Respiratory syncytial virus induces oxidative stress by modulating antioxidant enzymes

Abstract

Oxidative stress plays an important role in the pathogenesis of lung inflammation. Respiratory syncytial virus (RSV) infection induces reactive oxygen species (ROS) production in vitro and oxidative injury in lungs in vivo; however, the mechanism of RSV-induced cellular oxidative stress has not been investigated. Therefore, we determined whether RSV infection of airway epithelial cells modified the expression and/or activities of antioxidant enzymes (AOE). A549 cells, a human alveolar type II-like epithelial cell line, and small airway epithelial (SAE) cells, normal human cells derived from terminal bronchioli, were infected with RSV and harvested at various time points to measure F(2)-8 isoprostanes by enzyme-linked immunosorbent assay and total and reduced glutathione (GSH and GSSG) by colorimetric assay. Superoxide dismutase (SOD) 1, 2, and 3, catalase, glutathione peroxidase (GPx), and glutathione S-transferase (GST) expression was determined by quantitative real-time PCR and Western blot, and their activity was measured by colorimetric assays. RSV infection induced a significant increase of lipid peroxidation products as well as a significant decrease in the GSH/GSSG ratio. There was a significant decrease in SOD 1, SOD 3, catalase, and GST expression with a concomitant increase of SOD 2 in RSV-infected cells, compared with uninfected cells. Total SOD activity was increased, but catalase, GPx, and GST activities were decreased, after RSV infection. Our findings suggest that RSV-induced cellular oxidative damage is the result of an imbalance between ROS production and antioxidant cellular defenses. Modulation of oxidative stress represents a potential novel pharmacologic approach to ameliorate RSV-induced acute lung inflammation.

Figure 1.

Evidence of oxidative stress in A549 cells. (A) A549 or (B) small airway epithelial (SAE) cells were infected with respiratory syncytial virus (RSV) (solid bars) and harvested at 6, 15, 24, and 48 hours after infection to measure F2-isoprostanes and GSH/GSSG ratio (C). Open bars, control. *P < 0.05 relative to uninfected cells. The figure is representative of two different experiments run in triplicate.

Figure 2.

RSV infection modifies the expression of AOE in A549 cells. Total cell lysates, prepared from A549 cells uninfected or infected with RSV for 6, 15, 24, and 48 hours, were resolved on 10% SDS-PAGE, and Western blot was performed using antibodies against superoxide dismutase (SOD) 1, 2, 3, catalase, and glutathione-S-transferase (GST) proteins. Membranes were stripped and reprobed for β-actin as an internal control for protein integrity and loading. (A) Lanes 1, 3, 5, and 7: uninfected control cells for 6, 15, 24, and 48 hours after infection; lanes 2, 4, 6, and 8: RSV-infected cells for corresponding time points of infection. (B) Densitometric analysis of Western blot bands. Open bars, control; solid bars, RSV. The figure is representative of two independent experiments.

Figure 2.

RSV infection modifies the expression of AOE in A549 cells. Total cell lysates, prepared from A549 cells uninfected or infected with RSV for 6, 15, 24, and 48 hours, were resolved on 10% SDS-PAGE, and Western blot was performed using antibodies against superoxide dismutase (SOD) 1, 2, 3, catalase, and glutathione-S-transferase (GST) proteins. Membranes were stripped and reprobed for β-actin as an internal control for protein integrity and loading. (A) Lanes 1, 3, 5, and 7: uninfected control cells for 6, 15, 24, and 48 hours after infection; lanes 2, 4, 6, and 8: RSV-infected cells for corresponding time points of infection. (B) Densitometric analysis of Western blot bands. Open bars, control; solid bars, RSV. The figure is representative of two independent experiments.

Figure 3.

RSV infection down-regulates AOE gene expression in A549 and SAE cells. (A) A549 or (B) SAE cells were infected with RSV (solid bars) and harvested to prepare total RNA at various time points after infection. SOD 1, 2, 3 and catalase gene expression was measured by Q-RT-PCR. Open bars, control. *P < 0.05 relative to uninfected cells. The figure represents three independent experiments.

Figure 3.

RSV infection down-regulates AOE gene expression in A549 and SAE cells. (A) A549 or (B) SAE cells were infected with RSV (solid bars) and harvested to prepare total RNA at various time points after infection. SOD 1, 2, 3 and catalase gene expression was measured by Q-RT-PCR. Open bars, control. *P < 0.05 relative to uninfected cells. The figure represents three independent experiments.

Figure 4.

Effect of RSV infection on AOE activities in A549 and SAE cells. Total cell lysates were prepared from uninfected (open bars) and RSV-infected (solid bars) (A) A549 cells or (B) SAE cells for 6, 15, 24, and 48 hours to measure total SOD, catalase, GPx, and GST enzyme activities. The figure is representative of two independent experiments.

Figure 4.

Effect of RSV infection on AOE activities in A549 and SAE cells. Total cell lysates were prepared from uninfected (open bars) and RSV-infected (solid bars) (A) A549 cells or (B) SAE cells for 6, 15, 24, and 48 hours to measure total SOD, catalase, GPx, and GST enzyme activities. The figure is representative of two independent experiments.

Figure 5.

Effect of catalytic scavengers on RSV-induced chemokine secretion. A549 cells were infected with RSV in the absence or presence of 400 μM EUK-134 or EUK-163. Culture supernatants, from uninfected and infected cells, were assayed 24 hours later for RANTES and IL-8 production by ELISA. *P < 0.05 relative to untreated, RSV-infected cells. The figure is representative of two independent experiments run in triplicate.

Figure 6.

RSV infection down-regulates Nrf2 expression. A549 cells were infected with RSV for various lengths of time and harvested to prepare nuclear extracts. (A) Nuclear amounts of Nrf2 protein were determined by Western blot. Nrf2 gene expression after RSV infection was quantified in (B) A549 cells or (C) SAE cells by real-time PCR. Open bars, control; solid bars, RSV. Data are presented as fold changes and are representative of two independent experiments. *P < 0.05 relative to uninfected cells.

Figure 7.

Schematic representation of the proposed mechanism(s) of oxidative cell damage in RSV infection.