Effects Of IFN-α, Entecavir And Thymosin α1 On The Function Of Dendritic Cells Derived From Chronic Hepatitis B Patients In Vitro

Background and ObjectivesHepatitis B virus (HBV) infection is a global public health problem. This infection is endemic in China. Considerable numbers of chronic HBV carriers suffer from progressive liver diseases, even develop serious complications like liver cirrhosis and hepatocellular carcinoma. The immune response of the host plays an important role in the pathogenesis of chronic HBV infection. One of the important mechanisms for chronic HBV infection is immune tolerance and the host can not produce specific cytotoxic T lymphocyte (CTL) immune response to viral antigens, which is critical for complete elimination of virus. One of the important reasons responsive for the immune tolerance in chronic hepatitis B (CHB) is impaired function of dendritic cell (DC) which can not efficiently present HBV antigens to host immune system. Therefore, dendritic cell -based therapeutic vaccine has recently been a potential approach to treat CHB.Dendritic cells are crucial antigen-presenting cells responsible for initiating immunity in naive T lymphocytes and play an important role in the induction of antiviral immune response. Enough peripheral blood monocyte-derived DCs (MoDC) can be generated from the peripheral-blood monocytes induced by granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin 4 (IL-4). CD1a is a characteristic marker of human DC, which mainly expresses in thymus gland cell and DC. CD83 is an important marker of mature DC. CD80 and HLA-DR are importmant costimulatory molecules on DC membrane.Registered antiviral treatments for chronic hepatitis B currently consist of interferon and nucleoside analogues. Besides directly inhabiting the replication ofHBV, IFN-αalso play an indirectly antivirus role by regulating the host's immunesystem. Entecavir (ETV), a new nucleoside analogue, specifically inhibits the hepadnaviral DNA polymerase by competing with the corresponding dNTPs for incorporation into nascent DNA and by acting as a chain terminator after incorporation. Thymosinα1 can enhance host non-specific immunity and is used totreat CHB combined with antivirus drug. The influence of ETV, IFN-αand thymosinα1 on DC of CHB has not been fully investigated.We therefore pulsed DCs derived from peripheral blood monocyte of CHB patients with constrained concentration of ETV, IFN-αand thymosinα1 in virto and observed their effects on DCs phenotype and function. This research provides new support for the application of these medicine and dendritic cell-based immunotherapy in clinical practice of CHB therapy.MethodsPeripheral blood mononuclear cells (PBMC) were collected from heparinized fresh blood of CHB patients and healthy volunteer by centrifuging on a column of Ficoll-Conray in vitro. PBMC were suspended in RPMI 1640 plus 10% fetal bovine serum (FBS) and seeded in 24-well plastic plates for 2h. The nonadherent cells were gently washed out and the adherent cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 10ng/ml rhGM-CSF, 5ng/ml rhIL-4 in incubator with humidified atmosphere and 5%CO₂ at 37°C. Half of the medium in each well was refreshed every 2 day. On the fifth day DCs from CHB patients were treated with or without IFN (300U/ml), ETV (0.05μg /ml) and thymosinα1 (0.1μg /ml) as IFN treated group, ETV treated group, thymosinα1 treated group, IFN+thymosinα1 treated group and CHB control group. DCs from healthy volunteer were as healthy control group without any intervention above. On 8^th day, the following experiments were performed:1. DCs morphology was monitored under inverted microscope.2. DCs surface molecules including HLA-DR, CD80, CD83, CD1a were assayed by flow cytometry.3. The functional capacity of the isolated DCs was checked in allogenic mixed leukocyte reaction (MLR).4. The concentrations of IL-6 and IL-12 in the supernatant were detected by an ELISA method using the commercial kit according to the instruction of the manufacturer.Results1. On 8^th day, more dendritic cell clusters were observed suspending in the medium in healthy control groups. More dendritic cells were also observed in all drug-treated groups. More polygonal cells were observed in CHB control groups. The differentiation of DCs in all drug-treated groups and healthy control groups was better than that in CHB control groups.2. The expressions of surface molecules were different from each group. The expression levels of CD1a, CD80, CD83 and HLA-DR on DC in healthy control group were significantly higher than those in other experimental groups (PO.05). The expression levels of CD1a, CD80, CD83 and HLA-DR on DC in CHB control group were significantly lower than those in other experimental groups. There was no significant difference in the expression level of CD1a, CD80, CD83 and HLA-DR on DC among IFN-α, thymosinα1 and IFN+thymosinα1 treated groups (P>0.05).3. The stimulatory capacity of the drug-treated DC in the allogeneic mixed leukocyte reaction (MLR) was markedly enhanced compared with the DC from CHB control group (P<0.05). There was no significant difference for the stimulatory capacity among IFN-α, thymosinα1 and IFN+thymosinα1 treated groups (P>0.05).4. The secretion of IL-12 was significantly increased in DC culture supernatant in drug-treated groups in comparison with that in CHB control group (P<0 .05), while IL-6 was significantly decreased (P<0. 05).Conclusions1. Dendritic cells can be generated from the peripheral-blood monocytes induced by GM-CSF and IL-4. These dendritic cells can be used for further research.2. Dendritic cells in CHB patient are impaired according to the lower expressions of CD1a, CD80, CD83 and HLA-DR.3. IFN-α, ETV and thymosinα1 can increase the expressions of CD1a, CD80, CD83 and HLA-DR and enhance the stimulatory capacity of DC from CHB patient.4. There is no significant difference among the effect of IFN-α, thymosinα1 and IFN-α+ thymosinα1 on enhancing the function of DC of CHB patient, which is more effective than ETV.