Background: Decreased activity of forkhead transcription factor class O (FoxO)3A, a negative regulator of NF-κB-mediated chemokine expression, is implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Previously, we showed that quercetin reduces lung inflammation in a murine model of COPD. Here, we examined the mechanisms underlying decreased FoxO3A activation and its modulation by quercetin in COPD human airway epithelial cells and in a COPD mouse model. Methods: Primary COPD and normal human airway epithelial cells were treated with quercetin, LY294002 or erlotinib for two weeks. IL-8 was measured by ELISA. FoxO3A, Akt, and EGF receptor (EGFR) phosphorylation and nuclear FoxO3A levels were determined by Western blot analysis. Effects of quercetin on lung chemokine expression, nuclear FoxO3A levels and phosphorylation of EGFR and Akt were determined in COPD mouse model. Results: Compared to normal, COPD cells showed significantly increased IL-8, which negatively correlated with nuclear FoxO3A levels. COPD bronchial biopsies also showed reduced nuclear FoxO3A. Decreased FoxO3A in COPD cells was associated with increased phosphorylation of EGFR, Akt and FoxO3A and treatment with quercetin, LY294002 or erlotinib increased nuclear FoxO3A and decreased IL-8 and phosphorylation of Akt, EGFR and FoxO3A, Compared to control, elastase/LPS-exposed mice showed decreased nuclear FoxO3A, increased chemokines and phosphorylation of EGFR and Akt. Treatment with quercetin partially reversed these changes. Conclusions: In COPD airways, aberrant EGFR activity increases PI 3-kinase/Akt-mediated phosphorylation of FoxO3A, thereby decreasing nuclear FoxO3A and increasing chemokine expression. Quercetin restores nuclear FoxO3A and reduces chemokine expression partly by modulating EGFR/PI 3-kinase/Akt activity.

Barrier dysfunction of airway epithelium may increase the risk for acquiring secondary infections or allergen sensitization. Both rhinovirus (RV) and polyinosinic-polycytidilic acid [poly(I·C)], a double-stranded RNA (dsRNA) mimetic, cause airway epithelial barrier dysfunction, which is reactive oxygen species (ROS) dependent, implying that dsRNA generated during RV replication is sufficient for disrupting barrier function. We also demonstrated that RV or poly(I·C)-stimulated NADPH oxidase 1 (NOX-1) partially accounts for RV-induced ROS generation. In this study, we identified a dsRNA receptor(s) contributing to RV-induced maximal ROS generation and thus barrier disruption. We demonstrate that genetic silencing of the newly discovered dsRNA receptor Nod-like receptor X-1 (NLRX-1), but not other previously described dsRNA receptors, abrogated RV-induced ROS generation and reduction of transepithelial resistance (R T ) in polarized airway epithelial cells. In addition, both RV and poly(I·C) stimulated mitochondrial ROS, the generation of which was dependent on NLRX-1. Treatment with Mito-Tempo, an antioxidant targeted to mitochondria, abolished RV-induced mitochondrial ROS generation, reduction in R T , and bacterial transmigration. Furthermore, RV infection increased NLRX-1 localization to the mitochondria. Additionally, NLRX-1 interacts with RV RNA and poly(I·C) in polarized airway epithelial cells. Finally, we show that NLRX-1 is also required for RV-stimulated NOX-1 expression. These findings suggest a novel mechanism by which RV stimulates generation of ROS, which is required for disruption of airway epithelial barrier function. IMPORTANCE Rhinovirus (RV), a virus responsible for a majority of common colds, disrupts the barrier function of the airway epithelium by increasing reactive oxygen species (ROS). Poly(I·C), a double-stranded RNA (dsRNA) mimetic, also causes ROS-dependent barrier disruption, implying that the dsRNA intermediate generated during RV replication is sufficient for this process. Here, we demonstrate that both RV RNA and poly(I·C) interact with NLRX-1 (a newly discovered dsRNA receptor) and stimulate mitochondrial ROS. We show for the first time that NLRX-1 is primarily expressed in the cytoplasm and at the apical surface rather than in the mitochondria and that NLRX-1 translocates to mitochondria following RV infection. Together, our results suggest a novel mechanism for RV-induced barrier disruption involving NLRX-1 and mitochondrial ROS. Although ROS is necessary for optimal viral clearance, if not neutralized efficiently, it may increase susceptibility to secondary infections and alter innate immune responses to subsequently inhaled pathogens, allergens, and other environmental factors.

