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Molecular characterization of woodchuck interleukin-10 receptor and enhanced function of specific T cells from chronically infected woodchucks following its blockade
Comparative Immunology, Microbiology and Infectious Diseases 35 (2012) 563–573
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Comparative Immunology, Microbiology and Infectious Diseases journal homepage: www.elsevier.com/locate/cimid
Molecular characterization of woodchuck interleukin-10 receptor and enhanced function of specific T cells from chronically infected woodchucks following its blockade Min Jiang a,d , Jia Liu b , Ejuan Zhang a,b , Zhongji Meng b , Baoju Wang c , Michael Roggendorf b , Dongliang Yang c , Mengji Lu a,b , Yang Xu a,∗ a b c d
Department of Microbiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Institute of Virology, University Hospital of Essen, Essen, Germany Department of Infectious Disease, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Shenzhen Centre for Disease Control and Prevention, Shenzhen, China
a r t i c l e
i n f o
Article history: Received 21 January 2012 Received in revised form 16 June 2012 Accepted 19 June 2012
Keywords: Interleukin 10 receptor Woodchuck
a b s t r a c t Interleukin 10 (IL-10) is a pleiotropic cytokine acting on a variety of immune cells through the cell surface receptor (IL-10R). It has been suggested to resuscitate antiviral immunity by interfering with IL-10/IL-10R pathway. The woodchuck model infected by woodchuck hepatitis virus (WHV) represents an informative animal model to study hepatitis B virus (HBV) infection. In this study, the woodchuck IL-10R (wIL-10R) was molecularly cloned and characterized, showing high similarity of its nucleotide and amino acid sequences to that of other mammalian species. The expression level of wIL-10R mRNA in woodchuck peripheral blood mononuclear cells was significantly increased in acute WHV infection but down-regulated during chronic WHV infection. Specific rabbit antibodies against wIL-10R were prepared and showed the ability to enhance the proliferation and degranulation of specific T-cells from chronically WHV-infected woodchucks in vitro. The present work on wIL-10R provided a good basis for future preclinical studies on therapeutic approaches for chronic HBV infection. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction IL-10 is an anti-inflammatory cytokine with a crucial role in preventing inflammatory cytokine and autoimmune pathologies [1,2]. IL-10 is expressed by many cells of the adaptive immune system, including T regulatory cells (Treg), Th1, Th2, CD8+ cells and B cells. And IL-10 production is also associated with cells of the innate immune
∗ Corresponding author at: Tongji Medical College, Huazhong University of Science and Technology, Hangkong Road 13, 430030 Wuhan, China. Tel.: +86 27 83657829; fax: +86 27 83662894. E-mail addresses: [email protected], [email protected] (Y. Xu). 0147-9571/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cimid.2012.06.003
system, including dendritic cells (DCs), macrophages and natural killer (NK) cells [2]. IL-10 exerts its crucial role as a feedback regulator of diverse immune responses by interacting with the specific IL-10 receptor (IL-10R). IL-10R is expressed on the surface of a variety of cells, especially immune cells. The functional IL-10R consists of a dimmer of the heterodimer of IL-10R1 and IL-10R2 [3]. The IL-10R1 binds IL-10 with a high affinity, and the recruitment of IL-10R2 enables signal transduction [4]. The functions of IL-10 have been studies in a large number of studies. For example, IL-10 suppresses macrophages and DCs function, thereby limiting Th1 and Th2 effector responses [3,5]. IL-10 production is of potential benefit to both the host (preventing autoimmunity) and the pathogen (allowing persistent infection) [6]. It was reported that the up-regulation of
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Fig. 1. (Continued)
IL-10 induced impaired T-cell responses and benefited the persistent infection of lymphocytic choriomeningitis virus (LCMV). In vivo blockade of IL-10R with a neutralizing antibody resulted in rapid resolution of LCMV persistence [7,8]. The results suggested that blockade of IL-10R could break the negative regulatory loop mediated by IL-10, and may represent a new therapeutic strategy to treat chronic viral infection. According to World Health Organization, more than two billion people have been infected with hepatitis B virus (HBV) and 350 million suffer from chronic HBV infection [9]. Currently two types of antiviral therapies are approved for clinical use: pegylated interferon alpha 2a (PEG-IFN␣) and nucleos(t)ide analogs (NA) [10]. However, only one third of patients treated with PEG-IFN-␣ have a sustained antiviral response. On the other hand, the therapy with NA significantly suppresses HBV replication. But the rebound of viremia occurs after withdrawal of NA and drug-resistant mutants frequently arise with long-term treatment [11]. Therefore, new therapeutic approaches for the treatment of chronic hepatitis B (CHB) are intensely required. In CHB, one of the defining characteristics is the loss or functional inactivation of antiviral effector T cells [12]. The increased production of IL-10 was observed in patients with CHB [13], which hints that blockade of IL-10R might become a feasible therapeutic approach for CHB. Woodchuck (Marmota monax) is the natural host of woodchuck hepatitis virus (WHV) and represents a highly valuable animal model to investigate immune response, pathogenesis,
and therapy of HBV infection [11]. In the present work, we cloned and aligned the complete coding sequence of woodchuck IL-10R (wIL-10R). The extracellular region of wIL-10R (wIL-10Re) was expressed and purified. The blockade of IL-10R with the prepared antibody against wIL-10Re in vitro could enhance the proliferation and degranulation of WHV-specific T-cells from chronically WHV-infected woodchucks.
2. Materials and methods 2.1. Animals and cell cultures Woodchucks were purchased from Northwest Wildlife (Ithaca, NY) and maintained in the animal facilities of Tongji Medical College and University Hospital of Essen, in accordance with the Guide for the Care and Use of Laboratory Animals. The peripheral blood mononuclear cells (PBMCs) of woodchuck were separated by Ficoll-Paque (Pharmacia, Freiburg, Germany) centrifugation and resuspended in RPMI 1640 medium (Gibco BRL, Neu-Isenburg, Germany) supplemented with 10% fetal calf serum (FCS) (Gibco). PBMCs were cultured at a density of 106 cells per ml in flat-bottom 24-well microtiter plates (Falcon, Becton Dickinson, NJ) at 37 ◦ C and 5% CO2 [14]. Baby hamster kidney (BHK) cells (provided by American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% FCS.
Fig. 1. Alignment and phylogenetic tree analysis of wIL-10R sequences. The GenBank accession numbers of the sequences used in the study: woodchuck (JN122322), human (BC028082), chimpanzee (XM 508785), horse (XM 001917543), cattle (NM 001205757), dog (XM 848380), mouse (L12120), rat (NM 057193). (A) Alignment of the deduced aa sequence of wIL-10R with homologues from different species by ‘MCLONE’ software. Dots resemble conserved amino acids and gaps (−) were inserted to maximize alignment. In ECD, three potential glycosylation sites (NQS, NVT and NWT) and two cystein residues (C203 and C224) are indicated in bold and boxes. In ICD, highly conserved GYXXQ sequences (GYLKQ and GYVKQ) are indicated in bold and underlines. (B) The predicted secondary structure of wIL-10R. The precursor of wIL-10R consists of a 18 aa signal peptide and a 558 aa mature protein, which was composed of ECD, TM and ICD. (C) The phylogenetic tree based on the nucleotide sequences of wIL-10R and different mammalian species. The phylogenetic tree was designed in the software ClustalW.
