Respiratory syncytial virus activates innate immunity through Toll-like receptor 2

Abstract

Respiratory syncytial virus (RSV) is a common cause of infection that is associated with a range of respiratory illnesses, from common cold-like symptoms to serious lower respiratory tract illnesses such as pneumonia and bronchiolitis. RSV is the single most important cause of serious lower respiratory tract illness in children <1 year of age. Host innate and acquired immune responses activated following RSV infection have been suspected to contribute to RSV disease. Toll-like receptors (TLRs) activate innate and acquired immunity and are candidates for playing key roles in the host immune response to RSV. Leukocytes express TLRs, including TLR2, TLR6, TLR3, TLR4, and TLR7, that can interact with RSV and promote immune responses following infection. Using knockout mice, we have demonstrated that TLR2 and TLR6 signaling in leukocytes can activate innate immunity against RSV by promoting tumor necrosis factor alpha, interleukin-6, CCL2 (monocyte chemoattractant protein 1), and CCL5 (RANTES). As previously noted, TLR4 also contributes to cytokine activation (L. M. Haynes, D. D. Moore, E. A. Kurt-Jones, R. W. Finberg, L. J. Anderson, and R. A. Tripp, J. Virol. 75:10730-10737, 2001, and E. A. Kurt-Jones, L. Popova, L. Kwinn, L. M. Haynes, L. P. Jones, R. A. Tripp, E. E. Walsh, M. W. Freeman, D. T. Golenbock, L. J. Anderson, and R. W. Finberg, Nat. Immunol. 1:398-401, 2000). Furthermore, we demonstrated that signals generated following TLR2 and TLR6 activation were important for controlling viral replication in vivo. Additionally, TLR2 interactions with RSV promoted neutrophil migration and dendritic cell activation within the lung. Collectively, these studies indicate that TLR2 is involved in RSV recognition and subsequent innate immune activation.

FIG. 1.

RSV activates murine macrophages through TLR2 and TLR6. Macrophages were harvested 4 days after thioglycolate treatment from wild-type, TLR2 KO, and TLR4 KO mice. (a to c) Macrophages were stimulated with RSV (MOI of 0.3), UV-inactivated RSV (MOI equivalent of 0.3 MOI), LPS (TLR4 ligand; 100 ng/ml), Pam₂CSK₄ (TLR2 ligand; 100 ng/ml), Vero cell lysate (50 μl), or UV-inactivated Vero cell lysate (50 μl) for 24 h in the presence of brefeldin A as indicated. (d) Wild-type macrophages were stimulated with sucrose-purified RSV (MOI of 0.02) or UV-inactivated sucrose purified RSV (MOI equivalent of 0.02). Cells were stained for CDllb, permeabilized, and stained for intracellular TNF-α. Values indicate the percentages of CDllb⁺ macrophages producing TNF-α. TNF-α production (black line) was compared to that of the isotype control (gray line).

FIG. 2.

RSV induces inflammatory mediators through TLR2 and TLR6. Macrophages were stimulated with RSV (MOI of 0.3) or medium control for 24 h. Culture supernatants were harvested and tested for IL-6 (a), CCL2 (b), and CCL5 (c) production using ELISA. Error bars indicate ± standard deviations. Macrophages were stimulated with RSV (MOI of 2), poly(I:C) (50 μg/ml), LPS (100 ng/ml), or medium alone for 24 h. (d) Supernatants were tested for type I IFN production.

FIG. 3.

TLR2 and TLR6 are required for controlling RSV replication in vivo. Wild-type (n = 8), TLR4 KO (n = 8), TLR2 KO (n = 6), and TLR6 KO (n = 4) mice were infected by i.n. inoculation with RSV strain A2 (2.4 × 10⁶ PFU/mouse). Lungs were harvested 4 days after infection, and plaques were enumerated using a Vero cell immunoplaque assay. Titers are represented as log₁₀ PFU/gram of lung tissue. Solid lines represent the mean 1og₁₀ value for each group. Error bars indicate ± SEM. Mice that were negative for RSV by plaque assay were scored at 0.5 times the lower limit of detection.

FIG. 4.

TLR2 signaling influences early innate cellular response against RSV in vivo. Wild-type, TLR2 KO, and MyD88 KO mice were infected by i.n. inoculation with RSV strain A2 (2.4 × 10⁶ PFU/mouse). Uninfected mice (n = 3) were used to control for lung cytokine production and BAL infiltrate for each group. At 24, 48, and 96 h after infection, BAL and lung tissue were harvested from mice. (a) Lung tissue was homogenized and tested for CCL2 production by ELISA (wild-type and TLR2 KO mice, n = 7; MyD88 KO mice, n = 6). Data are represented as picograms/gram of lung tissue. Solid lines represent the means for each group. Error bars are ± SEM. BAL from each group (n = 3 per group) was pooled, and neutrophils (b) and activated DCs (c) were enumerated by flow cytometry. Neutrophil (7.4+ F4/80⁻) and DC (CD11b⁺ CD11c⁺ CD86⁺) populations are indicated as the percent positive cells of the total number of gated cells.