Bacterial infections following rhinovirus (RV), a common cold virus, are well documented, but pathogenic mechanisms are poorly understood. We developed animal and cell culture models to examine the effects of RV on subsequent infection with non-typeable Hemophilus influenzae (NTHi). We focused on NTHI-induced neutrophil chemoattractants expression that is essential for bacterial clearance. Mice infected with RV1B were superinfected with NTHi and lung bacterial density, chemokines and neutrophil counts determined. Human bronchial epithelial cells (BEAS-2B) or mouse alveolar macrophages (MH-S) were infected with RV and challenged with NHTi, TLR2 or TLR5 agonists. Chemokine levels were measured by ELISA and expression of IRAK-1, a component of MyD88-dependent TLR signaling, assessed by immunoblotting. While sham-infected mice cleared all NTHi from the lungs, RV-infected mice showed bacteria up to 72 h post-infection. However, animals in RV/NTHi cleared bacteria by day 7. Delayed bacterial clearance in RV/NTHi animals was associated with suppressed chemokine levels and neutrophil recruitment. RV-infected BEAS-2B and MH-S cells showed attenuated chemokine production after challenge with either NTHi or TLR agonists. Attenuated chemokine responses were associated with IRAK-1 protein degradation. Inhibition of RV-induced IRAK-1 degradation restored NTHi-stimulated IL-8 expression. Knockdown of TLR2, but not other MyD88-dependent TLRs, also restored IRAK-1, suggesting that TLR2 is required for RV-induced IRAK-1 degradation.In conclusion, we demonstrate for the first time that RV infection delays bacterial clearance in vivo and suppresses NTHi-stimulated chemokine responses via degradation of IRAK-1. Based on these observations, we speculate that modulation of TLR-dependent innate immune responses by RV may predispose the host to secondary bacterial infection, particularly in patients with underlying chronic respiratory disorders.

Psychological distress is associated with increased lung cancer incidence and mortality. We have shown that non small cell lung cancer (NSCLC) cells in vitro are stimulated by the cAMP-dependent activation of CREB and ERK downstream of beta-adrenergic receptors and that this pathway is inhibited by the neurotransmitter γ-aminobutyric acid (GABA). Because the stress neurotransmitters noradrenalin and adrenaline are beta-adrenergic agonists, the current study has tested the hypothesis that social stress stimulates NSCLC growth in vivo and that GABA inhibits this effect. Social stress was induced in mice carrying xenografts from two NSCLC cell lines in the presence and absence of treatment with GABA. Xenograft sizes were measured after 30 days. Noradrenalin, adrenalin, cortisol, GABA and cAMP were measured in blood and tumor tissues by immunoassays. Expression of nicotinic receptors in the xenografts was assessed by real-time PCR and Western blotting. Protein expression of p-CREB, CREB, p-ERK, ERK and glutamate decarboxylase (GAD) 65 and 67 were determined by Western blotting. Xenograft sizes in stress-exposed mice were significantly increased. Nicotinic acetylcholine receptor (nAChR) subunits α3, α4, α5, and α7 in xenograft tissues showed posttranscriptional induction. Noradrenalin, adrenalin and cortisol were elevated in serum and xenograft tissue while GABA was suppressed. Levels of cAMP, p-CREB and p-ERK were increased while GAD 65 and GAD 67 were suppressed in tumor tissue. Treatment with GABA reversed the effects of stress. Our findings suggest that social stress stimulates NSCLC by increasing nAChR-mediated stress neurotransmitter signaling and that GABA is a promising novel agent for NSCLC intervention.