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Table 1 Primers used for cloning in the present study. Primers
Position of 5 base
Nucleotide sequences
Referencea
IL-10R-P1 IL-10R-P2 IL-10R-P3 IL-10R-P4 IL-10R-P5 IL-10R-P6 IL-10R-P7 IL-10R-P8
38 1854 57 713 1 1728 588 708
5 -atg ctg tcg cgc ctg gta gt-3 5 -gcc cag gtc act cat tcg ag-3 5 -cgg ggt accb gaa ctg cct agc cct cca-3 5 -ccg gaa ttc tca ata ctg ctt ggt gag gac-3 5 -ccg gaa ttc atg ctg tcg cgc ctg gta gt-3 5 -acg cgt cga cct cat tcg agt gca ggc tgg-3 5 -agg aga ggt ggg aaa gtt-3 5 -gtt ggt cgc agt gaa ata c-3
L12120 L12120 JN122322 JN122322 JN122322 JN122322 JN122322 JN122322
a b
Reference: the GenBank accession numbers of the used reference sequences are given. The 5 extensions were KpnI, EcoRI and SalI restriction sites and are indicted as italic letters.
2.2. Cloning and alignment of woodchuck IL-10R sequence cDNA fragment coding for the full length of wIL-10R (1731 base pair, bp) was amplified by reverse transcriptionpolymerase chain reaction (RT-PCR) with the primers of IL-10R-P1 and -P2 (Table 1), which were designed according to the aligned sequences and chosen from regions conserved among human and other mammalian species. For RT-PCR, total RNA was prepared from woodchuck PBMCs with TRIzol reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. The PCR product was cloned into pMD18-T TA vector (TaKaRa, Dalian, China), named as pMD18-wIL-10R, and sequenced by commercial services (Yingjun Biotechnology Company, Shanghai, China). The phylogenetic analysis of wIL-10R was performed online with the software ClustalW at the website of European Bioinformatics Institute (http://www.ebi.ac.uk). The deduced amino acid (aa) sequence of wIL-10R was aligned with homologues from different species by ‘MCLONE’ software. And the secondary structure of wIL-10R residues was predicted by an online service of Swiss Institute of Bioinformatics (http://swissmodel.expasy.org).
2.3. Expression of recombinant wIL-10R protein in E. coli and BHK cells The coding sequence of the extracellular region of wIL10R (wIL-10Re, nucleotide 57-713) was amplified by PCR from pMD18-IL-10R with the primers of IL-10R-P3 and -P4 (Table 1), and subcloned into the prokaryotic expression vector of pET30a (Merck, Germany) at KpnI and EcoRI sites resulting in the plasmid of pET30a-wIL-10Re, which express the extracellular region of wIL-10R fused with His tag (His-wIL-10Re). The recombinant protein of His-wIL-10Re expressed in E. coli BL21 (DE plus) was purified with HisTrap Kit (Amersham, Braunschweig, Germany) under denaturation conditions according to the manufacturer’s instructions and analyzed by 15% SDS-PAGE. The full length of wIL-10R was amplified by PCR from pMD18-wIL-10R with the primers of IL-10R-P5 and -P6 (Table 1), and subcloned into the mammalian expression vector of pXF3H (kindly provided by Prof. Xinghua Feng) resulting in the plasmid of pXF3H-wIL-10R. The eukaryotic expression of the full length of wIL-10R fused with hemagglutinin (HA) tag was under the control of cytomegalovirus
immediate-early promoter [15]. The transient transfection of pXF3H-wIL-10R in BHK cells was performed by using Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) as described previously [16].
2.4. Generation of polyclonal antibody against the extracellular region of wIL-10R (anti-wIL-10R) and determination of the antibody titer by ELISA New Zealand white rabbits were immunized by subcutaneous injection with 200 g of purified His-wIL-10Re in Freund’s incomplete adjuvant at 2 weeks interval. The antiserum was collected 14 days after the third immunization. The antibody titer of anti-wIL-10R was measured by indirect enzyme-linked immunosorbent analysis (ELISA). His-wIL-10Re was diluted to 1 g/ml, and coated on plates at 100 l aliquot per well in 96-well immunoplate at 4 ◦ C overnight. The coated wells were blocked with 100 l of 10% FCS for 2 h at 37 ◦ C, and then incubated with 100 l of anti-wIL-10R with serial dilutions of 1:2 (from 1:16,000 to 1:1,024,000). The bound anti-wIL-10R was detected by HRP-conjugated goat-anti-rabbit IgG (dilution 1:1000, DB Biosciences, CA). The serum from the pre-immune rabbit was used as a negative control. The development of color was read at 490 nm. The cut-off value was set as 3 times over the negative control. The titers of anti-wIL-10R were calculated by extrapolation of ELISA values of serially diluted samples and corresponded to the reciprocal values of the highest dilutions that were regarded as positive.
2.5. Immunofluorescence (IF) staining of wIL-10R expressed in BHK cells and on woodchuck PBMCs To test the recognition of wIL-10R protein by the prepared anti-wIL-10R, IF staining was performed in transiently transfected BHK cells with pXF3H-wIL-10R. Forty eight hours after transfection, BHK cells were fixed with 50% methanol and stained with the prepared anti-wIL10R (dilution 1:100) and a fluorescein isothiocyanate (FITC) labeled goat anti-rabbit IgG (dilution 1:200, DAKO, Hamburg, Germany) [15]. The serum from the pre-immune rabbit was used as a negative control. To detect the expression level of wIL-10R on woodchuck PBMCs, PBMCs were plated on microscope slides at a density of 105 cells per slide, and fixed with 50% methanol. Cells were stained with the prepared
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Fig. 2. Expression of His-wIL-10Re fusion protein and determination of the titer of anti-wIL-10R by ELISA. (A) Expression of His-wIL-10Re in E. coli was analyzed by 15% SDS-PAGE. M: protein MW standards; lane 1: the whole lysate of the transformed E. coli without IPTG induction; lanes 2 and 3: the whole lysate of the transformed E. coli with 2 mM of IPTG induction for 2 and 4 h, respectively. (B) The purified wIL-10Re was analyzed by 15% SDS-PAGE. Lane 1: the unpurified protein; lanes 2 and 3: the purified His-wIL-10Re. (C) The antibody titer of the prepared anti-wIL-10R was determined as 1:512,000 by ELISA.
anti-wIL-10R (dilution 1:40) and the FITC labeled goat anti-rabbit IgG (dilution 1:200, DAKO).
2.6. Treatment of woodchuck PBMCs by TLR ligands and detection of wIL-10R expression on PBMCs by real time RT-PCR Woodchuck PBMCs were treated with the ligands of TLR3 (poly I:C, 12.5 g/ml), TLR7 (imiquimod, 10 g/ml), TLR9 (CpG ODN, 12.5 g/ml) by transfection with Lipofectamin 2000, and direct administration of TLR1/2 (Pam3Cysk4, 2 g/ml), TLR2/6 (Pam2Cysk4, 2 g/ml), TLR4 (lipopolysaccharid, 12.5 g/ml), TLR5 (flagellin, 10 g/ml) and woodchuck IFN-␣ and -␥ (500 U/ml) for 6 h. TLR19 ligands were purchased from Invitrogen (Karlsruhe, Germany). Woodchuck IFN-␣ and IFN-␥ were produced by eukaryotic transfection.
Total RNA was extracted from woodchuck PBMCs with TRIzol (Invitrogen) as described previously [17]. The mRNA expression of wIL-10R was quantified by real time RTPCR with primers of IL-10R-P7 and -P8 (Table 1), using QuantiFast SYBR Green RT-PCR Kit (Invitrogen) on a Light CyclerTM (Roche Diagnostics, Mannheim, Germany). All samples were analyzed in triplicate. The copy number of woodchuck -actin mRNA was determined for normalization [18].
2.7. Detection of WHV DNA in serum WHV DNAs in woodchuck sera were extracted with a QIAamp DNA blood kit (Qiagen Düsseldorf, Germany) and quantified by real time PCR with primers of wc1 and wc149s, using DNA Master SYBR Green Kit (Roche) on a Light CyclerTM [16].
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Fig. 3. Detection of wIL-10R with the prepared anti-wIL-10R by IF staining. (A) BHK cells were transiently transfected with pXF3H-wIL-10R. Transfected BHK cells were stained by pre-immune rabbit serum (a) or anti-wIL-10R (b). (B) Woodchuck PBMCs were stained by pre-immune rabbit serum (a) or anti-wIL-10R (b). The positively stained PBMCs are indicated by arrows.
