Expression Of Type Ⅰ Interferon In Peripheral Blood Monocyte-derived Dendritic Cells(MoDC) After TLR3 Triggered In Patients With Chronic Hepatitis B

ObjectiveTo detect the expression of typeⅠinterferon in monocyte-derived dendritic cells(MoDC) after TLR3 triggered in patients with chronic hepatitis B,and analyze the functional status of DCs in patients with chronic hepatitis B virus(HBV) infection and its roles in the mechanism of persistent infection of HBV.MethodsPeripheral blood mononuclear cells(PBMCs) were isolated from 18 healthy volunteers(Control Group) and 26 chronically HBV-infected patients(CHB Group). Their plasma was isolated.Meanwhile,peripheral blood mononuclear cells were separated using a Ficoll gradient,and CD14~+ cells were enriched by using human CD14 Microbeads.Then dendritic cells(DCs) were induced and proliferated in a culture medium with rhGM-CSF and rhlL-4.DCs were stimulated with different concentration of purified HBV associated antigen(HBsAg,HBeAg and HBcAg) and poly I:C,then the supernatant were collected at 0h,1h,3h,8h,16h,24h and 48h after stimulation.DCs were also collected and processed for further examination of IFN-βmRNA.Along with plasma,the protein level of typeⅠinterferon was examined by ELISA,and the mRNA level of IFN-βwas detected by Real-time PCR.And CCK8 was used to measure the function of DCs from part of the individuals.ResultsThe method of enriching high purity of CD14~+ cells by using human CD14 Microbeads can meet the need of acquiring quantity of DCs and carrying the following study of DCs in vitro.The expression of typeⅠinterferon after HBV associated antigen stimulation wasn't upregulated and there were no significant differences between CHB and Control at every time point after stimulated with every concentration of HBV associated antigen.The level of IFN-αand IFN-βexpression in plasma had no significant difference between Control group and CHB group.IFN-βexpression in supernatant after 8h of Poly I:C stimulation were(38.02±7.52, 49.16±10.42)pg/ml,respectively in CHB patients and Control,and had significant difference between them(t=-4.124,P＜0.05).And the expression of IFN-βin supernatant at 16h after stimulation,were(58.12±6.58,132.37±51.31)pg/ml,and had also significant difference between them(t=-7.330,P＜0.05).And 24h after stimulation,were(34.24±6.74,48.85±19.42)pg/ml,and the difference between them was also significant(t=-3.555,P＜0.05).As to IFN-α,there were no significant differences at every time point after stimulation between CHB group and Control group.And the IFN-βmRNA level of Control group were(0.89±0.27,5.53±1.40), respectively before and after stimulation of poly I:C,which had statistical difference (t=-3.252,P＜0.05).However,there was no difference in CHB patients.The capacity of DCs to stimulate the allogeneic lymphocytes was weakened in patients with chronic hepatitis B at three different DCs/T ratio of 1:10,1:20 and 1:40,and there were statistical differences between CHB and Control Group(P＜0.01).The stimulation index were(1.01±0.11,1.17±0.23),respectively in CHB patients and Control at the ratio of 1:80 and there was no difference between them.At the ratio of 1:40,the stimulation index were(1.08±0.07,1.97±0.32),separately in CHB patients and Control,and had statistical difference(t=8.6476,P＜0.01).And at the ratio of 1:20, were(1.04±0.07,2.67±0.08),and had also statistical difference(t=42.8129,P＜0.01). And at the ratio of 1:10,were(1.05±0.09,3.05±0.02),and the difference was also significant(t=52.9503,P＜0.01).ConclusionsOur results suggested that the expression of IFN is lower in CHB patients than Control after HBV associated antigen stimulation,but has no statistical difference between them.And the leve of IFN is higher after stimulation with Poly I:C than HBV antigen.IFN-βis the main subtype of typeⅠinterferon after TLR3 trigged in MoDC from patients with chronic hepatitis B,and also suggested that the function of DCs to stimulate the allogeneic lymphocytes is weakened in patients with chronic hepatitis B.The peripheral blood monocyte-derived DCs have functional impairment and impaired maturation.The abnormal expression of typeⅠinterferon on both protein and mRNA level may play an important role in impaired maturation of DCs and relate to the persistence of HBV infection.