Immunobiology 219 (2014) 944–949
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Hepatitis B virus e antigen (HBeAg) may have a negative effect on dendritic cell generation Ibrahim Hatipoglu a,∗ , Duygu Ercan a , Ceyda Acilan a , Aynur Basalp a,1 , Deniz Durali a,b , Ahmet Tarik Baykal b a b
TUBITAK Marmara Research Center, Genetic Engineering and Biotechnology Institute, P.O. Box 21, 41470 Gebze, Kocaeli, Turkey Istanbul Medipol University, Department of Medical Biochemistry, School of Medicine, Ataturk Bulvari No. 27, 34083 Unkapani, Fatih-Istanbul, Turkey
a r t i c l e
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Article history: Received 25 February 2014 Received in revised form 6 June 2014 Accepted 29 July 2014 Available online 7 August 2014 Keywords: Dendritic cell HBeAg Hepatitis B virus LC–MS/MS Proteomics
a b s t r a c t Hepatitis B virus (HBV) continues to be a serious worldwide health problem despite the use of protective HBV vaccines and therapeutic regimens against chronic HBV infection. Chronic HBV patients cannot induce sufficient immune responses against the virus. HBV and its antigens are believed to suppress immune responses during chronic infection. Hence, studying the role of HBV in immune suppression is very important for the development of alternative therapeutic strategies for HBV infections. In the present study, we investigated the effect of Hepatitis B virus e antigen (HBeAg) on the generation of bone marrow derived dendritic cells (BMDCs) and the stimulation of plasmacytoid DCs (pDCs). In the presence of HBeAg, the ratio of BMDCs was decreased, but the ratio of CD11b+ Ly6G+ immature myeloid cells was increased. The expression of 47 proteins was also changed during HBeAg treatment; however, CpG-induced MHC-II expression on pDCs was not affected. Our results indicate that HBeAg may have a negative effect on the generation of DCs from bone morrow precursors. © 2014 Elsevier GmbH. All rights reserved.
Introduction Hepatitis B virus (HBV) infection is a persistent global health problem. According to the World Health Organization (WHO), 350 million people are chronically infected with the virus (Dienstag, 2008; Lai et al., 2003). Chronic HBV patients cannot induce an effective immune response against HBV; hence, studying the virus and its host immune system interactions is essential to understanding its chronicity and to developing therapies (Maini et al., 2000; Boonstra et al., 2008). Dendritic cells (DCs) stimulate T cells and regulate the immune system (Banchereau and Steinman, 1998; Ardavin et al., 2001). DCs have impaired function in chronic HBV patients compared with healthy individuals, and the virus and/or
Abbreviations: HBV, Hepatitis B virus; HBeAg, Hepatitis B virus e antigen; IFN, interferon; DC, dendritic cell; LC–MS/MS, high-performance liquid chromatography coupled with electrospray tandem mass spectrometry; MDSC, myeloid-derived suppressor cells; MHC-II, major histocompatibility complex class II; pDC, plasmacytoid dendritic cell; PDCA-1, plasmacytoid dendritic cell antigen-1; OVA, ovalbumin. ∗ Corresponding author at: TUBITAK MRC, Genetic Engineering and Biotechnology Institute, P.O. Box 21, 41470 Gebze, Kocaeli, Turkey. Tel.: +90 262 677 33 63; fax: +90 262 641 23 09. E-mail address: [email protected] (I. Hatipoglu). 1 Current address: Mehmet Akif Ersoy Univesity Health Scholl Istiklal Campus, Burdur, Turkey. http://dx.doi.org/10.1016/j.imbio.2014.07.020 0171-2985/© 2014 Elsevier GmbH. All rights reserved.
its antigens may be responsible for this effect (Xu et al., 2009; van der Molen et al., 2004; Woltman et al., 2011). HBV e antigen (HBeAg) is a marker of viral replication and one of the three factors for the treatment algorithm (Nguyen and Keeffe, 2008; Gitlin, 1997). The infection relapse rate of HBeAg-positive chronic patients is higher than HBeAg-negative chronic patients (Song et al., 2000). Moreover, HBeAg has a negative effect on the expression of toll-like receptors (TLRs) and the pro-inflammatory cytokine tumor necrosis factor-␣ (TNF-␣) in peripheral blood mononuclear cells (PBMCs). Furthermore, highly concentrated HBeAg may induce T cell anergy against HBV (Riordan et al., 2006; Visvanathan et al., 2007; Lang et al., 2011; Chen et al., 2005). In this study, we evaluated the effects of HBeAg on the generation of bone marrow derived DCs (BMDCs) and the stimulation of spleen resident plasmacytoid dendritic cells (pDCs). Depending on its concentration, HBeAg affected CD11c+ DC generation and the ratio of the immature myeloid cell population, but it did not have any effect on MHC-II expression on CpG-induced pDCs. Materials and methods Isolation and generation of dendritic cells Eight- to ten-week-old C57BL/6J×BALB/c mice were housed at TUBITAK, Marmara Research Center, Genetic Engineering
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and Biotechnology Institute (TUBITAK MRC GEBI) under specific pathogen-free organism (SPF) conditions. Animal experiments were approved by the local Ethics Committee of TUBITAK MRC GEBI. BMDCs were generated as described previously, with minor modifications (Shimizu et al., 1998). Briefly, bone marrow cells were removed from the femurs and tibias of mice, and erythrocytes were depleted using 0.14 M ammonium chloride. The cells (1 × 106 /ml) were seeded on 60-mm Petri dishes in RPMI-1640 medium (Gibco Life Sciences, USA) supplemented with 25 mM N2-Hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES), 2 g/l sodium bicarbonate, 100 U/ml penicillin, 100 g/ml streptomycin, 10% fetal bovine serum (FBS), 1% MEM nonessential amino acids, 50 M -mercaptoethanol, 20 ng/ml human recombinant GMCSF and 200 mIU/ml IL-4 (Gibco Life Sciences, USA). The cells were incubated with different concentrations (5 g/ml, 0.5 g/ml, 0.05 g/ml) of HBeAg (Fitzgerald, USA, Cat. no. 30-AH18), 5 g/ml Hepatitis B core antigen ((HBcAg), Fitzgerald, USA, Cat. no. 30AH39) or 5 g/ml OVA (Sigma, USA, Cat. no. A3059) at 37 ◦ C and 5% CO2 . Every 3 days, 2/3 of the medium was replaced with fresh media. On day 6, cells were harvested for CD11c expression analysis, proteomics and the WST-1 assay. pDCs were isolated from the spleen of mice using the pDC Isolation Kit II (Miltenyi Biotec, Germany, Cat. no. 130-092-786), and the purity of pDCs was more than 70%. After isolation, pDCs were cultured in 96-well U-bottom culture plates at a concentration of 5 × 104 cells/well in 200 l medium, treated with 5 g/ml HBeAg for 4 h and then stimulated with 10 g/ml CpG ODN 1826 (Invivogen, USA, Cat. no. tlrl-modn) overnight at 37 ◦ C and 5% CO2 . The control groups were not treated or treated with HBeAg or CpG. After incubation, cells were suspended and stained with specific antibodies for flow cytometry analysis. WST-1 assay The effect of HBeAg on BMDC viability was measured by WST-1 assay. BMDCs (harvested at day 6 as indicated in section “Isolation and generation of dendritic cells”) were seeded on 96-well plates (105 cells/well) and treated with 5 g/ml HBeAg. The control group was given only medium. After incubation for 24 h and 48 h, cell viability was detected by formazan product formation via the WST-1 assay (Roche, Germany, Cat. no. 11644807001) following the manufacturer’s instructions. The absorbance was measured on a microplate reader (Bio-Rad, USA, Cat. no. 3550) at 450 nm with a reference wavelength of 655 nm.