2.8. Proliferation assay and CD107a degranulation assay of woodchuck PBMCs after blockade of wIL-10R by the prepared anti-wIL-10R Triplicates samples of PBMCs from naïve and chronically WHV-infected woodchucks were cultured at a density of 5 × 104 per well in 96-well microtiter plates (Falcon), and treated with different dilutions of the prepared anti-wIL10R (5, 10, 20 g/ml) and WHcAg at a final concentration of 1 g/ml for 5 days. The pre-immune rabbit serum was included as a negative control. Woodchuck PBMCs were labeled with 1 Ci of [2-3 H] adenine (Amersham, Braunschweig, Germany) for 18 h and collected by a cell harvester (Packard Instrument Company, Calif). The incorporation of [2-3 H] adenine into proliferating cells was read by a Top counter (Packard Instrument). The results for triplicate cultures are presented as the mean stimulation index (SI) and SI is calculated with the formula: (stimulated cpm-blank cpm)/(unstimulated cpm-blank cpm). SIs greater than 2 were considered as positive [16]. In CD107a degranulation assay, PBMCs from chronically infected woodchucks were cultured at a density of 1 × 106 per well in 96-well microtiter plates, and incubated with 20 g/ml of anti-wIL-10R for 2 days. A WHcAgderived peptide (aa 96–110, 2 g/ml), which was identified as a CD8 epitope, was used for stimulation. The FITCconjugated anti-mouse CD107a antibody (dilution 1:100,
BDPharmingen, Heidelberg, Germany) was added to the stimulated cells. Due to the lack of available anti-CD8 antibody, woodchuck PBMCs were stained with cross-reactive antibodies to CD3 and CD4. The CD3+ CD4− lymphocytes were gated and regarded as mainly CD8+ cells, with minor fractions of CD3+ CD4− CD8− cells. Data were acquired with a FACS-Calibur flow cytometer (Becton, Dickinson, Heidelberg, Germany), and analyzed with FlowJo software (Tree Star, Ashland, Oregon) [19]. 3. Results 3.1. Molecular cloning and characterization of wIL-10R The complete coding region of wIL-10R (1731 bp) was amplified by RT-PCR, and showed high similarity to the IL-10R sequences of other mammalian species, ranging from 69.67% to 81.11% (Table 2). The deduced aa sequence of wIL-10R has a length of 576 residues, and showed high homology to other mammalian species, ranging from 56.08% to 71.53% (Table 2). The putative precursor of wIL10R consists of a signal peptide with 18 aa residues and a mature protein with 558 aa residues, as inferred from sequences of other species (Fig. 1A and B). Like mouse IL-10R, wIL-10R may be glycosylated at three potential sites: NQS, NVT and NWT in the extracellular domain (ECD, aa 19–237) (Fig. 1A) [20]. Two cysteine residues
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Fig. 4. Detection of wIL-10R expressions on PBMCs by real time RT-PCR. (A) The expression levels of wIL-10R on PBMCs from naïve woodchucks were up-regulated by the stimulations of different TLR ligands. (B) In the acutely WHV-infected woodchuck (WH70093), wIL-10R expression on PBMCs fluctuated in the correlation with viral loads during viremic phase. (C) The wIL-10R expression levels in naïve and chronically WHV-infected woodchucks were 4852 and 3246 copies/105 w-actin on average, respectively. wIL-10R expression was suppressed in chronically infected woodchucks. The statistical significance of the results was given as P values (P < 0.05).
of C203 and C224 in ECD of wIL-10R, which form an intramolecular disulfide bridge linking -strands [21], are highly conserved in the identified IL-10R proteins. The transmembrane region (TM, aa 238–261) consists of a stretched hydrophobic segment. Within the intracellular
Table 2 Homology of woodchuck IL-10R to the respective sequences of other species on the nucleotide residues and amino acid level.
Human Chimpanzee Horse Cattle Dog Mouse Rat
Nucleotide residues (%)
Amino acid (%)
81.11 80.82 80.24 78.57 77.24 70.60 69.67
71.53 71.01 68.92 64.54 64.75 56.42 56.08
domain (ICD, aa 262–576) several residues of wIL-10R are highly conserved, which are implicated in receptor function. For instance, two redundant GYXXQ sequences (GYLKQ and GYVKQ) are required for the recruitment of STAT3 [22] (Fig. 1A). The phylogenetic tree analysis of IL10R sequences showed the close genetic distance among species, indicating the conservation of IL-10R during the evolution (Fig. 1C). 3.2. Generation of the neutralizing antibody to wIL-10R The recombinant protein of His-wIL-10Re containing the extracellular region of wIL-10R fused with His tag was produced. The purified His-wIL-10Re with the expected molecular weight (MW) of 27 kDa was detected by 15% SDS-PAGE (Fig. 2B). Rabbits were immunized with the purified His-wIL-10Re to generate the polyclonal antibody of
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Fig. 5. Blockade of the IL-10/IL-10R pathway by the specific anti-wIL-10R antibody enhanced the proliferation of specific T-cells from chronically WHVinfected woodchucks in vitro. PBMCs from naïve (A) or chronically WHV-infected (B) woodchucks were incubated with different dilutions of anti-wIL-10R and pre-immune rabbit serum as a negative control in the presence or absence of WHcAg in vitro for 5 days. Proliferation of PBMCs was measured by [2-3 H] adenine incorporation. PBMCs from two chronically WHV-infected woodchucks WH35034 and WH41321 showed enhanced proliferation.
anti-wIL-10R, which was tested at different dilutions for the reactivity with His-wIL-10Re in ELISA. The titer of antiwIL-10R was determined as 1:512,000 (Fig. 2C). The specificity of anti-wIL-10R was proven by IF staining, in which the transfected BHK cells expressing the full length wIL-10R protein showed positively stained (Fig. 3A). The expression of wIL-10R on woodchuck PBMCs were detected by IF staining with anti-wIL-10R. A small portion of woodchuck PBMCs was strongly positively stained (Fig. 3B), consistent with the knowledge that IL-10R is expressed on peripheral blood cells.
and -␥. The transcripts of wIL-10R were detected by real time RT-PCR, in which a fragment of 293 bp (nt 588–708) was amplified. The base line expression of wIL-10R on unstimulated PBMC was about 2251 copies/105 w-actin on average. The expression of wIL-10R was up-regulated by TLR3 and TLR7 (6 fold) ligands, TLR2 and TLR4 (2.8 fold) ligands, and TLR5 (1.6 fold) ligand. However, IFN-␣ and IFN-␥ had no effect on wIL-10R expression (Fig. 4A).
3.3. Induction of wIL-10R expression on PBMCs by TLR ligands
To establish the relationship to WHV infection, wIL10R expression on PBMCs in naïve and WHV-infected woodchucks was examined by real time RT-PCR. In acutely WHV-infected woodchucks (n = 4), wIL-10R expression highly increased during the viremic phase and returned to the baseline after viral clearance. We showed a representative result in Fig. 4B. wIL-10R expression in
PBMCs from naïve woodchucks were treated with TLR ligands or IFNs: TLR1/2 (Pam3Cysk4), TLR2/6 (Pam2Cysk4), TLR3 (poly I:C), TLR4 (LPS), TLR5 (flagellin), TLR7 (imiquimod), TLR9 (CpG ODN), woodchuck IFN-␣
3.4. wIL-10R expression on PBMCs in naïve, acutely or chronically WHV-infected woodchucks
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Fig. 6. Blockade of the IL-10/IL-10R pathway by the specific anti-wIL-10R antibody improved the degranulation of specific T-cells from chronically WHVinfected woodchucks in vitro. PBMCs from chronically infected woodchucks were treated with anti-wIL-10R in the presence of WHcAg derived peptide in vitro for 2 days. The degranulation of specific T-cells was detected by CD107a staining. Control antibody (ab) is from normal rabbit serum. In the chronically infected woodchucks: WH35034 (A) and WH41321 (B), the frequencies of CD107a+ CD3+ CD4− T cells were increased, indicating an improved degranulation activity.
