Immunobiology 219 (2014) 880–887

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The hepatitis B virus e antigen suppresses the respiratory burst and mobility of human monocytes and neutrophils Chuen-Miin Leu a,b,∗ , Yong-Chen Lu a,1 , Wei-Li Peng c , Hsin-Tzu Chu c , Cheng-po Hu c,d,∗∗ a

Institute of Microbiology and Immunology, National Yang-Ming University, Taipei City, Taiwan Infection and Immunity Research Center, National Yang-Ming University, Taipei City, Taiwan Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei City, Taiwan d Department of Life Science, Tunghai University, Taichung City, Taiwan b c

a r t i c l e

i n f o

Article history: Received 27 February 2014 Received in revised form 29 April 2014 Accepted 15 July 2014 Available online 23 July 2014 Keywords: Hepatitis B virus e antigen Innate immune response Chemokinesis Monocytes Neutrophils Respiratory burst

a b s t r a c t The Hepatitis B virus (HBV) e antigen (HBeAg) is a secretory, non-structural protein, and associated with persistent infection of HBV. Previous studies indicate that HBeAg is able to regulate T cell-mediated responses, however, the interaction between HBeAg and the innate immune system is poorly understood. In this study, we demonstrated that recombinant HBeAg (rHBe) bound to human peripheral blood monocytes, neutrophils, and B lymphocytes but not to T lymphocytes. We focused on investigating the effects of HBeAg on monocytes and neutrophils and found that rHBe decreased the respiratory burst in both types of cells. Furthermore, we observed that cell migration in monocytes and neutrophils was suppressed by rHBe in a transwell assay. The attenuation of rHBe was not caused by a general cytotoxic effect because rHBe treatment stimulated low levels of cytokine and chemokine production by monocytes and it promoted neutrophil survival. Since the recruitment of monocytes and neutrophils to the infected site is crucial for the initiation of inflammation, HBeAg may modulate innate immune responses by diminishing the respiratory burst and migration of monocytes and neutrophils, which might interfere with the subsequent innate and adaptive immune responses against HBV, leading to the establishment of chronic infection. © 2014 Elsevier GmbH. All rights reserved.

Introduction The hepatitis B virus (HBV) causes acute and chronic liver diseases, and chronic HBV infection may lead to the development of liver cirrhosis and hepatocellular carcinoma. The HBV genome contains four open reading frames (ORFs), and the core ORF encodes for both the core protein (HBcAg) and the e protein (HBeAg). The core protein ORF is preceded by an in-framed initiation codon positioned 29 codons upstream (Fig. 1A). The sequence of these 29

Abbreviations: DHR, dihydrorhodamine 123; HBV, hepatitis B virus; HBc, HBV core protein; HBe, HBV e protein; ORF, open reading frame; ROS, reactive oxygen species; TPA, 12-o-tetradecanoyl phorbol-13-acetate; WHV, woodchuck hepatitis virus. ∗ Corresponding author at: Institute of Microbiology and Immunology, National Yang-Ming University, 155 Sec. 2, Li-Nong Street, Taipei City 11221, Taiwan Tel.: +886 2 2826 7296; fax: +886 2 2821 2880. ∗∗ Corresponding author. E-mail addresses: [email protected] (C.-M. Leu), [email protected] (C.-p. Hu). 1 Current address: Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. http://dx.doi.org/10.1016/j.imbio.2014.07.008 0171-2985/© 2014 Elsevier GmbH. All rights reserved.

codons has been termed the “precore” sequence, and the protein translated from the upstream initiation codon has been termed the “precore–core” protein (Ou et al. 1986). The precore–core protein contains a leader peptide that directs the protein to the endoplasmic reticulum, where the protein undergoes N- and C-terminal cleavages within the secretory pathway and forms the secretory HBeAg (Ou et al. 1986; Ou 1997; Roossnick et al. 1986). HBeAg is a non-structural protein of the virus, and viruses that lack the precore ATG are competent in viral infection, replication, virion assembly and secretion in vitro (Carman et al. 1989; Chang et al. 1987; Chen et al. 1992; Schlicht et al. 1987). Because HBeAg is secretory and maintains a high concentration in the circulation of certain chronic hepatitis B (CHB) infection patients, HBeAg has been proposed to modulate the immune system and may play a role in the establishment of chronic infection. To date, the biological functions of the HBe protein in the life cycle of HBV remain elusive (Ou et al. 1986; Will 1991). Because the precore sequence and HBeAg secretion are revolutionarily conserved among hepadnaviruses (Carlier et al. 1994; Ou et al. 1986; Schlicht 1991), it likely plays an important role in the HBV life cycle. In this regard, secreted HBeAg has been suggested to modify the

