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Originally published online as doi:10.1189/jlb.0207128 on June 26, 2007

Published online before print June 26, 2007
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(Journal of Leukocyte Biology. 2007;82:479-487.)
© 2007 by Society for Leukocyte Biology

Toll-like receptors 1 and 6 are involved in TLR2-mediated macrophage activation by hepatitis C virus core and NS3 proteins

Serena Chang, Angela Dolganiuc and Gyongyi Szabo1

Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA

1 Correspondence: Department of Medicine, University of Massachusetts Medical School, LRB 215, 364 Plantation Street, Worcester, MA 01605-2324, USA. E-mail: gyongyi.szabo{at}umassmed.edu

ABSTRACT

Hepatitis C virus (HCV) is a leading cause of end-stage liver disease through sustained inflammation of the liver produced by the host’s immune system. The mechanism for HCV evasion or activation of the immune system is not clear. TLRs are cellular activators of the innate immune system. We recently reported that TLR2-mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo- or heterodimerization with TLR1 or TLR6. Here, we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing small interfering RNAs targeted to TLR2, TLR1, and TLR6, we showed that knockdown of each of these receptors impairs pro- and anti-inflammatory cytokine activation by TLR-specific ligands as well as by HCV core and NS3 proteins in human embryonic kidney-TLR2 cells and in primary human macrophages. We found that HCV core and NS3 proteins induced TNF-{alpha} and IL-10 production in human monocyte-derived macrophages, which was impaired by TLR2, TLR1, and TLR6 knockdown. Contrary to human data, results from TLR2, TLR1, or TLR6 knockout mice indicated that the absence of TLR2 and its coreceptor TLR6, but not TLR1, prevented the HCV core and NS3 protein-induced peritoneal macrophage activation. In conclusion, TLR2 may use TLR1 and TLR6 coreceptors for HCV core- and NS3-mediated activation of macrophages and innate immunity in humans. These results imply that multiple pattern recognition receptors could participate in cellular activation by HCV proteins.

Key Words: TNF-{alpha} • IL-10 • IL-6 • siRNA

INTRODUCTION

Hepatitis C virus (HCV)-induced liver inflammation and cirrhosis is one of the leading causes of liver transplantations. Unlike other hepatitis viruses, HCV leads to a high rate of chronic infection in approximately 80% of the 170 million people infected worldwide [1 ]. Approximately 50% of the chronically infected individuals can eliminate HCV after an IFN-{alpha}-based Ribavirin-combined therapy, while nonresponders develop potentially fatal chronic disease [1 2 3 ]. A major symptom and possibly a vital participant in persistent infection of HCV is chronic inflammation in and outside the liver, mediated by cytokines such as TNF-{alpha} [4 ], IL-12 [5 ], and IL-6 [6 , 7 ]. Chronic infection can cause liver cirrhosis and/or hepatocellular carcinoma, ultimately leading to liver failure as a result of inflammatory cell-mediated liver damage [8 ]. Although the current thought is that immune-mediated mechanisms can inactivate or maintain this process, the mechanisms for continued inflammation are still ambiguous.

HCV is a positive, single-stranded RNA virus from the Flaviviridae family [9 ]. It is composed of three structural proteins: core and envelopes 1 and 2; a small integral membrane protein p7; and six nonstructural proteins, NS2, NS3, NS4A, NS4B, NS5A, and NS5B [10 11 12 ]. HCV core and NS3 proteins can stimulate the host’s innate immune system and trigger inflammatory cytokine production; therefore, identifying receptors activated by HCV proteins could be potential therapeutic targets [13 , 14 ]. HCV NS3 is a serine protease, which also has the ability to act as an RNA helicase [15 , 16 ]. Current research revealed that NS3 protein could inhibit the innate immune system by blocking the TLR3-mediated type 1 IFN-induction pathway [17 ] and stimulate the innate immune system by activating the TLR2-mediated inflammatory cytokine pathway [14 ]. HCV core protein is a highly basic RNA-binding protein, which can also stimulate the innate immune inflammatory pathways via TLR2 and plays an important role in the formation of viral nucleocapsids and interactions with the viral genome [14 , 18 ]. The core protein is located intracellularly in infected hepatocytes; however, there are relatively high levels of core protein detected in patients’ blood [19 ]. HCV core and NS3 proteins interact with many host proteins [17 , 20 ]; however, the mechanisms by which HCV proteins modulate the innate immune system to contribute to disease are unanswered.

TLRs, an important arm of the innate immune system, activate pathways that produce pro- and anti-inflammatory cytokines when stimulated by pathogen-derived ligands. Some TLRs, such as TLR2, -4, -3, -7, and -9, are involved in detection of viral proteins [17 , 21 22 23 ]. We have identified in a previous study TLR2-specific activation by HCV NS3 and core proteins but not HCV E2 [17 ]. Existing research illustrates that TLR2 homo- or heterodimerizes with TLR1 or TLR6 [24 ]. Therefore, to further identity the receptor recognition of TLR2-specific HCV protein activation, we created a knockdown system using RNA interference (RNAi) in TLR-expressing human cells and used TLR-specific knockout mice. Our results revealed that in human cells, knockdown of TLR1 or TLR6 caused a significant reduction in cytokine production upon HCV protein stimulation, suggesting that in macrophages, TLR2 exploits TLR1 or TLR6 in innate immune recognition of HCV proteins.

MATERIALS AND METHODS

Reagents
DMEM and RPMI 1640 were from Gibco (Grand Island, NY, USA), and FCS was from HyClone (Logan, UT, USA). Phenol-purified (p)LPS was from List Biological Laboratories (Campbell, CA, USA), Staphylococcus aureus peptidoglycan (PGN) was from Fluka (Milwaukee, WI, USA), and PAM2CSK4 and PAM3CSK4 were from EMC Microcollections GmbH (Germany). ß-Galactosidase, expressed and purified identically to HCV core and NS3 proteins (kindly provided by Biodesign, Saco, ME, USA), was used as a control where indicated. Recombinant HCV core and NS3 proteins were purchased from Biodesign and contained 0 EU endotoxin at 10 ug/ml, as detected using the Limulus amoebocyte lysate (LAL) assay. The minimum value of detection by the LAL assay is 12 pg endotoxin.

Cells
Human embryonic kidney (HEK)293T cells stably transfected with TLR2/yellow fluorescent protein (HEK/TLR2) were maintained in DMEM with 10% heat-inactivated FCS and selective antibiotics and subcultured three times/week.