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naïve woodchucks (n = 9) was 4852 copies/105 w-actin on average. In chronically WHV-infected woodchucks (n = 9), wIL-10R expression at a value of 3246 copies/105 w-actin on average, was apparently suppressed and lower than those in naïve woodchucks (P < 0.05) (Fig. 4C). 3.5. Enhancement of proliferation and degranulation of specific T-cells by blockade of wIL-10R The recent reports demonstrated that blockade of IL10R may restore T-cell functions in persistently infected individuals [7,8]. In this study, we addressed whether in vitro blockade of wIL-10R by neutralizing antibody of anti-wIL-10R may enhance T-cell functions in chronic WHV infection. Fresh PBMCs were prepared from woodchucks including naïve (n = 4) and chronically WHVinfected animals (n = 4), and stimulated with WHcAg or WHcAg-derived peptides in the presence or absence of anti-wIL-10R. The proliferation and CD107a degranulation of specific T-cells were determined. The stimulation fold change post blockade was calculated and the fold change more than 2 was regarded as a positive result. PBMCs from naïve woodchucks did not show any response to WHcAg with or without anti-wIL-10R (Fig. 5A). In 2 of 4 chronically infected woodchucks (WH35034 and WH41321), the proliferations of WHcAg-specific T-cells were enhanced in the presence of different concentrations of anti-wIL-10R (Fig. 5B). Furthermore, WHcAg-specific CD3+ CD4− T cells from 2 of 4 chronically infected woodchucks (WH35034 and WH41321) showed a improved CD107a degranulation in the presence of anti-wIL-10R and WHcAg peptide (Fig. 6A and B). A 3.9-fold increment of the frequency of CD107a+ CD3+ CD4− T cells was measured in WH35034 (Fig. 6A). 4. Discussion Virus-specific CD4+ and CD8+ T cells become functionally unresponsive during persistent viral infections by human immunodeficiency virus (HIV), HBV and hepatitis C virus (HCV), indicating that conserved mechanisms of immunosuppression may downregulate T-cell activity [7]. The levels of systemic IL-10 production were upregulated during chronic HIV, HBV and HCV infections [13,23–27]. It is likely that IL-10-induced immunosuppression is active during these persistent infections. Treg mediate their regulatory function in vivo through IL-10 [5,28]. Treg are possibly involved in the mechanism of HBV persistence. It was reported that Treg accumulated in the liver during chronic severe HBV infection and the frequency of Treg correlated with viral load in CHB [29]. Besides the immunosuppression of specific T-cell response, IL-10 plays an important role in the induction of immune tolerance in liver. Liver DCs generated more suppressive Treg and IL4-producing Th2 cells via an IL-10-dependent mechanism [30]. Liver Kupffer cells (KCs) release substantial amounts of IL-10 during the induction of Treg-mediated tolerance [31]. In addition, IL-10 down-regulated T-cell activation through liver sinusoidal endothelial cells (LSEC) [32]. In vivo
blockade of IL-10R with a neutralizing antibody resulted in extremely low viral load and enhancement of IFN-␥ secreting CD8+ T cells and specific memory T cell response in LCMV clone-13 persistently infected mice [8]. It is likely that neutralization of IL-10 activity may boost antiviral immune responses, restore T-cell function, and enhance the treatment of the persistent infections. However, we should notice that IL-10 as a negatively regulatory cytokine drives the generation of Treg and downregulates a pathological immune response [33,34]. The blockade of IL-10 signaling might lead the overexpression of inflammatory cytokines and induce autoimmune diseases. Therefore, the blockade of IL-10R as a therapeutic approach in chronic viral infection needs to be careful and more balanced [5,35]. In our study, the nucleotide sequence of wIL-10R presented close genetic distance to human IL-10R. The critical residues of wIL-10R are highly conserved for the receptor function. The expression of wIL-10R was detectable on a small portion of PBMCs from naïve woodchucks, and the expression level of wIL-10R could be up-regulated by various TLR ligands. In acutely WHVinfected woodchucks the expression levels of wIL-10R fluctuated, correlating with the viral load and the activation of T-cell responses. However, in chronically WHV-infected woodchucks the expressions of wIL-10R were downregulated and lower than those in naïve woodchucks. The blockade of wIL-10R with the neutralizing antibody of anti-wIL-10R in vitro could enhance the proliferation of WHV-specific T-cells and improve the degranulation of WHV-specific T-cells from chronically infected woodchucks. However, not every chronic woodchucks was responsive to the blockade, which indicated that the wIL-10/IL10R system may be not the only cause for low T-cell responses in chronic WHV infection. Our previous study showed that the programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) system played a role in the negative regulation of T-cell functions in chronic HBV infection [36]. Recent publications indicated that the expression of PDL1 and PD-L2 on human macrophages is differentially modulated by IL-10. IL-10 induced up-regulation of PDL1 expression but not of PD-L2, and blockade of IL-10 enhanced only PD-L2 expression [37]. Therefore, the combined approaches of antibodies against such inhibitory molecules with antiviral drugs would be useful to restore T-cell functions in chronically infected individuals. Here we found that in vitro blockade of the wIL-10/IL-10R pathway has the potence to enhance specific T-cell functions for a short time. Future experiments in vivo will clarify whether blockade of the IL-10/IL-10R signaling may break immune tolerance and restore antiviral immune responses in chronic HBV infection. Our work provided a basis for further studies to investigate immunotherapeutic approaches based on IL-10R in CHB.
Conflict of interest statement The authors declare that they have no conflict of interest related to the manuscript.