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Fig. 1. rHBe binds to monocytes, neutrophils, and B cells but not to T cells. (A) HBV core and e proteins share the same open reading frame. (B) The purity of the recombinant HBeAg (rHBe) was determined using Coomassie blue staining (lane 1), or by immunoblotting using rabbit anti-HBc/e antibody (lane 2) or mouse anti-HBe mAb (lane 3). (C) rHBe binds to monocytes, neutrophils, and B cells. Biotinylated rHBe (10 ␮g/ml) was incubated with peripheral blood mononuclear cells or purified neutrophils on ice for 30 min. After washing, the cells were stained with streptavidin-FITC and with PE-conjugated anti-CD14, anti-CD3 or anti-CD19 mAbs. The binding of rHBe was analyzed by gating specific lineage of cells. Gray peak, biotinylated rHBe; thin line, streptavidin-FITC control. One representative of at least four independent experiments is shown. T cells (n = 4), monocytes (n = 8), B cells (n = 7), neutrophils (n = 5).

host immune response during perinatal transmission. Studies of babies born to HBeAg-negative and -positive mothers suggest that HBeAg may be important for establishing persistent infection following neonatal infection (Milich et al. 1990; Okada et al. 1976). Similar results were observed in the studies of the related woodchuck hepatitis virus (WHV) (Chen et al. 1992; Miller et al. 1989; Ou et al. 1986). Seventy percent of neonatal woodchucks receiving the HBeAg-positive WHV became chronically infected, and in contrast, all control animals receiving the HBeAg-negative WHV developed transient viremia and were able to recover from the infection (Chen et al. 1992). Thus, HBeAg may play an essential role in the natural infection in vivo. Still unsolved are the mechanisms by which HBeAg helps establishing persistent infection. In animal models, circulating HBeAg has been shown to have a potential to deplete HBeAg- and HBcAg-specific Th1 cells and to suppress anti-HBc antibody response (Chen et al. 2004, 2005; Milich et al. 1998), suggesting that HBeAg may regulate T cellmediated immune responses. However, the mechanisms by which HBeAg affects T cells remain unclear. Because a proper activation of the innate immune responses is important for the initiation of T cell responses, it is possible that HBeAg may modulate T cell activities by disturbing the innate immune system. Since the initial discovery of HBeAg in 1972, the interaction between HBeAg and the innate immune system is largely unknown. One study demonstrated that the Toll-like receptor (TLR) 2 expression on Kupffer cells and blood monocytes is significantly reduced in patients with HBeAg-positive CHB infection in comparison to those with HBeAg-negative CHB infection (Visvanathan et al. 2007). Furthermore, HBeAg- but not HBcAg-containing conditioned medium suppresses the p38 MAPK phosphorylation induced by TLR2- or TLR4- agonist in blood CD14+ monocytes (Visvanathan et al. 2007). These observations imply that HBeAg may have a potential to reduce TLR signaling in monocytes. Monocytes are members in the innate immunity and they

transmigrate across endothelium into tissues or inflammation sites, where they further differentiate into macrophages or dendritic cells (DCs), two major antigen presenting cells. At the early stage of viral infection, macrophages and DCs sense invading pathogens and rapidly produce large amount of proinflammatory cytokines and chemokines that limit viral replication and promote the initiation of adaptive immune responses. Thus, many viruses may escape immune surveillance and establish infection by attenuating the innate immunity. After phagocytosis or encountering a variety of stimulation, phagocytes such as neutrophils and macrophages produce a large amount of reactive oxygen species (ROS) via a membrane bound multicomponent enzyme complex termed the NADPH oxidase. This process is called “respiratory burst” because phagocytes exhibits a striking increase in oxygen consumption (Fialkow et al. 2007; Forman and Torres 2002). It is worthy to note that ROS generated inside phagocytes not only involve in direct killing of ingested microorganisms, but also serve as signal molecules mediating signal pathways leading to many physiological responses, including transcriptional activation, apoptosis (Fialkow et al. 2007; Forman and Torres 2002), and neutrophil chemotaxis (Hattori et al. 2010). During inflammation, ROS have been shown to activate transcription factors such as NF-␬B, c-Fos, and c-Jun (Lo and Cruz 1995; Schreck et al. 1991), and therefore regulate gene expression. In addition, ROS production can lead to cell apoptosis in many cell types including neutrophils (Gardai et al. 2002). This self-limited mechanism plays an important role in the removal of leukocytes and in the maintenance of leukocyte homeostasis after inflammation ends. Because HBeAg is secretory and associated with persistent infection, we hypothesize that it may interact with peripheral blood cells and modulate their functions. To test this, we produced highly purified recombinant HBe protein (rHBe) and studied its effect on blood

