| ?BACKGROUND?Hepatitis B virus(HBV)infection,with approximately 400 million chronic cases worldwide,is a global health problem,with China being one of the high prevalence countries Hepatitis B surface antigen(HBs Ag)loss is considered as the ultimate endpoint of therapy for chronic hepatitis B(CHB).Pegylated interferon alfa-2a or nucleos(t)ide analogue(NUC)treatments are currently available in clinical practice.In our study,we found HBs Ag loss and seroconversion rates are higher in pegylate interferon-alpha-2a(PEG-IFN?-2a)treatment when compared to nucleos(t)ide analogue therapy,in which therole of natural killer(NK)cell has not been fully elucidated.?OBJECTIVE?The objective of this study is to investigate the role of NK cells in the decline and clearance of HBs Ag in entecavir suppressed patients when switching to Peg IFN,and to explore possible novel immunological mechanisms involved.?METHODS?1?Patients enrollment and charateristis.CHB patients who had received ETV for 9–36months,with HBe Ag <100 PEIU/ml and HBV DNA < 1000 copies/ml,were randomized1:1 to receive ETV 0.5 mg/day for 48 weeks B Cohort)or peginterferon alfa-2a 180?g/week(A Cohort).2?CD56 bright natural killer cell and receptors(activation receptors NKP30,NKP46,NKG2 C and NKG2 D,inhibition receptor NKG2A),were evaluated dynamically in 55 patients by flow cytometry at week 4,12,24 and 48.Viral responder was defined as any decline of HBs Ag levels and/or seroconversion.The absolute number and rate of CD56 bright natural killer cells in NK cells and those receptors were analysed between viral responder and non-responder patients.NK cells were analyzed for the expression of tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)in 15 patients from Peg IFN alfa by flow cytometry3?NK cells were analyzed for the release of TNF-alpha and IFN-gamma in 15 patients from Peg IFN alfa and the ETV group individually by flow cytometry as intracellularcytokine,4?In vitro,NK cells were prepared from PBMCs of 4 Peg IFN treated patients and 4matched ETV treated patients by immunomagnetic beads(MACS).MACS-sorted natural killer cells were cocultured with Hep AD38 cell lines for one week,HBs Ag and ccc DNAlevels were measured at 1 day,2days and 3 days.5?The expressions of APOBEC3 A an APOBEC3 B in Hep AD38 cell after co-culturing were measured by Western Blot.APOBEC3 A an APOBEC3 B in Hep AD38 cell,which could be blocked by anti-IFN? and anti-TNF-?.?RESULTS?1?HBs Ag loss and seroconversion rates are higher in PEG-IFN?-2a treatment when compared to nucleos(t)ide analogue therapy,HBs Ag levels significantly decline at week 24 and week 48(P=0.0175 and 0.0143,respectively)in patients who switched to peginterferon alfa-2a vs.those who continued ETV.Peg IFN alfa responders exhibited a significant decline in HBs Ag levels at week 24 and 48(P=0.033 and <0.001,respectively).2?Accordingly the number of CD56 bright NK cell significantly elevated at week 24 and 48(P< 0.001 and P= 0.0010,respectively),the number of CD56 dim NK cell significantly decreased at week 24 and 48(P< 0.05),Moreover,Compared with Peg IFN alfa non-responders,Peg IFN alfa responders exhibited a significant increase in CD56 bright natural killer cell at week 12 and week 24(P=0.036 0.0039 and 0.004,respectively)and the number of CD56 dim NK cell significantly decreased at week 12 and week 24,(P<0.05),there were not significant differences in number and proportions of NK cells between ETV patients and Peg IFNa-2a patients.3?In Peg IFN treated patients,the percentage of activate receptor NKp30,NKp46,but not NKG2 C,NKG2D or inhibitory receptor NKG2 A,significantly increased at week4,week12,week24 and week48,when compared with ETV patients.Compared with Peg IFN non-responders,Peg IFN responders exhibited a significant increase in NKP30 and NKp46+CD56 brightnatural killer cells at week 24(P=0.016 and 0.0173,respectively)4?Compared with ETV treated patients,Peg IFN treated patients had higher TRAIL+expression in CD56 bright NK cells,and TNF-alpha and interferon ? production at week 24.(P< 0.001 P=0.036 and 0.004,respectively).5?In Peg IFN treated patients,percentage of CD56 bright NK cells positively correlated with HBs Ag decline at week 24(P=0.043,R = 0.63).percentage of CD56 dim NK cells negatively correlated with HBs Ag decline at week 24(P=0.043,R = 0.63).percentage of NK cells and NKp30+NK cell were uncorrelated with HBs Ag decline at week 24((P=0.229 R= 0.262,and P=0.504,R=0.163).6?In vitro,NK cells from Peg IFN patients cocultued with Hep AD38 exert significant decline of HBs Ag levels in supernatant when compared the ones from ETV treated patients.This effect was significantly inhibited in vitro when human anti-TRAIL Abs,individually,or in combination with anti-TNF-a,anti-IFN-r Abs?CONCLUSION?1.The number and function of CD56 bright NK cells was recovered during PEG-IFN?treatment in Entecavir suppressed CHB patients.The percentage of CD56 bright NK cells at week 24 positively correlated with HBs Ag decline,suggesting CD56 bright NK cells play important role in the process of HBs Ag clearance.2.An elevation of activation receptors NKp30 and NKp46 in CD56 bright NKs was observed at week 24 in Peg IFN responders.This phenomena suggests early functional restoration of NK cells may be critical for HBs Ag decline and is important for reconstitution of immune function.3.In parallel with early immune restoration at week 24,CD56 bright NK cells from Peg IFN treated patients subsequently exert its effect(HBs Ag decline)via direct TRAIL/TRAIL-R pathway or indirectly APOBEC3A/B pathway through secretion of IFN-r and TNF-a. |
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