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. 2003 Apr;77(8):4588-96.
doi: 10.1128/jvi.77.8.4588-4596.2003.

Human cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like receptor 2

Teresa Compton  1 , Evelyn A Kurt-Jones, Karl W Boehme, John Belko, Eicke Latz, Douglas T Golenbock, Robert W Finberg
Affiliations

Human cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like receptor 2

Teresa Compton et al. J Virol. 2003 Apr.

Abstract

Human cytomegalovirus (CMV) is a ubiquitous opportunistic pathogen that causes significant morbidity and mortality in immunocompromised people. An understanding of how CMV induces and circumvents host immunity is of critical importance in efforts to design effective therapeutics. It was recently discovered that mere cell contact by CMV particles leads to profound modulation of cellular gene expression, including induction of inflammatory cytokines and interferon-stimulated genes characteristic of innate immune detection. These findings suggest that a membrane receptor recognizes a CMV envelope protein(s), leading to innate immune activation. Here, we show that the pattern recognition receptors Toll-like receptor 2 (TLR2) and CD14 recognize CMV virions and trigger inflammatory cytokine production. Induction of inflammatory cytokines is mediated via TLR2-dependent activation of NF-kappa B. Since many of the pathological processes associated with CMV disease are facilitated or directly mediated by inflammatory cytokines, identification of the host membrane detection machinery may ultimately lead to improved therapeutics.

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Figures

FIG. 1.
FIG. 1.
CMV activation of human PBMC. (A) CMV stimulates cytokine secretion from normal human PBMC. Human PBMC (5 × 105/well) were incubated with no stimulus (Medium) or with UV-inactivated CMV (2.5 × 103 to 2 × 104 PFU/well), phenol reextracted LPS (pLPS; 100 ng/ml; TLR4 ligand), yeast zymosan (Zym; 10 μg/ml; TLR2 ligand), or IL-1β (100 ng/ml; TLR- and CD14-independent positive control ligand). The supernatants were harvested 18 h later, and IL-8 levels were determined by ELISA. The error bars indicate standard deviations. (B) CMV DBs induce both IL-6 and IL-8 cytokine secretion from human PBMC. CMV DBs (0.01 to 5 μl/well) were added to human PBMC as described for panel A. The supernatants were harvested 18 h later, and IL-6 and IL-8 levels were determined by ELISA.
FIG. 2.
FIG. 2.
IL-8 secretion from human monocytes is CD14 dependent. Human PBMC (5 × 105/well) were incubated with no stimulus (Medium) or with UV-inactivated CMV (2.5 × 103 to 2 × 104 PFU/well) or DBs (0.01 to 5 μl/well). The supernatants were harvested 18 h later, and IL-8 levels were determined by ELISA. Anti-CD14 monoclonal antibody (MY4; 5 μg/ml) or isotype control antibody was added to the cultures 30 min prior to the addition of stimulants. The error bars indicate standard deviations.
FIG. 3.
FIG. 3.
CMV-induced cytokine secretion is CD14 and TLR2 dependent. (A) HEK293 cells stably transfected with CD14 alone or in combination with TLR2 or TLR4 were incubated with no stimulus (Mock) or with IL-1β (100 ng/ml; TLR- and CD14-independent positive control ligand), phenol-reextracted LPS (pLPS; 10 ng/ml; TLR4 ligand), yeast zymosan (10 μg/ml; TLR2 ligand), or virions, UV-inactivated virions, or DBs. The supernatants were harvested 18 h later, and IL-8 levels were determined by ELISA. The error bars indicate standard deviations. (B) PECs (105/well) isolated from thioglycolate-injected wild-type (wt), TLR2−/−, or TLR4−/− mice were incubated with no stimulus (Medium) or with pLPS (10 ng/ml; TLR4 ligand), yeast zymosan (10 μg/ml; TLR2 ligand), or UV-inactivated CMV (1 × 103 to 3 × 104 PFU/well). The supernatants were harvested 18 h later, and IL-6 levels were determined by ELISA.
FIG. 4.
FIG. 4.
TLR2-dependent responses require CD14. HEK293 cell lines stably transfected with CD14, TLR2 alone, or a combination of TLR2 and CD14 were incubated with no stimulus (Medium) or with UV-inactivated HCMV (102 to 104 PFU/well), zymosan (10 μg/ml; TLR2 ligand), or IL-1β (100 ng/ml; TLR- and CD14-independent positive control ligand). The supernatants were harvested 18 h later, and IL-8 levels were determined by ELISA. The error bars indicate standard deviations.
FIG. 5.
FIG. 5.
TLR2 is required for NF-κB activation in response to CMV. (A and B) CHO/CD14 (A) or CHO/CD14/TLR2 (B) reporter cells were mock infected, treated with zymosan A (10 μg/ml), or infected with CMV (multiplicity of infection, 0.1), and NF-κB activity was measured using the NF-κB-driven CD25 reporter. At 18 h posttreatment-infection, cell surface expression of CD25 was measured by FACS. For each histogram, the vertical axis represents the relative cell numbers, and the horizontal axis represents the intensity of fluorescence staining. The shaded areas represent the isotype control, the dotted lines represent mock-infected cells, the thin solid lines represent zymosan A-treated cells, and the thick solid lines represent CMV-infected cells. IgG, immunoglobulin G; FITC, fluorescein isothiocyanate. (C) The percentages of CD25-positive cells from the data in panels A and B are presented graphically.
FIG. 6.
FIG. 6.
NF-κB transcriptional responses to CMV are mediated by TLR2 and enhanced by CD14. HEK293 cells stably transfected with TLR2, TLR4, or a combination of TLR4 and MD2 were transiently transfected with an NF-κB-driven firefly luciferase construct and a herpes simplex virus thymidine kinase-driven Renilla luciferase construct (for normalization). Where indicated, the cells were also transfected with CD14 or vector control DNA. Eighteen hours later, the cells were incubated with no stimulus (Medium) or with UV-inactivated HCMV (102 to 104 PFU/well). Following a 6-h stimulation, the cells were lysed and luciferase activity was determined using a Dual-Glo Luciferase Assay. The data are expressed as the mean (plus standard deviation) relative light units (RLU) of triplicate wells. The NF-κB luciferase activity of each well was normalized for transfection efficiency using the Renilla luciferase activity.
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