Human monocyte-derived macrophages (hMDM) were differentiated from PBMCs of HCV-negative, healthy human blood donors using Ficoll-Paque density gradient centrifugation. Monocytes were separated from PBMCs by adherence to flasks coated for 2 h with 2% sterile, endotoxin-free gelatin and dried overnight in a 37°C, 5% CO2 incubator. Before addition of PBMCs, the flasks were coated additionally for 1 h with platelet-free autologous human serum and rinsed with HBSS (Gibco). PBMCs were seeded into the gelatin and serum-coated flasks for 1 h at 37°C, 5% CO2. Adherent monocytes were washed three times with RPMI and removed from the flasks using a mixture of RPMI with 10% FCS and 10 mM EDTA in a 1:1 dilution. Isolated monocytes were washed three times with RPMI and plated at 5 x 105/ml in RPMI supplemented with 18% autologous serum for 8 days.

C57BL/6J (wild-type), TLR2, TLR1, and TLR6 knockout mice were a gift from Dr. Robert Finberg at the University of Massachusetts Medical School (Worcester, MA, USA). Mice were injected with a 4% thioglycollate medium i.p., and peritoneal macrophages were extracted from peritoneal exudates 5 days later. Macrophages were plated in 96-well plates (BD Biosciences, Franklin Lakes, NJ, USA) at 106/ml.

Small interfering (si)RNA
TLR2, TLR1, TLR6, and control siRNA oligonucleotides were purchased from Xeragon (Valencia, CA, USA). The full mRNA sequence was located from GeneBank, and siRNA sequences were identified as 21 nucleotides, starting with AA and ending with TT after the first 150 bp. Sequences with G/C content between 30% and 70% were used. Sequence alignment tests confirmed specificity to corresponding TLR.

HEK/TLR2 cells were plated in a 24-well plate at 106/ml and grown to yield 70% confluency. siRNAs (final concentration 300 nM/well) were combined with Mirus TransIT transfection reagent and Opti-MEM medium for 15 min. The culture medium was changed prior to transfection, and the siRNA mix was added to each well and incubated for 8 h at 37°C. After transfection, 1 ml fresh culture medium was added to each well, and the cells were grown for an additional time up to 72–96 h.

hMDM were transfected with siRNAs using Lipofectamine 2000. Briefly, 3 µl Lipofectamine 2000, 350 µl DMEM, and 60 pmol siRNA/well were incubated for 15 min at room temperature. The macrophages were washed with serum-free DMEM and incubated at 37°C, 5% CO2, for 3 h with siRNA transfection mixture as described above. After incubation, the cells were washed with serum-free DMEM and maintained in RPMI with 18% autologous serum for an additional up to 72–96 h. Knockdown of a specified gene was confirmed by real-time PCR analysis for the mRNA expression and by Western blot analysis for protein expression.

Protein quantification by Western blot
Twenty-four and 72 h after siRNA transfection, the cells were counted and lysed for 10 min on ice with lysis solution: [1% Nonidet P-40, 0.5% docetaxel, 1.5 mM NaCl, 0.5 M EDTA, Complete Mini® protease inhibitor cocktail (Roche, Indianapolis, IN, USA)]. The lysate was spun at 4°C for 10 min at 14,000 rpm, collected, and mixed with SDS sample buffer. Protein samples were heated at 95°C for 2 min, loaded onto a 10% SDS-PAGE, and transferred to a nitrocellulose membrane. Precision Plus protein standard Bio-Rad (Hercules, CA, USA) was used for the molecular weight ladder. Membranes were blocked overnight with 5% nonfat milk in PBS and then probed with anti-TLR6 (Zymed, San Francisco, CA, USA) or anti-ß-actin (Abcam, Cambridge, MA, USA) antibodies at 1:10,000 dilution in PBS for 1 h at room temperature. Secondary HRP-labeled goat anti-mouse antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and ECL [LumiGlo kit, Cell Signaling Technology (Danvers, MA, USA)] were used to identify the immunoreactive bands. The densitometry analysis of the immunoreactive bands was performed using the Fuji ultrasonic-doppler velocity profile (UVP) system and LabWorks program.

mRNA quantification by RT and PCR
RNA was extracted from cells collected 48 and 72 h after siRNA transfection using the RNeasy kits (Qiagen, Valencia, CA, USA), as the manufacturer instructed. RT was performed using the RT System® from Promega (Madison, WI, USA), according to the manufacturer’s instructions. The sequences for the primers used were as follows: TLR2 (5'-ATCAGCAGGAACAGAGCACA-3', 5'-ACTCAGGAGCAGCAAGCAC-3'), TLR1 (5'-GGGTCAGCTGGACTTCAGAG-3', 5'-AAAATCCAAATGCAGGAACG-3'), TLR6 (5'-GCTGTTCTGTGGAATGGGTT-3', 5'-GAACATGATTCTGCCTGGGT-3'), all supplied by IDT (Coralville, IA, USA). 18S primers (5'-GGAACTGAGGCCATGATTAA-3', 5'-TCGGAACTACGACGGTATCT-3') were from Ambion (Austin, TX, USA). The PCR protocol consisted of 95°C for 5 min, 30 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, and 72°C for 10 min. PCR was conducted using the Bio-Rad I-cycler. The PCR products were separated in agarose gel, stained with 0.01% ethidium bromide, detected with UV light, and quantified using the Fuji UVP system and LabWorks program.

Flow cytometry analysis
HEK/TLR2 cells were detached from plates using trypsin and washed with cell culture medium. Control and siRNA-treated cells were incubated with anti-human TLR2 antibody (a kind gift from Dr. R. Finberg, University of Massachusetts Medical School) and secondary APC-conjugated antibody (Caltag Laboratories, Burlingame, CA, USA). APC-conjugated antibody alone was used as a background control. Cell fluorescence was analyzed using an LSR II (BD Biosciences) flow cytometer and FlowJo FACS analysis program (TreeStar, Ashland, OR, USA).

Cytokine quantification by ELISA
siRNA-transfected cells and mouse peritoneal macrophages were stimulated for 10 h with TLR-specific ligands or HCV core or NS3 recombinant proteins. Cell culture supernatants were collected and analyzed using cytokine-specific ELISAs [IL-6, IL-8, and TNF-{alpha} ELISAs from BD Bioscience; IL-10 ELISA from eBioscience (San Diego, CA, USA)], all performed according to the manufacturer’s instructions.

