Monocytes from Chronic HBV Patients React In Vitro to HBsAg and TLR by Producing Cytokines Irrespective of Stage of Disease

et al. (2014) Monocytes from Chronic HBV Patients React In Vitro to HBsAg and TLR by Producing Cytokines Irrespective of Stage of Disease. PLoS ONE 9(5): e97006. doi:10.1371/journal.pone.0097006 Monocytes from Chronic HBV Patients React In Vitro to HBsAg and TLR by Producing Cytokines Irrespective of Stage of Disease Arjan Boltjes 0 Zwier M. Groothuismink 0 Gertine W. van Oord 0 Harry L. A. Janssen 0 Andrea M. Woltman 0 Andre Boonstra 0 Isabelle A. Chemin, CRCL-INSERM, France 0 1 Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center , Rotterdam , The Netherlands , 2 Liver Clinic University Health Network, Division of Gastroenterology, University of Toronto , Toronto, ON , Canada Individuals who are chronically infected with the hepatitis B virus (HBV) are highly heterogenous with respect to serum levels of HBV DNA, HBV particles and viral proteins. Since circulating leukocytes, such as monocytes, are constantly exposed to these viral components, it is likely that the functionality of these cells is affected. However, at present, little information is available on the consequences of the interaction between monocytes and viral components. Therefore, we examined the in vitro effects of HBV surface antigen (HBsAg) on monocytes and evaluated whether these effects were reflected in vivo. We observed that in vitro HBsAg exposure of monocytes induced robust production of IL-6 and TNF. However, between chronic HBV patients with distinct levels of serum HBsAg, HBV early antigen (HBeAg), and HBV DNA, TLR-induced monocyte cytokine production did not differ. Importantly, HBsAg-induced cytokine production by monocytes was similar between patients and healthy controls showing that earlier in vivo exposure to HBsAg does not affect the in vitro response. Additionally, we show that IL-10 is able to inhibit cytokine production by HBsAg-induced monocytes. In conclusion, we demonstrate that monocytes can recognize and respond to HBsAg, resulting in vigorous pro-inflammatory cytokine production in vitro. However, phenotype and function of the monocyte compartment in chronic HBV patients are not influenced by differences in levels of serum viral components, suggesting that regulatory mechanisms are active to avoid excessive in vivo monocyte activation. - Funding: This study was supported by the Virgo consortium, funded by the Dutch government project number FES0908, by the Netherlands Genomics Initiative (NGI; http://www.genomics.nl/) project number 050-060-452, and by The Netherlands Organization for Scientific Research (NWO VIDI Grant 91712329 to A.M.W.; http://www.nwo.nl/en/funding/our-funding-instruments/nwo/innovational-research-incentives-scheme/vidi/index.html). 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. Hepatitis B virus (HBV) infection is a major health problem. Although the majority of infected individuals clear the virus spontaneously, a fraction of patients is unable to clear the virus and develops a chronic form of hepatitis. Their numbers have already reached over 240 million people [1]. In time, persistence of HBV can lead to progressive liver damage, which increases the patients risk of developing liver cirrhosis, liver failure and liver cancer. Chronicity of HBV is the result of a complex interaction between the replicating virus and an inadequate immune response [24]. After infection, viral replication takes place inside hepatocytes, and the secretion of infectious virions can take place for decades at high rates, and consequently HBV DNA, as well as viral proteins, like HBV early antigen (HBeAg) and HBV surface antigen (HBsAg), can be easily detected in serum. The levels of these clinical markers may fluctuate over time and are a reflection of disease activity and commonly used to define the patients disease stage [3,4]. Although circulating monocytes represent about 10% of leukocytes in human blood, relatively little is known on the consequences of chronic viral infections on monocytes. In HIV infections impaired monocyte functions have been reported [5,6], and we recently demonstrated altered Toll-like receptor (TLR) responsiveness of monocytes obtained from patients with chronic HCV infections [7,8]. Monocytes can be divided into two distinct subpopulations that are discerned based on their surface expression of CD14 and CD16. CD14highCD162 monocytes make up the majority (8090%) of blood monocytes, and have been reported to produce relatively high IL-10 and weak TNF levels, whereas the CD14+CD16+ subpopulation produces higher levels of pro-inflammatory cytokines, such as TNF and IL-1b [9,10]. Also in chronic HBV, some studies reported modulation of the monocyte compartment as a result of the disease. Depending on the clinical phase of the chronic HBV infection altered monocyte subsets frequencies were reported [11,12]. Moreover, PBMC from HBeAg-positive patients produced less TNF and IL-6 upon stimulation with TLR2 agonists as compared to HBeAg-negative patients [13], which was explained by lower expression of TLR2 in HBeAg-positive patients [14]. Furthermore, exposure of monocytes to HBsAg suppressed LPS-induced TNF and IL-1b production [15], while others reported that HBsAg has an immunostimulatory effect by inducing TNF and IL-10 production [16]. Since the consequences of constant exposure of peripheral monocytes to viral particles and the viral proteins HBeAg and HBsAg are still not completely understood, we here studied the in vitro and in vivo effects of these molecules on the phenotype and function of peripheral monocytes. Materials and Methods Patients and ethics statement Peripheral blood was collected from patients chronically infected with HBV who visited the outpatient clinic of the Erasmus Medical Center. Patients eligible for the study were positive for HBsAg, and were not on-treatment before blood samples were taken for this study. Patients co-infected with human immunodeficiency virus, hepatitis A virus, hepatitis C virus or hepatitis D virus were excluded. Patient characteristics are presented in Table 1. The medical ethical committee of the Erasmus MC University Medical Center approved the study and all patients gave written informed consent before inclusion. Laboratory measurements HBsAg levels and HBeAg levels were measured in sera from a total of 45 chronic HBV patients using the Architect HBsAg assay (Abbott Laboratories, Abbott Park, IL, USA; range 0.05250 IU/ ml) or HBeAg assay (Abbott Laboratories; interpreted using a ratio of the sample relative light unit (RLU) rate to the cut-off RLU (S/ CO)). HBV DNA levels were measured in serum using the Cobas TaqMan (Roche Diagnostics; lower limit of quantification, 20 IU/ ml). ALT was measured as part of standard diagnostic procedures. HBV genotype was determined by means of the INNO-LiPA assay ( (...truncated)