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phase A (0.1% formic acid in LC–MS grade water; Merck, Germany), and the column temperature was set to 45 ◦ C. Peptides were separated from the trap column (Symmetry C18 5 m, 180 m i.d. × 20 mm; Waters, USA) by gradient elution onto an analytical column (BEH C18, 1.7 m, 75 m i.d. × 250 mm; Waters, USA) at a 300 nl/min flow rate with a linear gradient from 5 to 40% mobile phase B (0.1 formic acid in hypergrade acetonitrile; Merck, Germany) over 90 min All samples were analyzed in triplicate. The instrument was operated in data-independent acquisition mode (MSE ) by using the positive ion V mode and applying the MS and MS/MS functions over 1.5-s intervals with 6 V low-energy and 15–40 V high-energy collusion. Glu-fibrinopeptide (internal mass calibrant) was infused at a 300 nl/min flow rate. The m/z values over 50–1600 were analyzed. Tandem mass data extraction, charge state deconvolution and deisotoping were performed with ProteinLynx Global Server v2.5 (Waters), and peptides were searched with the IDENTITYE algorithm with a fragment ion mass tolerance of 0.025 Da and a parent ion tolerance of 0.0100 Da against the reviewed mouse protein database from Uniprot (March 30th 2012, 30419 entries). The amino acid sequence of the internal standard (yeast enolase, Uniprot accession no. P00924) was included in the FASTA file of the database. The following parameters were used: carbamidomethyl-cysteine fixed modification and acetyl Nterm, deamidation of asparagine and glutamine, and oxidation of methionine variable modifications. Scaffold software (version Scaffold 3.6.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm (Keller et al., 2002). Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Only the protein IDs that satisfied the above criteria were accepted, and the corresponding information regarding peptide sequences and protein identification probabilities are provided in the supplementary file (Supp. Table 1). The false positive rate for the identified proteins was calculated to be 0.9% based on the Protein Prophet algorithm in Scaffold. Progenesis LC–MS software V4.0 (Nonlinear Dynamics) was used to quantify protein expression changes. Normalization across samples was based on total ion intensity. Similar proteins were grouped, and the quantitative value was assigned for the one with the highest score. Only proteins with non-conflicting peptide features were quantified.
Proteomics analysis For trypsin digestion of proteins, cells were washed twice with cold 50 mM ammonium bicarbonate and lysed by boiling at 100 ◦ C in UPX Buffer (Protein discovery, USA). Tryptic peptides were generated according to the Filter Aided Sample Preparation Protocol (FASP) (Wisniewski et al., 2009). Briefly, 50 g protein was washed with 6 M urea in a 30 kDa cut-off spin column, alkylated with 10 mM iodoactamide (IAA) in the dark for 20 min at room temperature and trypsinized overnight (1:100 trypsin-to-protein ratio). Peptides were eluted from the column, and their concentration was measured with a Nanodrop spectrometer. A total of 500 ng tryptic peptides were spiked with 50 fmol internal standard (MassPREP Enolase Digestion Standard, Waters, USA) and injected to the LC–MS/MS system. LC–MS/MS analysis and database searches for protein identification were based on our previously published protocol (Hacariz et al., 2012). Briefly, 500 ng tryptic peptides were analyzed by the LC–MS/MS system (nanoACQUITY UPLC column (Waters) and SYNAPT high definition mass spectrometer with a NanoLockSpray ion source (Waters)). Columns were equilibrated with 97% mobile
Flow cytometry analysis BMDCs were harvested from Petri dishes on day 6 and washed with PBS. The phenotype of the cells was evaluated using the following fluorochrome-conjugated antibodies: 7AAD, anti-CD11cPE (BD, USA), anti-Ly6G-PE-Cy5 (Abcam, UK), anti-CD11b-FITC (ebioscience, USA), anti-PDCA-1 (Miltenyi Biotec, Germany), antiMHC-II (Biolegend, USA) and relevant isotype controls. Cells were stained with labeled antibodies at 4 ◦ C for 20 min or 10 min for 7AAD, washed with cold PBS and then re-suspended in PBS. Data was acquired with FACScan and FACSCanto II instruments (BD Biosciences, USA) and analyzed using BD CellQuest and BD FACSDiva.
Statistical analysis Differences between the groups were analyzed by one-way ANOVA (Tukey’s test) using SPSS 15.0 (IBM, USA). Values were considered statistically significant when p < 0.05.
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Fig. 1. Effect of HBeAg on BMDC generation. The frequency of CD11c+ cells was determined in HBeAg-treated groups. HBcAg-treated, OVA-treated and the untreated group were used as controls. HBeAg decreased the generation of BMDCs. The means ± SE of at least three independent experiments are shown (*p < 0.05, **p < 0.001).
Fig. 3. Cell viability of BMDC after treatment with 5 g/ml HBeAg for 24 h and 48 h. HBeAg was non-cytotoxic to BMDC, as determined by WST-1 assay. Data represents the mean ± SD of three independent experiments (n = 4).
control and HBeAg-treated cells was 96 ± 1% and 94 ± 2%, respectively. In the second protocol, BMDCs were treated with HBeAg for 24 h and 48 h after generation. DC viability was determined by WST-1 assay (Fig. 3); HBeAg did not have any cytotoxic effect on BMDCs. Thus, HBeAg did not reveal any cytotoxicity on DCs even at the highest concentration used (5 g/ml). Effect of HBeAg on protein expression level
Fig. 2. The CD11b+ Ly6G+ cell population during BMDC differentiation. Untreated cells and cells treated with HBeAg (5 g/ml) were collected on day 0, 3 and 6. FACS showed that the percentage of CD11b+ Ly6G+ cells was affected by the presence of HBeAg. The numbers on contour plots represent the means ± SE of at least three independent experiments.