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Acknowledgements We thank Thekla Kemper for excellent technical assistance. This work was sponsored by the Science and Technology Project of Wuhan (201052399653), the Fundamental Research Funds for the Central Universities (HUST: 2012QN140) to Y. Xu and grants of Deutsche Forschungsgemeinschaft to M. Lu (GRK1045/2 and Transregio TRR60). References [1] Ouyang W, Rutz S, Crellin NK, Valdez PA, Hymowitz SG. Regulation and functions of the IL-10 family of cytokines in inflammation and disease. Annual Review of Immunology 2011;29:71–109. [2] Saraiva M, O’Garra A. The regulation of IL-10 production by immune cells. Nature Reviews Immunology 2010;10:170–81. [3] Moore KW, de Waal Malefyt R, Coffman RL, O’Garra A. Interleukin10 and the interleukin-10 receptor. Annual Review of Immunology 2001;19:683–765. [4] Mosser DM, Zhang X. Interleukin-10: new perspectives on an old cytokine. Immunological Reviews 2008;226:205–18. [5] Hawrylowicz CM, O’Garra A. Potential role of interleukin-10secreting regulatory T cells in allergy and asthma. Nature Reviews Immunology 2005;5:271–83. [6] Couper KN, Blount DG, Riley EM. IL-10: the master regulator of immunity to infection. Journal of Immunology 2008;180:5771–7. [7] Brooks DG, Trifilo MJ, Edelmann KH, Teyton L, McGavern DB, Oldstone MB. Interleukin-10 determines viral clearance or persistence in vivo. Nature Medicine 2006;12:1301–9. [8] Ejrnaes M, Filippi CM, Martinic MM, Ling EM, Togher LM, Crotty S, et al. Resolution of a chronic viral infection after interleukin-10 receptor blockade. Journal of Experimental Medicine 2006;203:2461–72. [9] Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection. Nature Reviews Immunology 2005;5:215–29. [10] Dienstag JL. Hepatitis B virus infection. New England Journal of Medicine 2008;359:1486–500. [11] Lu M, Menne S, Yang D, Xu Y, Roggendorf M. Immunomodulation as an option for the treatment of chronic hepatitis B virus infection: preclinical studies in the woodchuck model. Expert Opinion on Investigational Drugs 2007;16:787–801. [12] Guidotti LG, Chisari FV. Immunobiology and pathogenesis of viral hepatitis. Annual Review of Pathology 2006;1:23–61. [13] Rico MA, Quiroga JA, Subira D, Castanon S, Esteban JM, Pardo M, et al. Hepatitis B. virus-specific T-cell proliferation and cytokine secretion in chronic hepatitis B e antibody-positive patients treated with ribavirin and interferon alpha. Hepatology 2001;33:295–300. [14] Menne S, Maschke J, Lu M, Grosse-Wilde H, Roggendorf M. T-cell response to woodchuck hepatitis virus (WHV) antigens during acute self-limited WHV infection and convalescence and after viral challenge. Journal of Virology 1998;72:6083–91. [15] Tian Y, Xu Y, Zhang Z, Meng Z, Qin L, Lu M, et al. The amino acid residues at positions 120 to 123 are crucial for the antigenicity of hepatitis B surface antigen. Journal of Clinical Microbiology 2007;45:2971–8. [16] Lu M, Isogawa M, Xu Y, Hilken G. Immunization with the gene expressing woodchuck hepatitis virus nucleocapsid protein fused to cytotoxic-T-lymphocyte-associated antigen 4 leads to enhanced specific immune responses in mice and woodchucks. Journal of Virology 2005;79:6368–76. [17] Liu J, Hu B, Yang Y, Ma Z, Yu Y, Liu S, et al. A new splice variant of the major subunit of human asialoglycoprotein receptor encodes a secreted form in hepatocytes. PLoS One 2010;5:e12934. [18] Zhang X, Meng Z, Qiu S, Xu Y, Yang D, Schlaak JF, et al. Lipopolysaccharide-induced innate immune responses in primary hepatocytes downregulates woodchuck hepatitis virus replication via interferon-independent pathways. Cellular Microbiology 2009;11:1624–37.
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Contents lists available at SciVerse ScienceDirect
Comparative Immunology, Microbiology and Infectious Diseases journal homepage: www.elsevier.com/locate/cimid
Molecular characterization of woodchuck interleukin-10 receptor and enhanced function of specific T cells from chronically infected woodchucks following its blockade Min Jiang a,d , Jia Liu b , Ejuan Zhang a,b , Zhongji Meng b , Baoju Wang c , Michael Roggendorf b , Dongliang Yang c , Mengji Lu a,b , Yang Xu a,∗ a b c d
Department of Microbiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Institute of Virology, University Hospital of Essen, Essen, Germany Department of Infectious Disease, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Shenzhen Centre for Disease Control and Prevention, Shenzhen, China
a r t i c l e
i n f o
Article history: Received 21 January 2012 Received in revised form 16 June 2012 Accepted 19 June 2012
Keywords: Interleukin 10 receptor Woodchuck
a b s t r a c t Interleukin 10 (IL-10) is a pleiotropic cytokine acting on a variety of immune cells through the cell surface receptor (IL-10R). It has been suggested to resuscitate antiviral immunity by interfering with IL-10/IL-10R pathway. The woodchuck model infected by woodchuck hepatitis virus (WHV) represents an informative animal model to study hepatitis B virus (HBV) infection. In this study, the woodchuck IL-10R (wIL-10R) was molecularly cloned and characterized, showing high similarity of its nucleotide and amino acid sequences to that of other mammalian species. The expression level of wIL-10R mRNA in woodchuck peripheral blood mononuclear cells was significantly increased in acute WHV infection but down-regulated during chronic WHV infection. Specific rabbit antibodies against wIL-10R were prepared and showed the ability to enhance the proliferation and degranulation of specific T-cells from chronically WHV-infected woodchucks in vitro. The present work on wIL-10R provided a good basis for future preclinical studies on therapeutic approaches for chronic HBV infection. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction IL-10 is an anti-inflammatory cytokine with a crucial role in preventing inflammatory cytokine and autoimmune pathologies [1,2]. IL-10 is expressed by many cells of the adaptive immune system, including T regulatory cells (Treg), Th1, Th2, CD8+ cells and B cells. And IL-10 production is also associated with cells of the innate immune
∗ Corresponding author at: Tongji Medical College, Huazhong University of Science and Technology, Hangkong Road 13, 430030 Wuhan, China. Tel.: +86 27 83657829; fax: +86 27 83662894. E-mail addresses: [email protected], [email protected] (Y. Xu). 0147-9571/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cimid.2012.06.003
system, including dendritic cells (DCs), macrophages and natural killer (NK) cells [2]. IL-10 exerts its crucial role as a feedback regulator of diverse immune responses by interacting with the specific IL-10 receptor (IL-10R). IL-10R is expressed on the surface of a variety of cells, especially immune cells. The functional IL-10R consists of a dimmer of the heterodimer of IL-10R1 and IL-10R2 [3]. The IL-10R1 binds IL-10 with a high affinity, and the recruitment of IL-10R2 enables signal transduction [4]. The functions of IL-10 have been studies in a large number of studies. For example, IL-10 suppresses macrophages and DCs function, thereby limiting Th1 and Th2 effector responses [3,5]. IL-10 production is of potential benefit to both the host (preventing autoimmunity) and the pathogen (allowing persistent infection) [6]. It was reported that the up-regulation of
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Fig. 1. (Continued)
IL-10 induced impaired T-cell responses and benefited the persistent infection of lymphocytic choriomeningitis virus (LCMV). In vivo blockade of IL-10R with a neutralizing antibody resulted in rapid resolution of LCMV persistence [7,8]. The results suggested that blockade of IL-10R could break the negative regulatory loop mediated by IL-10, and may represent a new therapeutic strategy to treat chronic viral infection. According to World Health Organization, more than two billion people have been infected with hepatitis B virus (HBV) and 350 million suffer from chronic HBV infection [9]. Currently two types of antiviral therapies are approved for clinical use: pegylated interferon alpha 2a (PEG-IFN␣) and nucleos(t)ide analogs (NA) [10]. However, only one third of patients treated with PEG-IFN-␣ have a sustained antiviral response. On the other hand, the therapy with NA significantly suppresses HBV replication. But the rebound of viremia occurs after withdrawal of NA and drug-resistant mutants frequently arise with long-term treatment [11]. Therefore, new therapeutic approaches for the treatment of chronic hepatitis B (CHB) are intensely required. In CHB, one of the defining characteristics is the loss or functional inactivation of antiviral effector T cells [12]. The increased production of IL-10 was observed in patients with CHB [13], which hints that blockade of IL-10R might become a feasible therapeutic approach for CHB. Woodchuck (Marmota monax) is the natural host of woodchuck hepatitis virus (WHV) and represents a highly valuable animal model to investigate immune response, pathogenesis,
and therapy of HBV infection [11]. In the present work, we cloned and aligned the complete coding sequence of woodchuck IL-10R (wIL-10R). The extracellular region of wIL-10R (wIL-10Re) was expressed and purified. The blockade of IL-10R with the prepared antibody against wIL-10Re in vitro could enhance the proliferation and degranulation of WHV-specific T-cells from chronically WHV-infected woodchucks.
2. Materials and methods 2.1. Animals and cell cultures Woodchucks were purchased from Northwest Wildlife (Ithaca, NY) and maintained in the animal facilities of Tongji Medical College and University Hospital of Essen, in accordance with the Guide for the Care and Use of Laboratory Animals. The peripheral blood mononuclear cells (PBMCs) of woodchuck were separated by Ficoll-Paque (Pharmacia, Freiburg, Germany) centrifugation and resuspended in RPMI 1640 medium (Gibco BRL, Neu-Isenburg, Germany) supplemented with 10% fetal calf serum (FCS) (Gibco). PBMCs were cultured at a density of 106 cells per ml in flat-bottom 24-well microtiter plates (Falcon, Becton Dickinson, NJ) at 37 ◦ C and 5% CO2 [14]. Baby hamster kidney (BHK) cells (provided by American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% FCS.
