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HBV core and nuclear antigen can enhance the maturition of human dendritic cells and the secretion of IFN-and IL-12p70 Keywords: dendritic cell; HBcAg, HBeAg Abstract:Background Hepatitis B virus（HBV） can induce severe liver damage and lead to hepatic cirrhosis or hepatoma. Monocyte-derived dendritic cells (MoDCs) are a promising cellular adjuvant for effector immune responses against tumors and chronic viral infections, including HBV. Several factors can induce DC maturation and activation including microorganisms, bacterial and viral products and cytokines. As everyone knows, HBV core and nuclear antigen(HBcAg and HBeAg) are the most important antigens of HBV. We thus endeavored to analyze the ability of these two antigens to enhance the maturition of human DCs and the secretion of IFN-and IL-12p70 by activated DCs, and to evaluate the influence of HBcAg and HBeAg on Human DCs in the anti-HBV pathoimmunological mechanisms.Methods The DC populations were generated from peripheral blood mononuclear cells(PBMCs) derived from healthy donors and then pulsed with HBcAg and HBeAg respectively.Cell surface molecules, including CD83, CD86 and HLA-ABC, were analyzed with flow cytometry. Concentrations of IFN-,IL-12p70 in the supernatant were assayed by ELISA methods.Results Upregulation of maturation-associated phenotypic markers（human leucocyte antigen HLA-ABC, CD83, CD86）on Maturity Dendritic Cell(mDC) after 72 h HBcAg/HBeAg pulsing was observed. The lymphocyte proliferation, the cytotoxic activity,as well as the IFN-and IL-12p70 secretion, were observed more sronger in the HBcAg and HBeAg pulsed DCs than in the mDCs non-stimulated. And, HBcAg is more powerful than HBeAg.Conclusion HBcAg and HBeAg can enhance the maturation and secretion of human dendritic cells,and can improve lympholeukocyte multiplication and cytotoxic T lymphocyte activity.Hepatitis B virus(HBV) is a global public health problem. Among the approximately 2 billion people who have been infected worldwide, more than 400 million are chronic carriers of HBV. Considerable numbers of chronic HBV carriers suffer from progressive liver diseases. HBV infection possibly lead to liver cirrhosis and hepatoma. In addition, all HBV carriers are permanent source of this virus. Until nowdays no curative therapy for chronic HBV carriers can be achieved. Antiviral drugs are recommended for about 10% patients, however, these drugs are costly, have limited efficacy, and possess considerable side effects. Recent studies have shown that immune responses of the host to the HBV are critically involved at every stage of chronic HBV infection1. A dendritic cell-based therapeutic vaccine has been developed for treating chronic HBV infection2.Monocyte-derived dendritic cells (MoDCs) are a promising cellular adjuvant for effector immune responses against tumours and chronic viral infections, including HBV3.They are distributed widely in non-lymphoid tissues,where they exist in an immature state. When they are exposed to pathogens or inflammatory stimuli, DCs mature,up-regulate co-stimulatory molecules and acquire the capacity to secrete immunomodulatory cytokines, such as IFN- and IL-12p70.Several factors may induce DC maturation and activation including microorganisms, bacterial and viral products and cytokines4-7. In this study, we endeavered to observe the influence of HBcAg and HBeAg on DC maturation and the secretion of IFN-, IL-12p70, and to study the pathogenesis mechanism of HBV infection and DC vaccines .METHODSvolunteersTwenty volunteers (12 men and 8 women) received from Aug 2006 to Aug 2007 in our hospital, aged from 19 to 36 years，normal hepatic function, excluding infection of HAV, HBV, HCV, HDV, HEV, HIV, without diabetes mellitus and other chonic diseases. The protocol of this research was approved by the ethics committee of our hospital.MaterialsRecombinate human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), recombinant human interleukin(rhIL)-4, human tumor necrosis factor(TNF-) was purchased from Peprotech Co.,USA; fetal bovine serum from Hyclone Co.,USA; RPMI-1640 from Gibco Co.,USA; HBcAg and HBeAg from Genescript Co.,USA;FITC labeled anti-human CD83,FITC labeled anti-human HLA-ABC, FITC labeled anti-human CD86 and their isotype（FITC mouse IgG1）from BD pharmingen Co.,USA; IL-12p70 and IFN-ELISA kit from Biosource Co.,USA. CytoTox 96 Non-Radioactive Cytotoxicity Assay from Promega Co.,USA. HepG2 cells preserved by our research institute.Cell Preparation and CulturePeripheral blood was obtained from normal donors in heparinized Vacutainer tubes.DCs were prepared as described previously. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated by sedimentation through Histopaque and allowed to adhere to tissue culture plastic (24 well cell culture cluster, Corning, USA) for 2 h. The non-adherent cells were removed. The adherent cells were cultured in RPMI-1640 medium supplemented with 15% fetal calf serum,penicillin and streptomycin. The adherent PBMCs were cultured for 7 days in the presence of IL-4 at a concentration of 100ng/ml and granulocyte macrophage-colony stimulating factor (GM-CSF) at a concentration of 100ng/ml. Half nutrient solution were changed and half cytokines were added on days 2, 4 and 6. All DCs used in experiments were at day 7 of culture.mDCs were generated from PBMCs as previously described by culturing adhering cells for 7 days,on day 7, supplemented with 100 ng/mL of TNF- The cells were then separated into three groups. 5g/ml of HBcAg, 5g/ml of HBeAg and RPMI-1640（10l）were added respectively into different group.The DC populations were obtained on day 10. And the supernatants were also harvested and stored at -70C for enzyme-linked immunosorbent assay (ELISA). Measurement of mDC phenotype The dendritic cells were washed twice with PBS containing 1% bovine serum albumin,and then re-suspended into different tubes with 5 105 cells each tube.15 l of FITC labeled anti-human CD83,FITC labeled anti-human HLA-ABC,FITC labeled anti-human CD86 and their isotype（FITC mouse IgG1）were added into different tubes, respectively, blending, and cultured for 30 min at 4C. The expression of cell surface molecules was measured by direct immunofluorescent flow cytometric analysis using a FACS Calibur (BD Immunocytometry Systems) flow cytometer and CellQuest software.Measurement of Cytokine Production HBcAg and HBeAg induced IL-12p70 and IFN- secretion in DCs was determined by adding HBcAg and HBeAg(5g/ml respectively) to the purified DC cultures, followed by the testing of the supernatants at 3 day intervals. Cytokine production was quantified in culture supernatants by specific sandwich ELISAs for IL-12p70 and IFN- according to the manufacturers protocol. The optical density of each well was determined within 30 min using a microplate reader (450 nm).DC-induced lymphocyte proliferation in vitroDCs were generated as previously described. On day 8, 5g/ml of HBcAg, 5g/ml of HBeAg and RPMI-1640（10l）were added respectively into the DC groups, and then co-incubated with autologous lymphocytes in 96-well plates for 5 days, at 37C, 5% CO2 conditions.12 hours before harvesting, 1Ci/hole of 3H-thymidine were added, the supernatants were harvested for the multiplier effect detection using liquid scintillation counter.Lymphocytotoxicity by DC in vitroDCs were generated as above. On the eighth day, the HBcAg pulsed DCs、HBeAg pulsed DCs and mDCs alone were mixed and incubated respectively with autologous lyphocytes at ratio of 1:20 for another 5 days, in the presence of rhIL-2(100 U/mL), as effctors. The effector cells and the target HepG2 cells were then co-cultured for 4 hours at E/T ratio of 40:1, the supernatants were harvested and the cytotoxicity was assayed using CytoTox 96 Non-Radioactive Cytotoxicity Assay kit.Statistical analysis Data were analyzed with software SPSS for Windows 12.0 and expressed as meanstandard deviation (SD). Statistical significance was evaluated by students t-test.Difference was assumed to be statistically significant when P values were less than 0.01.RESULTSHBcAg and HBeAg influence on DCDCs were stained and analysed by flow cytometry. Aggregation and apoptosis of DCs are the most significant events in in HBcAg pulsed DC group after 3-day treatment. Expression of CD83, CD86 and HLA-ABC molecules on the DCs treated with HBcAg and HBeAg is dramatically stronger than the DCs without HBcAg/HBeAg treatment(P0.01). The expression of the above three molecules on the DCs induced with HBcAg is more vigorous than that treated with HBeAg (P0.01).Fig.1 Cellular Morphology of mutural Dendritic Cells. DCs treated with HBcAg（left）,mDCs alone（middle）, DCs treated with HBeAg（right）(Induced 10 Days，400 Magnmcation)Table 1 Expression of molecules on differant groups of DCs (%)Table 2 Expression of IFN- and IL-12p70 in differant groups of DCsTable 3 The percentages of target cell lysis (%)lymphocyte proliferation induced by DCs in vitroHBcAg and HBeAg treated DCs can induce the auto-mixed lymphocytes