAg and HBeAg was detected by ELISA. To further explore the role of CD8+T cells in HBV inhibition by dual vector, we depleted them from HBV+mice with an anti-CD8β mAb. And we also adoptively transferred splenic CD8+T cells or CD8+T cell-depleted splenic lymphocytes from wild-type mice into HBV+Rag-1-/-mice. Then serum HBV DNA and HBsAg level was detected. We further detected the dual vector-mediated activation of CD8+T cells and inhibition of HBV replication when blocking type I IFN signaling in vivo with a neutralizing antibody against the IFN-α/β receptor or inhibiting TLR7signal activation using the TLR7inhibitor.On the other hand, we compared the PD-L1level on HepG2.2.15and HepG2; HBV+and HBV-primary murine hepatocytes, using FACS. In the mouse model established by hydrodynamic injection of pAAV/HBV1.2or pEGFP-mPD-L1, the PD-L1level in liver tissue was detected by IH, and the activation of CD8+T cells was detected by FACS. To further explore the role of PD-1/PD-L1in HBV induced immunotolerance, the activation of CD8+T cells was detected by FACS when PD-1was blocking by α-PD-1. Then we predicted the microRNAs that target the3’-UTR of PD-L1using miRNAs, and PD-L1on cells transfected with synthetic microRNA mimics or inhibitor was detected. And the dual luciferace reporter assay was used to confirm that miR-200c directly targets3’-UTR of PD-L1. Finally, the activation of CD8+T cells and the HBV DNA level was detected when miR-200c was over-expressed.Result:Part I:Therapeutic recovery of HBV-induced hepatocyte-intrinsic immune defect reverses systemic adaptive immune tolerance 1HBV induces cell-intrinsic and systemic immunotolerance.By qPCR assay, we found that HBV persistence inhibits the expression of type I IFNs and IFN-inducible genes (ISG15and MxA), but induces immunosuppressive cytokines (TGF-(3and IL-10) expression in HepG2.2.15(comparing with HepG2), HBV+HCC (comparing with HBV-HCC) and HBV+primary murine hepatocytes (comparing with HBV-primary murine hepatocytes). And the Western blot assay and ELISA assay also show that HBV persistence inhibits IFNs and IFN-inducible genes production. These results collectively indicate that HBV infection induces hepatocyte-intrinsic innate immunotolerance.In the HBV-persistent mice, we also found that the percentage and absolute number of hepatic CD8+T cellwas reduced, but PD-1on hepatic CD8+T cells was almost3-fold higher than in HBV-mice. And hepatic HBc-specific and IFN-y+CD8+T cells decreased significantly in HBV-persistent mice. And moreover, HBV-persistent mice did not produce anti-HBs Ab when it was inoculated with HBV vaccine (rHBs/CFA). All the results raised the possibility that impairing HBV-induced hepatocyte-intrinsic immune responses leads to systemic adaptive immunotolerance.2A dual-function immunostimulatory HBx-shRNA vector reverses HBV-induced intrinsic immunotolerance.We constructed a dually functional vector containing an immunostimulatory ssRNA and an HBx-gene-silencing shRNA. And the dual vector could induced higher IFN-α, IFN-β, ISG15, and MxA production, but reduced TGF-β and IL-10expression in HepG2.2.15, HBV+HCC and HBV+primary murine hepatocytes. The dual vector also more efficiently inhibited HBV replication and transcription. These results strongly suggest that the ssRNA-HBx-shRNA dual vector powerfully inhibits HBV replication and successfully reverses HBV-induced hepatocyte-intrinsic immunotolerance.3Anti-HBV recall immunity is recovered after reversing hepatocyte-intrinsic tolerance.The dual vector could regain anti-HBs Ab production in the HBV-persistent mice after vaccination. And it also up-regulated the percentage and absolute number of hepatic CD8+T cells and activating CD8+T cells, as well as HBV-specific hepatic CD8+T cells, HBV-specific CD107a+and IFN-y+CD8+T cells. All these indicated that reversing hepatocyte-intrinsic immunotolerance may recover anti-HBV adaptive immunity and reverse the HBV viral persistence.We depleted NK, CD4+T and CD8+T cells from HBV+mice with an anti-CD8β mAb. CD8+T cells, but not CD4+T, are critical for dual-vector-mediated inhibition of HBV replication. Then we adoptively transferred splenic CD8+T cells or CD8+T cell-depleted splenic lymphocytes from wild-type mice into HBV+Rag-1-/-mice. Adoptively transferring CD8+T cells but not non-CD8+T cells or IFN-γ-/-CD8+T cells inhibited HBV replication in HBV-persistent Rag-1-/-mice when dual vector treatment. These results suggest that recovering anti-HBV adaptive immunity by reversing hepatocyte-intrinsic tolerance is at least partially dependent on