Fig. 1. Alignment and phylogenetic tree analysis of wIL-10R sequences. The GenBank accession numbers of the sequences used in the study: woodchuck (JN122322), human (BC028082), chimpanzee (XM 508785), horse (XM 001917543), cattle (NM 001205757), dog (XM 848380), mouse (L12120), rat (NM 057193). (A) Alignment of the deduced aa sequence of wIL-10R with homologues from different species by ‘MCLONE’ software. Dots resemble conserved amino acids and gaps (−) were inserted to maximize alignment. In ECD, three potential glycosylation sites (NQS, NVT and NWT) and two cystein residues (C203 and C224) are indicated in bold and boxes. In ICD, highly conserved GYXXQ sequences (GYLKQ and GYVKQ) are indicated in bold and underlines. (B) The predicted secondary structure of wIL-10R. The precursor of wIL-10R consists of a 18 aa signal peptide and a 558 aa mature protein, which was composed of ECD, TM and ICD. (C) The phylogenetic tree based on the nucleotide sequences of wIL-10R and different mammalian species. The phylogenetic tree was designed in the software ClustalW.
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Table 1 Primers used for cloning in the present study. Primers
Position of 5 base
Nucleotide sequences
Referencea
IL-10R-P1 IL-10R-P2 IL-10R-P3 IL-10R-P4 IL-10R-P5 IL-10R-P6 IL-10R-P7 IL-10R-P8
38 1854 57 713 1 1728 588 708
5 -atg ctg tcg cgc ctg gta gt-3 5 -gcc cag gtc act cat tcg ag-3 5 -cgg ggt accb gaa ctg cct agc cct cca-3 5 -ccg gaa ttc tca ata ctg ctt ggt gag gac-3 5 -ccg gaa ttc atg ctg tcg cgc ctg gta gt-3 5 -acg cgt cga cct cat tcg agt gca ggc tgg-3 5 -agg aga ggt ggg aaa gtt-3 5 -gtt ggt cgc agt gaa ata c-3
L12120 L12120 JN122322 JN122322 JN122322 JN122322 JN122322 JN122322
a b
Reference: the GenBank accession numbers of the used reference sequences are given. The 5 extensions were KpnI, EcoRI and SalI restriction sites and are indicted as italic letters.
2.2. Cloning and alignment of woodchuck IL-10R sequence cDNA fragment coding for the full length of wIL-10R (1731 base pair, bp) was amplified by reverse transcriptionpolymerase chain reaction (RT-PCR) with the primers of IL-10R-P1 and -P2 (Table 1), which were designed according to the aligned sequences and chosen from regions conserved among human and other mammalian species. For RT-PCR, total RNA was prepared from woodchuck PBMCs with TRIzol reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. The PCR product was cloned into pMD18-T TA vector (TaKaRa, Dalian, China), named as pMD18-wIL-10R, and sequenced by commercial services (Yingjun Biotechnology Company, Shanghai, China). The phylogenetic analysis of wIL-10R was performed online with the software ClustalW at the website of European Bioinformatics Institute (http://www.ebi.ac.uk). The deduced amino acid (aa) sequence of wIL-10R was aligned with homologues from different species by ‘MCLONE’ software. And the secondary structure of wIL-10R residues was predicted by an online service of Swiss Institute of Bioinformatics (http://swissmodel.expasy.org).
2.3. Expression of recombinant wIL-10R protein in E. coli and BHK cells The coding sequence of the extracellular region of wIL10R (wIL-10Re, nucleotide 57-713) was amplified by PCR from pMD18-IL-10R with the primers of IL-10R-P3 and -P4 (Table 1), and subcloned into the prokaryotic expression vector of pET30a (Merck, Germany) at KpnI and EcoRI sites resulting in the plasmid of pET30a-wIL-10Re, which express the extracellular region of wIL-10R fused with His tag (His-wIL-10Re). The recombinant protein of His-wIL-10Re expressed in E. coli BL21 (DE plus) was purified with HisTrap Kit (Amersham, Braunschweig, Germany) under denaturation conditions according to the manufacturer’s instructions and analyzed by 15% SDS-PAGE. The full length of wIL-10R was amplified by PCR from pMD18-wIL-10R with the primers of IL-10R-P5 and -P6 (Table 1), and subcloned into the mammalian expression vector of pXF3H (kindly provided by Prof. Xinghua Feng) resulting in the plasmid of pXF3H-wIL-10R. The eukaryotic expression of the full length of wIL-10R fused with hemagglutinin (HA) tag was under the control of cytomegalovirus
immediate-early promoter [15]. The transient transfection of pXF3H-wIL-10R in BHK cells was performed by using Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) as described previously [16].
2.4. Generation of polyclonal antibody against the extracellular region of wIL-10R (anti-wIL-10R) and determination of the antibody titer by ELISA New Zealand white rabbits were immunized by subcutaneous injection with 200 g of purified His-wIL-10Re in Freund’s incomplete adjuvant at 2 weeks interval. The antiserum was collected 14 days after the third immunization. The antibody titer of anti-wIL-10R was measured by indirect enzyme-linked immunosorbent analysis (ELISA). His-wIL-10Re was diluted to 1 g/ml, and coated on plates at 100 l aliquot per well in 96-well immunoplate at 4 ◦ C overnight. The coated wells were blocked with 100 l of 10% FCS for 2 h at 37 ◦ C, and then incubated with 100 l of anti-wIL-10R with serial dilutions of 1:2 (from 1:16,000 to 1:1,024,000). The bound anti-wIL-10R was detected by HRP-conjugated goat-anti-rabbit IgG (dilution 1:1000, DB Biosciences, CA). The serum from the pre-immune rabbit was used as a negative control. The development of color was read at 490 nm. The cut-off value was set as 3 times over the negative control. The titers of anti-wIL-10R were calculated by extrapolation of ELISA values of serially diluted samples and corresponded to the reciprocal values of the highest dilutions that were regarded as positive.
2.5. Immunofluorescence (IF) staining of wIL-10R expressed in BHK cells and on woodchuck PBMCs To test the recognition of wIL-10R protein by the prepared anti-wIL-10R, IF staining was performed in transiently transfected BHK cells with pXF3H-wIL-10R. Forty eight hours after transfection, BHK cells were fixed with 50% methanol and stained with the prepared anti-wIL10R (dilution 1:100) and a fluorescein isothiocyanate (FITC) labeled goat anti-rabbit IgG (dilution 1:200, DAKO, Hamburg, Germany) [15]. The serum from the pre-immune rabbit was used as a negative control. To detect the expression level of wIL-10R on woodchuck PBMCs, PBMCs were plated on microscope slides at a density of 105 cells per slide, and fixed with 50% methanol. Cells were stained with the prepared
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Fig. 2. Expression of His-wIL-10Re fusion protein and determination of the titer of anti-wIL-10R by ELISA. (A) Expression of His-wIL-10Re in E. coli was analyzed by 15% SDS-PAGE. M: protein MW standards; lane 1: the whole lysate of the transformed E. coli without IPTG induction; lanes 2 and 3: the whole lysate of the transformed E. coli with 2 mM of IPTG induction for 2 and 4 h, respectively. (B) The purified wIL-10Re was analyzed by 15% SDS-PAGE. Lane 1: the unpurified protein; lanes 2 and 3: the purified His-wIL-10Re. (C) The antibody titer of the prepared anti-wIL-10R was determined as 1:512,000 by ELISA.
anti-wIL-10R (dilution 1:40) and the FITC labeled goat anti-rabbit IgG (dilution 1:200, DAKO).
