HBsAg Inhibits IFN-α Production in Plasmacytoid Dendritic Cells through TNF-α and IL-10 Induction in Monocytes

et al. (2012) HBsAg Inhibits IFN-a Production in Plasmacytoid Dendritic Cells through TNF-a and IL-10 Induction in Monocytes. PLoS ONE 7(9): e44900. doi:10.1371/journal.pone.0044900 HBsAg Inhibits IFN-a Production in Plasmacytoid Dendritic Cells through TNF-a and IL-10 Induction in Monocytes Bisheng Shi 0 Guangxu Ren 0 Yunwen Hu 0 Sen Wang 0 Zhanqing Zhang 0 Zhenghong Yuan 0 Volker Thiel, Kantonal Hospital St. Gallen, Switzerland 0 1 Shanghai Public Health Clinical Center, Fudan University , Shanghai , People's Republic of China, 2 Laboratory of Molecular Virology, Shanghai Medical College, Fudan University , Shanghai , People's Republic of China Type I Interferon (IFN) is one of the first lines of defense against viral infection. Plasmacytoid dendritic cells (pDCs) are professional IFN-a-producing cells that play an important role in the antiviral immune response. Previous studies have reported that IFN-a production is impaired in chronic hepatitis B (CHB) patients. However, the mechanisms underlying the impairment in IFN-a production are not fully understood. Here, we report that plasma-derived hepatitis B surface antigen (HBsAg) and HBsAg expressed in CHO cells can significantly inhibit toll like receptor (TLR) 9-mediated Interferon-a (IFN-a) production in peripheral blood mononuclear cells (PBMCs) from healthy donors. Further analysis indicated that monocytes participate in the inhibitory effect of HBsAg on pDCs through the secretion of TNF-a and IL-10. Furthermore, TLR9 expression on pDCs was down-regulated by TNF-a, IL-10 and HBsAg treatment. This down-regulation may partially explain the inhibition of IFN-a production in pDCs. In conclusion, we determined that HBsAg inhibited the production of IFN-a by pDCs through the induction of monocytes that secreted TNF-a and IL-10 and through the down-regulation of TLR9 expression on pDCs. These data may aid in the development of effective antiviral treatments and lead to the immune control of the viral infections. - Funding: This work was supported by the National Program on Key Basic Research Project (2012CB519005), Shanghai Clinical New Frontier Project (SHDC12010106), National Natural Science Foundation of China (81101242) and grants from Shanghai Science and Technology Commission (09ZR1426500). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. More than 350 million people worldwide are chronically infected with hepatitis B virus (HBV), and chronic infection with HBV causes significant morbidity and mortality [1,2]. It is estimated that most neonates and approximately 5% of adults progress to chronic HBV infection following acute infection. Antiviral therapy can suppress HBV, but it cannot eliminate HBV infection entirely. Additionally, interferon-a (IFN-a), which is one of the most commonly used treatment for HBV infection, is only effective in half of all chronic HBV-infected patients who received the IFN-a treatment. Accumulating evidence indicates that an inadequate immune response to HBV is responsible for viral persistence [3,4]. Plasmacytoid dendritic cells (pDCs) contribute to at least 95% of all of the IFN-a production among peripheral blood mononuclear cells (PBMCs). pDCs constitutively express the pattern recognition receptors toll like receptor (TLR) 7 and TLR9 [5,6,7]. TLR ligand stimulation or the detection of pathogen associated molecular patterns (PAMPs) can activate the myeloid differentiation factor 88 (MyD88)-interferon regulatory factor 7 (IRF-7) signaling pathway and result in high amounts of IFN-a secretion by pDCs [8]. Previous reports have indicated that both a reduced frequency and an impaired function of pDCs are observed in CHB patients. Following lamivudine treatment, a reduced HBV DNA level was observed to accompany an increased pDCs amounts. This suggested that HBV is important in the observed deficiency in the function of pDCs [9,10]. However, the mechanism underlying the impairment of pDC function has not been fully elucidated. It has been reported that HBV and the HBV surface antigen can enter dendritic cells, but the cells do not support viral replication [11]. This suggests a possible role of the HBV surface antigen in the impairment of pDC function. HBV surface antigen (HBsAg) is the subviral form of HBV envelope protein, and it contains multiple lipid modifications from the host hepatocyte and o-glycosylation [12,13]. HBsAg consists of a small (S), a middle (M) and a large (L) protein that are all encoded by the same open reading frame (ORF), but translation is initiated at three distinct start codons. It has been reported that HBsAg is found in excess concentrations relative to the concentration of the HBV virion in CHB patients [14]. In some CHB patients, HBsAg concentrations have been reported to be as high as 100 mg/ml. However, there are still conflicting reports regarding the role of HBV and HBsAg in the inhibition of pDC function. Our previous study demonstrated that HBsAg could inhibit the IFN-a production of pDCs through the binding of HBsAg to the inhibitory receptor BDCA-2, thus leading to the down-regulation of IRF-7 nuclear translocation [15]. Woltman and colleagues have reported that HBV and HBsAg could abrogate the CpG-A/TLR9-induced mammalian target of rapamycin (mTOR)-mediated S6 phosphorylation, leading to IRF7 phosphorylation and IFN-a gene transcription. Additionally, HBV/HBsAg was also shown to inhibit the upregulation of co-stimulatory molecules, the production of TNF-a, IP-10 and IL-6 and pDC-induced NK cell function [16]. However, Vincent, I.E et al. have reported that HBV, but not HBsAg, can inhibit IFN-a production and TLR9 expression in pDCs [17]. HBsAg has also been reported to inhibit the TLR4mediated cytokine production and CD80 upregulation in mDCs [18]. Herein, we further demonstrate that HBsAg can inhibit the production of IFN-a by pDCs in an indirect manner. HBsAg induced the secretion of TNF-a and IL-10 in monocytes, and these cytokines down-regulated the expression of TLR9 in pDCs to subsequently inhibit IFN-a production by pDCs. Materials and Methods Blood Donors Five milliliters of EDTA-treated anti-coagulated peripheral blood specimens were collected from each chronic hepatitis B (CHB) patient at the Shanghai Public Health Clinical Center (SHAPHC) of Fudan University. CHB patients that were coinfected with HIV, HCV, HDV or HAV were excluded from this study. Patients PBMCs were isolated on recipient and stored in liquid nitrogen for further analysis. Plasma samples were stored at 280uC until required. Buffy coat from healthy donors were collected from the Shanghai (Red Cross) Blood Center. Ethics Statement This study was reviewed and approved by the Ethics Committee of SHAPHC and all participants were given written informed consent before inclusion in the study. Cells and (...truncated)