functional rescue by IFN-y-producing CD8+T cells.4Type I IFN is important for reversing CD8+T cells tolerance in a TLR7-dependent manner.Both blocking type I IFN signaling in vivo with a neutralizing antibody against the IFN-α/β receptor, and inhibiting TLR7signal pathway with the TLR7inhibitor IRS661, could attenuate CD8+T cell activation and HBV inhibition. These data suggest that type I IFN signaling and TLR7is required for recovering CD8+T cell function and HBV clearance after dual vector-reversed hepatocyte-intrinsic tolerance.Part II:HBV induces T cell exhaustion through down-regulation of miR-200c1HBV induced T exhaustion through up-regulating PD-L1Firstly, we compared the PD-L1levels on HepG2, HepG2.2.15and pAAV/HBV1.2transfected HepG2cells, and the FACS data show that the presence of HBV could promote the PD-L1expression on hepatocytes. Then we also found PD-L1on primary hepatocytes was augmented in HBV-persistent mice than that in HBV-negative mice. All these results show that HBV persistence induced PD-L1expression on hepatocytes.Blockage of PD-L1/PD-1signal pathway in HBV persistent mice increased the percentages of CD69+CD8+, and IFN-γ+CD8+T cells. But PD-L1overexpression could decrease the percentages of CD69+CD8+, and IFN-γ+CD8+T cells. These results show that the elevated PD-L1on hepatocytes indeed impaired the activation of CD8+T cells.2miR-200c targets PD-L1at3’-UTRPD-L1was detected by FACS on HepG2.2.15and HepG2which are both transfected with the mimics or inhibitor of predicted miRNAs. And we found miR-200c could down-regulate PD-L1level in a dose-dependent manner. And the dual luciferase assay show that a significant decrease of luciferase activity was detected in cells with miR-200c mimics transfection. Our results indicated that miR200c indeed directly targets PD-L1at the3’-UTR.3HBV persistence induced PD-L1expression through down-regulation of miR200cBy qPCR, we found that HBV and HBs/x could down-regulate miR-200c expression. And moreover, miR-200c overexpression significantly attenuated HBV-induced upregulation of PD-L1expression on hepatocytes in vivo. These data showed that HBV could induce PD-L1expression on hepatocytes through down-regulation of miR-200c.4MiR-200c over-expression reversed CD8+T cell exhaustionmiR-200c over-expression significantly increased the percentages of CD69+CD8+and IFN-γ+CD8+T cells. And it also decreased the apoptosis rates of CD8+T cells. Further， miR-200c over-expression increased the percentages of HBc-specific CD8+T cells, in particular the HBc-specific IFN-γ+CD8+T cells, as well as the CD44+CD127+memory CD8+T cells. These results show that miR-200c overexpression not only increased the percentages of memory and HBV-specific CD8+T cells but also enhanced the function of HBV-specific CD8+T cells.5MiR-200c overexpression efficiently inhibited HBV replication in HBV persistent miceWe have found that the function of CD8+T cells was restored by miR-200c. Then we found that miR-200c overexpression significantly reduce the serum levels of HBV DNAand HBsAg. Similarly, the protein levels of HBsAg and HBcAg in hepatocytes also reduced detected by immunochemistry. These results suggest that miR-200c could restore the function of CD8+T cells and reduce HBV load.Conclusions:1HBV could induce cell-intrinsic and systemic immunotolerance, and finally promoted HBV replication and persistence.2A dual-function immunostimulatory HBx-shRNA vector could recover HBV-induced systemic immunotolerance by reversing HBV-induced intrinsic immunotolerance. And during the dual vector inhibit HBV replication and transcription, CD8+T cells play an important role in TLR7-IFN-I manner.3We firstly construted a TLR7-dependent dual-function immunostimulatory HBx-shRNA vector for HBV therapy. And we are the first to propose that reversing hepatocyte-intrinsic immunotolerance by dually functional immunostimulatory HBx-shRNA therapy can induce the recovery of systemic immunotolerance. This bifunctional therapeutic strategy shows promise for treating other persistent viral infections (such as HCV and HPV) and associated cancers, including HCC.4PD-L1was a target gene of miR-200c. HBV could down-regulate miR-200c expression, leading to up-regulation of PD-L1. And evaluated PD-L1provided inhibitory signal for CD8+T cells and promoted CD8+T cell apoptosis. MiR-200c overexpression could restore the function of CD8+T cells and reduce HBV load. Moreover, Becsuse miR-200c could interrupt PD-1/PD-L1signaling pathway, miR-200c also is a good therapeutic candidate for other viral infection and tumor.