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cells in vitro. We focused on investigating the impacts of rHBe on monocytes and neutrophils in this study because little is known about the interaction between HBeAg and the innate immune system.

isothiocyanate (FITC) (BD Biosciences, CA, USA) and lineage specific antibodies were added. The cells were washed and analyzed by flow cytometer (FACSCalibur, Becton Dickinson, CA, USA). Measurement of respiratory burst

Materials and methods Expression and purification of recombinant HBe To generate rHBe in E. coli, a DNA fragment containing the mature form of HBeAg (adw subtype, a.a. −10 to 149 of the core open reading frame, Fig. 1A) was amplified using PCR and the pA3cp1744HBV Pst/3zf plasmid as a template. For the PCR, the forward primer 5 ATTACCATGGCTTCCAAGCTGTGCCTTGGGTG-3 and the reverse primer 5 -GCGAAGCTTTCAAACAACAGTAGTTTCCGGAAG-3 were used. The HBeAg DNA fragment was digested using the restriction enzymes NcoI and HindIII, purified, and cloned into the NcoI/HindIII digested pET-32a vector (Novagen, WI, USA). The expression vector was named pET32aHBeAg, which includes a Trx-Tag, a His-Tag, an S-Tag, and an enterokinase cleavage site at the N-terminus of HBeAg. The rHBe was expressed in E. coli BL21 and purified as follows. In brief, the bacteria transformed with pET32aHBeAg were homogenized and the soluble fraction in the lysate was incubated with pre-equilibrium Ni2+ resin (ProBond resin, Invitrogen, CA, USA). The rHBe was eluted from the Ni2+ column and then treated with EnterokinaseMAX (Invitrogen) to cut at the N-terminal enterokinase cleavage site. The N-terminal tags and EnterokinaseMAX were removed by the addition of Ni2+ and EKAway resins (Invitrogen). Contaminated LPS in the rHBe was removed by Endotrap® blue (Profos AG, Regensburg, Germany). The endotoxin content in the purified rHBe was 30 pg/␮g rHBe, as determined by the Limulus amebocyte lysate assay (Pyrochrome® Chromogenic Endotoxin Testing Reagents, Associates of Cape Cod, Inc., East Falmouth, USA). An equivalent or higher amount of LPS (from E. coli, serotype 055:B5, Sigma–Aldrich) was included as a control in our assays. Cells, antibodies, and chemicals Cells were cultured in RPMI1640 medium containing 100 U/ml penicillin, 100 ␮g/ml streptomycin, 2 mM l-glutamine, and 10% fetal calf serum (Life Technologies, Grand Island, NY). The human blood was collected in accordance with the policies established by the Taipei Veterans General Hospital Institutional Review Board. Peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) were isolated using FicollHypaque gradient centrifugation. Contaminating RBCs in PMNs were removed using RBC lysis buffer (155 mM NH4 Cl, 10 mM KHCO3 , and 1 mM EDTA). CD14+ monocytes were purified using antibody-conjugated microbeads (Miltenyi Biotec, CA, USA). Cell purity was determined by staining with phycoerythrin (PE) conjugated anti-CD14 antibody and it was >90% in all experiments in this study. The PE-conjugated anti-human-CD3, -CD14, and -CD19 antibodies were purchased from Serotec (Oxford, UK). Western blot was performed using a rabbit anti-HBc/eAg antibody or mouse antiHBeAg antibody (Dako, Denmark). The signals were detected by enhanced chemiluminescence (PerkinElmer, MA, USA). HBeAg binding assay rHBe was biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Pierce, IL, USA). To perform the binding assay, 106 cells were suspended in 50 ␮l binding buffer (0.5% BSA in PBS with 10 mM HEPES) and incubated with 10 ␮g/ml biotinylated-rHBe on ice for 30 min. After washing, streptavidin-conjugated with fluorescein