Statistical analysis
All data derived from cell lines were analyzed with t-test; those from mouse macrophages used ANOVA; and those from hMDM used nonparametric Wilcoxon statistical analysis methods. A P < 0.05 was considered statistically significant.

RESULTS

Previous work showed that HCV proteins, core and NS3, stimulated human monocytes in a TLR2-specific manner and induced production of TNF-{alpha} and IL-10 [14 ]. HCV infection targets the liver, where Kupffer cells, resident liver macrophages, are the major cell type involved in innate immune pathogen surveillance and proinflammatory cytokine production. Similar to human monocytes and hMDMs, Kupffer cells express TLR2, TLR1, TLR6, TLR4, and TLR9 [25 , 26 ]. We found that normal human monocytes and hMDMs express TLR2, TLR1, and TLR6 (Fig. 1A ). To determine the effect of HCV proteins on hMDMs, we stimulated hMDMs with recombinant HCV core or NS3 protein. We found that as little as 10 ng/ml HCV protein induced a dose-dependent induction of TNF-{alpha} in hMDMs (Fig. 1B) . hMDMs contribute to not only the generation of inflammatory responses triggered by pathogen-derived ligands but also to the generation of anti-inflammatory cytokines, such as IL-10, which can inhibit pathogen-specific T cell activation. It is unknown whether TLR2 alone and/or in combination with TLR1 or TLR6 participates in HCV-specific anti-inflammatory production during stimulation with HCV core and NS3 protein. Here, we established that hMDMs produced IL-10 when stimulated with HCV core or NS3 proteins in a dose-dependent manner (Fig. 1C) . It is important that the levels of IL-10 induced with as little as 76 ng/ml HCV proteins were closely comparable with cytokine and HCV core protein levels reported in the serum of HCV-infected patients [13 , 27 ].


Figure 1
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  		Figure 1. HCV core and NS3 proteins induce TNF-{alpha} and IL-10 production in a dose-dependent manner in human monocytes and macrophages. (A) Equal amounts of mRNA extracted from normal human monocytes (upper gel) and macrophages (lower gel) were transcribed into cDNA and amplified using TLR1-, TLR2-, TLR6-, and 18S-specific primers in RT-PCR. The results from one individual out of n = 3 with similar results are shown. Normal hMDM were stimulated with recombinant HCV core (solid bars) and NS3 (open bars) proteins for 10 h. Culture supernatants were analyzed for TNF-{alpha} (B) and IL-10 (C) using specific ELISA. Data are shown as an average ± SD ng/ml from three or more experiments.

Before performing siRNA knockdown, we determined that the HCV core and NS3 recombinant proteins were TLR2-specific and were void of endotoxin. Murine peritoneal macrophages were extracted from wild-type C57BL/6 (Fig. 2A ), TLR2–/– (Fig. 2B) , TLR4 mutant (Fig. 2C) , and TLR4–/– mice (Fig. 2D) and stimulated with TLR2 and TLR4 ligands, HCV core, and NS3 proteins. IL-6 production from peritoneal macrophages showed that stimulation with HCV core and NS3 proteins rendered no response in the TLR2–/– mice as opposed to the wild-type, TLR4–/–, or TLR4 mutant mice. The pLPS was used as a TLR4-specific ligand, as the PGN was used as a TLR2-specific ligand. ß-Galactosidase and IL-1ß were used as controls. In human cells overexpressing TLR2 or TLR4 (Fig. 2E) , stimulation with TLR2 or TLR4 ligands, HCV core, or NS3 proteins showed the same pattern of TLR2 specificity. In HEK/TLR4 cells, PGN, HCV core, and NS3 proteins did not elicit NF-{kappa}B activation, as opposed to HEK/TLR2 cells, which showed significant activation of NF-{kappa}B. To further investigate the specificity to TLR2, we used TLR2-blocking antibodies in human monocytes stimulated with the same ligands and proteins as in Figure 2E and measured TNF-{alpha} production (Fig. 2F) . The blocking antibodies resulted in a significant reduction of cytokine production compared with the isotype antibody control but did not inhibit cytokine output completely. The remaining cytokine production was most likely a result of the incomplete blocking by the antibody and not to TLR2 contaminants in the HCV core and NS3 proteins, as the anti-TLR2 antibody also failed to achieve full inhibition of the classical TLR2 ligands (Fig. 2F) .


Figure 2
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  		Figure 2. Ligand specificity to TLR2 by HCV core and NS3 proteins. Peritoneal macrophages from (A) wild-type C57BL/6, (B) TLR2–/–, (C) TLR4 mutant, and (D) TLR4–/– were stimulated with pLPS (100 ng/ml), PGN (10 ug/ml), HCV core and NS3 proteins (5 ug/ml), and ß-galactosidase (5 ug/ml). After 12 h, the culture supernatants were analyzed for IL-6 content using ELISA. Data are shown as an average ± SD ng/ml from n = 3. (E) HEK293/TLR2 or HEK293/TLR4 cells were stimulated with pLPS (100 ng/ml), PGN (10 ug/ml), HCV core and NS3 proteins (5 ug/ml), ß-galactosidase (5 ug/ml), and IL-1ß (20 ug/ml). After 12 h, the cells were lysed, and luciferase NF-{kappa}B levels were measured. Data are shown as an average ± SD ng/ml from n = 3. *, TLR4; #, TLR2 represent P values <0.05 compared with the controls calculated by Student’s t-test. (F) Mouse peritoneal macrophages were stimulated with LPS, PGN, HCV core and NS3 proteins, and ß-galactosidase in the same concentrations as in E. Culture supernatants were analyzed for TNF-{alpha} by ELISA. Blocking antibodies, isotype, and TLR2 were added 2 h before the 16 h stimulation. *, P values <0.05 compared with the controls calculated by Student’s t-test.

