Human respiratory syncytial virus (RSV), a member of the Paramyxoviridae family, is the most important viral agent of pediatric respiratory tract disease worldwide. Human airway epithelial cells (AEC) are the primary targets of RSV. AEC are responsible for the secretion of a wide spectrum of cytokines and chemokines that are important mediators of the exacerbated airway inflammation triggered by the host in response to RSV infection. NF-κB is a key transcription factor responsible for the regulation of cytokine and chemokine gene expression and thus represents a potential therapeutic target. In the present study, we sought to delineate the role of RSV-induced reactive oxygen species in the regulation of the signaling pathways leading to NF-κB activation. First, we demonstrate that besides the well-characterized IκBα-dependent pathway, phosphorylation of p65 at Ser536 is an essential event regulating NF-κB activation in response to RSV in A549. Using antioxidant and RNA-interference strategies, we show that a NADPH oxidase 2 (NOX2)-containing NADPH oxidase is an essential regulator of RSV-induced NF-κB activation. Molecular analyses revealed that NOX2 acts upstream of both the phosphorylation of IκBα at Ser32 and of p65 at Ser536 in A549 and normal human bronchial epithelial cells. Similar results were obtained in the context of infection by Sendai virus, thus demonstrating that the newly identified NOX2-dependent NF-κB activation pathway is not restricted to RSV among the Paramyxoviridae. These results illustrate a previously unrecognized dual role of NOX2 in the regulation of NF-κB in response to RSV and Sendai virus in human AEC.

Airway epithelial cells are key initial innate immune responders in the fight against respiratory viruses, primarily via the secretion of antiviral and proinflammatory cytokines that act in an autocrine/paracrine fashion to trigger the establishment of an antiviral state. It is currently thought that the early antiviral state in airway epithelial cells primarily relies on IFNβ secretion and the subsequent activation of the interferon-stimulated gene factor 3 (ISGF3) transcription factor complex, composed of STAT1, STAT2 and IRF9, which regulates the expression of a panoply of interferon-stimulated genes encoding proteins with antiviral activities. However, the specific pathways engaged by the synergistic action of different cytokines during viral infections, and the resulting physiological outcomes are still ill-defined. Here, we unveil a novel delayed antiviral response in the airways, which is initiated by the synergistic autocrine/paracrine action of IFNβ and TNFα, and signals through a non-canonical STAT2-and IRF9-dependent, but STAT1-independent cascade. This pathway ultimately leads to the late induction of the DUOX2 NADPH oxidase expression. Importantly, our study uncovers that the development of the antiviral state relies on DUOX2-dependent H 2 O 2 production. Key antiviral pathways are often targeted by evasion strategies evolved by various pathogenic viruses. In this regard, the importance of the novel DUOX2-dependent antiviral pathway is further underlined by the observation that the human respiratory syncytial virus is able to subvert DUOX2 induction.