Dihydrorhodamine 123 (DHR, Invitrogen) was used as an indicator because it is converted to fluorescent rhodamine 123 after oxidation. Cells were suspended in HBSS containing 0.1% BSA and were seeded onto 96-well culture plates. rHBe or HBSAg (adw subtype, yeast recombinant protein, kindly provided by Dr. Wei-Kuang Chi at the Development Center for Biotechnology, New Taipei City, Taiwan) was added to the cells and incubated at 37 ◦ C for 30 min. After washing, DHR (2 mM) and catalase (50 U/␮l) were added to each well and incubated at 37 ◦ C for another 30 min. The spontaneous (in monocytes and PMNs) or activation-induced (in PMNs) respiratory burst was determined using flow cytometric analysis. The mean fluorescent intensity in each data set was normalized to that in untreated (medium only) control. Chemotaxis and chemokinesis assay 106 monocytes were seeded on 6-well culture plates. After 2 h, the cells were stimulated with rHBe for 16 h. The monocytes were re-suspended in the in MEM␣ medium containing 0.5% BSA and 10 mM HEPES and transferred into the upper chamber of a 6.5 mm Transwell (5 ␮m pore size, Coring Costar Corp., MA, USA) at a density of 5 × 106 cells/ml. CCL2/MCP-1 purchased from R&D Systems was loaded in the lower chamber. After incubation at 37 ◦ C for 60 min, monocytes migrated to the lower chamber were collected and counted using a CyQUANT cell proliferation assay kit (Molecular Probes, OR, USA). The chemokinesis assay was similar to the chemotaxis assay, except that none or 100 ng/ml CCL2/MCP-1 was included in both the upper and lower chambers. Enzyme-linked immunosorbent assay (ELISA) 106 monocytes were seeded on 6-well culture plates. After 16 h, fresh medium was supplemented with or without various concentrations of rHBe or LPS. The supernatant was collected after 24 h, and cytokine level was measured using ELISA kits (R&D System, Minneapolis, MN, USA). Statistical analysis The two-tailed Student’s t test was used for the statistical analysis of the results. The differences were considered significant when p < 0.05. * p < 0.05; ** p < 0.01; *** p < 0.005. Results HBe binds to monocytes, neutrophils, and B cells Because HBeAg is a secretory protein, we hypothesized that HBeAg may interact with human peripheral blood cells. To test this, we produced rHBe (adw subtype) with little contaminated LPS (Fig. 1B, Coomassie blue staining in lane 1). Western blot analysis of rHBe using rabbit anti-HBc/e antibody and mouse anti-HBe mAb confirmed that the purified protein contains HBeAg epitopes (Fig. 1B, lane 2 and lane 3). The residual endotoxin content in the purified rHBe was 30 pg/␮g rHBe as determined by the Limulus amebocyte lysate assay. Equal or higher amounts of LPS were included as controls in our assays. We first tested whether rHBe binds peripheral blood mononuclear cells (PBMCs). BiotinylatedrHBe was incubated with PBMCs on ice, followed by incubation with streptavidin-conjugated FITC and lineage specific markers.