Based on these findings, we hypothesized that the TLR2 coreceptors, TLR1 and/or TLR6, are involved in TLR2-mediated recognition and activation by HCV core and NS3 proteins. We specifically knocked down TLR2, TLR1, and TLR6 receptor-specific mRNA in HEK/TLR2 cells and primary human macrophages using siRNA technology to evaluate which TLR2 coreceptor participates in TLR2-mediated cell activation by HCV core and NS3 proteins. First, to determine the efficiency in the knockdown of the siRNAs, we examined the expression of target mRNA over a 3-day time course after siRNA transfection. For each target TLR, there were two separate sequence siRNAs: sequence (seq)-A and seq-B. We found a significant reduction (>50%) of mRNA in at least one or both of the two separate siRNA sequences for TLR2, TLR1, and TLR6 on Day 2 after transfection (Fig. 3A ). To determine whether mRNA reduction correlated with loss of protein expression, we examined surface protein levels by FACS (Fig. 3B) and cellular protein levels by Western blot analysis (Fig. 3C) . By Day 2, TLR2 surface expression levels decreased dramatically in TLR2 siRNA but not in control siRNA-transfected HEK/TLR2 cells (Fig. 3B) . By Day 3, there was a >75% decrease in TLR6 protein expression using siRNA knockdown in HEK/TLR2 cells (Fig. 3C) . As a result of the lack of efficient Western blot- or FACS-suitable antibody, TLR1 protein levels could not be determined. These results suggest that the designed siRNAs were specific and functional for knockdown, portrayed by the significant decrease in mRNA and/or protein expression levels of TLR2, TLR1, and TLR6 in HEK/TLR2 cells.


Figure 3
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  		Figure 3. TLR-specific siRNAs knock down TLR mRNA and protein expression in HEK293/TLR2 cells. (A) Two different RNAi sequences, Seq-a and Seq-b, were constructed to target TLR2, TLR1, and TLR6 and transfected in HEK/TLR2 cells, as described in Materials and Methods. The control (Ctrl) represents the baseline expression of the corresponding TLR in negative control, siRNA-transfected cells. At 24 h (Day 1) and 48 h (Day 2) after siRNA transfection, the mRNA from HEK/TLR2 cells was extracted, and equal amounts of mRNA were transcribed into cDNA. The levels of TLR1 (top gel), TLR2 (middle gel), and TLR6 (bottom gel) were determined using specific primers and PCR. 18S was included as an internal control for all experiments. PCR products were separated on a 1% agarose gel, stained with ethidium bromide, and detected upon exposure to UV light. Shown are representative gels from one out of n = 3 experiments with similar results. (B) HEK/TLR2 cells were transfected with siRNA specific to TLR2. The cells were stained with secondary antibody alone (Negative Control) or anti-TLR2 antibody followed by secondary APC-labeled antibody. The fluorescence was analyzed by flow cytometry. The TLR2 expression in cells transfected with the negative control siRNA, as described in Materials and Methods, was considered as a positive control. TLR2 expression was determined over a 5-day time course; shown are representative fluorescence histograms from one experiment analyzed on Day 2 (top), Day 3 (middle panel), and Day 4 (bottom panel) out of n = 4 with similar results. (C) HEK/TLR2 cells were transfected with siRNA specific to TLR6. Control represents cells transfected with the negative control siRNA, as described in Materials and Methods. On Day 1 and Day 3 after siRNA transfection, the cells were lysed, and equal amounts of proteins from each experimental group were separated in SDS-PAGE, transferred to a membrane, and probed with specific anti-TLR6 or anti-ß-actin antibodies followed by secondary HRP-labeled antibodies. The expression of TLR6 (upper blot) or ß-actin (lower blot) and the densitometric analysis of TLR6 expression adjusted to ß-actin from one experiment out of n = 3 with similar results are shown.

To evaluate if the receptor knockdown correlated with reduced receptor function, we stimulated siRNA-transfected cells with TLR-specific ligands, including the TLR2 ligand, PGN, TLR2/1 ligand Pam3CSK4, and TLR2/6 ligand Pam2CSK4, and measured cell activation by means of cytokine production. TLR-specific siRNAs knocked down IL-8 cytokine production in HEK/TLR2 cells when compared with control siRNAs (Fig. 4 ). TLR2 RNAi transfection resulted in a significant loss of function indicated by a decrease in IL-8 production (50–75%) compared with control siRNA after stimulation with all three TLR2 ligands: PGN (TLR2/6), Pam2CSK4 (TLR2/6), and Pam3CSK4 (TLR2/1; Fig. 4A ). TLR1 silencing resulted in significant IL-8 reduction (~57%) after stimulation with TLR2/1 ligand Pam3CSK4 and not with the TLR2/6 ligand Pam2CSK4, indicating specific TLR1 silencing (Fig. 4B) . TLR6 silencing also showed specificity in functional knockdown, as significant reduction occurred with Pam2CSK4 (TLR2/6) and not with Pam3CSK4 (TLR2/1) ligand (Fig. 4C) . These results implied that knockdown of mRNA transcripts and protein expression by the RNA interference method correlates with a significant reduction in TLR receptor function and inhibition of ligand-stimulated cytokine production. We used IL-1ß as a stimulation control, which was unaffected by transfection of any of the siRNAs (Fig. 4D) , as IL-1ß induces cell activation via IL-1ß receptor and not via TLRs.


Figure 4
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  		Figure 4. Specific siRNAs for TLR2, TLR1, and TLR6 affect functional outcome of these receptors in HEK/TLR2 cells, which were transfected with siRNAs specific for TLR2 (A), TLR1 (B), and TLR6 (C) receptors. Control represents cells transfected with a negative control siRNA, as described in Materials and Methods. On Day 3 after siRNA transfection, the cells were stimulated for 10 h with PGN (TLR2/TLR6 ligand) at 5 ug/ml, Pam3CSK4 (TLR2/TLR1 ligand) at 50 ng/ml, Pam2CSK4 (TLR2/TLR6 ligand) at 5 ug/ml, or recombinant IL-1ß at 50 ng/ml (D), and the IL-8 production in culture supernatants was ascertained using a specific ELISA.

After we confirmed the specificity of siRNAs to TLR2, TLR1, and TLR6, we intended to establish which TLR2 coreceptor participates in recognition of HCV proteins, core and NS3. In HEK/TLR2 cells, knockdown of all three targeted siRNAs, TLR2 (Fig. 5A ), TLR1 (Fig. 5B) , and TLR6 (Fig. 5C) individually, resulted in significant inhibition of HCV core (P<.009, P<.02, P<.005, respectively)- and NS3 (P<.003, P<.03, P<.002, respectively)- induced IL-8 production compared with a control siRNA (Fig. 5A 5B 5C) . The absence of TLR2 or its coreceptors, TLR1 or TLR6, greatly diminished cellular activation by HCV NS3 and core proteins. These results suggest that TLR1 and TLR6 participate in TLR2 activation by HCV NS3 and core.