Results HBeAg downregulates DC generation To determine the effect of HBeAg on the generation of BMDCs, three different HBeAg concentrations (5, 0.5, 0.05 g/ml) were added to the culture medium. HBcAg (5 g/ml), OVA (5 g/ml) and non-supplemented medium were used as controls. On day 6, the frequency of CD11c+ DCs was decreased proportionally to the concentration of HBeAg compared with the HBcAg, OVA and no antigen control groups (Fig. 1). Furthermore, CD11b+ Ly6G+ immature myeloid cell frequency was analyzed during DC generation. The percentage of CD11b+ Ly6G+ cells was analyzed on days 0, 3 and 6 after 5 g/ml HBeAg treatment. The ratio of CD11b+ Ly6G+ immature myeloid cells in the total cell population increased in comparison to the untreated control group (Fig. 2). Investigation of the HBeAg cytotoxic effect Two different protocols were used to distinguish whether the diminished generation of BMDCs was due to HBeAg (5 g/ml) cytotoxicity. First, a BMDC generation protocol was used in the presence and absence of HBeAg. At day 6, cell viability was monitored using the 7-amino-actinomycin D (7-AAD) stain, which penetrates nonviable cell membranes and stains DNA. There was no significant difference in the viability of cells in either group. The viability of the
To evaluate the molecular pathways in DC generation affected by HBeAg treatment, we determine global proteomic changes by nLC–MS/MS analysis. Cells were either treated with HBeAg or with a vehicle control and were collected at day 6. A total of 464 proteins were identified, and 47 of these were differentially regulated with a 40% cut-off (p < 0.05). Table 1 shows the complete list of differentially regulated proteins. Four of these 47 proteins (H-2 class II histocompatibility antigen A-D beta chain, H-2 class II histocompatibility antigen A-S alpha chain, ganglioside GM2 activator, cathepsin S) were directly related to DC generation and function. The MHC II protein complex is important for antigen presentation on DCs, and its chains were downregulated in the HBeAg-treated group (Table 1). Ganglioside GM2 activator protein, which has an immunosuppressive role during the differentiation of monocytes to DCs, was also downregulated (Wright et al., 2005). Cathepsin S, which regulates MHC II–peptide complex formation and expression, was downregulated by HBeAg (Magister et al., 2012). Additionally, the expression levels of S100A8 and S100A9, Ca+2 binding proteins, were increased in the presence of HBeAg (Table 1). Ingenuity Pathway Analysis of differentially regulated proteins To understand which molecular pathways and their associated proteins were affected by the differentially regulated proteins detected in this study, an Ingenuity Pathway Analysis (IPA, Spring 2013 version) was performed. IPA is knowledge-based software that models and analyzes complex biological and chemical systems; it relies on a repository of expertly curated biological interactions and functional annotations based on relationships between proteins, genes, complexes, cells, tissues, drugs, and diseases. As a result of our IPA analyses, the top biological function associated with HBeAg treatment was “cell-to-cell signaling and interaction” (Supplemental Fig. 1). The category of “cell growth and proliferation” was also prominent, with 23 molecules (p = 6.06 × 10−6 ), whereas the “cellular movement” category was associated with 17 molecules (p = 1.25 × 10−6 ). “Immune cell trafficking” was represented by 12 molecules (p = 1.47 × 10−6 ), indicating that there were HBeAg-dependent changes in host cell-immune system signaling pathways. Furthermore, the “viral
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Table 1 Proteins that were significantly and >40% differentially expressed during DC generation in response to HBeAg. The arrows indicate up- or downregulation. Accession number
Anova (p)
Description
Function
Fold change
DC related proteins P01921; P06342
0.00111064 0.00164758
Antigen processing and presentation of exogenous peptide antigen via MHC class II Peptide antigen binding
↓ 2.1
P14437 Q60648
0.00112813
H-2 class II histocompatibility antigen, A-D beta chain H-2 class II histocompatibility antigen, A-S alpha chain Ganglioside GM2 activator
↓ 2.1
O70370
2.19E−05
Cathepsin S
Binds gangliosides and stimulates ganglioside GM2 degradation Key protease responsible for the removal of the invariant chain from MHC class II molecules
Other proteins P31725 P27005 P68134; P68033; P68135
3.56E−08 1.07E−06 0.03051749
Protein S100-A9 Protein S100-A8 Actin, alpha skeletal muscle
P48036 O89053
0.00709654 0.00989136
Annexin A5 Coronin-1A
P43275
0.02653709
Histone H1.1
Q62465
0.03330329
P08905; P17897
0.00032004
Synaptic vesicle membrane protein VAT-1 homolog Lysozyme C-2
P30044
8.95E−06
Q9FJE8 P10810
0.00308165 0.00035253
Q8R180 P97449 P41216
0.00185244 0.01261818 0.02514271
P41377
0.00770115
Q05144
0.03373887
Q9JII6
0.01096198
Q05816
0.0021051
P02088; P02057; P11758; P02089 Q99P72
0.00926448
Fatty acid-binding protein, epidermal Hemoglobin subunit beta-1
0.0103125
Reticulon-4
P35175; P35173 P62702; P47836 Q9D154
0.02080033 0.0292214 0.03340597
Stefin-1 40S ribosomal protein S4 Leukocyte elastase inhibitor A
Q01768
0.01820937
P09602
4.35E−05
P37040
0.03848945
P47955
0.04781885
Nucleoside diphosphate kinase B Non-histone chromosomal protein HMG-17 NADPH – cytochrome P450 reductase 60S acidic ribosomal protein P1
P10639 P29391; P49945
0.00542218 0.01831099
Thioredoxin Ferritin light chain 1
P12265
0.02857571
Beta-glucuronidase
P68040
0.03456048
P34884
0.00233358
Q9ZPX5; Q8K2B3
0.04841603
P28667 Q9Z183
0.01599274 0.01613393
P54987
0.0237136
Guanine nucleotide-binding protein subunit beta-2-like 1 Macrophage migration inhibitory factor Succinate dehydrogenase [ubiquinone] flavoprotein subunit 2, MARCKS-related protein Protein-arginine deiminase type-4 Immune-responsive gene 1 protein
Peroxiredoxin-5, mitochondrial Probable histone H2A.7 Monocyte differentiation antigen CD14 ERO1-like protein alpha Aminopeptidase N Long-chain-fatty-acid – CoA ligase 1 Eukaryotic initiation factor 4A-2 Ras-related C3 botulinum toxin substrate 2 Alcohol dehydrogenase [NADP(+)]
A calcium- and zinc-binding protein A calcium- and zinc-binding protein Highly conserved proteins that are involved in various types of cell motility An anticoagulant protein May be a crucial component of the cytoskeleton of highly motile cells, Binds to linker DNA between nucleosomes forming the macromolecular structure Plays a part in calcium-regulated keratinocyte activation in epidermal repair mechanisms Lysozymes have primarily a bacteriolytic function Reduces hydrogen peroxide and alkyl hydroperoxides Core component of nucleosome Leading to NF-kappa-B activation, cytokine secretion and the inflammatory response Essential oxidoreductase Broad specificity aminopeptidase Activation of long-chain fatty acids for both synthesis of cellular lipids, and degradation via beta-oxidation ATP-dependent RNA helicase
↓ 2.8
↓ 2.2
↑ 2.9 ↑ 3.6 ↓ 2.8 ↓ 1.5 ↑ 1.7 ↑ 1.5 ↓ 1.4 ↓ 1.8 ↑ 1.8 ↑ 5.2 ↑ 2.9 ↑ 1.6 ↓2 ↑ 1.5
↓ 1.9
Plasma membrane-associated small GTPase
↓ 1.6
Catalyzes the NADPH-dependent reduction of a variety of aromatic and aliphatic aldehydes to their corresponding alcohols High specificity for fatty acids
↓ 1.5
Involved in oxygen transport from the lung to the various peripheral tissues Developmental neurite growth regulatory factor Intracellular thiol proteinase inhibitor Structural constituent of ribosome Regulates the activity of the neutrophil proteases Major role in the synthesis of nucleoside triphosphates other than ATP. Binds to the inner side of the nucleosomal DNA Required for electron transfer from NADP to cytochrome P450 in microsomes Plays an important role in the elongation step of protein synthesis Participates in various redox reactions Stores iron in a soluble, non-toxic, readily available form Plays an important role in the degradation of dermatan and keratan sulfates Involved in the recruitment, assembly and/or regulation of a variety of signaling molecules Pro-inflammatory cytokine
↓ 1.8 ↓ 1.7 ↑ 1.6 ↑2 ↑ 2.1 ↑ 1.5 ↓ 1.5 ↑6 ↑ 1.8 ↓ 1.4 ↓ 1.4 ↑ 1.6 ↓ 1.7 ↓ 1.8 ↓ 1.4
Flavoprotein (FP) subunit of succinate dehydrogenase (SDH)
↓ 1.5
Controls cell movement Catalyzes the citrullination/deimination of arginine residues of proteins 2-methylcitrate dehydratase activity
↑ 4.7 ↑ 4.9 ↑ 2.1
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Table 1 (Continued) Accession number
Anova (p)
Description
Function
Fold change
P42746
0.00267475
Accepts the ubiquitin from the E1 complex
↑ 34.1
Q9JJW6
0.01273248
Acts as chaperone
↑ 5.5
Q60749
0.00041957
Recruited and tyrosine phosphorylated by several receptor systems
↑ 8.4
P35174 Q8QZT1
9.99E−05 0.00167717
Ubiquitin-conjugating enzyme E2 3 RNA and export factor-binding protein 2 KH domain-containing, RNA-binding, signal transduction-associated protein 1 Stefin-2 Acetyl-CoA acetyltransferase, mitochondrial
intracellular thiol proteinase inhibitor Plays a major role in ketone body metabolism
Infinity ↓ 3.2
Random4313 Random14989
0.03443238 0.00548945
Random Sequence 4313 Random Sequence 14,989
↓ 1.6 ↓ 1.6
Fig. 4. MHC-II expression on PDCA-1-positive cells. pDCs were treated with HBeAg for 4 hours and then stimulated with CpG overnight. HBeAg had no effect on MHC-II expression on PDCA-1-positive cells. The mean fluorescence intensity (MIF) of MHC-II- and PDCA-1-positive cells is indicated. The data represent the mean ± SE of at least three independent experiments.
