Resveratrol-mediated gamma interferon reduction prevents airway inflammation and airway hyperresponsiveness in respiratory syncytial virus-infected immunocompromised mice

Abstract

Respiratory syncytial virus (RSV) is the most important cause of severe, lower respiratory tract infections in infants, and RSV infections have been associated with chronic wheezing and asthma during childhood. However, the mechanism of RSV-induced airway inflammation and airway hyperresponsiveness (AHR) is poorly understood. Furthermore, there are presently neither effective vaccines nor drugs available for the prevention or treatment of RSV infections. In this study, we investigated the effect of the plant extract resveratrol as a means of preventing airway inflammation and attenuating RSV-induced AHR. Our data showed that resveratrol reduced RSV lung titers and the number of infiltrating lymphocytes present in bronchoalveolar lavage fluid (BALF) and reduced inflammation. Furthermore, resveratrol attenuated airway responses to methacholine following RSV infection and significantly decreased gamma interferon (IFN-γ) levels in BALF of RSV-infected mice. Data presented in this report demonstrated that resveratrol controlled Toll-like receptor 3 (TLR3) expression, inhibited the TRIF signaling pathway, and induced M2 receptor expression following RSV infection. These data support a role for the use of resveratrol as a means of reducing IFN-γ levels associated with RSV-mediated airway inflammation and AHR, which may be mediated via TLR3 signaling.

Fig. 1.

Effect of resveratrol on pulmonary virus titers. Lungs harvested from resveratrol (RES)-treated (30 mg/kg/day) and RSV-infected mice (n = 6/group) were individually homogenized, and virus titers were determined by use of a plaque assay. Data are expressed as PFU per gram of lung tissue. Values are expressed as means ± SD on day 3 (P < 0.01 for the resveratrol-treated group versus the RSV-infected group), day 4 (P < 0.05 for the resveratrol-treated group versus the RSV-infected group), and day 5 (P < 0.01 for the resveratrol-treated group versus the RSV-infected group) postinfection.

Fig. 2.

Effect of resveratrol on the recruitment of inflammatory cells recoverable from BALF. Cells were isolated by cytospin and stained with DiffQuik. Cell numbers were assessed by use of a hemocytometer. Control, PBS control; RSV, PBS-treated RSV-infected mice; RSV+RES, resveratrol (RES)-treated RSV-infected mice. Values are expressed as the means ± SD (n = 6/group). (A) Total cells present in the BALF of the respective treatment groups. (B) Differences in cell types between treatment groups. **, P < 0.01 versus control and resveratrol-treated mice.

Fig. 3.

Effect of resveratrol on the recruitment of leukocytes into lung tissues. (A) Histological examination of lung tissues performed at 5 days post-resveratrol treatment. Lung tissues were fixed, and 5-μm-thick sections were stained with H&E (magnifications, ×200 [I, III, and V] and ×400 [II, IV, and VI]). Panels I and II, control mice; panels III and IV, RSV-infected mice; panels V and VI, resveratrol-treated mice. (B) Lung tissue inflammatory cell infiltration scores. *, P < 0.05 versus the control group; **, P < 0.01 versus the control and resveratrol-treated mice.

Fig. 4.

Effect of resveratrol on RSV-induced AHR. AHR was measured at 5 days post-resveratrol treatment in mice treated with increasing methacholine concentrations (3.125 to 50.0 mg/ml) by plethysmography. Control, untreated mice; RSV, RSV-infected mice; RSV+RES, resveratrol (RES)-treated RSV-infected mice. Values are expressed as means ± SD (n = 8/group). *, P < 0.01 versus the control and resveratrol-treated RSV-infected groups; **, P < 0.01 versus the control group.

Fig. 5.

Effect of resveratrol on BALF cytokine levels. BALF was collected at 5 days post-resveratrol treatment. Each sample was analyzed by an ELISA (n = 8/group). IFN-γ (A), IL-4 (B), IL-6 (C), IL-10 (D), and IL-17 (E) levels in BALF are shown. Control, untreated mice; RSV, RSV-infected mice; RSV+RES, resveratrol-treated mice. Values are expressed as means ± SD (n = 8/group). **, P < 0.01 versus the control and resveratrol-treated groups.

Fig. 6.

Effect of IFN-γ on airway inflammation and hyperresponsiveness. (A) Total cells present in BALF. (B) Leukocyte percentages before and after anti-IFN-γ treatment. (C) Penh levels. (D) M2R mRNA levels. (E) IFN-γ levels after anti-IFN-γ treatment. Values are expressed as means ± SD (n = 8/group). *, P < 0.05 versus the RSV group; **, P < 0.001 versus the RSV group with between 12.5 and 50 mg/ml methacholine.

Fig. 7.

Effect of resveratrol on TLR3, TRIF, and M2 expression levels. Lung tissues were harvested at 5 days post-resveratrol treatment. (A) TLR3 gene expression in lung tissue. (B) M2R gene expression in lung tissue. Resveratrol decreased the TLR3 level and increased the M2 gene expression level. Each sample was analyzed by RT-PCR (n = 7/group). (C) Relative TRIF protein expression was inhibited by resveratrol treatment. Each sample was analyzed by Western blotting (n = 5/group). *, P < 0.05 versus the RSV group.