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We found that rHBe bound the monocytes, neutrophils, and B lymphocytes but not the T lymphocytes (Fig. 1C). To test the specificity of HBeAg binding, we pre-incubated purified monocytes or neutrophils with excess amount (50-fold) of un-labled rHBe and performed the binding assay. Our analysis showed that un-labeled rHBe reduced 50% of the binding of biotinylated-rHBe (our unpublished observation), suggesting that the binding is specific. Because TLR-2 and TLR-4 are expressed on monocytes, neutrophils, and B cells, but not on T cells, we speculated that rHBe may bind to TLR-2 and TLR-4. To test this, expression vectors containing TLR2, TLR4, and MD-2 were co-transfected into 293T cells and similar rHBe binding assay was conducted. Although TLR2 and TLR4 were expressed on the surface of the transfected 293T cells, the rHBe binding to these cells did not increase (data not shown). Therefore, expression of TLR2 or TLR4 alone on the cell surface may not be sufficient for rHBe binding. Furthermore, pre-incubation of 1 mg/ml anti-TLR4 antibody did not prevent the binding of rHBe to monocytes (data not shown). These observations imply that TLR2 and TLR4 may not be the rHBe receptor. HBe suppresses respiratory burst in monocytes and neutrophils Next, we studied whether rHBe modulates the functions of monocytes and neutrophils. After recognition of invading microbes, signals delivered from surface receptors on monocytes and neutorphils will trigger the production of ROS and inflammatory mediators. We first examined the respiratory burst in monocytes using DHR as an indicator because DHR becomes fluorescent rhodamine 123 after oxidation. PBMCs were incubated with rHBe, HBsAg (adw subtype, yeast recombinant protein), or LPS at 37 ◦ C for 30 min, and then reacted with DHR and catalase for another 30 min before flow cytometric analysis. We found a spontaneous respiratory burst in monocytes after cultured with DHR (Fig. 2A, gray peak), and the respiratory burst in monocytes was suppressed by rHBe in a dose-dependent manner (Fig. 2A). In average, 7.5 ␮g/ml rHBe reduced the respiratory burst by 54.7 ± 20.7% (Fig. 2B, n = 8). In contrast, no significant change was observed following the treatment with 7.5 ␮g/ml HBs, 0.0005, or 0.01 ␮g/ml LPS (Fig. 2B). Higher doses of LPS were also included in the experiments but no obvious effect was observed (data not shown). This result indicates that rHBe specifically suppresses respiratory burst in monocytes. Similarly, rHBe reduced the respiratory burst in neutrophils in the absence or presence of TPA (phorbol-12myristate-13-acetate, Fig. 2C and D, n = 4∼11), which is used to activate neutrophils. Without TPA, 5 ␮g/ml rHBe decreased the respiratory burst by 37.5 ± 12.5% (n = 11), and 7.5 ␮g/ml rHBe suppressed by 48.0 ± 10.8% (Fig. 2D, left panel, n = 4). In the presence of TPA, 5 ␮g/ml rHBe reduced the respiratory burst by 40.7 ± 22.2% (n = 7), and 7.5 ␮g/ml rHBe suppressed by 56.7 ± 19.0% (Fig. 2D, right panel, n = 7). These results indicate that rHBe attenuates respiratory burst in both monocytes and neutrophils. HBe inhibits chemokinesis of monocytes and neutrophils Monocytes extravasate from the blood to tissues and differentiate into macrophages and DCs, two major antigen presenting cells. To examine whether HBeAg modulates monocyte migration, a transwell assay was conducted. As shown in Fig. 3A, in the presence of 50 ng/ml MCP-1, 1 ␮g/ml rHBe reduced monocyte migation by 69.8 ± 23.0% (n = 4), and 5 ␮g/ml rHBe decreased by 80.0 ± 16.3% (n = 3). We noticed that rHBe affected the mobility of monocytes in the absence of a chemokine gradient (which is so called chemokinesis, Fig. 3A). One ␮g/ml rHBe suppressed monocyte migration by 66.6 ± 24.6% (n = 4), while 5 ␮g/ml rHBe reduced by 76.0 ± 18.7% (n = 3). To rule out the role of contaminated LPS in the suppression, 30 pg/ml LPS was added to the cells and no effect

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on monocyte migration was observed (data not shown). Furthermore, we examined the effect of rHBe on neutrophil chemokinesis and found that rHBe worked only at higher concentrations (Fig. 3B, n = 5). Five ␮g/ml rHBe reduced by 58.1 ± 12.7%, and 2.5 ␮g/ml rHBe decreased by 51.7 ± 11.3%. In contrast, 150 or 75 pg/ml LPS exhibited marginal effects on chemokinesis (18.2 ± 16.7% or 23.2 ± 17.5%, respectively). In summary, we found that HBe impairs chemokinesis of monocytes and neutrophils. The effect of rHBe on the production of cytokines and chemokines by monocytes Another important function of monocytes is to secrete cytokines and chemokines. To test whether rHBe regulates cytokine secretion, purified monocytes were incubated with rHBe, HBS, or LPS for 24 h, and the cytokines/chemokines in the supernatants were detected using ELISA. Our results show that the production of IL-6, CXCL8/IL-8, IL-10, and CCL2/MCP-1 was slightly increased following the treatment with rHBe (Fig. 4). Five ␮g/ml rHBe stimulated a 2.6-fold increase in IL-6, a 2.5-fold increase in IL-8, a 1.6-fold increase in IL-10, and a 4.6-fold increase in CCL2. In contrast, 30 pg/ml LPS or 5 ␮g/ml HBS had no effect. As a positive control, 100 ng/ml LPS induced a 5.7- to 14.4-fold increase in the levels of cytokines and chemokines produced by monocytes. Similarly, rHBe induced a ∼2-fold increase in IL-8 production by neutrophils, but it did not induce the production of IL-6, CCL2, or CCL4 by neutrophils (data not shown). It is worthy to note that IL-12 and TNF-␣ could not be detected before or after the treatment of rHBe in monocytes and neutrophils (data not shown). Our results indicate that in contrast to 100 ng/ml LPS, which potently stimulate cytokine and chemokine production, rHBe elicits only low levels of cytokine and chemokine production by monocytes. These observations suggest that the rHBe does not have a general inhibitory effect on monocyte metabolism. HBe prevents neutrophil apoptosis in vitro In the above experiments, we found that rHBe attenuated the respiratory burst and chemokinesis in neutrophils. Because neutrophils undergo spontaneous apoptosis fast in vitro, it is possible that rHBe modulates neutrophil functions by inducing cell death. To test this possibility, purified neutrophils were incubated with HBS, rHBe, or LPS in vitro, and stained with Annexin V and propidium iodide (PI) at different time points. We found that the percentage of live neutrophils (Annexin V− PI− ) was increased by the treatment with rHBe and this effect was most obvious at 16 h after in vitro culture (Fig. 5 and data not shown). We incubated neutrophils with 0.2, 1, and 5 ␮g/ml rHBe and found that 1 ␮g/ml rHBe already had a maximal protection effect (Fig. 5 and data not shown). The treatment with rHBe increased the percentage of survival cells by an average of 2-fold, however, HBS or LPS had little effect on neutrophil survival (Fig. 5, n = 4 ∼ 5). This result indicates that the suppression of rHBe on neutrophils is not due to a cytotoxic effect. Discussion Previous studies demonstrated that circulating HBeAg has a potential to deplete HBeAg- and HBcAg-specific Th1 cells and it blocks anti-HBc antibody response in vivo (Chen et al. 2004, 2005; Milich et al. 1998). Although these findings reveal the ability of HBeAg to modulate adaptive responses, however, whether HBeAg affects the innate immune responses remains largely unknown. In this study, we discovered that rHBe suppressed the respiratory burst and chemokinesis in monocytes and neutrophils. The suppression of respiratory burst may cause a reduction of ROS production, subsequently attenuate ROS-induced biological functions

