Differential role for TLR3 in respiratory syncytial virus-induced chemokine expression

Abstract

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in young infants worldwide. Previous studies have reported that the induction of interleukin-8/CXCL8 and RANTES/CCL5 correlates with disease severity in humans. The production of these chemokines is elicited by viral replication and is NF-kappaB dependent. RSV, a negative-sense single-stranded RNA virus, requires full-length positive-sense RNA for synthesis of new viral RNA. The aim of our studies was to investigate whether active viral replication by RSV could evoke chemokine production through TLR3-mediated signaling pathways. In TLR3-transfected HEK 293 cells, live RSV preferentially activated chemokines in both a time- and dose-dependent manner compared to vector controls. RSV was also shown to upregulate TLR3 in human lung fibroblasts and epithelial cells (MRC-5 and A549). Targeting the expression of TLR3 with small interfering RNA decreased synthesis of IP-10/CXCL10 and CCL5 but did not significantly reduce levels of CXCL8. Blocking the expression of the adapter protein MyD88 established a role for MyD88 in CXCL8 production, whereas CCL5 synthesis was found to be MyD88 independent. Production of CCL5 by RSV was induced directly through TLR3 signaling pathways and did not require interferon (IFN) signaling through the IFN-alpha/beta receptor. TLR3 did not affect viral replication, since equivalent viral loads were recovered from RSV-infected cells despite altered TLR3 expression. Taken together, our studies indicate that TLR3 mediates inflammatory cytokine and chemokine production in RSV-infected epithelial cells.

FIG. 1.

RSV selectively upregulates chemokines in HEK 293 cells transfected with TLR3. HEK 293 cells stably transfected with TLR3 and vector controls were infected with live RSV or UV-inactivated RSV (MOI of 0.1) or incubated with media alone for 8, 24, and 48 hpi. At each time point, RNA levels were analyzed by QPCR and expressed as the increase (n-fold) relative to media controls (A), and protein levels in the supernatant were determined by ELISA (B and C). HEK 293 cells expressing TLR3 versus vector controls differed significantly in their production of CXCL8 and CCL5 message and protein at 48 hpi following RSV infection. Data are representative of three experiments, with n = 3 per treatment per experiment. Each time point represents the mean ± standard error of the mean. *, P < 0.05.

FIG. 2.

TLR3 expression has no effect on viral replication in epithelial cells. HEK 293 TLR3 and vector control cells were infected with RSV at an MOI of 0.1. Cells were harvested at various times postinfection, and a plaque assay was performed. Data are representative of three experiments, with n = 3 per cell line per experiment.

FIG. 3.

TLR3 RNAi impairs CCL5 and CXCL10 production but has no effect on CXCL8 synthesis or viral replication in MRC-5 cells. TLR3 siRNA and random siRNA controls were transfected into MRC-5 cells and infected with RSV (MOIs of 0.5 to 1). Expression levels of TLR3 (A), RSV mRNA (B), and chemokines (D to F) were determined at 24 and 48 hpi. MRC-5 cells transfected with TLR3 siRNA versus random siRNA controls differed significantly in TLR3 expression and production of CCL5 at 48 hpi (*, P < 0.05). At 48 hpi, a plaque assay was performed to determine the viral load in cells with altered TLR3 expression (C). Data are representative of three experiments, with n = 3 per treatment per experiment. Each time point represents the mean ± standard error of the mean. TLR3i, TLR3 siRNA.

FIG. 4.

TLR3 RNAi impairs CCL5 and CXCL10 production but has no effect on CXCL8 synthesis or viral replication in A549 cells. TLR3 siRNA and random siRNA controls were transfected into MRC-5 cells and infected with RSV (MOI of 1). Levels of TLR3 expression (A), viral replication (B), and chemokines (C to E) were determined at 24 and 48 hpi. A549 cells transfected with TLR3 siRNA versus random siRNA controls differed significantly in TLR3 expression and synthesis of CCL5 and CXCL10 at 48 hpi (*, P < 0.05). Cells were harvested at 48 hpi postinfection, and a plaque assay was performed (B). Data are representative of three experiments, with n = 3 per treatment per experiment. Each time point represents the mean ± standard error of the mean. TLR3i, TLR3 siRNA.

FIG. 5.

MyD88-dependent and -independent signaling linked to RSV-induced chemokine production. A549 cells were transfected with random siRNA or siRNA toward TLR3 and MyD88. Twelve hours after transfection, cells were infected with RSV at an MOI of 1. At 48 hpi, RNA and protein expression levels of chemokines were determined. Data are representative of three experiments, with n = 3 per treatment per experiment. Each time point represents the mean ± standard error of the mean. The asterisk indicates statistically significant (P < 0.05) decreases compared to RSV-infected cells transfected with random siRNA.

FIG. 6.

IFN-α/β signaling is not required for RSV-induced chemokine production. A549 cells were pretreated for 1 h before RSV infection with anti-IFN-α/β receptor (aIFNR) antibody (10 μg/ml), immunoglobulin G (IgG; 10 to 20 μg/ml), or medium. The antibody was left in the medium and cells were infected at an MOI of 0.5. At 48 hpi, RNA levels of CXCL8 and CCL5 were analyzed.