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.

Human metapneumovirus (hMPV) is a leading cause of acute respiratory tract infection in infants, as well as in the elderly and immunocompromised patients. No effective treatment or vaccine for hMPV is currently available. A recombinant hMPV lacking the G protein (rhMPV-ΔG) was recently developed as a potential vaccine candidate and shown to be attenuated in the respiratory tract of a rodent model of infection. The mechanism of its attenuation, as well as the role of G protein in modulation of hMPV-induced cellular responses in vitro, as well as in vivo, is currently unknown. In this study, we found that rhMPV-ΔG-infected airway epithelial cells produced higher levels of chemokines and type I interferon (IFN) compared to cells infected with rhMPV-WT. Infection of airway epithelial cells with rhMPV-ΔG enhanced activation of transcription factors belonging to the nuclear factor (NF)-κB and interferon regulatory factor (IRF) families, as revealed by increased nuclear translocation and/or phosphorylation of these transcription factors. Compared to rhMPV-WT, rhMPV-ΔG also increased IRF- and NF-κB-dependent gene transcription, which was reversely inhibited by G protein expression. Since RNA helicases have been shown to play a fundamental role in initiating viral-induced cellular signaling, we investigated whether retinoic induced gene (RIG)-I was the target of G protein inhibitory activity. We found that indeed G protein associated with RIG-I and inhibited RIG-I-dependent gene transcription, identifying an important mechanism by which hMPV affects innate immune responses. This is the first study investigating the role of hMPV G protein in cellular signaling and identifies G as an important virulence factor, as it inhibits the production of important immune and antiviral mediators by targeting RIG-I, a major intracellular viral RNA sensor.

Respiratory syncytial virus (RSV) produces intense pulmonary inflammation, in part, through its ability to induce chemokine synthesis in infected airway epithelial cells. RANTES (regulated upon activation, normal T-cells expressed and secreted) is a CC chemokine which recruits and activates monocytes, lymphocytes, and eosinophils, all cell types present in the lung inflammatory infiltrate induced by RSV infection. In this study we investigated the role of reactive oxygen species in the induction of RANTES gene expression in human type II alveolar epithelial cells (A549), following RSV infection. Our results indicate that RSV infection of airway epithelial cells rapidly induces reactive oxygen species production, prior to RANTES expression, as measured by oxidation of 2,7-dichlorofluorescein. Pretreatment of airway epithelial cells with the antioxidant butylated hydroxyanisol (BHA), as well a panel of chemically unrelated antioxidants, blocks RSV-induced RANTES gene expression and protein secretion. This effect is mediated through the ability of BHA to inhibit RSV-induced interferon regulatory factor binding to the RANTES promoter interferon-stimulated responsive element, that is absolutely required for inducible RANTES promoter activation. BHA inhibits de novo interferon regulator factor (IRF)-1 and -7 gene expression and protein synthesis, and IRF-3 nuclear translocation. Together, these data indicates that a redox-sensitive pathway is involved in RSV-induced IRF activation, an event necessary for RANTES gene expression. Respiratory syncytial virus (RSV)1 is an enveloped, negativesense single-stranded RNA virus (1). Since its isolation, RSV has been identified as a leading cause of epidemic respiratory tract illness in children in the United States and worldwide. In fact, RSV is so ubiquitous that it will infect 100% of children before the age of 3. It is estimated that 40 -50% of children hospitalized with bronchiolitis and 25% of children with pneumonia are infected with RSV, resulting in 100,000 hospital admissions annually in the United States alone (1). In addition to acute morbidity, there are long-term consequences of RSV infection in infancy: RSV has been shown to predispose to the development of hyperreactive airway disease (2) and recurrent episodes of wheezing in asthmatic children are often precipitated by RSV infection. The mechanisms of RSV-induced airway disease and its long-term consequences are largely unknown, but the delicate balance between immunopathology and immunoprotection in the airway mucosa may be altered by an exuberant and unwanted local inflammatory response. Airway infiltration of monocytes and lymphocytes is typical of RSV infection (1), and activation of eosinophil and basophil leukocytes has been shown to correlate with the severity of acute RSV disease (3, 4). The composition of the cellular response at sites of tissue inflammation is controlled by gradients of chemokines, a family of small chemotactic cytokines, which direct leukocyte transendothelial migration and movement t...

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Human metapneumovirus (hMPV) is a major cause of upper and lower respiratory infections in children and adults. Recent work from our group demonstrated that hMPV G glycoprotein is an important virulence factor, responsible for inhibiting innate immune responses in airway epithelial cells. Myeloid dendritic cells are potent antigen presenting cells and play a major role in initiating and modulating the innate and adaptive immune responses. In this study, we found that TLR4 plays a major role in hMPV-induced activation of monocyte-derived dendritic cells (moDCs), as downregulation of its expression by siRNA significantly blocked hMPV-induced chemokine and type I interferon expression. Similar results were found in bone marrow derived-dendritic cells (BM-DCs) from TLR4 deficient mice. MoDCs infected with a virus lacking G protein expression (rhMPV-ΔG) produced higher levels of cytokines and chemokines, compared to cells infected with wild-type virus (rhMPV-WT), suggesting that G protein plays an inhibitory role in viral-induced cellular responses. Specifically, G protein affects TLR4-dependent signaling, as rhMPV-ΔG infection of moDCs inhibited LPS-induced production of cytokine and chemokines significantly less than rhMPV-WT, and treatment of moDCs with purified G protein resulted in a similar inhibition of LPS-dependent signaling. Our results demonstrate that hMPV G protein plays an important role in inhibiting host innate immune responses, likely affecting adaptive responses too.