Figure 5
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  		Figure 5. TLR2, TLR1, and TLR6 participate in recognition of HCV core and NS3 proteins in HEK/TLR2 cells, which were transfected with siRNA specific for TLR2 (top panel), TLR1 (middle panel), and TLR6 (bottom panel). Control represents cells transfected with a negative control siRNA, as described in Materials and Methods. Three days after siRNA transfection, the cells were stimulated for 10 h with HCV core and NS3 proteins at 5 ug/ml (A and B) and 10 ug/ml (C), and the IL-8 production in culture supernatants was ascertained using IL-8-specific ELISA. Data are shown as an average ± SD ng/ml from n = 3.

To assess the involvement of TLR1 or TLR6 in HCV protein stimulation in macrophages, we stimulated TLR-specific, siRNA-transfected hMDMs with HCV proteins. There was a significant reduction in TNF-{alpha} (Fig. 6A ) and IL-6 (Fig. 6B) production in all three TLR-specific, siRNA-transfected macrophages stimulated with Pam2CSK4 (TLR2/TLR6), Pam3CSK4 (TLR2/TLR1), or PGN (TLR2/TLR6), suggesting a loss of function. Upon sole transfection of siRNA (medium control), there were no cytokines detected (Fig. 6A and 6B) . Silencing of TLR2, TLR1, or TLR6 did not affect TLR4-induced (pLPS) IL-6 or TNF-{alpha} production in hMDMs, confirming the lack of cross-reactivity. It is more important that TLR1 and TLR6 silencing caused reduced levels of inflammatory cytokines, TNF-{alpha} (Fig. 6A) and IL-6 (Fig. 6B) , when stimulated with HCV core or NS3 proteins. TLR2, TLR1, and TLR6 knockdowns in hMDM cells followed the same pattern as in the HEK/TLR2 cells (Fig. 4) , supporting the hypothesis that TLR1 and TLR6 are involved in conjunction with TLR2 in cellular activation by HCV core and NS3.


Figure 6
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  		Figure 6. Targeting of TLR2, TLR1, and TLR6 with specific siRNA conditions reduced cytokine production in hMDMs, which were transfected with siRNAs specific for negative control, TLR2, TLR1, and TLR6, or treated with transfection reagent alone (media). On Day 3 after siRNA transfection, the cells were stimulated with Pam3CSK4 (TLR2/TLR1 ligand) at 50 ng/ml, Pam2CSK4 (TLR2/TLR6 ligand) at 5 ug/ml, PGN (TLR2/TLR6 ligand) at 10 ug/ml, phenol extraction-pLPS (TLR4 ligand) at 100 ng/ml, or HCV core and HCV NS3 proteins at 5 ug/ml, as indicated. TNF-{alpha} (A), IL-6 (B), and IL-10 (C) were measured in culture supernatants using specific ELISA. Data are shown as an average ± SD ng/ml from n = 5. *, P < 0.05, calculated using Student’s t-test in siRNA-transfected samples compared with corresponding transfection reagent-treated controls.

To evaluate whether the anti-inflammatory response from HCV protein stimulation in macrophages is dependent on TLR2, TLR1, or TLR6, we measured IL-10 levels after TLR-specific knockdown in hMDMs stimulated with HCV core or NS3 proteins. There was a significant attenuation in IL-10 induction in TLR2 siRNA-transfected hMDMs after stimulation with core or NS3 proteins, suggesting that TLR2 was important for IL-10 production (Fig. 6C) . TLR1 and TLR6 siRNA attenuated IL-10 induction by HCV core and NS3 proteins in macrophages, but only TLR1 knockdown resulted in significant inhibition of NS3-induced IL-10 (Fig. 6C) . These results suggest that TLR2 is involved in IL-10 induction by HCV core and NS3 stimulation.

Our method of knockdown by siRNA transfection was neither complete nor stable and left residual cytokine production during stimulation. Thus, we used peritoneal macrophages isolated from TLR-specific knockout mice as an alternate approach to determine the roles of TLR2, TLR1, or TLR6 receptors in HCV protein-induced stimulation. In accordance with previous findings [14 ], the absence of TLR2 prevented TNF-{alpha} induction by HCV core, NS3, or TLR2 ligand stimulation (Fig. 7A ). Macrophages from TLR1–/– mice showed no significant decrease in TNF-{alpha} production when stimulated by recombinant HCV core or NS3 proteins (Fig. 7B) . Contrary to the TLR1–/–, macrophages from TLR6–/– mice had a significantly attenuated TNF-{alpha} production upon HCV core or NS3 protein stimulation (Fig. 7C) compared with wild-type animals. These results imply that in mouse peritoneal macrophages, TLR2 and TLR6, but not TLR1, are involved substantially in HCV core or NS3 protein-induced activation.


Figure 7
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  		Figure 7. HCV core and NS3 protein use TLR2 and TLR6 to activate mouse peritoneal macrophages, and the peritoneal macrophages of TLR2 (A), TLR1 (B), and TLR6 (C) knockout mice or wild-type mice were stimulated with Pam3CSK4 (Pam3; TLR2/TLR1 ligand) at 200 ng/ml, Pam2CSK4 (Pam2; TLR2/TLR6 ligand) at 10 ug/ml, or phenol extraction-pLPS (TLR4 ligand) at 200 ng/ml. HCV core and HCV NS3 proteins were used at serial dilution concentrations between 10 and 0.1 µg/ml, as indicated. After 10 h, the culture supernatants were analyzed for TNF-{alpha} content using ELISA. Data are shown as an average ± SD ng/ml from n = 3. *, P < 0.05, calculated using Student’s t-test in cells with siRNA-transfected samples compared with corresponding stimulation in wild-type cells.

DISCUSSION

We established the identity and involvement of TLR2 and its coreceptors in cellular activation by HCV NS3 and core proteins in human and mouse cells. Here, we show that HCV core and NS3 proteins activate not only monocytes [14 ] but also hMDMs to produce TNF-{alpha} and IL-10. We confirmed that TLR2 siRNA knockdown reduced activation significantly by HCV core and NS3 proteins. Innate immune cells in the liver, which are rich in TLRs include resident macrophages, NK cells, and NKT cells but also, recruited monocytes and macrophages [28 ]. As Kupffer cells, the resident hepatic macrophages, compose ~20% of the liver, we used macrophages derived from human monocytes to parallel Kupffer cells and to examine the effects of HCV core and NS3. Kupffer cells produce the majority of TNF-{alpha} in the liver, which can mediate liver injury [29 ]. Here, we show for the first time that TLR1 and TLR6 are involved in macrophage activation by HCV core and NS3 proteins.