infections” category contained 16 molecules (p = 4.74 × 10−6 ), confirming that our proteomic analyses identified the cellular response to treatments based on protein expression changes. Effect of HBeAg on pDC stimulation MHC-II expression on PDCA-1-positive cells was examined to investigate the effect of HBeAg on spleen pDC stimulation. PDCA1 is an essential marker of pDCs. pDCs were isolated from spleen tissue and treated sequentially with HBeAg and CpG. HBeAg did not significantly affect MHC-II expression on PDCA-1 positive cells compared with the control group (Fig. 4). Discussion Although HBeAg is not required for virus assembly, infection or replication, it may play a critical role in suppressing immune responses to HBV (Schlicht et al., 1987; Milich and Liang, 2003). HBeAg induces anergy in HBeAg- and HBcAg-specific T cells, inhibits innate immune responses by interacting with TLRs, reduces interferon alpha (IFN␣) production and reduces NF-B activation (Lang et al., 2011; Chen et al., 2005). In this study, we determined the potential effects of HBeAg on DC differentiation and stimulation. The postulated HBV-dependent decrease in DC populations is controversial (van der Molen et al., 2004; Duan et al., 2005). To our knowledge, there has not been a study explaining the HBeAgmediated decrease in DCs. We showed that HBeAg reduced the generation of DCs from bone marrow progenitors, but although HBeAg and HBcAg share a large part of their amino acid sequences, HBcAg did not have the same effect. This result also indicates another functional difference between these two antigens; the decrease of DC generation could be related to the NF-B signaling pathway. HBeAg inhibits NF-B activation by inhibiting the TIR:TIR
interaction in TLR signaling, which essential for GM-CSF-driven DC generation (Visvanathan et al., 2007; Lang et al., 2011; van de Laar et al., 2010; Ouaaz et al., 2002). In contrast, the receptor that may have a role in DC differentiation, TLR-2, is downregulated in the presence of HBeAg. A TLR-2 agonist induces DC generation from human bone marrow CD34+ progenitors (Sioud and Floisand, 2007). We performed nLC–MS/MS-based proteomic analysis to investigate the effects of HBeAg on protein expression during DC generation. The proteomic results confirmed the flow cytometry data, as the expression of some proteins that are important for DC generation and physiology (MHC II complex proteins, cathepsin S, ganglioside GM2 activator protein) were decreased in the presence of HBeAg (Wright et al., 2005; Magister et al., 2012). Moreover, the S100A8 and S100A9 proteins were increased in the presence of HBeAg. These Ca2+ -binding proteins have functions in inflammation, cancer and autoimmune diseases (Vogl et al., 2012; Leukert et al., 2006). The upregulation of S100A9 and S100A8 inhibits DC differentiation (Cheng et al., 2008). Furthermore, we found that in the presence of HBeAg (5 g/ml), the immature myeloid cell population (CD11b+ Ly6G+ ) frequency was approximately threefold higher than the control group. CD11b+ Ly6G+ immature myeloid cells represent different cell populations (Yang et al., 2011). Tumor associated neutrophils (TANs) and myeloid derived suppressor cells (MDSCs) are CD11b+ Ly6G+ , and these cells are potentially immunosuppressive. In contrast, increased S100A9 and S100A8 expression is positively correlated with an increased frequency of MDSCs (Zhao et al., 2012). Thus, HBeAg may induce immune suppression. Virus antigens have the potential to induce MDSCs. For instance, hepatitis C virus (HCV) extracellular core protein induces the expansion of MDSCs, and HIV-positive patients exhibit a higher rate of MDSCs than healthy controls (Tacke et al., 2012; Vollbrecht et al., 2012).pDCs have a vital role in anti-viral immunity because they produce type I IFNs (Colonna et al., 2004). HBeAg-positive patients have limited production of IFN␣ from pDCs, but HBV-induced HLA-DR expression in
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stimulated pDCs is not significant (Woltman et al., 2011). There has been no study investigating the specific effects of HBeAg on MHCII expression. Here, we showed that HBeAg did not significantly affect MHC-II expression on CpG-stimulated and non-stimulated pDCs. In conclusion, our results indicated that HBeAg inhibits BMDC generation. Further clinical studies are needed to investigate the correlation between HBeAg concentration and the ratio of DCs and/or the immature myeloid cell population. Furthermore, the effect of HBeAg on MDSC and TAN generation need clarification. This study illustrated the effect of HBV on the immune system and strengthened the potential for HBeAg as a new therapeutic target in HBeAg-positive chronic HBV patients. Conflict of interest statement The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Acknowledgments This study was supported by grants nos. 108S300, 110S497 and 105G0656 from The Scientific and Technological Research Council of Turkey (TUBITAK). We thank Dr. Betul Guloglu for critically reviewing the manuscript and Duygu OZCELIK for her excellent technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.imbio. 2014.07.020. References Ardavin, C., del Hoyo, G., Martin, P., Anjuere, F., Arias, C., Marin, A., Ruiz, S., Parrillas, V., Hernandez, H., 2001. Origin and differentiation of dendritic cells. Trends Immunol. 22, 691. Banchereau, J., Steinman, R.M., 1998. Dendritic cells and the control of immunity. Nature 392, 245. Boonstra, A., Woltman, A.M., Janssen, H.L., 2008. Immunology of hepatitis B and hepatitis C virus infections. Best Pract. Res. Clin. Gastroenterol. 22, 1049. Chen, M., Sällberg, M., Hughes, J., Jones, J., Guidotti, L.G., Chisari, F.V., Billaud, J.N., Milich, D.R., 2005. Immune tolerance split between hepatitis B virus precore and core proteins. J. Virol. 79, 3016. Cheng, P., Corzo, C.A., Luetteke, N., Yu, B., Nagaraj, S., Bui, M.M., Ortiz, M., Nacken, W., Sorg, C., Vogl, T., Roth, J., Gabrilovich, D.I., 2008. Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein. J. Exp. Med. 205, 2235. Colonna, M., Trinchieri, G., Liu, Y.J., 2004. Plasmacytoid dendritic cells in immunity. Nat. Immunol. 5, 1219. Dienstag, J.L., 2008. Hepatitis B virus infection. N. Engl. J. Med. 359, 1486. Duan, X.Z., Zhuang, H., Wang, M., Li, H.W., Liu, J.C., Wang, F.S., 2005. Decreased numbers and impaired function of circulating dendritic cell subsets in patients with chronic hepatitis B infection (R2). J. Gastroenterol. Hepatol. 20, 234. Gitlin, N., 1997. Hepatitis B: diagnosis, prevention, and treatment. Clin. Chem. 43, 1500. Hacariz, O., Sayers, G., Baykal, A., 2012. A proteomic approach to investigate the distribution and abundance of surface and internal Fasciola hepatica proteins during the chronic stage of natural liver fluke infection in cattle. J. Proteome Res. 11, 3592. Keller, A., Nesvizhskii, A., Kolker, E., Aebersold, R., 2002. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 74, 5383. Lai, C.L., Ratziu, V., Yuen, M.F., Poynard, T., 2003. Viral hepatitis B. Lancet 362, 2089.
