HBsAg Inhibits IFN-α Production In Plasmacytoid Dendritic Cells Via Inducing TNF-α And IL-10 Production In Monocytes

Worldwide more than 350 million people are chronically infected with Hepatitis B virus (HBV) and chronic hepatitis B causes significant morbidity and mortality. It's estimated that most neonates and about 5% adults develop into chronic HBV infection after acute infection. Antiviral therapy suppresses but does not eliminate chronic HBV infection, and the most commonly used Interferon-a (IFN-a) is effective in only half of all chronic HBV-infected patients.Accumulating evidence shows that inadequate immune responses to HBV are responsible for viral persistence,which has led to the search for immune escape strategies of these viruses, thus therapeutic enhancement of immune responses has been proposed as alternative or supplementary therapy for chronic infection. Plasmacytoid dendritic cells (pDCs) are professional IFN-a producing cells and they contribute to most of the IFN-a production among peripheral blood mononuclear cells (PBMC).Previouse literatures have shown that reduced frequency and impaired function of pDCs in CHB patients and after Lamivudine treatment, reduced HBV DNA level was accompanied with elevated pDCs population, which suggests HBV is important in pDCs deficiency. However, the mechanisms underlying pDCs' impairment are not fully elucidated and our work intends to understand how pDCs interact with the virus, and what the functional consequences are of this interaction.In our study, we first confirmed that the IFN-a production in PBMCs from CHB patients was impaired as compared to healthy donors. Since hepatitis B virus surface antigen (HBsAg) is a kind of noninfectious subviral particles abundant in periphery and liver in HBV infected individuals, it may play some special role in virus-host immune interaction. We examined the IFN-a production in PBMCs and isolated pDCs from healthy donors after HBsAg pretreatment and TLR7, TLR9 ligand stimulation. Results showed that HBsAg inhibited TLR7, TLR9 mediated IFN-a production in PBMCs in a dose dependent manner, but not in isolated pDCs. Since monocyte are one of the major cells and antigen presenting cells in PBMCs, we propose that HBsAg indirectly inhibits pDCs IFN-a production through interaction with other cells, such as monocytes in PBMCs. We isolated monocytes and co-cultured them with pDCs in different ratio.Results showed that HBsAg inhibited pDCs IFN-a production in the existence of certain amount of monocytes, and the inhibition became more significant as more monocytes were co-cultured with pDCs.Based on the above results, further studies were carried out to investigate how monocytes act on pDCs:via cell-to-cell contact or via cytokine secretion. Transwell assay was performed and the two cells were separated with a transwell chamber. Results showed that HBsAg inhibited IFN-a production either when pDCs co-cultured or separated with monocytes, indicating that cytokines secreted by monocytes played the inhibitory role in pDCs. Tumor necrosis factor-a (TNF-a) and Interleukin-10 (IL-10) are important cytokines secreted by monocytes and they have been reported to inhibit IFN-a production, so we further examined the two cytokines level in PBMCs, pDCs and the monocytes-pDCs co-cultured supernatant. Results showed that TNF-a and IL-10 level were both up-regulated in HBsAg treated PBMCs and the two cells co-cultured system but not in single pDCs. Besides, no significant up-regulation in the two cytokines was observed in the monocyte depleted PBMCs. Addition of recombinant TNF-a and IL-10 confirmed that the two cytokines played inhibitory role in IFN-a production by pDCs.Antibody neutralization assyas further support the role of HBsAg induced cytokines from monocytes in the inhibition to IFN-a production. Besides, TNF-a and IL-10 down-regulating TLR9 expression on pDCs might contribute to IFN-a inhibition.In conclusion, we found that HBsAg inhibited pDCs IFN-a production through inducing monocytes secreting TNF-a and IL-10.Our work helps to understand how HBV establish its persistent infection and the lack of strong antiviral immunity as observed in CHB patients, which may eventually aid in developing effective antiviral treatment leading to immune control of the viruses.