Bile Acids Inhibit Response To Interferon-? Therapy In Patients With Chronic Hepatitis B And Its Mechanism

Background and AimsPegylated interferon-alpha(Peg IFN?)therapy has limited effectiveness in hepatitis B e antigen(HBe Ag)-positive chronic hepatitis B(CHB)patients.However,the mechanism behind this failure has not yet been fully elucidated.Previous research has demonstrated that bile acids(BAs)promote hepatitis B virus(HBV)replication and inhibit IFN-?activity in cell models,but there is little relevant evidence from clinical data.Therefore,we aimed to investigate the influence of BAs on the response to Peg-IFN-?therapy in CHB patients and its regulatory mechanism.MethodsWe used an ultra-high-performance liquid chromatography/tandem mass spectrometry(UPLC-MS/MS)system to measure serum BA composition in 20healthy controls(HCs)and 110 patients with chronic HBV infection,including 18HBe Ag-positive sustained response(SR)CHB patients and 19 HBe Ag-positive non-response(NR)CHB patients receiving Peg-IFN-?therapy.We used the correlation analysis to investigate the correlation between BAs and HBV markers.We dynamically monitored the rate of HBe Ag and HBs Ag loss throughout the course of treatment to evaluate the influence of BAs on the response to Peg-IFN-?therapy in HBe Ag-positive CHB patients.We used flow cytometry to analyze peripheral blood lymphocyte subsets in 74patients with chronic HBV infection,including 49 patients with low serum TBA levels and 25 patients with high serum TBA levels.To further assess the effector functions of CD3~+CD8~+T and natural killer(NK)cells,we used flow cytometry to characterized the markers associated with cytokine(interferon-?[IFN-?]and tumor necrosis factor-?[TNF-?])and degranulation(granzyme B and perforin)production in 50 patients with chronic HBV infection,including 30 patients with low serum TBA levels and 20 patients with high serum TBA levels.To explore the effect of taurocholic acid(TCA)on the effector functions of CD3~+CD8~+T and NK cells in vitro,after freshly isolated peripheral blood mononuclear cells(PBMCs)were stimulated with TCA,we used flow cytometry to detect the proportion of CD3~+CD8~+T and NK cells,and the expression levels of cytokines and cytolytic granules secreted by CD3~+CD8~+T and NK cells.To further evaluate the influence of TCA on the immune effector functions of CD3~+CD8~+T and NK cells in vivo,we used flow cytometry to examine the frequency and effector functions of CD3~+CD8~+T and NK cells in peripheral blood of C57BL/6 mice(carrying r AAV8-1.3HBV)after 2 weeks of TCA gavage.To determine the effect of TCA on the immunomodulatory activity of IFN-?,after freshly isolated PBMCs were stimulated with IFN-?or TCA plus IFN-?,we used flow cytometry to measure the expression levels of cytokines and cytolytic granules secreted by CD3~+CD8~+T and NK cells.In order to investigate the effect of BAs on HBV replication,after cells were stimulated with BAs or IFN-?,we used the corresponding kits to detect the levels of supernatant HBs Ag,HBe Ag and HBV DNA in HepG2.2.15 and HepAD38 cells.To reveal the influence of BAs on the interferon signaling pathway,the phosphorylation of signal transducer and activator of transcription(STAT)and the expression of antiviral protein were validated by real-time fluorescence quantitative PCR(qRT-PCR)and western blot.ResultsSerum BA profile di ered significantly across the natural history of chronic HBV infection.Serum total bile acids(TBA)were significantly elevated in HBe Ag-positive CHB patients compared with HCs and patients in other phases of chronic HBV infection.Among these,only TCA,glycochenodeoxycholic acid(GCDCA)and glycocholic acid(GCA)were significantly elevated in HBe Ag-positive CHB patients compared with HCs and patients in other phases of chronic HBV infection.In particular,amongst the BAs,TCA levels most clearly differentiated between HBe Ag-positive CHB patients and HCs.TCA was correlated with HBV markers in chronic HBV infection.Moreover,serum BAs,particularly TCA,inhibited the response to Peg IFN?therapy in HBe Ag-positive CHB patients.In chronic HBV infection,we found that the high TBA group had a lower number and proportion of CD3~+CD8~+T and NK cells,and expression levels of IFN-?,TNF-?,granzyme B and perforin in CD3~+CD8~+T and NK cells was reduced in patients with high TBA levels compared with patients with low TBA levels,suggesting that BAs decrease the frequency,number,and effector functions of CD3~+CD8~+T and NK cells in chronic HBV infection.In particular,TCA had the greatest effect on the percentage of CD3~+CD8~+T and NK cells.TCA reduced the frequency and impaired the effector functions of CD3~+CD8~+T and NK cells in vitro and in vivo,and inhibited the immunoregulatory activity of IFN-?in vitro.Most BAs promoted HBV replication in HepG2.2.15 and HepAD38 cells,with chenodeoxycholic acid(CDCA)being the strongest promoter of HBV replication.In addition,CDCA compromised the anti-HBV effect of IFN-?by down-regulation the phosphorylation of STAT1 and STAT2 in HepG2.2.15 cells.ConclusionsTCA inhibits the Peg IFN?therapeutic response by impairing the effector functions of CD3~+CD8~+T and NK cells in HBe Ag-positive CHB patients.CDCA inhibits the anti-HBV effect of IFN-?by down-regulation of the phosphorylation of STAT1 and STAT2.Thus,our results reveal a novel mechanism involved in poor therapeutic response to Peg IFN?and suggest that targeting BAs,especially TCA,could be a promising approach for restoring the immunological functions of IFN-?during CHB treatment.