The Role Of ISG15/USP18 Signaling Pathway In Interferon Resistance Of HBV And The Underlying Molecular Mechanism

Background&Aims:As a major world health problem,chronic hepatitis B virus(CHB)infection has a prevalence of about 240 million wordwide,and approximately 20%-30%of those CHB patients may develop into cirrhosisor/and HCC(hepatocellular carcinoma).Despite the emerging viral resistance,type I interferon(IFN)treatment is still one of the most commonly usedantiviral therapies for chronic HBV patients.IFNs are not only used as antiviral treatment but also play important roles in modulating host immune activities.Type I IFN binds to cell surface receptors and activates JAK,which then phosphorylates the STAT family of proteins.Phosphorylated STAT proteins then form homodimer or heterodimers,migrate to the nucleus,and promote the transcription of IFN stimulated genes(ISGs),resulting in the upregulation of a few hundred ISG proteins which function in multiple cellular processes,such as host defense,apoptosis,and immune modulation.Our previous study showed that cell-type specific gene expression signature in liver predicts response to interferon therapy in chronic hepatitis B infection:elevated expression of ISGs,including USP18 and ISG15 which are involved in a same ubiquitin-like signaling pathway,was more pronounced in hepatocytes in treatment non-responders while in Kupffer cells in responders.Our previous analysis of macrophages from USP18-/-mouse showed a predominant M2 cytokines expression compared with a Ml cytokines expression in USP18 wide type macrophages.Moreover,macrophages from ISG 15-/-mouse experienced impaired phagocytosis ability.We therefore hypothesize that ISG15/USP18 signaling pathway may be involved in IFN resistance during HBV infection through both affecting HBV replication and regulating macrophages polarization(phenotype)and function.This study aimed to investigate:1)the effects of ISG15/USP18 signaling pathway on HBV replication;2)the effects of ISG15/USP18 on polarization and function of macrophage and 3)the effects of macrophages with different phenotypes(M1/M2)on type ? IFN signalling pathway.Methods:1.Expression of ISG15 or USP18 was upregulated by ISG15?USP18 or USP18 C64S protease inactive mutant plasmid transfection into HepG2.2.15 cells.HBV replication was evaluated by real-time PCR,western blot,ELISA,etc.2.Expression of ISG15 or USP18 was upregulated by ISG15 or USP18 plasmid transfection into PMA-differentiated MO THP-1 macrophages.Polarization was induced by either IFNy/LPS(for Ml polarization)or IL-4(for M2 polarization)treatment.Real-time PCR,flow cytometry,beads uptake experiment and neutral red uptake experiment were performed to analyze M1/M2 markers(e.g.cytokines,enzymes,surface markers and so on)expression,reactive oxygen species(ROS)production,phgocytosis and pinnocytosis ability,respectively.3.(1)Mouse primary hepatocytes were co-cultured with peritoneal exudate macrophages(PEMs)isolated from USP18-/-or UESP18+/+ mice,respectively.Cultured hepatocytes were stimulated by type ? IFN after 24h,48h or 72h co-culture.Real-time PCR was employed to analyze hepatic ISGs expression and ELISA was used to determin M1/M2 cytokines expression in co-culture medium.(2)Macrophages depletion induced by clodronate liposome and followed by USP18-/-PEMs supplement were performed in wide type mice.Mice were kept overnight and then stimulated by LPS.Twenty-four hours later,mice were injected with type ? IFN and then kept for another 24 hours.Serum was collected for ELISA to determine M1/M2 cytokines expression and liver function evaluation.Liver tissue was collected for real-time PCR to analyze hepatic ISGs expression and HE stain,respectively.Results:1.Overexpression of ISG15,USP18 or USP18 C64S mutant significantly promoted HBV DNA level in HepG2.2.15 cell culture medium in a dose dependent manner,although intracellular total HBV DNA,cccDNA,pgRNA,HBcAg,supernatant HBeAg and HBsAg were not affected?This pro-HBV DNA effect in the culture medium was abolished following inhibition of ISGylation by UBE1L siRNA knock-down.Results from real-time PCR showed that overexpression of ISG15 or USP18 did not affect the expression levels of endogenous IFN? and IFN?,although some ISGs were upregulated at mRNA level.2.M1 macrophage specific markers were upregulated by overexpression of ISG15 or USP18 in PMA-induced M0 THP-1 macrophage,while ROS production,phgocytosis and pinocytosis were inhibited.3.(1)M2 specific cytokines(GM-CSF and IL-10)were more predominant in the medium of USP18-/-PEMs,while M1 specific cytokines(TNF-? and IL-12)were more predominant in the medium of USP18+/+ PEMs.Hepatocytes co-cultured with USP18-/-PEMs experienced significantly higher expression levels of ISGs such as Mxl,ISG15 and USP18 following type ? IFN stimulation,compared to hepatocytes co-cultured with USP18+/+PEMs(2)Wide-type mice with macrophage depletion and replenishment by PEMs from USP18-/-mice(macrophage switch)experienced higher serum M2 cytokines and hepatic ISG expression following IFNa treatment compared with wild type control mice.Liver function analysis showed that ALT,AST and ALP are significantly lower in USP18-/-PEMs switch mice than those in control mice,although HE stain showed no significant inflammation following LPS treatment.Conclusions:1.Upregulation of ISG15 or USP18 promoted HBV production in HepG2.2.15 cells,depending on ISGylation but independent of USP18 protease activity and endogenous type ? IFN signaling pathway.2.Upregulation of ISG15 or USP18 promoted M1 polarization in PMA-induced M0 THP-1 macrophages.ROS production,phagocytosis and pinnocytosis function of these polarized macrophages were inhibited.3.PEMs from USP18-/-mice were predominantly M2 macrophages which enhanced the hepatic response to type ? IFN through regulating micro-environment by cytokine expression.In conclusion,ISG15/USP18 pathway may contribute to HBV resistance to type ?IFN,leading to persistant infection through at least two ways:1)upregulation of ISG15/USP18 in hepatocytes promoted HBV production independent of endogenous typy ? IFN signaling pathway;2)ISG15/USP18 could affect macrophage polarization and function:lower expression levels of ISG15/USP18 is associated with M2-like macrophages which could increase hepatic response to type ? IFN,resulting in over-activation of type ? IFN signaling pathway leading to subsequent poor response to IFN therapy;and higher expression levels of ISG15/USP18 is associated with M1-like macrophages which prevent over-activation of type ? IFN signaling pathway before treatment,making IFN therapy effective.