Regulation Of Anti-HBV Effect Of IFN-? By IL-17

ObjectivesIFN-? is one of the main anti-viral drugs for treating chronic hepatitis B.Many host and virus factors are related to its efficacy.Till now,there hasn't been any reports about whether pro-inflammatory cytokine IL-17 could regulate the anti-HBV effect of IFN-? and then affect the clinical efficacy of IFN-?.Therefore,this study intends to explore the effect of IL-17 on the anti-HBV effect of IFN-?,and finally provide some new data to illustrate the poor efficacy of IFN-? in some CHB patients.Methods1.A cell model was constructed by transfecting pcDNA3.1-HBV1.3 plasmid into HepG2 cells(HepG2-HBV1.3).In order to compare the effects of different concentrations of IL-17 on the anti-HBV effect of IFN-?,HepG2-HBV1.3 cells were pretreated with IL-17 at 0 ng/ml,0.2 ng/ml,1 ng/ml,25 ng/ml,50 ng/ml,or 100 ng/ml for 24 h,followed by 1000 IU/mL IFN-? for 48 h.HBsAg and HBeAg levels in the cell culture supernatants were detected by ELISA,expression of HBsAg and HBcAg protein were detected by western blotting,and HBV-DNA,HBsAg or HBcAg mRNA levels were detected by q RT-PCR.2.In order to analysis the effect of IL-17 on anti-HBV effect of IFN-? at different pre-treatment time,HepG2-HBV1.3 cells were pretreated with 50 ng/ml IL-17 for 6 h,12 h,24 h,48 h,and followed by 1000 IU/mL IFN-? for 48 h.HBsAg and HBeAg levels in the cell culture supernatants were detected by ELISA,expression of HBsAg and HBcAg protein were detected by western blotting,and HBV-DNA,HBs Ag or HBcAg mRNA levels were detected by q RT-PCR.3.In order to clarify the possible mechanism of IL-17 affecting the antiviral effects of IFN-?,HepG2-HBV1.3 cells were pretreated with 50 ng/ml IL-17 for 24 h and then treated with 1000 IU/mL IFN-? for 48 h.Expression of p-STAT1,p-STAT2 and IRF9 protein were detected by western blotting.Expression of ISG15,MX1,ISG20,IFI16,IRF9,SCOS1 and USP18 mRNA was detected by qRT-PCR.4.The experimental data were showed as the mean ± standard deviation.Statistical analysis was performed by SPSS 23.0 software.One-way analysis of variance(ANOVA)was used to compare multiple groups.P < 0.05 was considered statistically significant.Results1.Compared with control HepG2-HBV1.3 cells group,when treated with 1000 IU/mL IFN-? for 48 h or 72 h,cells showed significantly lower levels of HBsAg and HBeAg in culture supernatants,and lower expression of cellular HBsAg or HBcAg mRNA(P values were all <0.05).But there was no significant change in levels of HBsAg and HBeAg in cell supernatant when treated with IL-17 at different concentrations,.2.Compared with HepG2-HBV1.3 cells treated with 1000 IU/mL IFN-? alone for 48 h,when pre-treated with IL-17 at relatively high concentration(25,50 or 100 ng/ml)for 24 h,cells displayed significantly higherlevel of HBsAg,HBeAg or HBV-DNA in culture supernatants,and higher expression of cellular HBsAg,HBcAg mRNA and protein(P values were all <0.05).At the same time,HepG2-HBV1.3 cells pretreated with 50ng/ml IL-17 for 12 h or 24 h had significantly higher levels of HBsAg,HBeAg and HBV-DNA in the cell supernatant,and higher expresssion of cellular HBsAg and HBcAg mRNA or protein(P values were all <0.05).3.Compared with HepG2-HBV1.3 cells treated with 1000 IU/mL IFN-? alone for 48 h,when pre-treated with IL-17 at r25,50 for 24 h,cells displayed significantly lower level of phosphorylated STAT1 protein,phosphorylated STAT2 protein and IRF9 protein(P values were all <0.05).At the same time,Hep G2-HBV1.3 cells pretreated with 50 ng/ml IL-17 for 24 h had significantly lower mRNA expression of ISG15,ISG20,MX1,IFI16 or IRF9,but higher expression of USP18?SCOS1 mRNA(P values were all <0.05).Conclusions1.At certain concentration,IL-17 could significantly attenuate theanti-HBV effect of IFN-?2.This effect of IL-17 might be induced by: IL-17 could reduce the level of p-STAT1,p-STAT2 and IRF9,further decrease the expression of the interferon-stimulating genes ISG15,ISG20,MX1,,and finally weaken the anti-HBV effect of IFN-?.At the same time,IL-17 could increase the expression of IFN-? signaling pathway inhibitory factors USP18 and SCOS1 and then inhibit the antiviral effect of IFN-?.