Our results indicate that in human cells, the absence of TLR1 or TLR6 had a dramatic negative effect on HCV core and NS3 stimulation, suggesting the involvement of both of these TLR2 coreceptors. However, selective silencing of only one coreceptor did not result in the complete loss of cytokine induction by the HCV ligands. As a result of an incomplete knockdown using RNAi technology, we used TLR2, TLR1, and TLR6 knockout mice. In the knockout mouse model, our data demonstrate that HCV core and NS3 use the TLR2/TLR6 complex. The observation of only minimal inhibition of HCV core- or NS3-induced TNF-{alpha} in TLR1–/– suggests that in mice, recognition or activation by HCV core or NS3 proteins may not involve TLR1. As HCV does not infect mice, and their TLRs have slightly different sequences than humans, it is not surprising that there may be some differences in ligand activation between these two TLR2 coreceptors.

We also found that although there was a significant reduction in TNF-{alpha} in the HCV protein-stimulated TLR6 knockout macrophages, residual cytokine production remained. These results could imply alternate use of TLR1 or the possibility of another TLR2 coreceptor, such as CD36 (dectin-1) [30 , 31 ] or CD14 [32 ]. Alternatively, our results cannot rule out use of a different receptor in HCV core- and NS3-induced innate cell activation. Several groups demonstrated that the HCV core protein can inactivate T cells via gC1qR [33 34 35 ] and the implication of gC1qR-mediated, HCV-induced innate immune suppression awaits confirmation. Recent reports also indicate that HCV core protein may activate proinflammatory cell activation in an IFN-{gamma} receptor-dependent manner [36 , 37 ]. We and others [13 , 38 39 40 ] have reported previously that HCV core and NS3 proteins inhibit macrophage-derived dendritic cell differentiation and functional capacity.

Use of TLR1 or TLR6 as TLR2 coreceptors in macrophage activation by HCV core and NS3 proteins supports a potential for broad-range recognition and cell activation by these proteins. Recent reports show that a number of viruses, such as cytomegalovirus [41 , 42 ] and vaccinia virus [43 ], activate TLR2 and TLR1 or TLR6 inflammatory cytokine response. It is unclear as to whether specific TLR2, TLR1, and TLR6 receptor-mediated activation by HCV proteins is helpful or harmful to the host. These proteins elicit an inflammatory and anti-inflammatory response in hMDM. Considering that HCV core and NS3 proteins induce inflammatory cytokine production, TLR2-mediated signaling may represent a mechanism for nonspecific, inflammatory activation seen in chronic HCV. In support of this contention, patients with chronic HCV infection exhibit an activated phenotype of Kupffer cells [44 ]. However, it is not negligible that HCV core and NS3 proteins trigger production of IL-10, a potent anti-inflammatory cytokine. Such dual modulation of innate immunity by triggering pro- and anti-inflammatory pathways seems to be common for TLR2-signaling pathogens of different origin [45 ]. Further, in HCV-infected patients, there are reports of elevated levels of serum IL-10 [46 ] and increased IL-10 production in immune cells [47 ]. It is important that the modulation of expression of TLR2 and its coreceptors in immune and liver compartments is common for multiple liver diseases, including chronic HCV infection [4 , 48 ].

With recent research progress, the possibility of cytokine regulation and the modulation of TLR function for therapeutic purposes become realistic [49 ]. Our data provide novel insight into the mechanisms of HCV protein-induced activation of immune cells and indicate a new, potential direction in managing the imbalanced immune functions during chronic infection with HCV.

Received February 25, 2007; revised May 29, 2007; accepted May 30, 2007.

