Viral-mediated inhibition of antioxidant enzymes contributes to the pathogenesis of severe respiratory syncytial virus bronchiolitis

Abstract

Rationale: Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in children, for which no specific treatment or vaccine is currently available. We have previously shown that RSV induces reactive oxygen species in cultured cells and oxidative injury in the lungs of experimentally infected mice. The mechanism(s) of RSV-induced oxidative stress in vivo is not known.

Objectives: To measure changes of lung antioxidant enzymes expression/activity and activation of NF-E2-related factor 2 (Nrf2), a transcription factor that regulates detoxifying and antioxidant enzyme gene expression, in mice and in infants with naturally acquired RSV infection.

Methods: Superoxide dismutase 1 (SOD 1), SOD 2, SOD 3, catalase, glutathione peroxidase, and glutathione S-transferase, as well as Nrf2 expression, were measured in murine bronchoalveolar lavage, cell extracts of conductive airways, and/or in human nasopharyngeal secretions by Western blot and two-dimensional gel electrophoresis. Antioxidant enzyme activity and markers of oxidative cell injury were measured in either murine bronchoalveolar lavage or nasopharyngeal secretions by colorimetric/immunoassays.

Measurements and main results: RSV infection induced a significant decrease in the expression and/or activity of SOD, catalase, glutathione S-transferase, and glutathione peroxidase in murine lungs and in the airways of children with severe bronchiolitis. Markers of oxidative damage correlated with severity of clinical illness in RSV-infected infants. Nrf2 expression was also significantly reduced in the lungs of viral-infected mice.

Conclusions: RSV infection induces significant down-regulation of the airway antioxidant system in vivo, likely resulting in lung oxidative damage. Modulation of oxidative stress may pave the way toward important advances in the therapeutic approach of RSV-induced acute lung disease.

Figures

Figure 1.

Antioxidant enzymes are reduced in bronchoalveolar lavage (BAL) of respiratory syncytial virus (RSV)-infected mice. Groups of mice were infected with RSV or sham inoculated with saline (Control) and BAL was collected at Days 1, 3, 5, and 9. BAL proteins were resolved on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blots were performed using antibodies against superoxide dismutase (SOD) 1, SOD 2, SOD 3, catalase, and glutathione S-transferase (GST)-mu. Membranes were stripped and reprobed for β-actin as an internal control for protein integrity and loading. Lanes 1 to 4 are BAL from four control and 5 to 8 from four RSV-infected mice at each time point. Densitometric analysis of Western blot band intensities is presented in Figure E1. The figure is representative of three independent experiments, each experiment with four mice per group at each time point.

Figure 2.

Superoxide dismutase (SOD) 1, SOD 2, and SOD 3 in conductive airway epithelial cells. (A) Proteins of conductive airway epithelial cells were obtained by lysis-lavage from respiratory syncytial virus (RSV)-infected or control mice (Day 1 after infection). Proteins were analyzed by Western blot for content of SOD 1, SOD 2, and SOD 3 as in Figure 1. (B) Densitometric analysis of Western blot band intensities was performed using Alpha Ease software, version 2200 (2.2d) (Alpha Innotech Co., San Leandro, CA). Bands in RSV-infected samples were normalized to uninfected control sample background and are presented as mean ± SEM of n = 4. *P < 0.05; ***P < 0.001 relative to control mice.

Figure 3.

Respiratory syncytial virus (RSV) infection inhibits antioxidant enzyme activity in the lung. Specific biochemical assays were used to determine total superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione S-transferase (GST) activity in bronchoalveolar lavage of groups of mice that were RSV infected or sham inoculated as in Figure 1. The figure is representative of three independent experiments, each experiment with four to five mice per group at each time point. Data are presented as mean ± SEM of four mice per group at each time point. *P < 0.05; **P < 0.01 and ***P < 0.001 relative to control mice.

Figure 4.

Two-dimensional gel electrophoresis (2DE) of bronchoalveolar lavage (BAL) proteins reveals a global reduction in antioxidant enzyme expression after respiratory syncytial virus (RSV) infection. SYPRO Ruby–stained 2DE of BAL from (A) control and (B) RSV-infected mouse at Day 3 after infection Desalted and albumin-depleted proteins (200 μl at 1 mg/ml concentration) were fractionated over immobilized pH gradients from 3 to 10 in the horizontal dimension, followed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in the vertical dimension. Left, migration of molecular mass standards (kDa). The spot volumes were normalized based on the total spot volume for each gel, and the control and RSV-infected samples were compared. The numbers indicate differentially expressed spots identified by tryptic peptide mass fingerprinting and listed in C.

Figure 5.

Respiratory syncytial virus (RSV) infection is associated with decreased levels of nuclear Nrf2 in the lung. Mice were infected with RSV or sham inoculated and lungs were harvested at 12 and 24 hours to isolate nuclear proteins. Equal amounts of nuclear proteins were analyzed by Western blot using anti-Nrf2 antibody. Membranes were stripped and reprobed for Lamin B, as control for equal loading of the samples. Lanes 1 to 3 are lung nuclear proteins from control and 4 to 6 from RSV-infected mice at each time point. The figure is representative of three independent experiments, each experiment with four to five mice per group at each time point. Densitometric analysis of Western blot band intensities was performed using Alpha Ease software presented as mean ± SEM of n = 3. *P < 0.05; **P < 0.01 relative to control mice.

Figure 6.

Concentrations of the oxidative stress markers in nasopharyngeal secretions (NPS) of infants with naturally acquired respiratory syncytial virus (RSV) infections. NPS collected from infants and young children with RSV-proven upper respiratory tract infections (URTI) and bronchiolitis were tested for (A) F2-isoprostane or (B) malondialdehyde (MDA) concentrations. Horizontal lines indicate the mean concentration. **P < 0.01 and ***P < 0.001 compared with URTI.

Figure 7.

Antioxidant enzyme expression in nasopharyngeal secretions (NPS) of infants with naturally acquired respiratory syncytial virus (RSV) infections. (A) Western blot analysis of superoxide dismutase (SOD) 1, SOD 2, and SOD 3, catalase, and glutathione S-transferase (GST)-mu in NPS of children with upper respiratory tract infections (URTI), bronchiolitis (BR), bronchiolitis with hypoxia (BR + H), and patients on ventilatory support (VS). β-actin was used as a control for protein integrity and equal loading of the samples. Densitometric analysis of Western blot band intensities was performed using Alpha Ease software. *P < 0.05; **P < 0.01, compared with URTI.