2.6. Treatment of woodchuck PBMCs by TLR ligands and detection of wIL-10R expression on PBMCs by real time RT-PCR Woodchuck PBMCs were treated with the ligands of TLR3 (poly I:C, 12.5 g/ml), TLR7 (imiquimod, 10 g/ml), TLR9 (CpG ODN, 12.5 g/ml) by transfection with Lipofectamin 2000, and direct administration of TLR1/2 (Pam3Cysk4, 2 g/ml), TLR2/6 (Pam2Cysk4, 2 g/ml), TLR4 (lipopolysaccharid, 12.5 g/ml), TLR5 (flagellin, 10 g/ml) and woodchuck IFN-␣ and -␥ (500 U/ml) for 6 h. TLR19 ligands were purchased from Invitrogen (Karlsruhe, Germany). Woodchuck IFN-␣ and IFN-␥ were produced by eukaryotic transfection.
Total RNA was extracted from woodchuck PBMCs with TRIzol (Invitrogen) as described previously [17]. The mRNA expression of wIL-10R was quantified by real time RTPCR with primers of IL-10R-P7 and -P8 (Table 1), using QuantiFast SYBR Green RT-PCR Kit (Invitrogen) on a Light CyclerTM (Roche Diagnostics, Mannheim, Germany). All samples were analyzed in triplicate. The copy number of woodchuck -actin mRNA was determined for normalization [18].
2.7. Detection of WHV DNA in serum WHV DNAs in woodchuck sera were extracted with a QIAamp DNA blood kit (Qiagen Düsseldorf, Germany) and quantified by real time PCR with primers of wc1 and wc149s, using DNA Master SYBR Green Kit (Roche) on a Light CyclerTM [16].
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Fig. 3. Detection of wIL-10R with the prepared anti-wIL-10R by IF staining. (A) BHK cells were transiently transfected with pXF3H-wIL-10R. Transfected BHK cells were stained by pre-immune rabbit serum (a) or anti-wIL-10R (b). (B) Woodchuck PBMCs were stained by pre-immune rabbit serum (a) or anti-wIL-10R (b). The positively stained PBMCs are indicated by arrows.
2.8. Proliferation assay and CD107a degranulation assay of woodchuck PBMCs after blockade of wIL-10R by the prepared anti-wIL-10R Triplicates samples of PBMCs from naïve and chronically WHV-infected woodchucks were cultured at a density of 5 × 104 per well in 96-well microtiter plates (Falcon), and treated with different dilutions of the prepared anti-wIL10R (5, 10, 20 g/ml) and WHcAg at a final concentration of 1 g/ml for 5 days. The pre-immune rabbit serum was included as a negative control. Woodchuck PBMCs were labeled with 1 Ci of [2-3 H] adenine (Amersham, Braunschweig, Germany) for 18 h and collected by a cell harvester (Packard Instrument Company, Calif). The incorporation of [2-3 H] adenine into proliferating cells was read by a Top counter (Packard Instrument). The results for triplicate cultures are presented as the mean stimulation index (SI) and SI is calculated with the formula: (stimulated cpm-blank cpm)/(unstimulated cpm-blank cpm). SIs greater than 2 were considered as positive [16]. In CD107a degranulation assay, PBMCs from chronically infected woodchucks were cultured at a density of 1 × 106 per well in 96-well microtiter plates, and incubated with 20 g/ml of anti-wIL-10R for 2 days. A WHcAgderived peptide (aa 96–110, 2 g/ml), which was identified as a CD8 epitope, was used for stimulation. The FITCconjugated anti-mouse CD107a antibody (dilution 1:100,
BDPharmingen, Heidelberg, Germany) was added to the stimulated cells. Due to the lack of available anti-CD8 antibody, woodchuck PBMCs were stained with cross-reactive antibodies to CD3 and CD4. The CD3+ CD4− lymphocytes were gated and regarded as mainly CD8+ cells, with minor fractions of CD3+ CD4− CD8− cells. Data were acquired with a FACS-Calibur flow cytometer (Becton, Dickinson, Heidelberg, Germany), and analyzed with FlowJo software (Tree Star, Ashland, Oregon) [19]. 3. Results 3.1. Molecular cloning and characterization of wIL-10R The complete coding region of wIL-10R (1731 bp) was amplified by RT-PCR, and showed high similarity to the IL-10R sequences of other mammalian species, ranging from 69.67% to 81.11% (Table 2). The deduced aa sequence of wIL-10R has a length of 576 residues, and showed high homology to other mammalian species, ranging from 56.08% to 71.53% (Table 2). The putative precursor of wIL10R consists of a signal peptide with 18 aa residues and a mature protein with 558 aa residues, as inferred from sequences of other species (Fig. 1A and B). Like mouse IL-10R, wIL-10R may be glycosylated at three potential sites: NQS, NVT and NWT in the extracellular domain (ECD, aa 19–237) (Fig. 1A) [20]. Two cysteine residues
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Fig. 4. Detection of wIL-10R expressions on PBMCs by real time RT-PCR. (A) The expression levels of wIL-10R on PBMCs from naïve woodchucks were up-regulated by the stimulations of different TLR ligands. (B) In the acutely WHV-infected woodchuck (WH70093), wIL-10R expression on PBMCs fluctuated in the correlation with viral loads during viremic phase. (C) The wIL-10R expression levels in naïve and chronically WHV-infected woodchucks were 4852 and 3246 copies/105 w-actin on average, respectively. wIL-10R expression was suppressed in chronically infected woodchucks. The statistical significance of the results was given as P values (P < 0.05).
of C203 and C224 in ECD of wIL-10R, which form an intramolecular disulfide bridge linking -strands [21], are highly conserved in the identified IL-10R proteins. The transmembrane region (TM, aa 238–261) consists of a stretched hydrophobic segment. Within the intracellular
Table 2 Homology of woodchuck IL-10R to the respective sequences of other species on the nucleotide residues and amino acid level.
Human Chimpanzee Horse Cattle Dog Mouse Rat
Nucleotide residues (%)
Amino acid (%)
81.11 80.82 80.24 78.57 77.24 70.60 69.67
71.53 71.01 68.92 64.54 64.75 56.42 56.08
domain (ICD, aa 262–576) several residues of wIL-10R are highly conserved, which are implicated in receptor function. For instance, two redundant GYXXQ sequences (GYLKQ and GYVKQ) are required for the recruitment of STAT3 [22] (Fig. 1A). The phylogenetic tree analysis of IL10R sequences showed the close genetic distance among species, indicating the conservation of IL-10R during the evolution (Fig. 1C). 3.2. Generation of the neutralizing antibody to wIL-10R The recombinant protein of His-wIL-10Re containing the extracellular region of wIL-10R fused with His tag was produced. The purified His-wIL-10Re with the expected molecular weight (MW) of 27 kDa was detected by 15% SDS-PAGE (Fig. 2B). Rabbits were immunized with the purified His-wIL-10Re to generate the polyclonal antibody of
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Fig. 5. Blockade of the IL-10/IL-10R pathway by the specific anti-wIL-10R antibody enhanced the proliferation of specific T-cells from chronically WHVinfected woodchucks in vitro. PBMCs from naïve (A) or chronically WHV-infected (B) woodchucks were incubated with different dilutions of anti-wIL-10R and pre-immune rabbit serum as a negative control in the presence or absence of WHcAg in vitro for 5 days. Proliferation of PBMCs was measured by [2-3 H] adenine incorporation. PBMCs from two chronically WHV-infected woodchucks WH35034 and WH41321 showed enhanced proliferation.
anti-wIL-10R, which was tested at different dilutions for the reactivity with His-wIL-10Re in ELISA. The titer of antiwIL-10R was determined as 1:512,000 (Fig. 2C). The specificity of anti-wIL-10R was proven by IF staining, in which the transfected BHK cells expressing the full length wIL-10R protein showed positively stained (Fig. 3A). The expression of wIL-10R on woodchuck PBMCs were detected by IF staining with anti-wIL-10R. A small portion of woodchuck PBMCs was strongly positively stained (Fig. 3B), consistent with the knowledge that IL-10R is expressed on peripheral blood cells.
and -␥. The transcripts of wIL-10R were detected by real time RT-PCR, in which a fragment of 293 bp (nt 588–708) was amplified. The base line expression of wIL-10R on unstimulated PBMC was about 2251 copies/105 w-actin on average. The expression of wIL-10R was up-regulated by TLR3 and TLR7 (6 fold) ligands, TLR2 and TLR4 (2.8 fold) ligands, and TLR5 (1.6 fold) ligand. However, IFN-␣ and IFN-␥ had no effect on wIL-10R expression (Fig. 4A).