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Fig. 2. rHBe suppresses the respiratory burst in monocytes and neutrophils. (A) and (B) rHBe suppresses the respiratory burst of monocytes. Purified PBMCs were seeded onto 96-well plates at a density of 106 ml–1 in 100 ␮l. Various concentrations of rHBe, HBs or LPS were added and incubated at 37 ◦ C for 30 min. Dihydrorhodamine (DHR, 2 mM) and catalase (50 U/␮l) were added to the cells and incubated at 37 ◦ C for another 30 min. The spontaneous respiratory burst of monocytes was determined using flow cytometry by gating the monocyte area in FSC–SSC dot plot as shown in panel (A). Gray peak in A, DHR signal in untreated cells; thin line, the cells without the addition of DHR; dotted lines and thick line are cells treated with rHBe. One representative result is shown. (B) the mean fluorescent intensity of DHR in each dataset was normalized to that in the untreated (medium only) control. A summary of all donors in more than 4 independent experiments is shown as mean ± SEM. rHBe (n = 8); HBs (n = 2); LPS (n = 2). (C) and (D) rHBe suppresses the respiratory burst of neutrophils. The purity of purified neutrophils is shown in (C). Neutrophils were treated and analyzed as described in (A), except TPA was used to activate cells. Donor numbers in panel (D): HBe 2.5 ␮g/ml (n = 8); HBe 5 ␮g/ml (n = 11), HBe 7.5 ␮g/ml (n = 4); HBS and LPS (n = 2). Panel C: HBe (n = 7); HBS (n = 2); LPS (n = 2). * p < 0.05; ** p < 0.01; *** p < 0.005.

Fig. 3. rHBe reduces the chemokinesis in human monocytes and neutrophils. (A) rHBe suppresses monocyte migration. Purified CD14+ monocytes were incubated with PBS, 1 ␮g/ml rHBe, or 5 ␮g/ml rHBe for 16 h. CCL2/MCP-1 was loaded in the lower chamber and the cells were into the upper insert. The number of migrated cells to the lower chamber was determined using CyQUNAT GR dye 1 h after incubation. The result from each dataset was normalized to the medium control A summary of 3–4 donors in 4 independent experiments is shown. (B) rHBe reduces neutrophil chemokinesis. The chemokinesis assay was similar to chemotaxis assay, except no chemokine was included in either the upper or lower chamber. The mean ± SEM from 5 donors in 5 independent experiments is shown.

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Fig. 4. The effect of rHBe on cytokine and chemokine production by monocytes. Purified monocytes were treated with various concentrations of HBe, HBs or LPS for 24 h. The concentration of cytokines and chemokines in the supernatant was determined using ELISA kits. One hundred ng/ml LPS was used as positive control, while 0.03 ng/ml LPS and 5 ␮g/ml HBS were included as negative controls. The mean ± SEM from three independent experiments is shown.

