Abstract Viral pneumonia has a significant global morbidity rate, necessitating the development of novel treatment and prevention strategies. Because the lung epithelium is accessible to inhalation therapies, aerosolized innate immune ligands can be employed to activate local antimicrobial defenses. By inhaling the synergistic agents Pam2CSK4, a TLR2/6 ligand, and ODN M362, a TLR9 ligand (collectively, "Pam2ODN"), we have shown that therapeutic immunomodulation to enhance lung intrinsic defenses protects mice from bacterial and viral infection models by producing pathogen-killing reactive oxygen species (ROS) from various sources of the lung epithelium, including dual oxidase 2 (DUOX2). Our lab has previously shown that lung epithelial cells treated with Pam2ODN exhibit increased expression of numerous transcription factor genes, including the antiviral transcription factor interferon regulatory factor 9 (IRF9) gene irf9. Although Irf9 and Duox2, which is an antiviral IRF9-dependent gene, are both driven by Pam2ODN, it is unclear if Pam2ODN-induced Duox2 results from Pam2ODN-induced IRF9 activation. To elucidate the function of Pam2ODN-induced IRF9 in the Pam2ODN-dependent DUOX2-induced reactive oxygen species generation and pathogen killing, we employed the western blot, qPCR analysis, siRNA, and shRNA techniques in conjunction with an in vitro influenza infection model using human bronchial epithelial cells (HBEC3kt) and mouse lung epithelial (MLE-15) cell lines. Here, we report that the IRF9 protein expression increases after Pam2ODN treatment of lung epithelial cells. IRF9 knockdown cells treated with Pam2ODN showed decreased DUOX2 expression and increased viral burden compared to control scrambled cells. To further investigate the involvement of all components of antiviral ISGF3 (interferon-stimulated gene factor 3) complex, which is constituted of IRF9, STAT1, and STAT2, we knocked down STAT1 and STAT2 in lung epithelial cells. Likewise, when STAT2, whose phosphorylation is necessary for antiviral IRF9 activation, is silenced, DUOX2 expression decreases and viral burden increases. Conversely, in STAT1 knockdown epithelial cells, the antiviral effects of Pam2ODN and DUOX2 induction remained maintained. These results imply that Pam2ODN-induced lung epithelial antiviral DUOX2 expression is driven by STAT2-IRF9 interaction rather than the activation of the entire ISGF3 complex. Altogether, our results offer a molecular explanation for how inhaled pattern recognition receptors (PRRs) induce the antiviral DUOX2 in the lung epithelium. This abstract is funded by: None
Ntita et al. (Fri,) studied this question.