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Immunobiology journal homepage: www.elsevier.com/locate/imbio
Hepatitis B virus e antigen (HBeAg) may have a negative effect on dendritic cell generation Ibrahim Hatipoglu a,∗ , Duygu Ercan a , Ceyda Acilan a , Aynur Basalp a,1 , Deniz Durali a,b , Ahmet Tarik Baykal b a b
TUBITAK Marmara Research Center, Genetic Engineering and Biotechnology Institute, P.O. Box 21, 41470 Gebze, Kocaeli, Turkey Istanbul Medipol University, Department of Medical Biochemistry, School of Medicine, Ataturk Bulvari No. 27, 34083 Unkapani, Fatih-Istanbul, Turkey
a r t i c l e
i n f o
Article history: Received 25 February 2014 Received in revised form 6 June 2014 Accepted 29 July 2014 Available online 7 August 2014 Keywords: Dendritic cell HBeAg Hepatitis B virus LC–MS/MS Proteomics
a b s t r a c t Hepatitis B virus (HBV) continues to be a serious worldwide health problem despite the use of protective HBV vaccines and therapeutic regimens against chronic HBV infection. Chronic HBV patients cannot induce sufficient immune responses against the virus. HBV and its antigens are believed to suppress immune responses during chronic infection. Hence, studying the role of HBV in immune suppression is very important for the development of alternative therapeutic strategies for HBV infections. In the present study, we investigated the effect of Hepatitis B virus e antigen (HBeAg) on the generation of bone marrow derived dendritic cells (BMDCs) and the stimulation of plasmacytoid DCs (pDCs). In the presence of HBeAg, the ratio of BMDCs was decreased, but the ratio of CD11b+ Ly6G+ immature myeloid cells was increased. The expression of 47 proteins was also changed during HBeAg treatment; however, CpG-induced MHC-II expression on pDCs was not affected. Our results indicate that HBeAg may have a negative effect on the generation of DCs from bone morrow precursors. © 2014 Elsevier GmbH. All rights reserved.
Introduction Hepatitis B virus (HBV) infection is a persistent global health problem. According to the World Health Organization (WHO), 350 million people are chronically infected with the virus (Dienstag, 2008; Lai et al., 2003). Chronic HBV patients cannot induce an effective immune response against HBV; hence, studying the virus and its host immune system interactions is essential to understanding its chronicity and to developing therapies (Maini et al., 2000; Boonstra et al., 2008). Dendritic cells (DCs) stimulate T cells and regulate the immune system (Banchereau and Steinman, 1998; Ardavin et al., 2001). DCs have impaired function in chronic HBV patients compared with healthy individuals, and the virus and/or
Abbreviations: HBV, Hepatitis B virus; HBeAg, Hepatitis B virus e antigen; IFN, interferon; DC, dendritic cell; LC–MS/MS, high-performance liquid chromatography coupled with electrospray tandem mass spectrometry; MDSC, myeloid-derived suppressor cells; MHC-II, major histocompatibility complex class II; pDC, plasmacytoid dendritic cell; PDCA-1, plasmacytoid dendritic cell antigen-1; OVA, ovalbumin. ∗ Corresponding author at: TUBITAK MRC, Genetic Engineering and Biotechnology Institute, P.O. Box 21, 41470 Gebze, Kocaeli, Turkey. Tel.: +90 262 677 33 63; fax: +90 262 641 23 09. E-mail address: [email protected] (I. Hatipoglu). 1 Current address: Mehmet Akif Ersoy Univesity Health Scholl Istiklal Campus, Burdur, Turkey. http://dx.doi.org/10.1016/j.imbio.2014.07.020 0171-2985/© 2014 Elsevier GmbH. All rights reserved.
its antigens may be responsible for this effect (Xu et al., 2009; van der Molen et al., 2004; Woltman et al., 2011). HBV e antigen (HBeAg) is a marker of viral replication and one of the three factors for the treatment algorithm (Nguyen and Keeffe, 2008; Gitlin, 1997). The infection relapse rate of HBeAg-positive chronic patients is higher than HBeAg-negative chronic patients (Song et al., 2000). Moreover, HBeAg has a negative effect on the expression of toll-like receptors (TLRs) and the pro-inflammatory cytokine tumor necrosis factor-␣ (TNF-␣) in peripheral blood mononuclear cells (PBMCs). Furthermore, highly concentrated HBeAg may induce T cell anergy against HBV (Riordan et al., 2006; Visvanathan et al., 2007; Lang et al., 2011; Chen et al., 2005). In this study, we evaluated the effects of HBeAg on the generation of bone marrow derived DCs (BMDCs) and the stimulation of spleen resident plasmacytoid dendritic cells (pDCs). Depending on its concentration, HBeAg affected CD11c+ DC generation and the ratio of the immature myeloid cell population, but it did not have any effect on MHC-II expression on CpG-induced pDCs. Materials and methods Isolation and generation of dendritic cells Eight- to ten-week-old C57BL/6J×BALB/c mice were housed at TUBITAK, Marmara Research Center, Genetic Engineering
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and Biotechnology Institute (TUBITAK MRC GEBI) under specific pathogen-free organism (SPF) conditions. Animal experiments were approved by the local Ethics Committee of TUBITAK MRC GEBI. BMDCs were generated as described previously, with minor modifications (Shimizu et al., 1998). Briefly, bone marrow cells were removed from the femurs and tibias of mice, and erythrocytes were depleted using 0.14 M ammonium chloride. The cells (1 × 106 /ml) were seeded on 60-mm Petri dishes in RPMI-1640 medium (Gibco Life Sciences, USA) supplemented with 25 mM N2-Hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES), 2 g/l sodium bicarbonate, 100 U/ml penicillin, 100 g/ml streptomycin, 10% fetal bovine serum (FBS), 1% MEM nonessential amino acids, 50 M -mercaptoethanol, 20 ng/ml human recombinant GMCSF and 200 mIU/ml IL-4 (Gibco Life Sciences, USA). The cells were incubated with different concentrations (5 g/ml, 0.5 g/ml, 0.05 g/ml) of HBeAg (Fitzgerald, USA, Cat. no. 30-AH18), 5 g/ml Hepatitis B core antigen ((HBcAg), Fitzgerald, USA, Cat. no. 30AH39) or 5 g/ml OVA (Sigma, USA, Cat. no. A3059) at 37 ◦ C and 5% CO2 . Every 3 days, 2/3 of the medium was replaced with fresh media. On day 6, cells were harvested for CD11c expression analysis, proteomics and the WST-1 assay. pDCs were isolated from the spleen of mice using the pDC Isolation Kit II (Miltenyi Biotec, Germany, Cat. no. 130-092-786), and the purity of pDCs was more than 70%. After isolation, pDCs were cultured in 96-well U-bottom culture plates at a concentration of 5 × 104 cells/well in 200 l medium, treated with 5 g/ml HBeAg for 4 h and then stimulated with 10 g/ml CpG ODN 1826 (Invivogen, USA, Cat. no. tlrl-modn) overnight at 37 ◦ C and 5% CO2 . The control groups were not treated or treated with HBeAg or CpG. After incubation, cells were suspended and stained with specific antibodies for flow cytometry analysis. WST-1 assay The effect of HBeAg on BMDC viability was measured by WST-1 assay. BMDCs (harvested at day 6 as indicated in section “Isolation and generation of dendritic cells”) were seeded on 96-well plates (105 cells/well) and treated with 5 g/ml HBeAg. The control group was given only medium. After incubation for 24 h and 48 h, cell viability was detected by formazan product formation via the WST-1 assay (Roche, Germany, Cat. no. 11644807001) following the manufacturer’s instructions. The absorbance was measured on a microplate reader (Bio-Rad, USA, Cat. no. 3550) at 450 nm with a reference wavelength of 655 nm.
