Interaction Between Interleukin-35 And Hepatitis B Virus Infection And Its Antiviral Mechanism

Backgrouds: Chronic hepatitis B(CHB)is an infectious disease that poses a serious threat to human health.It is particularly important for antiviral treatment of CHB patients to suppress HBV replication and delay the progression of liver pathological process.As we know that interferons(common interferon and pegylated interferon)and nucleoside(acid)analogues(lamivudine,telbivudine,adefovir,entecavir,tenofovir,etc.)are clinical first-line drugs for CHB.However,the resistance of HBV to interferon and nucleoside(acid)analogue resistance have affected the antiviral efficacy.Therefore,there is an urgent need to find more effective antiviral agents.It is well established that several cytokines of immune system,including IFN,IL-12,IL-15,IL-29,IL-27 and IL-32γ,exert anti-HBV activities.Among them,IL-35,which belongs to the IL-12 family,is a newly discovered cytokine composed of two heterologous subunits,IL-27 β chain EBI3 and IL-12 α chain P35.In several animal models of autoimmune diseases,it is found that IL-35 could reduce inflammation and then alleviate disease symptoms.Moreover,recent studies showed that IL-35 exerts a broad spectrum of antiviral activity through promoting cells secretion of interferon,including influenza A virus,human enterovirus 71,and vesicular stomatitis virus,etc.Also,our previous study found that IL-35 inhibits HBV replication and antigen secretion in vitro,indicating that IL-6 probably have potential anti-HBV activity.Objective: To investigate the possible relationship between HBV infection and IL-35 expression and to explore the role for IL-35 in antiviral treatment as well as to reveal the mechanism by which IL-35 affects viral replication and IFN-α antiviral activity.Methods: 169 patients with untreated chronic HBV infection and 54 healthy subjects were enrolled in the study.Serum IL-35 levels were analyzed by ELISA.The expression of IL-35 in liver tissues were analyzed by immunohistochemistry.During antiviral treatment,the changes in serum levels of IL-2,IFN-γ,TNF-β,IL-5,IL-10,IL-6 and IL-35 were determined.The patients were divided into sustained response(SR)and non-responder(NR)patients,and further analyzed the baseline level and dynamic changes of serum IL-35 in SR patients and NR patients.HepG2,HepG2.2.15,Huh7,LO2 and PLC/PRF/5 cells were used as models to transfect HBV whole gene plasmids pHBV1.3 or HBV-encoded protein expression plasmids(pEGFP-HBs,pEGFP-HBc,pEGFP-HBx and pEGFP-HBp),the mRNA and protein levels of IL-35 were analyzed by real-time PCR and ELISA.The promoter reporter plasmids(pGL3-EBI3-promoter and pGL3-p35-promoter)and HBV-encoded protein expression plasmid were co-transfected into cells that treatment with NF-κB inhibitor SN50,the luciferase activities were measured by photometer.Cells transfected with HBV gene were treated with IL-35 or combined with IFN-α,the levels of intracellular HBV-DNA and supernatant HBV antigen were analyzed by real-time quantitative PCR and ELISA,respectively.After co-transfection with pcDNA3.1/EBI3 or pcDNA3.1/P35 and HBV whole gene plasmids into cells,the levels of intracellular HBV-DNA were determined.Western-blotting and cellular immunofluorescence techniques were used to analyze the expression of IFN-α-JAK-STAT signaling pathway and the ISRE activity.pGL3-IL-6-promoter was co-transfected into hepatocytes with HBV whole gene plasmid,and luciferase activity was analyzed after IL-35 treatment.Then,the supernatants were assayed for IL-6 after cells treated with siRNA interference(siRNA-STAT1,siRNA-STAT2 and siRNA-IRF-9).Furthermore,cells treated with IL-6 alone or in combination with IFN-α,and the expression levels of SOCS3 were determined.Finally,the effects of SOCS3 on the expression of antiviral proteins and HBV replication were assessed.Finally,hepatocytes were co-transfected with siRNA-SOCS3 and pHBV1.3 followed by addition of IL-35 to analyze the possible antiviral mechanism of IL-35.Results: Compared to healthy controls,the serum and liver of IL-35 levels were increased in the patients with chronic HBV infection,especially in the patients with HBeAg-negative HBV infection.Other than hepatocytes,immune cells also up-regulated IL-6 expression in liver of the patients.During antiviral treatment,the changes in serum levels of IL-2,IFN-γ,TNF-β,IL-10,IL-6 and IL-35 were obvious.The levels of IL-35 in SR patients were higher than that in NR patients at baseline and 12 weeks after treatment.For predicting IFN-α antiviral efficacy,area under the receiver operating characteristic curve(AUC)of IL-35 levels at baseline and 12-week were 0.74 and 0.85,respectively.The expression of IL-35 was higher in HepG2.2.15 cells than HepG2 cells.Similar results were obtained when transient transfection hepatocytes were substituted for stable transfection hepatocytes.Upregulation of IL-35 expression in hepatocytes was mediated by the HBx/NF-κB pathway.In turn,IL-35 inhibits HBV replication in three type hepatocytes(HepG2,LO2 and PLC/PRF/5 cells).Also,IL-35 and IFN-α were coordinated to regulate HBV.Interestingly,P35 alone also had the effect of inhibiting HBV replication.IL-35 had no significant effect on the expression of JAK-STAT pathway molecules and the activity of ISRE,but down-regulated IL-6 promoter activity to decrease IL-6 secretion.IFN-α induces IL-6 expression in HepG2 and LO2 cells in a time-and concentration-dependent manner,and the addition of exogenous IL-6 inhibits the anti-HBV effect of IFN-α.siRNA-IRF-9 significantly inhibits the expression of IL-6 in hepatocytes treated with IFN-α,while pcDNA3.1/IRF-9 promoted induction of IL-6 by IFN-α.Both IL-6 and IFN-α could induce the expression of SOCS3 in hepatocytes,whereas IL-35 inhibited that and upregulated the expression of MxA,OAS and PKR.Conclusion:(1)HBV infection leads to elevated IL-35 levels in patients with chronic HBV infection.(2)Enhanced IL-35 contribute to anti-HBV efficacy of IFN-α therapy.(3)Upregulation of IL-35 expression in hepatocytes was mediated by the HBx/NF-κB pathway.(4)Both HBV and IFN-α could induce the expression of IL-6 in hepatocytes.Induction of IL-6 by IFN-α was mediated by IRF-9,which is an important molecule of JAK-STAT signaling pathway.(5)IL-35 reduced secretion of IL-6 by inhibiting IL-6/SOCS3 signaling pathway,thereby exerting direct antiviral activity and synergistic antiviral effect with IFN-α.