proliferation：989421cpm and 1032375cpm respectively，higher than 663258cpm in mDCs alone group.508143cpm and 491162cpm respectively of HBcAg and HBeAg treated auto-lymphocytes, higher than 440105cpm of auto-lymphocytes alone(P0.01).Lymphocytotoxicity induced by DCs in vitroThe lymphocytes induced in vitro by DCs stimulated with HBcAg and HBeAg show cytotoxicity to the target HepG2 cells(P0.01)(Table 3).DISCUSSIONDCs are bone marrow-derived cells specialized to present antigens to B and T cells in order to initiate a primary immune response, and its the unique antigen presenting cells(APCs) that can activate naive T cells in vivo. During the maturation of DCs, the ability of antigen capturing and processing decreased while the ability of antigen presenting arose. The immunomodulatory functions of DCs are highly associated with DC mature states. Immature DCs have the high phagocytic capacity and less-express surface adhesion molecules and co-stimulation molecules, thereby can induce T cell anergy or immune tolerance. On the contrary, mature DCs can highly express adhesion molecules,co-stimulation molecules, and massive MHC classand molecules, can present exogenous antigen or autoantigen and activate T cells, thus permitting establishment of immune-inflammatory responses. Thereby, intervention and inhibition of DC maturation or antigen-presenting functions become an important therapeutic intervention approach in immune-inflammatory diseases8-10. But on the other hand, DCs have plasticity and functional specialization in spite of its own bionomics. Microenvironment plays an important role in maintainting DCs function. Many materials demonstrated that both microbial and T cell-derived stimuli could synergize to induce DC expansion and displaying a characteristic mature DC phenotype,and the production of high levels of IL-12p7011,12. Furthermore, inflammatory cytokines such as IL-1 and type I interferon (IFN) released early during an immune response were found to be potent co-factors for CD40L-mediated IL-12p70 production by DCs. If without exogenous antigenic stimulus, DCs keep in quiescent condition. DCs can also differentiate into tolerance DCs by anti-inflammation factors. These DCs can induce T cell to Th2 polarization, and thus lead to immunological unresponsiveness or immunological tolerance, and functional inhibition of DCs and downregulation of some co-stimulation molecules did exist in HBV and HCV patients13-16. It has been reported that mature DCs secrete IL-12p70. IL-12p70 is a key cytokine in some aspects, such as inducing CD4+T cell differentiation to Th1 and the following immune-inflammatory responses. Therefore, scientists hypothesize that IL-12p70 may play a crucial role in Th1 immunological response. To some extent, IL-12p70 has such function as antianaphylaxis and extension of transplanting survival, but its mechanism hasnt been clear17-19.Thefore immunodepressiveness has considerate significance in severe hepatitis. But removement of immunological unresponsiveness or immunological tolerance plays an important role in the treatment of chronic HBV infection20.As an important anti-inflammatory factor, IL-12p70 also palys a role in regulating the major histocompatibility complex (MHC)-II and CD83, CD86. In this study, we found that CD83,CD86 and HLA-ABC (marks of APCs) on the surface of DCs upregulated after HBcAg and HBeAg stimulation. It indicated that HBcAg and HBeAg could induce immature DCs mature and hereby activate T cells. Results also demonstrated that secretion of IL-12p70 by mDCs was much higher after HBcAg and HBeAg stimulation. This proved that the secretion of IL-12p70 responded to the state of activation of mDCs, and excessive activation of mDCs possibly influence on the tissue and serum levels of IL-12p70.In this report, we found that the mDC apoptosis was significantly higher after HBcAg treatment, and the numbers of mDCs dramatically decreased after 3 days of stimulation. We presume that excessive activation of mDCs and expression of IL-12p70 may induce vigorous cellular immune response and humoral immune response, and hence lead to inflammation. In a way, the process of inflammation in the liver or the organism might be abnormal. Therefore, the appropriate selection of suitable adjuvants, antigens and co-response antibodies can initiate or lower the immunological responses in HBV infection, and thereby exert influence on the removement of viruses or mitigate the immuno-inflammatory reaction. 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