Fig. 5. rHBe prevents neutrophil from apoptosis in vitro. Neutrophils were treated with HBS, HBe, or LPS for 16 h and stained with Annexin V-FITC and propidium iodide (PI). The percentage of survival cells (Annexin V− PI− ) was analyzed by flow cytometry and the result was normalized to untreated control from individual donor. Bar graph is a summary of 4 (LPS 0.3 ng/ml) to 5 (the rest data set) donors in at least 4 independent experiments and is shown as mean ± SEM.

of monocytes and neutrophils, which might interfere the generation of proper immune responses. The impairment of monocytes and neutrophils migration by HBeAg may modulate the anti-viral responses in two ways. First, the number of antigen presenting cells, such as macrophages and DCs, might be reduced at infection sites, leading to a delayed or weak adaptive immune response. Second, local inflammation response might be attenuated since macrophages and neutrophils are key players in inflammation and they produce IL-12 to stimulate natural killer (NK) cells to secrete interferon-␥. The presence of IL-12 and interferon-␥ will promote Th1 differentiation, which in turn enhances cytotoxic T cell activity. Thus, through disturbing the functions of monocytes and neutrophils, HBeAg may modulate the innate response and might delay viral clearance, eventually leading to the establishment of chronic infection. Consistent with our findings, recent reports demonstrate that HBeAg may regulate the functions of monocytes and macrophages. The surface expression of TLR2 and response to TLR2 or TLR4 agonist in monocytes are significantly reduced in HBeAg-positive CHB patients when compared with HBeAg-negative CHB patients (Visvanathan et al. 2007), indicating that HBeAg contributes to the impaired responses in monocytes. In another study, the cytosolic-form HBeAg suppresses TLR-mediated NF-␬B activation by targeting the Toll/IL-receptor containing proteins TRAM and Mal (Lang et al. 2011). These reports together with our observations support the hypothesis that HBeAg modulates monocyte responses. While the importance of the adaptive immunity in

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controlling HBV infection has been widely accepted, the contribution of the innate system in viral clearance is starting to get more attentions. In HBV transgenic mice models, the activation of NK cells or antiviral responses by TLR ligands efficiently inhibits HBV replication (Isogawa et al. 2005; Kakimi et al. 2000; Wieland et al. 2000). However, the involvement of the innate system during natural HBV infection is still under debate. The studies in patients during early stage of HBV infection demonstrate a striking lack of type-I interferon response (Dunn et al. 2009) and lower serum proinflammatory cytokines and chemokines levels (Stacey et al. 2009) compared with HIV and HCV. Although the weaker responses may be due to the unusual delayed amplification of HBV replication in natural infection, however, our results indicate that HBV may actively suppress the innate immune responses at least in part by HBeAg. Because monocytes and neutrophils are major cells mediating inflammation, our observations provide a possible explanation for the lower inflammatory responses in patients. An important function of macrophages is to secrete cytokines and chemokines. We observed that rHBe induced low levels of cytokine and chemokine production by monocytes (Fig. 4), and no IL-12 or TNF-␣ production could be detected (data not shown). Therefore, HBeAg itself may not induce a strong inflammatory response. In mouse macrophages, TNF-␣ production is regulated by a ROS-activated NF-␬B pathway (reviewed in Forman and Torres 2002). It is possibly that HBeAg may suppress ROS production, subsequently alleviates the activation of transcription factors (NF␬B and AP-1) and disturb cytokine production by monocytes. Because rHBe may only suppress ROS-mediated, but not other pathway-activated NF-␬B, this could explain why rHBe induced the production of some cytokines/chemokines (IL-6, IL-8, and CCL2) but not others (TNF-␣ and IL-12). Neutrophils are part of the innate immune system, and their importance in the clearance of bacterial and fungal infections is well-documented in humans and mice. Intriguingly, recent discoveries indicate the participation of neutrophils in the activation, regulation, and effector functions of innate and adaptive immune cells (Lee et al. 2009). For example, neutrophils are required for the maturation and function of NK cells in humans and mice (Mantovani et al. 2011), and activated neutrophils promote the maturation of monocyte-derived DCs and subsequently affect the proliferation and polarization of T cells toward Th1 and Th17 (Abi Abdallah et al. 2011; Jaeger et al. 2012; Megiovanni et al. 2006; van Gisbergen et al. 2005). The results in our study imply that HBeAg may impair neutrophil activation and migration to infection site. As a consequence, the interactions between neutrophils and DCs or NK cells would be reduced, which might influence DC maturation and NK cell functions, and lead to a weaker Th1 response. Because both Th1 cells and NK cells are critical for viral clearance, HBeAg might attenuate this process indirectly by impeding the function and recruitment of neutrophils to the site of infection. The ROS generated after neutrophil activation participate not only in killing of phagocytosed microbes, but also can serve as signal molecules to regulate apoptosis in neutrophils. An increase of intracellular oxidant associates with an enhanced neutrophil apoptosis (Gardai et al. 2002). We observed that rHBe reduced the respiratory burst in neutrophils after activation, implicating a reduction of ROS generation. Thus, spontaneous neutrophil apoptosis blocked by the treatment of rHBe may be resulted from a decrease of ROS production. Even though rHBe promotes neutrophil survival, it simultaneously reduces neutrophil migration (Fig. 3) and respiratory burst (Fig. 2). Therefore, the overall net effects of rHBe on neutrophils are most likely to reduce their activation and migration to infection sites. A number of intracellular molecules, including phosphatidylinositol 3-kinase (PI3-K), phospholipase C (PLC), Ras, Rac and the Rho small GTPase are able to coordinate cell migration (Jones 2000; Kinashi 2005). Rac regulates the formation of