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phase A (0.1% formic acid in LC–MS grade water; Merck, Germany), and the column temperature was set to 45 ◦ C. Peptides were separated from the trap column (Symmetry C18 5 m, 180 m i.d. × 20 mm; Waters, USA) by gradient elution onto an analytical column (BEH C18, 1.7 m, 75 m i.d. × 250 mm; Waters, USA) at a 300 nl/min flow rate with a linear gradient from 5 to 40% mobile phase B (0.1 formic acid in hypergrade acetonitrile; Merck, Germany) over 90 min All samples were analyzed in triplicate. The instrument was operated in data-independent acquisition mode (MSE ) by using the positive ion V mode and applying the MS and MS/MS functions over 1.5-s intervals with 6 V low-energy and 15–40 V high-energy collusion. Glu-fibrinopeptide (internal mass calibrant) was infused at a 300 nl/min flow rate. The m/z values over 50–1600 were analyzed. Tandem mass data extraction, charge state deconvolution and deisotoping were performed with ProteinLynx Global Server v2.5 (Waters), and peptides were searched with the IDENTITYE algorithm with a fragment ion mass tolerance of 0.025 Da and a parent ion tolerance of 0.0100 Da against the reviewed mouse protein database from Uniprot (March 30th 2012, 30419 entries). The amino acid sequence of the internal standard (yeast enolase, Uniprot accession no. P00924) was included in the FASTA file of the database. The following parameters were used: carbamidomethyl-cysteine fixed modification and acetyl Nterm, deamidation of asparagine and glutamine, and oxidation of methionine variable modifications. Scaffold software (version Scaffold 3.6.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm (Keller et al., 2002). Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Only the protein IDs that satisfied the above criteria were accepted, and the corresponding information regarding peptide sequences and protein identification probabilities are provided in the supplementary file (Supp. Table 1). The false positive rate for the identified proteins was calculated to be 0.9% based on the Protein Prophet algorithm in Scaffold. Progenesis LC–MS software V4.0 (Nonlinear Dynamics) was used to quantify protein expression changes. Normalization across samples was based on total ion intensity. Similar proteins were grouped, and the quantitative value was assigned for the one with the highest score. Only proteins with non-conflicting peptide features were quantified.
Proteomics analysis For trypsin digestion of proteins, cells were washed twice with cold 50 mM ammonium bicarbonate and lysed by boiling at 100 ◦ C in UPX Buffer (Protein discovery, USA). Tryptic peptides were generated according to the Filter Aided Sample Preparation Protocol (FASP) (Wisniewski et al., 2009). Briefly, 50 g protein was washed with 6 M urea in a 30 kDa cut-off spin column, alkylated with 10 mM iodoactamide (IAA) in the dark for 20 min at room temperature and trypsinized overnight (1:100 trypsin-to-protein ratio). Peptides were eluted from the column, and their concentration was measured with a Nanodrop spectrometer. A total of 500 ng tryptic peptides were spiked with 50 fmol internal standard (MassPREP Enolase Digestion Standard, Waters, USA) and injected to the LC–MS/MS system. LC–MS/MS analysis and database searches for protein identification were based on our previously published protocol (Hacariz et al., 2012). Briefly, 500 ng tryptic peptides were analyzed by the LC–MS/MS system (nanoACQUITY UPLC column (Waters) and SYNAPT high definition mass spectrometer with a NanoLockSpray ion source (Waters)). Columns were equilibrated with 97% mobile
Flow cytometry analysis BMDCs were harvested from Petri dishes on day 6 and washed with PBS. The phenotype of the cells was evaluated using the following fluorochrome-conjugated antibodies: 7AAD, anti-CD11cPE (BD, USA), anti-Ly6G-PE-Cy5 (Abcam, UK), anti-CD11b-FITC (ebioscience, USA), anti-PDCA-1 (Miltenyi Biotec, Germany), antiMHC-II (Biolegend, USA) and relevant isotype controls. Cells were stained with labeled antibodies at 4 ◦ C for 20 min or 10 min for 7AAD, washed with cold PBS and then re-suspended in PBS. Data was acquired with FACScan and FACSCanto II instruments (BD Biosciences, USA) and analyzed using BD CellQuest and BD FACSDiva.
Statistical analysis Differences between the groups were analyzed by one-way ANOVA (Tukey’s test) using SPSS 15.0 (IBM, USA). Values were considered statistically significant when p < 0.05.
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Fig. 1. Effect of HBeAg on BMDC generation. The frequency of CD11c+ cells was determined in HBeAg-treated groups. HBcAg-treated, OVA-treated and the untreated group were used as controls. HBeAg decreased the generation of BMDCs. The means ± SE of at least three independent experiments are shown (*p < 0.05, **p < 0.001).
Fig. 3. Cell viability of BMDC after treatment with 5 g/ml HBeAg for 24 h and 48 h. HBeAg was non-cytotoxic to BMDC, as determined by WST-1 assay. Data represents the mean ± SD of three independent experiments (n = 4).
control and HBeAg-treated cells was 96 ± 1% and 94 ± 2%, respectively. In the second protocol, BMDCs were treated with HBeAg for 24 h and 48 h after generation. DC viability was determined by WST-1 assay (Fig. 3); HBeAg did not have any cytotoxic effect on BMDCs. Thus, HBeAg did not reveal any cytotoxicity on DCs even at the highest concentration used (5 g/ml). Effect of HBeAg on protein expression level
Fig. 2. The CD11b+ Ly6G+ cell population during BMDC differentiation. Untreated cells and cells treated with HBeAg (5 g/ml) were collected on day 0, 3 and 6. FACS showed that the percentage of CD11b+ Ly6G+ cells was affected by the presence of HBeAg. The numbers on contour plots represent the means ± SE of at least three independent experiments.
