Antioxidant treatment ameliorates respiratory syncytial virus-induced disease and lung inflammation

Abstract

Rationale: Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in children. No treatment has been shown to significantly improve the clinical outcome of patients with this infection. Recent evidence suggests that oxidative stress could play an important role in the pathogenesis of acute and chronic lung inflammatory diseases. We do not known whether RSV induces pulmonary oxidative stress and whether antioxidant treatment can modulate RSV-induced lung disease.

Objectives: To investigate the effect of antioxidant administration on RSV-induced lung inflammation, clinical disease, and airway hyperreactivity (AHR).

Methods: BALB/c mice were infected with 10(7) plaque-forming units of RSV, in the presence or absence of orally administered butylated hydroxyanisole (BHA), an antioxidant. Malondialdehyde and 4-hydroxynonenal were measured in bronchoalveoar lavage (BAL) by colorimetric assay. Cytokines and chemokines were measured in BAL by Bio-Plex and leukotrienes were measured by enzyme-linked immunosorbent assay. AHR to methacholine challenge was measured by whole-body plethysmography.

Results: BHA treatment significantly attenuated RSV-induced lung oxidative stress, as indicated by the decrease of malondialdehyde and 4-hydroxynonenal content in BAL of RSV-infected mice. RSV-induced clinical illness and body weight loss were also reduced by BHA treatment, which inhibited neutrophil recruitment to the lung and significantly reduced pulmonary cytokine and chemokine production after RSV infection. Similarly, antioxidant treatment attenuated RSV-induced AHR.

Conclusion: Modulation of oxidative stress represents a potential novel pharmacologic approach to ameliorate RSV-induced acute lung inflammation and potentially prevent long-term consequences associated with RSV infection, such as bronchial asthma.

Figure 1.

Lipid peroxidation products in the bronchoalveolar lavage (BAL). malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were detected by a colorimetric assay in BAL of respiratory syncytial virus (RSV)– and sham-infected mice at various times postinfection. The bar in the scatter plot represents the mean of five different animals. The figure is representative of two different experiments. *p < 0.01 relative to sham-infected mice.

Figure 2.

Effect of butylated hydroxyanisole (BHA) administration on RSV-induced weight loss. Mice were treated with increasing concentrations of BHA for 3 d before RSV infection and during the first 7 d of infection. The following is a representative diagram of three different experiments. All data points represent the mean of at least four animals. Open circles, sham; cross marks, RSV; triangles, RSV + BHA 50 mg/kg; inverted triangles, RSV + BHA 100 mg/kg; solid squares, RSV + BHA 150 mg/kg; solid circles, RSV + BHA 250 mg/kg. *p < 0.05 relative to sham-infected mice; **p < 0.01 relative to sham-infected mice.

Figure 3.

Effect of BHA on clinical illness and airway hyperreactivity (AHR). Mice were infected with RSV and treated with BHA 250 mg/kg for the following 7 d. (A) Differences in the appearance of fur in RSV + BHA– versus RSV + vehicle–treated mice at Day 3 postinfection. (B) Clinical illness scores of RSV + BHA (open squares) and RSV + vehicle (solid squares) were measured from Day 1 to Day 5 postinfection. Sham-infected mice treated with either vehicle or BHA received a healthy illness score = 0 throughout the course of the experiment (data not shown). Data are expressed as mean ± SD of five different animals and are representative of three different experiments. *p < 0.01 relative to RSV-infected mice. (C) The effect of BHA treatment on RSV-induced AHR during methacholine challenge was measured at Day 4 postinfection by whole-body plethysmography. All treatment groups were given an initial dose of saline and subsequently challenged with increasing concentrations of methacholine (mg/ml). Penh is reported as a percentage increase from baseline saline challenge. Data are expressed as mean ± SD of eight different animals. Open squares, sham; RSV, solid triangles; cross marks, RSV + BHA. *p < 0.01 relative to RSV-infected mice.

Figure 4.

Effect of BHA on airway inflammation and viral replication. (A) Total number of cells was measured in BAL of sham and RSV-infected mice, either untreated or treated with BHA, at various times postinfection. Data are expressed as mean ± SD of five different animals and representative of three different experiments. *p < 0.01 relative to RSV-infected mice. (B) Differential cell count was measured in BAL of sham and RSV-infected mice, either untreated or treated with BHA, at Day 3 postinfection. *p < 0.01 relative to RSV-infected mice. (C) Mice were infected with RSV or sham-infected and treated with either vehicle or BHA. At various days postinfection, mice were killed and lungs were excised, fixed in 10% buffered formalin, and embedded in paraffin. Lung sections were stained with hemotoxylin and eosin, and peribronchial, perivascular inflammation was scored. Data are expressed as mean ± SD of four animals/group. Data shown are representative of three independent experiments. **p < 0.01 relative to RSV-infected mice; ***p < 0.001 relative to sham-infected mice. (D) Mice were infected with RSV and either treated with vehicle or BHA. At Day 5 postinfection, lungs were excised and viral load was determined by plaque assay. Data shown are representative of three independent experiments (n = 10, mean ± SD). *p < 0.05 relative to RSV-infected mice.

Figure 5.

Effect of BHA on lipid peroxidation. (A) MDA and 4-HNE or (B) 8-isoprostane were detected by either colorimetric assay or ELISA in BAL of mice infected with RSV and treated with either vehicle (solid bars) or BHA (open bars) at various times postinfection. Hatched bars, sham-treated mice. The bar in the scatter plot represents the mean of five different animals. ×, sham; solid diamonds, RSV; open circles, RSV + BHA. Data shown are representative of two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 relative to RSV-infected mice.

Figure 6.

Effect of BHA on chemokine release in the BAL. Chemokine production was measured by Bio-Plex bead suspension array in the BAL of mice infected with RSV and treated with either vehicle or BHA at various times postinfection. The bars in the scatter plot represent the mean of six different animals. Data shown are representative of three independent experiments. *p < 0.01 relative to RSV-infected mice.

Figure 7.

Effect of BHA on proinflammatory cytokines release in BAL. Cytokine production was measured by Bio-Plex bead suspension array in the BAL of mice infected with RSV and treated with either vehicle or BHA at various times postinfection. The bars in the scatter plot represent the mean of six different animals. Data shown are representative of three independent experiments. ×, sham; solid diamonds, RSV; open circles, RSV + BHA.*p < 0.01 relative to RSV-infected mice.

Figure 8.

Effect of BHA on immunomodulatory cytokines in BAL. Cytokine production was measured by Bio-Plex bead suspension array in the BAL of mice infected with RSV and treated with either vehicle or control at various times postinfection. The bars in the scatter plot represent the mean of six different animals. Data shown are representative of three independent experiments.×, sham; solid diamonds, RSV; open circles, RSV + BHA. *p < 0.01 relative to RSV-infected mice.

Figure 9.

Effect of BHA on RSV-induced leukotriene release. Leukotriene C4 production was measured by ELISA in BAL of RSV-infected mice treated with either vehicle or BHA at various time points after infection. The bars in the scatter plot represent the mean of five different animals. Data shown are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 relative to RSV-infected mice.