3.3. Induction of wIL-10R expression on PBMCs by TLR ligands
To establish the relationship to WHV infection, wIL10R expression on PBMCs in naïve and WHV-infected woodchucks was examined by real time RT-PCR. In acutely WHV-infected woodchucks (n = 4), wIL-10R expression highly increased during the viremic phase and returned to the baseline after viral clearance. We showed a representative result in Fig. 4B. wIL-10R expression in
PBMCs from naïve woodchucks were treated with TLR ligands or IFNs: TLR1/2 (Pam3Cysk4), TLR2/6 (Pam2Cysk4), TLR3 (poly I:C), TLR4 (LPS), TLR5 (flagellin), TLR7 (imiquimod), TLR9 (CpG ODN), woodchuck IFN-␣
3.4. wIL-10R expression on PBMCs in naïve, acutely or chronically WHV-infected woodchucks
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Fig. 6. Blockade of the IL-10/IL-10R pathway by the specific anti-wIL-10R antibody improved the degranulation of specific T-cells from chronically WHVinfected woodchucks in vitro. PBMCs from chronically infected woodchucks were treated with anti-wIL-10R in the presence of WHcAg derived peptide in vitro for 2 days. The degranulation of specific T-cells was detected by CD107a staining. Control antibody (ab) is from normal rabbit serum. In the chronically infected woodchucks: WH35034 (A) and WH41321 (B), the frequencies of CD107a+ CD3+ CD4− T cells were increased, indicating an improved degranulation activity.
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naïve woodchucks (n = 9) was 4852 copies/105 w-actin on average. In chronically WHV-infected woodchucks (n = 9), wIL-10R expression at a value of 3246 copies/105 w-actin on average, was apparently suppressed and lower than those in naïve woodchucks (P < 0.05) (Fig. 4C). 3.5. Enhancement of proliferation and degranulation of specific T-cells by blockade of wIL-10R The recent reports demonstrated that blockade of IL10R may restore T-cell functions in persistently infected individuals [7,8]. In this study, we addressed whether in vitro blockade of wIL-10R by neutralizing antibody of anti-wIL-10R may enhance T-cell functions in chronic WHV infection. Fresh PBMCs were prepared from woodchucks including naïve (n = 4) and chronically WHVinfected animals (n = 4), and stimulated with WHcAg or WHcAg-derived peptides in the presence or absence of anti-wIL-10R. The proliferation and CD107a degranulation of specific T-cells were determined. The stimulation fold change post blockade was calculated and the fold change more than 2 was regarded as a positive result. PBMCs from naïve woodchucks did not show any response to WHcAg with or without anti-wIL-10R (Fig. 5A). In 2 of 4 chronically infected woodchucks (WH35034 and WH41321), the proliferations of WHcAg-specific T-cells were enhanced in the presence of different concentrations of anti-wIL-10R (Fig. 5B). Furthermore, WHcAg-specific CD3+ CD4− T cells from 2 of 4 chronically infected woodchucks (WH35034 and WH41321) showed a improved CD107a degranulation in the presence of anti-wIL-10R and WHcAg peptide (Fig. 6A and B). A 3.9-fold increment of the frequency of CD107a+ CD3+ CD4− T cells was measured in WH35034 (Fig. 6A). 4. Discussion Virus-specific CD4+ and CD8+ T cells become functionally unresponsive during persistent viral infections by human immunodeficiency virus (HIV), HBV and hepatitis C virus (HCV), indicating that conserved mechanisms of immunosuppression may downregulate T-cell activity [7]. The levels of systemic IL-10 production were upregulated during chronic HIV, HBV and HCV infections [13,23–27]. It is likely that IL-10-induced immunosuppression is active during these persistent infections. Treg mediate their regulatory function in vivo through IL-10 [5,28]. Treg are possibly involved in the mechanism of HBV persistence. It was reported that Treg accumulated in the liver during chronic severe HBV infection and the frequency of Treg correlated with viral load in CHB [29]. Besides the immunosuppression of specific T-cell response, IL-10 plays an important role in the induction of immune tolerance in liver. Liver DCs generated more suppressive Treg and IL4-producing Th2 cells via an IL-10-dependent mechanism [30]. Liver Kupffer cells (KCs) release substantial amounts of IL-10 during the induction of Treg-mediated tolerance [31]. In addition, IL-10 down-regulated T-cell activation through liver sinusoidal endothelial cells (LSEC) [32]. In vivo
blockade of IL-10R with a neutralizing antibody resulted in extremely low viral load and enhancement of IFN-␥ secreting CD8+ T cells and specific memory T cell response in LCMV clone-13 persistently infected mice [8]. It is likely that neutralization of IL-10 activity may boost antiviral immune responses, restore T-cell function, and enhance the treatment of the persistent infections. However, we should notice that IL-10 as a negatively regulatory cytokine drives the generation of Treg and downregulates a pathological immune response [33,34]. The blockade of IL-10 signaling might lead the overexpression of inflammatory cytokines and induce autoimmune diseases. Therefore, the blockade of IL-10R as a therapeutic approach in chronic viral infection needs to be careful and more balanced [5,35]. In our study, the nucleotide sequence of wIL-10R presented close genetic distance to human IL-10R. The critical residues of wIL-10R are highly conserved for the receptor function. The expression of wIL-10R was detectable on a small portion of PBMCs from naïve woodchucks, and the expression level of wIL-10R could be up-regulated by various TLR ligands. In acutely WHVinfected woodchucks the expression levels of wIL-10R fluctuated, correlating with the viral load and the activation of T-cell responses. However, in chronically WHV-infected woodchucks the expressions of wIL-10R were downregulated and lower than those in naïve woodchucks. The blockade of wIL-10R with the neutralizing antibody of anti-wIL-10R in vitro could enhance the proliferation of WHV-specific T-cells and improve the degranulation of WHV-specific T-cells from chronically infected woodchucks. However, not every chronic woodchucks was responsive to the blockade, which indicated that the wIL-10/IL10R system may be not the only cause for low T-cell responses in chronic WHV infection. Our previous study showed that the programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) system played a role in the negative regulation of T-cell functions in chronic HBV infection [36]. Recent publications indicated that the expression of PDL1 and PD-L2 on human macrophages is differentially modulated by IL-10. IL-10 induced up-regulation of PDL1 expression but not of PD-L2, and blockade of IL-10 enhanced only PD-L2 expression [37]. Therefore, the combined approaches of antibodies against such inhibitory molecules with antiviral drugs would be useful to restore T-cell functions in chronically infected individuals. Here we found that in vitro blockade of the wIL-10/IL-10R pathway has the potence to enhance specific T-cell functions for a short time. Future experiments in vivo will clarify whether blockade of the IL-10/IL-10R signaling may break immune tolerance and restore antiviral immune responses in chronic HBV infection. Our work provided a basis for further studies to investigate immunotherapeutic approaches based on IL-10R in CHB.
Conflict of interest statement The authors declare that they have no conflict of interest related to the manuscript.
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