Results HBeAg downregulates DC generation To determine the effect of HBeAg on the generation of BMDCs, three different HBeAg concentrations (5, 0.5, 0.05 g/ml) were added to the culture medium. HBcAg (5 g/ml), OVA (5 g/ml) and non-supplemented medium were used as controls. On day 6, the frequency of CD11c+ DCs was decreased proportionally to the concentration of HBeAg compared with the HBcAg, OVA and no antigen control groups (Fig. 1). Furthermore, CD11b+ Ly6G+ immature myeloid cell frequency was analyzed during DC generation. The percentage of CD11b+ Ly6G+ cells was analyzed on days 0, 3 and 6 after 5 g/ml HBeAg treatment. The ratio of CD11b+ Ly6G+ immature myeloid cells in the total cell population increased in comparison to the untreated control group (Fig. 2). Investigation of the HBeAg cytotoxic effect Two different protocols were used to distinguish whether the diminished generation of BMDCs was due to HBeAg (5 g/ml) cytotoxicity. First, a BMDC generation protocol was used in the presence and absence of HBeAg. At day 6, cell viability was monitored using the 7-amino-actinomycin D (7-AAD) stain, which penetrates nonviable cell membranes and stains DNA. There was no significant difference in the viability of cells in either group. The viability of the
To evaluate the molecular pathways in DC generation affected by HBeAg treatment, we determine global proteomic changes by nLC–MS/MS analysis. Cells were either treated with HBeAg or with a vehicle control and were collected at day 6. A total of 464 proteins were identified, and 47 of these were differentially regulated with a 40% cut-off (p < 0.05). Table 1 shows the complete list of differentially regulated proteins. Four of these 47 proteins (H-2 class II histocompatibility antigen A-D beta chain, H-2 class II histocompatibility antigen A-S alpha chain, ganglioside GM2 activator, cathepsin S) were directly related to DC generation and function. The MHC II protein complex is important for antigen presentation on DCs, and its chains were downregulated in the HBeAg-treated group (Table 1). Ganglioside GM2 activator protein, which has an immunosuppressive role during the differentiation of monocytes to DCs, was also downregulated (Wright et al., 2005). Cathepsin S, which regulates MHC II–peptide complex formation and expression, was downregulated by HBeAg (Magister et al., 2012). Additionally, the expression levels of S100A8 and S100A9, Ca+2 binding proteins, were increased in the presence of HBeAg (Table 1). Ingenuity Pathway Analysis of differentially regulated proteins To understand which molecular pathways and their associated proteins were affected by the differentially regulated proteins detected in this study, an Ingenuity Pathway Analysis (IPA, Spring 2013 version) was performed. IPA is knowledge-based software that models and analyzes complex biological and chemical systems; it relies on a repository of expertly curated biological interactions and functional annotations based on relationships between proteins, genes, complexes, cells, tissues, drugs, and diseases. As a result of our IPA analyses, the top biological function associated with HBeAg treatment was “cell-to-cell signaling and interaction” (Supplemental Fig. 1). The category of “cell growth and proliferation” was also prominent, with 23 molecules (p = 6.06 × 10−6 ), whereas the “cellular movement” category was associated with 17 molecules (p = 1.25 × 10−6 ). “Immune cell trafficking” was represented by 12 molecules (p = 1.47 × 10−6 ), indicating that there were HBeAg-dependent changes in host cell-immune system signaling pathways. Furthermore, the “viral
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Table 1 Proteins that were significantly and >40% differentially expressed during DC generation in response to HBeAg. The arrows indicate up- or downregulation. Accession number
Anova (p)
Description
Function
Fold change
DC related proteins P01921; P06342
0.00111064 0.00164758
Antigen processing and presentation of exogenous peptide antigen via MHC class II Peptide antigen binding
↓ 2.1
P14437 Q60648
0.00112813
H-2 class II histocompatibility antigen, A-D beta chain H-2 class II histocompatibility antigen, A-S alpha chain Ganglioside GM2 activator
↓ 2.1
O70370
2.19E−05
Cathepsin S
Binds gangliosides and stimulates ganglioside GM2 degradation Key protease responsible for the removal of the invariant chain from MHC class II molecules
Other proteins P31725 P27005 P68134; P68033; P68135
3.56E−08 1.07E−06 0.03051749
Protein S100-A9 Protein S100-A8 Actin, alpha skeletal muscle
P48036 O89053
0.00709654 0.00989136
Annexin A5 Coronin-1A
P43275
0.02653709
Histone H1.1
Q62465
0.03330329
P08905; P17897
0.00032004
Synaptic vesicle membrane protein VAT-1 homolog Lysozyme C-2
P30044
8.95E−06
Q9FJE8 P10810
0.00308165 0.00035253
Q8R180 P97449 P41216
0.00185244 0.01261818 0.02514271
P41377
0.00770115
Q05144
0.03373887
Q9JII6
0.01096198
Q05816
0.0021051
P02088; P02057; P11758; P02089 Q99P72
0.00926448
Fatty acid-binding protein, epidermal Hemoglobin subunit beta-1
0.0103125
Reticulon-4
P35175; P35173 P62702; P47836 Q9D154
0.02080033 0.0292214 0.03340597
Stefin-1 40S ribosomal protein S4 Leukocyte elastase inhibitor A
Q01768
0.01820937
P09602
4.35E−05
P37040
0.03848945
P47955
0.04781885
Nucleoside diphosphate kinase B Non-histone chromosomal protein HMG-17 NADPH – cytochrome P450 reductase 60S acidic ribosomal protein P1
P10639 P29391; P49945
0.00542218 0.01831099
Thioredoxin Ferritin light chain 1
P12265
0.02857571
Beta-glucuronidase
P68040
0.03456048
P34884
0.00233358
Q9ZPX5; Q8K2B3
0.04841603
P28667 Q9Z183
0.01599274 0.01613393
P54987
0.0237136
Guanine nucleotide-binding protein subunit beta-2-like 1 Macrophage migration inhibitory factor Succinate dehydrogenase [ubiquinone] flavoprotein subunit 2, MARCKS-related protein Protein-arginine deiminase type-4 Immune-responsive gene 1 protein
Peroxiredoxin-5, mitochondrial Probable histone H2A.7 Monocyte differentiation antigen CD14 ERO1-like protein alpha Aminopeptidase N Long-chain-fatty-acid – CoA ligase 1 Eukaryotic initiation factor 4A-2 Ras-related C3 botulinum toxin substrate 2 Alcohol dehydrogenase [NADP(+)]
A calcium- and zinc-binding protein A calcium- and zinc-binding protein Highly conserved proteins that are involved in various types of cell motility An anticoagulant protein May be a crucial component of the cytoskeleton of highly motile cells, Binds to linker DNA between nucleosomes forming the macromolecular structure Plays a part in calcium-regulated keratinocyte activation in epidermal repair mechanisms Lysozymes have primarily a bacteriolytic function Reduces hydrogen peroxide and alkyl hydroperoxides Core component of nucleosome Leading to NF-kappa-B activation, cytokine secretion and the inflammatory response Essential oxidoreductase Broad specificity aminopeptidase Activation of long-chain fatty acids for both synthesis of cellular lipids, and degradation via beta-oxidation ATP-dependent RNA helicase
↓ 2.8
↓ 2.2
↑ 2.9 ↑ 3.6 ↓ 2.8 ↓ 1.5 ↑ 1.7 ↑ 1.5 ↓ 1.4 ↓ 1.8 ↑ 1.8 ↑ 5.2 ↑ 2.9 ↑ 1.6 ↓2 ↑ 1.5
↓ 1.9
Plasma membrane-associated small GTPase
↓ 1.6
Catalyzes the NADPH-dependent reduction of a variety of aromatic and aliphatic aldehydes to their corresponding alcohols High specificity for fatty acids
↓ 1.5
Involved in oxygen transport from the lung to the various peripheral tissues Developmental neurite growth regulatory factor Intracellular thiol proteinase inhibitor Structural constituent of ribosome Regulates the activity of the neutrophil proteases Major role in the synthesis of nucleoside triphosphates other than ATP. Binds to the inner side of the nucleosomal DNA Required for electron transfer from NADP to cytochrome P450 in microsomes Plays an important role in the elongation step of protein synthesis Participates in various redox reactions Stores iron in a soluble, non-toxic, readily available form Plays an important role in the degradation of dermatan and keratan sulfates Involved in the recruitment, assembly and/or regulation of a variety of signaling molecules Pro-inflammatory cytokine
↓ 1.8 ↓ 1.7 ↑ 1.6 ↑2 ↑ 2.1 ↑ 1.5 ↓ 1.5 ↑6 ↑ 1.8 ↓ 1.4 ↓ 1.4 ↑ 1.6 ↓ 1.7 ↓ 1.8 ↓ 1.4
Flavoprotein (FP) subunit of succinate dehydrogenase (SDH)
↓ 1.5
Controls cell movement Catalyzes the citrullination/deimination of arginine residues of proteins 2-methylcitrate dehydratase activity
↑ 4.7 ↑ 4.9 ↑ 2.1
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I. Hatipoglu et al. / Immunobiology 219 (2014) 944–949
Table 1 (Continued) Accession number
Anova (p)
Description
Function
Fold change
P42746
0.00267475
Accepts the ubiquitin from the E1 complex
↑ 34.1
Q9JJW6
0.01273248
Acts as chaperone
↑ 5.5
Q60749
0.00041957
Recruited and tyrosine phosphorylated by several receptor systems
↑ 8.4
P35174 Q8QZT1
9.99E−05 0.00167717
Ubiquitin-conjugating enzyme E2 3 RNA and export factor-binding protein 2 KH domain-containing, RNA-binding, signal transduction-associated protein 1 Stefin-2 Acetyl-CoA acetyltransferase, mitochondrial
intracellular thiol proteinase inhibitor Plays a major role in ketone body metabolism
Infinity ↓ 3.2
Random4313 Random14989
0.03443238 0.00548945
Random Sequence 4313 Random Sequence 14,989
↓ 1.6 ↓ 1.6
Fig. 4. MHC-II expression on PDCA-1-positive cells. pDCs were treated with HBeAg for 4 hours and then stimulated with CpG overnight. HBeAg had no effect on MHC-II expression on PDCA-1-positive cells. The mean fluorescence intensity (MIF) of MHC-II- and PDCA-1-positive cells is indicated. The data represent the mean ± SE of at least three independent experiments.
