Activation Of AIM2 By HBV Results In Antiviral Immunity That Suppresses HCV During Coinfection

Background and aimThe hepatitis B virus(HBV)and hepatitis C virus(HCV)infections are a leading cause of chronic hepatitis,fibrosis,cirrhosis,and hepatocarcinoma worldwide.Clinical observations indicated that only 5-10% of adults who contract acute HBV infection become chronic carriers,while most patients who suffer from acute HBV infection recover spontaneously.On the other hand,most patients with acute HCV infection experience chronic persistent infections.However,the mechanisms by which acute HBV and HCV infection lead to spontaneous clearance and chronic persistent infection,respectively,have not been fully elucidated.In addition,because two viruses shared modes of transmission(blood,sexual,and mother-to-child transmission),HBV and HCV frequently coexist in highly endemic areas.Clinical studies have revealed that the two viruses exhibit immunological crosstalk.HCV suppress the replication of HBV genome by inducing the expression of interferon(IFN)and interferon-stimulated genes(ISGs)in primary hepatocytes.In contrast,as a “stealth” virus,HBV infection does not cause strong innate immune activation in hepatocytes.The mechanism by which HBV induces anti-HCV immunity is not yet understood.Studies have shown that HBV does not directly inhibit HCV genome replication in hepatocytes in vitro.Therefore,we hypothesize that the spontaneous clearance of acute HBV infection and the effect of HBV inhibition of HCV replication observed in co-infected patients may be due to indirect effects mediated by host innate and/or adaptive immune responses.In this study,we aimed to investigate the differences between HBV and HCV-induced innate and/or adaptive immune cells,and the mechanism of HBV spontaneous clearance and inhibition of HCV replication.MethodsIn present study,human peripheral blood mononuclear cells(PBMCs)were isolated by density gradient centrifugation,and monocytes and NK cells from PBMCs were purified and depleted by magnetic bead sorting,and fluorescence activated cell sorting(FACS)was used to examine cell purity.Co-culture of healthy individuals’ PBMCs in trans-wells with the HepG2.2.15(HepG2 cells transfected with HBV replicons)and JFH-1 Huh7.5(Huh7.5 cells transfected with HCV replicons)cell lines to mimic HBV and HCV mono-infection in vivo.In addition,HepG2 and Huh7.5 as negative controls,HepG2.2.15 treatment with entecavir and JFH-1 Huh7.5 treatment with telaprevir as antiviral positive controls,respectively.Based on this,different concentrations of cell culture-derived HCV(HCVcc)or HBV(HBVcc)were added to the supernatant to mimic HBV and HCV coinfection in vivo.Real-time PCR(qPCR or qRT-PCR),Western blot,immunofluorescence(IF)and FACS were used to investigate the effects of HBV and HCV-induced PBMCs and NK cells on viral genome replication and expression of viral encoded proteins in HepG2.2.15 or JFH-1 Huh7.5 cell lines.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of PBMCs,NK cells and monocytes secret cytokines(IFN-γ,IFN-α and IL-18)induced by HBV and HCV.t-SNE and FACS were used to identify the cellular subsets that secrete IFN-γ in PBMCs.Western blot and ELISA were used to detect the effects of HBV and HCV on AIM2 and NLRP3 inflammasomes activation.IF,FACS,and fluorescence in situ hybridization(FISH)were used to examine whether monocytes could phagocytic HBV and HCV and the mechanism of releasing ds DNA after phagocytosis.The interaction between the AIM2 or NLRP3 recombinant proteins and different length HBV ds DNA and HCV RNA was detected using Immunoprecipitation(IP),DNA pulldown,GST pulldown and electrophoretic mobility shift assay(EMSA).Finally,the effect of HBV in inducing AIM2 inflammasome activation was validated using re-analyzed single-cell RNA sequencing data from HBV patients and clinical samples from patients with chronic HBV and HCV mono-infection and co-infection.ResultsThe co-culture of PBMCs with HepG2.2.15 or HBVcc resulted in antiviral effects,which inhibit HBV replication as well as HCV replication,whereas JFH-1 Huh7.5 cells or HCVcc did not induce such effects.Mechanically,the inhibition of HBV and HCV replication was triggered by the IFN-γ production of NK cells,which was activated by IL-18 secreted from monocytes.Furthermore,both HBV and HCV activated the NF-κB pathway,resulting in Pro-IL-18 expression in monocytes.However,only HBV ds DNA released via lysosome after being phagocytosed by monocytes activated the AIM2 inflammasome,which led to Caspase-1 activation,cleavage,and secretion of IL-18.These findings suggest that the HBV,but bot HCV,activated AIM2 inflammasome signaling cascade ultimately led to the synthesis of IL-18 by monocytes,which then induced IFN-γ production by NK cells to suppress viral replication.Finally,we confirmed the above results in clinical samples from patients with chronic HBV and HCV mono-infection and co-infection and single-cell RNA sequencing data from HBV infected patients.ConclusionOur findings provide a new mechanism for HBV to induce antiviral responses in HBV mono-infected and HBV/HCV co-infected individuals,which is through the activation of the AIM2 inflammasome in monocytes by HBV,inducing NK cells to secrete IFN-γ and exert antiviral activity.Our study provides a new insight into antiviral strategies for patients with chronic HBV and HCV co-infection.