lamellipodia, Rho regulates the formation of the uropod, and PI3K and PLC are critical for directional sensing. It is possible that rHBe may regulate the migration of monocytes and neutrophils by disturbing the activation of these intracellular molecules, and the exact mechanisms require further investigations. The observation that HBeAg associates with lower TLR2- or TLR4-induced responses in human monocytes (Visvanathan et al. 2007) prompted us to test whether TLR2 and TLR4 are HBe receptors. However, overexpression of surface TLR2 or TLR4 did not augment rHBe binding to 293T cells (our unpublished observation), implying that TLR2 and TLR4 are not HBeAg receptors. Therefore, the attenuated TLR responses in monocytes from HBeAg-positive patients are probably not because of HBeAg-mediated TLR endocytosis. Our result is consistent with the previous observation that recombinant HBeAg did not activate human TLR signaling in a luciferase reporter assay in 293T cells (Lee et al. 2009). Because luciferase assay is highly sensitive, the result indicates that HBe is not a TLR agonist. Further investigations are necessary to identify the HBe receptor(s) and the mechanism by which HBeAg reduces TLR2 surface level. In summary, our results suggest that HBeAg may reduce the responses and movement of monocytes and neutrophils, which are at the front-line of host defense and important players in inflammation· Because inflammation is important for cyotoxic T cell differentiation and effector cell recruitment, the attenuation of monocytes and neutrophils by HBeAg may delay viral clearance and eventually facilitate the establishment of persistent infection. Conflict of interest The authors declare no conflict of interest. Funding This work was supported by National Science Council [grant numbers NSC95-2320-B-029–001-MY3 and NSC98-2311-B-029001-MY3 to C.-p.H.], and by Taipei Veterans General Hospital [grant numbers V99S5-001 and V100E4-002 to C.-M.L.]. Author contributions C.-M.L., Y.-C.L., and C.-p.H designed research; C.-M.L., Y.-C.L., W.-L.P., and H.-T.J. performed experiments; C.-M.L. and C.-p.H. prepared the manuscript. References Abi Abdallah, D.S., Egan, C.E., Butcher, B.A., Denkers, E.Y., 2011. Mouse neutrophils are professional antigen-presenting cells programmed to instruct Th1 and Th17cell differentiation. Int. Immunol. 23, 317–326. Carlier, D., Jean-Jean, O., Rossignol, J.M., 1994. Characterization and biosynthesis of the woodchuck hepatitis virus e antigen. J. Gen. Virol. 75, 171–175. Carman, W.F., Jacyna, M.R., Hadziyannis, S., Karayiannis, P., McGarvey, M.J., et al., 1989. Mutations preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet 2, 588–591. Chang, C., Enders, G., Sprengel, R., Peters, N., Varmus, H.E., et al., 1987. Expression of the precore region of an avian hepatitis B virus is not required for viral replication. J. Virol. 61, 3322–3329. Chen, H.S., Kew, M.C., Hornbuckle, W.E., Tennat, B.C., Cote, P.J., et al., 1992. The precore gene of woodchuck hepatitis virus genome is not essential for viral replication in the natural host. J. Virol. 66, 5682–5684. Chen, M.T., Billaud, J.N., Sallberg, M., Guidotti, L.G., Chisari, F.V., et al., 2004. A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen. Proc. Natl. Acad. Sci. U.S.A. 101, 14913–14918. Chen, M.T., Sallberg, M., Hughes, J.L., Jones, J.E., Guidotti, L.G., et al., 2005. Immune tolerance split between hepatitis B virus precore and core protein. J. Virol. 79, 3016–3027. Dunn, C., Peppa, D., Khanna, P., Nebbia, G., Jones, M., et al., 2009. Temporal analysis of early immune responses in patients with acute hepatitis B virus infection. Gastroenterology 137, 1289–1300.

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