infections” category contained 16 molecules (p = 4.74 × 10−6 ), confirming that our proteomic analyses identified the cellular response to treatments based on protein expression changes. Effect of HBeAg on pDC stimulation MHC-II expression on PDCA-1-positive cells was examined to investigate the effect of HBeAg on spleen pDC stimulation. PDCA1 is an essential marker of pDCs. pDCs were isolated from spleen tissue and treated sequentially with HBeAg and CpG. HBeAg did not significantly affect MHC-II expression on PDCA-1 positive cells compared with the control group (Fig. 4). Discussion Although HBeAg is not required for virus assembly, infection or replication, it may play a critical role in suppressing immune responses to HBV (Schlicht et al., 1987; Milich and Liang, 2003). HBeAg induces anergy in HBeAg- and HBcAg-specific T cells, inhibits innate immune responses by interacting with TLRs, reduces interferon alpha (IFN␣) production and reduces NF-B activation (Lang et al., 2011; Chen et al., 2005). In this study, we determined the potential effects of HBeAg on DC differentiation and stimulation. The postulated HBV-dependent decrease in DC populations is controversial (van der Molen et al., 2004; Duan et al., 2005). To our knowledge, there has not been a study explaining the HBeAgmediated decrease in DCs. We showed that HBeAg reduced the generation of DCs from bone marrow progenitors, but although HBeAg and HBcAg share a large part of their amino acid sequences, HBcAg did not have the same effect. This result also indicates another functional difference between these two antigens; the decrease of DC generation could be related to the NF-B signaling pathway. HBeAg inhibits NF-B activation by inhibiting the TIR:TIR
interaction in TLR signaling, which essential for GM-CSF-driven DC generation (Visvanathan et al., 2007; Lang et al., 2011; van de Laar et al., 2010; Ouaaz et al., 2002). In contrast, the receptor that may have a role in DC differentiation, TLR-2, is downregulated in the presence of HBeAg. A TLR-2 agonist induces DC generation from human bone marrow CD34+ progenitors (Sioud and Floisand, 2007). We performed nLC–MS/MS-based proteomic analysis to investigate the effects of HBeAg on protein expression during DC generation. The proteomic results confirmed the flow cytometry data, as the expression of some proteins that are important for DC generation and physiology (MHC II complex proteins, cathepsin S, ganglioside GM2 activator protein) were decreased in the presence of HBeAg (Wright et al., 2005; Magister et al., 2012). Moreover, the S100A8 and S100A9 proteins were increased in the presence of HBeAg. These Ca2+ -binding proteins have functions in inflammation, cancer and autoimmune diseases (Vogl et al., 2012; Leukert et al., 2006). The upregulation of S100A9 and S100A8 inhibits DC differentiation (Cheng et al., 2008). Furthermore, we found that in the presence of HBeAg (5 g/ml), the immature myeloid cell population (CD11b+ Ly6G+ ) frequency was approximately threefold higher than the control group. CD11b+ Ly6G+ immature myeloid cells represent different cell populations (Yang et al., 2011). Tumor associated neutrophils (TANs) and myeloid derived suppressor cells (MDSCs) are CD11b+ Ly6G+ , and these cells are potentially immunosuppressive. In contrast, increased S100A9 and S100A8 expression is positively correlated with an increased frequency of MDSCs (Zhao et al., 2012). Thus, HBeAg may induce immune suppression. Virus antigens have the potential to induce MDSCs. For instance, hepatitis C virus (HCV) extracellular core protein induces the expansion of MDSCs, and HIV-positive patients exhibit a higher rate of MDSCs than healthy controls (Tacke et al., 2012; Vollbrecht et al., 2012).pDCs have a vital role in anti-viral immunity because they produce type I IFNs (Colonna et al., 2004). HBeAg-positive patients have limited production of IFN␣ from pDCs, but HBV-induced HLA-DR expression in
I. Hatipoglu et al. / Immunobiology 219 (2014) 944–949
stimulated pDCs is not significant (Woltman et al., 2011). There has been no study investigating the specific effects of HBeAg on MHCII expression. Here, we showed that HBeAg did not significantly affect MHC-II expression on CpG-stimulated and non-stimulated pDCs. In conclusion, our results indicated that HBeAg inhibits BMDC generation. Further clinical studies are needed to investigate the correlation between HBeAg concentration and the ratio of DCs and/or the immature myeloid cell population. Furthermore, the effect of HBeAg on MDSC and TAN generation need clarification. This study illustrated the effect of HBV on the immune system and strengthened the potential for HBeAg as a new therapeutic target in HBeAg-positive chronic HBV patients. Conflict of interest statement The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Acknowledgments This study was supported by grants nos. 108S300, 110S497 and 105G0656 from The Scientific and Technological Research Council of Turkey (TUBITAK). We thank Dr. Betul Guloglu for critically reviewing the manuscript and Duygu OZCELIK for her excellent technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.imbio. 2014.07.020. References Ardavin, C., del Hoyo, G., Martin, P., Anjuere, F., Arias, C., Marin, A., Ruiz, S., Parrillas, V., Hernandez, H., 2001. Origin and differentiation of dendritic cells. Trends Immunol. 22, 691. Banchereau, J., Steinman, R.M., 1998. Dendritic cells and the control of immunity. Nature 392, 245. Boonstra, A., Woltman, A.M., Janssen, H.L., 2008. Immunology of hepatitis B and hepatitis C virus infections. Best Pract. Res. Clin. Gastroenterol. 22, 1049. Chen, M., Sällberg, M., Hughes, J., Jones, J., Guidotti, L.G., Chisari, F.V., Billaud, J.N., Milich, D.R., 2005. Immune tolerance split between hepatitis B virus precore and core proteins. J. Virol. 79, 3016. Cheng, P., Corzo, C.A., Luetteke, N., Yu, B., Nagaraj, S., Bui, M.M., Ortiz, M., Nacken, W., Sorg, C., Vogl, T., Roth, J., Gabrilovich, D.I., 2008. Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein. J. Exp. Med. 205, 2235. Colonna, M., Trinchieri, G., Liu, Y.J., 2004. Plasmacytoid dendritic cells in immunity. Nat. Immunol. 5, 1219. Dienstag, J.L., 2008. Hepatitis B virus infection. N. Engl. J. Med. 359, 1486. Duan, X.Z., Zhuang, H., Wang, M., Li, H.W., Liu, J.C., Wang, F.S., 2005. Decreased numbers and impaired function of circulating dendritic cell subsets in patients with chronic hepatitis B infection (R2). J. Gastroenterol. Hepatol. 20, 234. Gitlin, N., 1997. Hepatitis B: diagnosis, prevention, and treatment. Clin. Chem. 43, 1500. Hacariz, O., Sayers, G., Baykal, A., 2012. A proteomic approach to investigate the distribution and abundance of surface and internal Fasciola hepatica proteins during the chronic stage of natural liver fluke infection in cattle. J. Proteome Res. 11, 3592. Keller, A., Nesvizhskii, A., Kolker, E., Aebersold, R., 2002. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 74, 5383. Lai, C.L., Ratziu, V., Yuen, M.F., Poynard, T., 2003. Viral hepatitis B. Lancet 362, 2089.
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