Hepatitis B Virus Lacks Immune Activating Capacity, but Actively Inhibits Plasmacytoid Dendritic Cell Function

but Actively Inhibits Plasmacytoid Dendritic Cell Function. PLoS ONE 6(1): e15324. doi:10.1371/journal.pone.0015324 Hepatitis B Virus Lacks Immune Activating Capacity, but Actively Inhibits Plasmacytoid Dendritic Cell Function Andrea M. Woltman 0 Marjoleine L. Op den Brouw 0 Paula J. Biesta 0 Cui C. Shi 0 Harry L. A. Janssen 0 Heiner Wedemeyer, Hannover Medical School, Germany 0 1 Department of Gastroenterology and Hepatology, Erasmus MC - University Medical Center , Rotterdam , The Netherlands , 2 Department of Infectious Disease, Rujin Hospital , Shanghai , China Chronic hepatitis B virus (HBV) infection is caused by inadequate anti-viral immunity. Activation of plasmacytoid dendritic cells (pDC) leading to IFNa production is important for effective anti-viral immunity. Hepatitis B virus (HBV) infection lacks IFNa induction in animal models and patients and chronic HBV patients display impaired IFNa production by pDC. Therefore, HBV and HBV-derived proteins were examined for their effect on human pDC in vitro. In addition, the in vitro findings were compared to the function of pDC derived from chronic HBV patients ex vivo. In contrast to other viruses, HBV did not activate pDC. Moreover, HBV and HBsAg abrogated CpG-A/TLR9-induced, but not Loxoribine/TLR7-induced, mTORmediated S6 phosphorylation, subsequent IRF7 phosphorylation and IFNa gene transcription. HBV/HBsAg also diminished upregulation of co-stimulatory molecules, production of TNFa, IP-10 and IL-6 and pDC-induced NK cell function, whereas TLR7-induced pDC function was hardly affected. In line, HBsAg preferentially bound to TLR9-triggered pDC demonstrating that once pDC are able to bind HBV/HBsAg, the virus exerts its immune regulatory effect. HBV not only directly interfered with pDC function, but also indirectly by interfering with monocyte-pDC interaction. Also HBeAg diminished pDC function to a certain extent, but via another unknown mechanism. Interestingly, patients with HBeAg-positive chronic hepatitis B displayed impaired CpG-induced IFNa production by pDC without significant alterations in Loxoribine-induced pDC function compared to HBeAg-negative patients and healthy controls. The lack of activation and the active inhibition of pDC by HBV may both contribute to HBV persistence. The finding that the interaction between pDC and HBV may change upon activation may aid in the identification of a scavenging receptor supporting immunosuppressive effects of HBV and also in the design of novel treatment strategies for chronic HBV. - . These authors contributed equally to this work. Hepatitis B virus (HBV) infects the liver as primary target and may elicit progressive liver injury leading to increased risk of developing liver cirrhosis, liver failure and liver cancer [1]. Chronic infection with HBV is the result of an ineffective anti-viral immune response towards the virus [13]. The exact mechanism by which HBV escapes immunity is still not known. In general, the immune system is alerted and evokes a number of mechanisms that are aimed at eradicating the viral attack immediately following viral infection. The initial response to viral infection is the rapid release of type I interferons (IFN), IFNa and IFNb, which is observed for most viruses studied [4]. These IFN enhance the first defense against viral infections and modulate both innate and adaptive immune cells. Indications of the role of type I IFN during HBV infection are mostly based on studies in chimpanzees, since this is the only animal that can be infected with HBV. In sharp contrast to other viruses including hepatitis C virus, chimpanzees infected with HBV showed a complete lack in the induction of type I IFN and in IFN-response genes during the early stages of infection [5]. It is difficult to study the early events of acute HBV infection in humans. Nevertheless, it was recently shown that type I IFN responses are also lacking in acute HBV patients [6]. Plasmacytoid dendritic cells (pDC) are the principal producers of type I IFN and play a central role in immune responses against viral infections [7,8]. pDC respond to viruses and other pathogens primarily through the recognition of pathogen-associated molecular patterns by two intracellular Toll-like receptors (TLR), TLR7 and TLR9, which recognizes single stranded RNA and unmethylated DNA motifs, respectively [9,10]. TLR-triggering activates pDC to rapidly produce high levels of type I interferons, but also other cytokines, including TNF-a and IL-6, and cell surface costimulatory molecules. In this way pDC exert a direct anti-viral effect by producing factors that inhibit viral replication, but they also activate natural killer (NK) cells and T lymphocytes allowing further priming and regulation of anti-viral immunity [7,11,12]. Circulating blood pDC numbers seem to be unaffected by HBV, but functional deficits in pDC from chronic HBV patients including impaired IFN-a production have been reported [13]. Recently, it was reported that patient-derived HBsAg binds to human pDC in vitro and impairs TLR9-induced IFNa production by pDC [14]. The presence of HBV-DNA in or on pDC in vivo in chronic HBV patients [15,16] indicates that at least the whole virus and not only HBsAg interacts with pDC. Whether also HBV particles and/or other HBV-derived proteins present in patients circulation interfere with pDC function is not known. Given the central role that pDC play in antiviral immune responses, understanding the mechanisms whereby pDC interact with and respond to HBV may provide fundamental insights into the regulation of HBV-specific immunity and the development of HBV chronicity. Therefore, the present study investigated the effect of HBV as whole particles and HBV-derived proteins, i.e. HBcAg, HBeAg and HBsAg, on direct and indirect anti-viral functions of pDC. HBV does not activate pDC HBV is a DNA virus that replicates via an RNA intermediate. In theory, HBV may thus be able to activate pDC via TLR7, TLR9 and/or cytosolic pattern recognition receptors. However, no evidence exists that HBV replicates in pDC, which makes the direct activation of pDC by HBV via TLR7 maybe not very likely. Known synthetic and viral TLR7 and TLR9 ligands including Influenza virus, HSV-1, CpG and to a lesser extent Lox induced pDC to produce IFNa (Fig. 1A). In contrast, HBV did not give rise to IFNa producing pDC (Fig. 1). Similar data were observed for TNFa. Though HBV did not induce cytokines, the virus might induce pDC maturation. Whereas HSV-1 and Influenza virus as well as the synthetic TLR7 and TLR9 ligands upregulated the expression of CD40, CD80, CD86, and to a minor extent HLA-DR, HBV only marginally increased the expression of HLA-DR without affecting the expression of CD40, CD80 and CD86 (Table 1). HBV impairs CpG-induced pDC maturation and function To determine whether HBV mainly behaves as a stealth virus for pDC, or that it has an active role in the regulation of pDC function. pDC were (...truncated)