The Effect of Chronic Hepatitis B Virus Infection on BDCA3+ Dendritic Cell Frequency and Function

RESEARCH ARTICLE The Effect of Chronic Hepatitis B Virus Infection on BDCA3+ Dendritic Cell Frequency and Function Evelyn van der Aa, Sonja I. Buschow, Paula J. Biesta, Harry L. A. Janssen¤, Andrea M. Woltman* Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands a11111 ¤ Current address: Toronto Centre for Liver Disease, University Health Network, University of Toronto, Toronto, Canada * Abstract OPEN ACCESS Citation: van der Aa E, Buschow SI, Biesta PJ, Janssen HLA, Woltman AM (2016) The Effect of Chronic Hepatitis B Virus Infection on BDCA3+ Dendritic Cell Frequency and Function. PLoS ONE 11(8): e0161235. doi:10.1371/journal.pone.0161235 Editor: Scott N Mueller, The University of Melbourne, AUSTRALIA Received: March 24, 2016 Accepted: August 2, 2016 Published: August 16, 2016 Copyright: © 2016 van der Aa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Chronic hepatitis B virus (HBV) infection results from inadequate HBV-specific immunity. BDCA3+ dendritic cells (DCs) are professional antigen presenting cells considered to be important for antiviral responses because of specific characteristics, including high interferon-λ production. BDCA3+ DCs may thus also have a role in the immune response against HBV, and immunotherapeutic strategies aiming to activate DCs, including BDCA3+ DCs, in patient livers may represent an interesting treatment option for chronic HBV. However, neither the effect of chronic hepatitis B (CHB) infection on the frequency and function of BDCA3+ DCs in liver and blood, nor the effect of the viral surface protein (HBsAg) that is abundantly present in blood of infected individuals are known. Here, we provide an overview of BDCA3+ DC frequency and functional capacity in CHB patients. We find that intrahepatic BDCA3+ DC numbers are increased in CHB patients. BDCA3+ DCs from patient blood are not more mature at steady state, but display an impaired capacity to mature and to produce interferon-λ upon polyI:C stimulation. Furthermore, in vitro experiments exposing blood and intrahepatic BDCA3+ DCs to the viral envelope protein HBsAg demonstrate that HBsAg does not directly induce phenotypical maturation of BDCA3+ DCs, but may reduce IFN-λ production via an indirect unknown mechanism. These results suggest that BDCA3+ DCs are available in the blood and on site in HBV infected livers, but measures may need to be taken to revive their function for DC-targeted therapy. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by The Netherlands Organization for Scientific Research (NWO VIDI grant 91712329 to A.M.W.). 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. Introduction Hepatitis B virus (HBV) specifically infects hepatocytes and can cause chronic liver infection, often leading to severe liver diseases [1]. Chronic viral infection results from inadequate antiviral immunity, however, the mechanisms underlying ineffective HBV-specific immunity remain poorly understood [2, 3]. Effective viral immunity includes induction of antiviral cytokines PLOS ONE | DOI:10.1371/journal.pone.0161235 August 16, 2016 1 / 15 BDCA3+ Dendritic Cells in Chronic Hepatitis B such as interferons (IFNs) and virus-specific CD8+ cytotoxic T lymphocytes (CTL). Dendritic cells (DCs) play a crucial role in this process because they can, uniquely, activate virus-specific naïve T cells and produce high type I and type III IFN levels [4, 5]. The DC family comprises several subsets, including plasmacytoid DCs (pDC) and the myeloid DC (mDC) subsets BDCA1+ DCs and BDCA3hiCLEC9A+ DCs [6–8]. These DC subsets differ in ontogeny, localization, phenotype and function. We and others have previously characterized the frequency and function of BDCA1+ DCs and pDCs in peripheral blood of chronic HBV patients [9]. We demonstrated that although DC frequencies in blood were unaffected, blood BDCA1+ DCs were impaired in their capacity to mature, to produce pro-inflammatory cytokines and to stimulate T cells, and that pDCs were impaired in IFNα-producing capacity [10, 11]. More recently, BDCA3hiCLEC9A+ DCs (further referred to as BDCA3+ DCs) were identified and shown to excel over other DC subsets in cross-presentation of cell-associated antigens (Ag) to CD8+ T cells [12–15]. In mice, the equivalents of BDCA3+ DCs (CD8α+ and CD103+ DCs) have been shown to be crucial for generating optimal virus-specific CD8+ T cell responses to influenza virus and West Nile virus [16, 17]. In addition, BDCA3+ DCs are the most potent producers of IFN-λ in response to viruses that induce TLR3 signaling, or in response to the synthetic RNA polyI:C [18–22]. IFN-λ is an important antiviral cytokine that supports T cell skewing towards Th1 responses and has antiviral activity against multiple viruses, including HBV [23, 24]. Although the effect of IFN-λ on HBV replication in in vitro and mouse studies was debatable [25, 26], a recent clinical trial showed that in HBeAg-positive patients, PEG-IFN-λ induced a clear reduction in HBV DNA and viral surface antigen (HBsAg) levels, indicating that this cytokine may be valuable to fight CHB, and we envision that this cytokine could be even more effective when secreted on site [27]. BDCA3+ DCs may thus be a viable target to induce an effective immune response against HBV. BDCA3+ DCs are known to be present in human liver [21, 28, 29], however, it is unknown whether this is altered upon HBV-infection. Furthermore, the actual localization of BDCA3+ DCs within healthy and diseased liver tissue, as well as their functional state in HBV patients, and their response to the abundantly circulating HBsAg remains elusive. We here assessed the presence of BDCA3+ DCs in liver and blood of HBV-infected patients, as well as the effect of chronic HBV infection and HBsAg on BDCA3+ DC phenotype and function in vitro and ex vivo. We found that although BDCA3+ DCs are present in the liver immune infiltrate of chronic HBV (CHB) patients, their function may be compromised. Materials and Methods Patients and controls Heparinized peripheral blood samples were obtained from CHB patients and control subjects of which the clinical characteristics are listed in Table 1. CHB patients and control subjects used for functional characterization of DCs were matched for age, gender and race. Liver tissue was obtained from HBV-infected livers and non-HBV-infected livers, i.e. donor livers that were used for transplantation, donor livers that were rejected for transplantation, or noncancerous peri-tumor tissue of dono (...truncated)