Oxidation-Dependent Activation of Src Kinase Mediates Epithelial IL-33 Production and Signaling during Acute Airway Allergen Challenge.

The respiratory epithelium forms the first line of defense against inhaled pathogens and acts as an important source of innate cytokine responses to environmental insults. One critical mediator of these responses is the IL-1 family cytokine IL-33, which is rapidly secreted upon acute epithelial injury as an alarmin and induces type 2 immune responses. Our recent work highlighted the importance of the NADPH oxidase dual oxidase 1 (DUOX1) in acute airway epithelial IL-33 secretion by various airborne allergens associated with H2O2 production and reduction-oxidation-dependent activation of Src kinases and epidermal growth factor receptor (EGFR) signaling. In this study, we show that IL-33 secretion in response to acute airway challenge with house dust mite (HDM) allergen critically depends on the activation of Src by a DUOX1-dependent oxidative mechanism. Intriguingly, HDM-induced epithelial IL-33 secretion was dramatically attenuated by small interfering RNA- or Ab-based approaches to block IL-33 signaling through its receptor IL1RL1 (ST2), indicating that HDM-induced IL-33 secretion includes a positive feed-forward mechanism involving ST2-dependent IL-33 signaling. Moreover, activation of type 2 cytokine responses by direct airway IL-33 administration was associated with ST2-dependent activation of DUOX1-mediated H2O2 production and reduction-oxidation-based activation of Src and EGFR and was attenuated in Duox1 -/- and Src +/- mice, indicating that IL-33-induced epithelial signaling and subsequent airway responses involve DUOX1/Src-dependent pathways. Collectively, our findings suggest an intricate relationship between DUOX1, Src, and IL-33 signaling in the activation of innate type 2 immune responses to allergens, involving DUOX1-dependent epithelial Src/EGFR activation in initial IL-33 secretion and in subsequent IL-33 signaling through ST2 activation.

Acrolein and thiol-reactive electrophiles suppress allergen-induced innate airway epithelial responses by inhibition of DUOX1 and EGFR.

Acrolein is a major thiol-reactive component of cigarette smoke (CS) that is thought to contribute to increased asthma incidence associated with smoking. Here, we explored the effects of acute acrolein exposure on innate airway responses to two common airborne allergens, house dust mite and Alternaria alternata, and observed that acrolein exposure of C57BL/6 mice (5 ppm, 4 h) dramatically inhibited innate airway responses to subsequent allergen challenge, demonstrated by attenuated release of the epithelial-derived cytokines IL-33, IL-25, and IL-1α. Acrolein and other anti-inflammatory thiol-reactive electrophiles, cinnamaldehyde, curcumin, and sulforaphane, similarly inhibited allergen-induced production of these cytokines from human or murine airway epithelial cells in vitro. Based on our previous observations indicating the importance of Ca2+-dependent signaling, activation of the NADPH oxidase DUOX1, and Src/EGFR-dependent signaling in allergen-induced epithelial secretion of these cytokines, we explored the impact of acrolein on these pathways. Acrolein and other thiol-reactive electrophiles were found to dramatically prevent allergen-induced activation of DUOX1 as well as EGFR, and acrolein was capable of inhibiting EGFR tyrosine kinase activity via modification of C797. Biotin-labeling strategies indicated increased cysteine modification and carbonylation of Src, EGFR, as well as DUOX1, in response to acrolein exposure in vitro and in vivo, suggesting that direct alkylation of these proteins on accessible cysteine residues may be responsible for their inhibition. Collectively, our findings indicate a novel anti-inflammatory mechanism of CS-derived acrolein and other thiol-reactive electrophiles, by directly inhibiting DUOX1- and EGFR-mediated airway epithelial responses to airborne allergens.

Downregulation of epithelial DUOX1 in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease characterized by small airway remodeling and alveolar emphysema due to environmental stresses such as cigarette smoking (CS). Oxidative stress is commonly implicated in COPD pathology, but recent findings suggest that one oxidant-producing NADPH oxidase homolog, dual oxidase 1 (DUOX1), is downregulated in the airways of patients with COPD. We evaluated lung tissue sections from patients with COPD for small airway epithelial DUOX1 protein expression, in association with measures of lung function and small airway and alveolar remodeling. We also addressed the impact of DUOX1 for lung tissue remodeling in mouse models of COPD. Small airway DUOX1 levels were decreased in advanced COPD and correlated with loss of lung function and markers of emphysema and remodeling. Similarly, DUOX1 downregulation in correlation with extracellular matrix remodeling was observed in a genetic model of COPD, transgenic SPC-TNF-α mice. Finally, development of subepithelial airway fibrosis in mice due to exposure to the CS-component acrolein, or alveolar emphysema induced by administration of elastase, were in both cases exacerbated in Duox1-deficient mice. Collectively, our studies highlight that downregulation of DUOX1 may be a contributing feature of COPD pathogenesis, likely related to impaired DUOX1-mediated innate injury responses involved in epithelial homeostasis.

Inhalation of the reactive aldehyde acrolein promotes antigen sensitization to ovalbumin and enhances neutrophilic inflammation.

Acrolein (ACR), an α,ß-unsaturated aldehyde and a major component of tobacco smoke, is a highly reactive electrophilic respiratory irritant implicated in asthma pathogenesis and severity. However, few studies have directly investigated the influence of ACR exposure on allergen sensitization and pulmonary inflammation. The present study was designed to examine the impact of ACR inhalation on allergic sensitization to the inhaled antigen ovalbumin (OVA), as well as pulmonary inflammation during subsequent OVA challenge. Adult male C57BL/6 mice were exposed to inhaled OVA (1%, 30 min/day, 4 days/week) and/or ACR (5 ppm, 4 h/day, 4 days/week) over 2 weeks and subsequently challenged with aerosolized OVA (1%, 30 min/day) over three consecutive days. Serum anti-OVA IgG1 levels were increased significantly in animals exposed to both OVA and ACR, compared to animals exposed to either OVA or ACR alone. In addition, differential cell counts and histological analysis revealed an increase in BAL neutrophils in animals exposed to both OVA and ACR. However, exposure to both OVA and ACR did not influence mRNA expression of the cytokines il5, il10, il13 or tnfa, but significantly increased mRNA expression of ccl20. Moreover, ACR exposure enhanced lung mRNA levels of il17f and tgfb1, suggesting development of enhanced inhalation tolerance to OVA. Overall, the findings indicate that ACR inhalation can promote airway-mediated sensitization to otherwise innocuous inhaled antigens, such as OVA, but also enhances immune tolerance, thereby favoring neutrophilic airway inflammation.