REFERENCES

    1
  1. Szabo, E., Lotz, G., Paska, C., Kiss, A., Schaff, Z. (2003) Viral hepatitis: new data on hepatitis C infection Pathol. Oncol. Res. 9,215-221[Medline]
    2. 2
  2. Fried, M. W., Shiffman, M. L., Reddy, K. R., Smith, C., Marinos, G., Goncales, F. L., Jr, Haussinger, D., Diago, M., Carosi, G., Dhumeaux, D., Craxi, A., Lin, A., Hoffman, J., Yu, J. (2002) Peginterferon {alpha}-2a plus ribavirin for chronic hepatitis C virus infection N. Engl. J. Med. 347,975-982[Abstract/Free Full Text]
    3. 3
  3. Manns, M. P., Wedemeyer, H., Cornberg, M. (2006) Treating viral hepatitis C: efficacy, side effects, and complications Gut 55,1350-1359[Free Full Text]
    4. 4
  4. Riordan, S. M., Skinner, N. A., Kurtovic, J., Locarnini, S., McIver, C. J., Williams, R., Visvanathan, K. (2006) Toll-like receptor expression in chronic hepatitis C: correlation with pro-inflammatory cytokine levels and liver injury Inflamm. Res. 55,279-285[CrossRef][Medline]
    5. 5
  5. Trinchieri, G. (1995) Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity Annu. Rev. Immunol. 13,251-276[Medline]
    6. 6
  6. Gabay, C. (2006) Interleukin-6 and chronic inflammation Arthritis. Res. Ther. 8(Suppl. 2),S3
    7. 7
  7. Machida, K., Cheng, K. T. H., Sung, V. M. H., Levine, A. M., Foung, S., Lai, M. M. C. (2006) Hepatitis C virus induces Toll-like receptor 4 expression, leading to enhanced production of ß interferon and interleukin-6 J. Virol. 80,866-874[Abstract/Free Full Text]
    8. 8
  8. Colombo, M., Kuo, G., Choo, Q. L., Donato, M. F., Del Ninno, E., Tommasini, M. A., Dioguardi, N., Houghton, M. (1989) Prevalence of antibodies to hepatitis C virus in Italian patients with hepatocellular carcinoma Lancet 2,1006-1008[CrossRef][Medline]
    9. 9
  9. Miller, R. H., Purcell, R. H. (1990) Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus super groups Proc. Natl. Acad. Sci. USA 87,2057-2061[Abstract/Free Full Text]
    10. 10
  10. Takeuchi, K., Kubo, Y., Boonmar, S., Watanabe, Y., Katayama, T., Choo, Q-L., Kuo, G., Houghton, M., Saito, I., Miyamura, T. (1990) Nucleotide sequence of core and envelope genes of the hepatitis C virus genome derived directly from human healthy carriers Nucleic Acids Res. 18,4626[Medline]
    11. 11
  11. Harada, S., Watanabe, Y., Takeuchi, K., Suzuki, T., Katayama, T., Takebe, Y., Saito, I., Miyamura, T. (1991) Expression of processed core protein of hepatitis C virus in mammalian cells J. Virol. 65,3015-3021[Abstract/Free Full Text]
    12. 12
  12. Takamizawa, A., Mori, C., Fuke, I., Manabe, S., Murakami, S., Fujita, J., Onishi, E., Andoh, T., Yoshida, I., Okayama, H. (1991) Structure and organization of the hepatitis C virus genome isolated from human carriers J. Virol. 65,1105-1113[Abstract/Free Full Text]
    13. 13
  13. Dolganiuc, A., Kodys, K., Kopasz, A., Marshall, C., Do, T., Romics, L., Jr, Mandrekar, P., Zapp, M., Szabo, G. (2003) Hepatitis C virus core and nonstructural protein 3 proteins induce pro- and anti-inflammatory cytokines and inhibit dendritic cell differentiation J. Immunol. 170,5615-5624[Abstract/Free Full Text]
    14. 14
  14. Dolganiuc, A., Oak, S., Kodys, K., Golenbock, D. T., Finberg, R. W., Kurt-Jones, E., Szabo, G. (2004) Hepatitis C core and nonstructural 3 proteins trigger Toll-like receptor 2-mediated pathways and inflammatory activation Gastroenterology 127,1513-1524[CrossRef][Medline]
    15. 15
  15. Eckart, M. R., Selby, M., Masiarz, F., Lee, C., Berger, K., Crawford, K., Kuo, C., Kuo, G., Houghton, M., Choo, Q. L. (1993) The hepatitis C virus encodes a serine protease involved in processing of the putative nonstructural proteins from the viral polyprotein precursor Biochem. Biophys. Res. Commun. 192,399-406[CrossRef][Medline]
    16. 16
  16. Kim, D. W., Gwack, Y., Han, J. H., Choe, J. (1995) C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity Biochem. Biophys. Res. Commun. 215,160-166[CrossRef][Medline]
    17. 17
  17. Li, K., Foy, E., Ferreon, J. C., Nakamura, M., Ferreon, A. C., Ikeda, M., Ray, S. C., Gale, M., Jr, Lemon, S. M. (2005) Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF Proc. Natl. Acad. Sci. USA 102,2992-2997[Abstract/Free Full Text]
    18. 18
  18. Tanaka, Y., Shimoike, T., Ishii, K., Suzuki, R., Suzuki, T., Ushijima, H., Matsuura, Y., Miyamura, T. (2000) Selective binding of hepatitis C virus core protein to synthetic oligonucleotides corresponding to the 5' untranslated region of the viral genome Virology 270,229-236[CrossRef][Medline]
    19. 19
  19. Clemens, J. M., Taskar, S., Chau, K., Vallari, D., Shih, J. W., Alter, H. J., Schleicher, J. B., Mimms, L. T. (1992) IgM antibody response in acute hepatitis C viral infection Blood 79,169-172[Abstract/Free Full Text]
    20. 20
  20. Hsieh, T. Y., Matsumoto, M., Chou, H. C., Schneider, R., Hwang, S. B., Lee, A. S., Lai, M. M. (1998) Hepatitis C virus core protein interacts with heterogeneous nuclear ribonucleoprotein K J. Biol. Chem. 273,17651-17659[Abstract/Free Full Text]
    21. 21
  21. Rolland, A., Jouvin-Marche, E., Viret, C., Faure, M., Perron, H., Marche, P. N. (2006) The envelope protein of a human endogenous retrovirus-W family activates innate immunity through CD14/TLR4 and promotes Th1-like responses J. Immunol. 176,7636-7644[Abstract/Free Full Text]
    22. 22
  22. Tabeta, K., Georgel, P., Janssen, E., Du, X., Hoebe, K., Crozat, K., Mudd, S., Shamel, L., Sovath, S., Goode, J., Alexopoulou, L., Flavell, R. A., Beutler, B. (2004) Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection Proc. Natl. Acad. Sci. USA 101,3516-3521[Abstract/Free Full Text]
    23. 23
  23. Melchjorsen, J., Jensen, S. B., Malmgaard, L., Rasmussen, S. B., Weber, F., Bowie, A. G., Matikainen, S., Paludan, S. R. (2005) Activation of innate defense against a paramyxovirus is mediated by RIG-I and TLR7 and TLR8 in a cell-type-specific manner J. Virol. 79,12944-12951[Abstract/Free Full Text]
    24. 24
  24. Ozinsky, A., Underhill, D. M., Fontenot, J. D., Hajjar, A. M., Smith, K. D., Wilson, C. B., Schroeder, L., Aderem, A. (2000) The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between Toll-like receptors Proc. Natl. Acad. Sci. USA 97,13766-13771[Abstract/Free Full Text]
    25. 25
  25. Thobe, B. M., Frink, M., Hildebrand, F., Schwacha, M. G., Hubbard, W. J., Choudhry, M. A., Chaudry, I. H. (2007) The role of MAPK in Kupffer cell Toll-like receptor (TLR)2-, TLR4-, and TLR9-mediated signaling following trauma-hemorrhage J. Cell. Physiol. 210,667-675[CrossRef][Medline]
