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. 2007 Aug;81(15):8016-24.
doi: 10.1128/JVI.00403-07. Epub 2007 May 23.

Epstein-Barr virus induces MCP-1 secretion by human monocytes via TLR2

Eric Gaudreault  1 , Stéphanie Fiola, Martin Olivier, Jean Gosselin
Affiliations

Epstein-Barr virus induces MCP-1 secretion by human monocytes via TLR2

Eric Gaudreault et al. J Virol. 2007 Aug.

Abstract

Epstein-Barr virus (EBV) is a gammaherpesvirus infecting the majority of the human adult population in the world. TLR2, a member of the Toll-like receptor (TLR) family, has been implicated in the immune responses to different viruses including members of the herpesvirus family, such as human cytomegalovirus, herpes simplex virus type 1, and varicella-zoster virus. In this report, we demonstrate that infectious and UV-inactivated EBV virions lead to the activation of NF-kappaB through TLR2 using HEK293 cells cotransfected with TLR2-expressing vector along with NF-kappaB-Luc reporter plasmid. NF-kappaB activation in HEK293-TLR2 cells (HEK293 cells transfected with TLR2) by EBV was not enhanced by the presence of CD14. The effect of EBV was abrogated by pretreating HEK293-TLR2 cells with blocking anti-TLR2 antibodies or by preincubating viral particles with neutralizing anti-EBV antibodies 72A1. In addition, EBV infection of primary human monocytes induced the release of MCP-1 (monocyte chemotactic protein 1), and the use of small interfering RNA targeting TLR2 significantly reduced such a chemokine response to EBV. Taken together, these results indicate that TLR2 may be an important pattern recognition receptor in the immune response directed against EBV infection.

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Figures

FIG. 1.
FIG. 1.
EBV activates TLR2 in transfected HEK293 cells. (A) HEK293 cells were transiently transfected with TLR2 and NF-κB luciferase reporter plasmid. Forty-eight hours posttransfection, cells were stimulated with infectious EBV (MOI of 0.5) for 0 (mock stimulated [M]), 2, 8, and 24 h followed by luciferase assay. (B) HEK293 cells were transiently transfected with TLR2 and an NF-κB luciferase reporter plasmid. Forty-eight hours later, cells were mock stimulated (M) or stimulated with infectious EBV (MOI of 0.1 to 1) for 8 h or stimulated with LTA (20 μg/ml), and luciferase assay was performed. (C) HEK293 cells were transiently transfected with TLR2 and NF-κB luciferase reporter plasmid. Forty-eight hours posttransfection, cells were stimulated with infectious EBV (MOI of 0.5) B95-8, Akata, or AGS preparations or with their respective mock controls for 8 h followed by luciferase assay. Cells were also treated with sonicates from Raji and BJAB cells as described in Materials and Methods. Assays were performed in triplicate, and results are expressed in relative light units (RLU) per microgram of protein (mean plus standard error of the mean [error bar]). Values that were significantly different (P < 0.05) from the value for the mock control (M) are indicated (*). NS, not stimulated.
FIG. 2.
FIG. 2.
Blocking experiments inhibit EBV activation of NF-κB in HEK293 cells transfected with TLR2. (A) TLR2/NF-κB-transfected HEK293 cells were preincubated for 1 h at 37°C with blocking anti-TLR2 antibodies (6 μg/ml) prior to mock stimulation or stimulation with infectious EBV for 8 h, and the luciferase assay was performed. (B) Transiently transfected HEK293 cells were mock stimulated or stimulated with infectious EBV or with EBV particles preincubated with F(ab′)2 fragments of 72A1 monoclonal antibodies (37°C, 60 min) prior to cell treatment. In both panels A and B, assays were performed in triplicate, and results are expressed in relative light units (RLU) per microgram of protein (mean plus standard error of the mean [error bar]). Values that were significantly different (P < 0.05) from the value for the mock control (M) are indicated (*). NS, not stimulated; BSA, bovine serum albumin.
FIG. 3.
FIG. 3.
EBV is not recognized by TLR4. (A) HEK293 cells were transiently transfected with human TLR4, CD14/MD-2, and NF-κB luciferase reporter plasmid and were mock stimulated (−) or stimulated with EBV (+) or LPS (1 μg/ml), a natural ligand of TLR4, for 8 h. (B) HEK293 cells were transiently transfected with TLR2 and pretreated with polymyxin B (+) (10 μg/ml) (30 min, 37°C) before stimulation. Assays were performed in triplicate, and results are expressed in relative light units (RLU) per microgram of protein (mean plus standard error of the mean [error bar]). In panel A, values that were significantly different (P < 0.05) from the values for the mock-treated and EBV-treated cells are indicated (*). In panel B, values that were significantly different (P < 0.05) from the values for the mock-treated cells and cells that were not stimulated (NS) are indicated (*).
FIG. 4.
FIG. 4.
TLR2 activation by EBV is dependent on cell binding. (A) HEK293 cells were transiently transfected with TLR2 and NF-κB luciferase reporter plasmid and mock stimulated or stimulated with different concentrations of UV-irradiated EBV (U.V.-EBV) preparations. (B) TLR2-transfected cells were treated with infectious EBV or UV-irradiated or inactivated EBV or pretreated with phosphonoacetic acid (PAA) for 30 min (200 μg/ml) before stimulation with infectious EBV (PAA-EBV). Luciferase assays were performed in triplicate, and results are expressed in relative light units (RLU) per microgram of protein (mean plus standard error of the mean [error bar]). Values that were significantly different (P < 0.05) from the value for the mock control are indicated (*).
FIG. 5.
FIG. 5.
CD14 is not essential for TLR2 activation by EBV. HEK293 cells were transiently cotransfected with TLR2 and/or CD14/MD-2 and NF-κB luciferase reporter plasmid. Cells were mock stimulated or stimulated for 8 h with infectious EBV or LTA (20 μg/ml). Luciferase assays were performed in triplicate, and results are expressed in relative light units (RLU) per microgram of protein (mean plus standard error of the mean [error bar]). Values that were significantly different (P < 0.05) from the values for the medium (M) and mock control are indicated (*).
FIG. 6.
FIG. 6.
EBV stimulation leads to TLR2-dependent secretion of MCP-1 by human monocytes. (A) Monocytes were isolated from peripheral blood samples from healthy donors and mock stimulated or stimulated with infectious EBV or UV-irradiated EBV (UV-EBV) (MOI of 0.5) for 4 h before total RNA was extracted. Chemokine mRNA levels were evaluated by RPAs. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) Monocytes were isolated from peripheral blood samples from healthy donors. Cells (2 × 106 cells) were seeded in 12-well plates and were transfected with either scrambled (control [CTL]) or TLR2 (100 nM) siRNAs. Forty-eight hours posttransfection, cells were mock stimulated or stimulated with infectious EBV (MOI of 0.5) or LTA (20 μg/ml) for 8 h. Cell-free supernatants were harvested, and MCP-1 levels were determined by ELISAs. (C) Monocytes from healthy donors were isolated and pretreated or not with anti-CD14 monoclonal antibodies for 1 h at 37°C before mock stimulation or stimulation with EBV (MOI of 0.5) or LPS (1 μg/ml). Twenty-four hours posttreatment, supernatants were harvested, and MCP-1 concentrations were measured by ELISAs. Assays were performed with monocytes isolated from three different donors (except for panel C in which monocytes from two donors were tested) tested in triplicate. Values that were significantly different (P < 0.05) from the values for the mock-stimulated cells and cells transfected with control (CTL) siRNA are indicated (*).
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