    26. 26
  26. Dahle, M. K., Øverland, G., Myhre, A. E., Stuestøl, J. F., Hartung, T., Krohn, C. D., Mathiesen, Ø., Wang, J. E., Aasen, A. O. (2004) The phosphatidylinositol 3-kinase/protein kinase B signaling pathway is activated by lipoteichoic acid and plays a role in Kupffer cell production of interleukin-6 (IL-6) and IL-10 Infect. Immun. 72,5704-5711[Abstract/Free Full Text]
    27. 27
  27. El-Kady, I. M., Lotfy, M., Badra, G., El-Masry, S., Waked, I. (2005) Interleukin (IL)-4, IL-10, IL-18 and IFN-{gamma} cytokines pattern in patients with combined hepatitis C virus and Schistosoma mansoni infections Scand. J. Immunol. 61,87-91[CrossRef][Medline]
    28. 28
  28. Lauzon, N. M., Mian, F., MacKenzie, R., Ashkar, A. A. (2006) The direct effects of Toll-like receptor ligands on human NK cell cytokine production and cytotoxicity Cell. Immunol. 241,102-112[CrossRef][Medline]
    29. 29
  29. Kolios, G., Valatas, V., Kouroumalis, E. (2006) Role of Kupffer cells in the pathogenesis of liver disease World J. Gastroenterol. 12,7413-7420[Medline]
    30. 30
  30. Triantafilou, M., Gamper, F. G., Haston, R. M., Mouratis, M. A., Morath, S., Hartung, T., Triantafilou, K. (2006) Membrane sorting of Toll-like receptor (TLR)-2/6 and TLR2/1 heterodimers at the cell surface determines heterotypic associations with CD36 and intracellular targeting J. Biol. Chem. 281,31002-31011[Abstract/Free Full Text]
    31. 31
  31. Yadav, M., Schorey, J. S. (2006) The ß-glucan receptor dectin-1 functions together with TLR2 to mediate macrophage activation by mycobacteria Blood 108,3168-3175[Abstract/Free Full Text]
    32. 32
  32. Kirschning, C. J., Wesche, H., Merrill Ayres, T., Rothe, M. (1998) Human Toll-like receptor 2 confers responsiveness to bacterial lipopolysaccharide J. Exp. Med. 188,2091-2097[Abstract/Free Full Text]
    33. 33
  33. Kittlesen, D. J., Chianese-Bullock, K. A., Yao, Z. Q., Braciale, T. J., Hahn, Y. S. (2000) Interaction between complement receptor gC1qR and hepatitis C virus core protein inhibits T-lymphocyte proliferation J. Clin. Invest. 106,1239-1249[Medline]
    34. 34
  34. Yao, Z. Q., Eisen-Vandervelde, A., Waggoner, S. N., Cale, E. M., Hahn, Y. S. (2004) Direct binding of hepatitis C virus core to gC1qR on CD4+ and CD8+ T cells leads to impaired activation of Lck and Akt J. Virol. 78,6409-6419[Abstract/Free Full Text]
    35. 35
  35. Eisen-Vandervelde, A. L., Waggoner, S. N., Yao, Z. Q., Cale, E. M., Hahn, C. S., Hahn, Y. S. (2004) Hepatitis C virus core selectively suppresses interleukin-12 synthesis in human macrophages by interfering with AP-1 activation J. Biol. Chem. 279,43479-43486[Abstract/Free Full Text]
    36. 36
  36. Iwata, K., Wakita, T., Okumura, A., Yoshioka, K., Takayanagi, M., Wands, J. R., Kakumu, S. (1995) Interferon {gamma} production by peripheral blood lymphocytes to hepatitis C virus core protein in chronic hepatitis C infection Hepatology 22,1057-1064[CrossRef][Medline]
    37. 37
  37. Anthony, D. D., Post, A. B., Valdez, H., Peterson, D. L., Murphy, M., Heeger, P. S. (2001) ELISPOT analysis of hepatitis C virus protein-specific IFN-{gamma}-producing peripheral blood lymphocytes in infected humans with and without cirrhosis Clin. Immunol. 99,232-240[CrossRef][Medline]
    38. 38
  38. Dolganiuc, A., Chang, S., Kodys, K., Mandrekar, P., Bakis, G., Cormier, M., Szabo, G. (2006) Hepatitis C virus (HCV) core protein-induced, monocyte-mediated mechanisms of reduced IFN-{alpha} and plasmacytoid dendritic cell loss in chronic HCV infection J. Immunol. 177,6758-6768[Abstract/Free Full Text]
    39. 39
  39. Szabo, G., Dolganiuc, A. (2005) Subversion of plasmacytoid and myeloid dendritic cell functions in chronic HCV infection Immunobiology 210,237-247[CrossRef][Medline]
    40. 40
  40. Kanto, T., Hayashi, N., Takehara, T., Tatsumi, T., Kuzushita, N., Ito, A., Sasaki, Y., Kasahara, A., Hori, M. (1999) Impaired allostimulatory capacity of peripheral blood dendritic cells recovered from hepatitis C virus-infected individuals J. Immunol. 162,5584-5591[Abstract/Free Full Text]
    41. 41
  41. Compton, T., Kurt-Jones, E. A., Boehme, K. W., Belko, J., Latz, E., Golenbock, D. T., Finberg, R. W. (2003) Human cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like receptor 2 J. Virol. 77,4588-4596[Abstract/Free Full Text]
    42. 42
  42. Boehme, K. W., Guerrero, M., Compton, T. (2006) Human cytomegalovirus envelope glycoproteins B and H are necessary for TLR2 activation in permissive cells J. Immunol. 177,7094-7102[Abstract/Free Full Text]
    43. 43
  43. Zhu, J., Martinez, J., Huang, X., Yang, Y. (2007) Innate immunity against vaccinia virus is mediated by TLR2 and requires TLR-independent production of IFN-ß Blood 109,619-625[Abstract/Free Full Text]
    44. 44
  44. Burgio, V. L., Ballardini, G., Artini, M., Caratozzolo, M., Bianchi, F. B., Levrero, M. (1998) Expression of co-stimulatory molecules by Kupffer cells in chronic hepatitis of hepatitis C virus etiology Hepatology 27,1600-1606[CrossRef][Medline]
    45. 45
  45. Pecora, N. D., Gehring, A. J., Canaday, D. H., Boom, W. H., Harding, C. V. (2006) Mycobacterium tuberculosis LprA is a lipoprotein agonist of TLR2 that regulates innate immunity and APC function J. Immunol. 177,422-429[Abstract/Free Full Text]
    46. 46
  46. Jia, H. Y., Du, J., Zhu, S. H., Ma, Y. J., Chen, H. Y., Yang, B. S., Cai, H. F. (2002) The roles of serum IL-18, IL-10, TNF-{alpha} and sIL-2R in patients with chronic hepatitis C Hepatobiliary Pancreat. Dis. Int. 1,378-382[Medline]
    47. 47
  47. Kakumu, S., Okumura, A., Ishikawa, T., Iwata, K., Yano, M., Yoshioka, K. (1997) Production of interleukins 10 and 12 by peripheral blood mononuclear cells (PBMC) in chronic hepatitis C virus (HCV) infection Clin. Exp. Immunol. 108,138-143[CrossRef][Medline]
    48. 48
  48. Xiong, J., Zhu, Z. H., Liu, J. S., Wang, Y., Wu, H. S. (2006) The expression of Toll-like receptor 2, 4 of livers in mice with systemic inflammatory response syndrome Hepatobiliary Pancreat. Dis. Int. 5,143-146[Medline]
    49. 49
  49. Horsmans, Y., Berg, T., Desager, J. P., Mueller, T., Schott, E., Fletcher, S. P., Steffy, K. R., Bauman, L. A., Kerr, B. M., Averett, D. R. (2005) Isatoribine, an agonist of TLR7, reduces plasma virus concentration in chronic hepatitis C infection Hepatology 42,724-731[CrossRef][Medline]



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