Diet-induced obesity worsens allergen-induced type 2/type 17 inflammation in airways by enhancing DUOX1 activation.

More than 50% of people with asthma in the United States are obese, and obesity often worsens symptoms of allergic asthma and impairs response to treatment. Based on previously established roles of the epithelial NADPH oxidase DUOX1 in allergic airway inflammation, we addressed the potential involvement of DUOX1 in altered allergic inflammation in the context of obesity. Intranasal house dust mite (HDM) allergen challenge of subjects with allergic asthma induced rapid secretion of IL-33, then IL-13, into the nasal lumen, responses that were significantly enhanced in obese asthmatic subjects (BMI >30). Induction of diet-induced obesity (DIO) in mice by high-fat diet (HFD) feeding similarly enhanced acute airway responses to intranasal HDM challenge, particularly with respect to secretion of IL-33 and type 2/type 3 cytokines, and this was associated with enhanced epithelial DUOX1 expression and was avoided in DUOX1-deficient mice. DIO also enhanced DUOX1-dependent features of chronic HDM-induced allergic inflammation. Although DUOX1 did not affect overall weight gain by HFD feeding, it contributed to glucose intolerance, suggesting a role in glucose metabolism. However, glucose intolerance induced by short-term HFD feeding, in the absence of adiposity, was not sufficient to alter HDM-induced acute airway responses. DIO was associated with enhanced presence of the adipokine leptin in the airways, and leptin enhanced DUOX1-dependent IL-13 and mucin production in airway epithelial cells. In conclusion, augmented inflammatory airway responses to HDM in obesity are associated with increases in airway epithelial DUOX1, and by increased airway epithelial leptin signaling.

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.

Downregulation of DUOX1 function contributes to aging-related impairment of innate airway injury responses and accelerated senile emphysema.

Aging is associated with a gradual loss of lung function due to increased cellular senescence, decreased regenerative capacity, and impaired innate host defense. One important aspect of innate airway epithelial host defense to nonmicrobial triggers is the secretion of alarmins such as IL-33 and activation of type 2 inflammation, which were previously found to depend on activation of the NADPH oxidase (NOX) homolog DUOX1, and redox-dependent signaling pathways that promote alarmin secretion. Here, we demonstrate that normal aging of C57BL/6J mice resulted in markedly decreased lung innate epithelial type 2 responses to exogenous triggers such as the airborne allergen Dermatophagoides pteronyssinus, which was associated with marked downregulation of DUOX1, as well as DUOX1-mediated redox-dependent signaling. DUOX1 deficiency was also found to accelerate age-related airspace enlargement and decline in lung function but did not consistently affect other features of lung aging such as senescence-associated inflammation. Intriguingly, observations of age-related DUOX1 downregulation and enhanced airspace enlargement due to DUOX1 deficiency in C57BL/6J mice, which lack a functional mitochondrial nicotinamide nucleotide transhydrogenase (NNT), were much less dramatic in C57BL/6NJ mice with normal NNT function, although the latter mice also displayed impaired innate epithelial injury responses with advancing age. Overall, our findings indicate a marked aging-dependent decline in (DUOX1-dependent) innate airway injury responses to external nonmicrobial triggers, but the impact of aging on DUOX1 downregulation and its significance for age-related senile emphysema development was variable between different C57BL6 substrains, possibly related to metabolic alterations due to differences in NNT function.

Profibrotic epithelial TGF-ß1 signaling involves NOX4-mitochondria cross talk and redox-mediated activation of the tyrosine kinase FYN.

Idiopathic pulmonary fibrosis (IPF) is characterized by a disturbed redox balance and increased production of reactive oxygen species (ROS), which is believed to contribute to epithelial injury and fibrotic lung scarring. The main pulmonary sources of ROS include mitochondria and NADPH oxidases (NOXs), of which the NOX4 isoform has been implicated in IPF. Non-receptor SRC tyrosine kinases (SFK) are important for cellular homeostasis and are often dysregulated in lung diseases. SFK activation by the profibrotic transforming growth factor-ß (TGF-ß) is thought to contribute to pulmonary fibrosis, but the relevant SFK isoform and its relationship to NOX4 and/or mitochondrial ROS in the context of profibrotic TGF-ß signaling is not known. Here, we demonstrate that TGF-ß1 can rapidly activate the SRC kinase FYN in human bronchial epithelial cells, which subsequently induces mitochondrial ROS (mtROS) production, genetic damage shown by the DNA damage marker γH2AX, and increased expression of profibrotic genes. Moreover, TGF-ß1-induced activation of FYN involves initial activation of NOX4 and direct cysteine oxidation of FYN, and both FYN and mtROS contribute to TGF-ß-induced induction of NOX4. NOX4 expression in lung tissues of IPF patients is positively correlated with disease severity, although FYN expression is down-regulated in IPF and does not correlate with disease severity. Collectively, our findings highlight a critical role for FYN in TGF-ß1-induced mtROS production, DNA damage response, and induction of profibrotic genes in bronchial epithelial cells, and suggest that altered expression and activation of NOX4 and FYN may contribute to the pathogenesis of pulmonary fibrosis.

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.

Internalized FGF-2-Loaded Nanoparticles Increase Nuclear ERK1/2 Content and Result in Lung Cancer Cell Death.

: Innovative cancer treatments, which improve adjuvant therapy and reduce adverse events, are desperately needed. Nanoparticles provide controlled intracellular biomolecule delivery in the absence of activating external cell surface receptors. Prior reports suggest that intracrine signaling, following overexpression of basic fibroblast growth factor (FGF-2) after viral transduction, has a toxic effect on diseased cells. Herein, the research goals were to 1) encapsulate recombinant FGF-2 within stable, alginate-based nanoparticles (ABNs) for non-specific cellular uptake, and 2) determine the effects of ABN-mediated intracellular delivery of FGF-2 on cancer cell proliferation/survival. In culture, human alveolar adenocarcinoma basal epithelial cell line (A549s) and immortalized human bronchial epithelial cell line (HBE1s) internalized ABNs through non-selective endocytosis. Compared to A549s exposed to empty (i.e., blank) ABNs, the intracellular delivery of FGF-2 via ABNs significantly increased the levels of lactate dehydrogenase, indicating that FGF-2-ABN treatment decreased the transformed cell integrity. Noticeably, the nontransformed cells were not significantly affected by FGF-2-loaded ABN treatment. Furthermore, FGF-2-loaded ABNs significantly increased nuclear levels of activated-extracellular signal-regulated kinase ½ (ERK1/2) in A549s but had no significant effect on HBE1 nuclear ERK1/2 expression. Our novel intracellular delivery method of FGF-2 via nanoparticles resulted in increased cancer cell death via increased nuclear ERK1/2 activation.

The Transient Receptor Potential Channel Vanilloid 1 Is Critical in Innate Airway Epithelial Responses to Protease Allergens.

The airway epithelium plays a critical role in innate responses to airborne allergens by secreting IL-1 family cytokines such as IL-1α and IL-33 as alarmins that subsequently orchestrate appropriate immune responses. Previous studies revealed that epithelial IL-33 secretion by allergens such as Alternaria alternata or house dust mite involves Ca2+-dependent signaling, via initial activation of ATP-stimulated P2YR2 (type 2 purinoceptor) and subsequent activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase DUOX1. We sought to identify proximal mechanisms by which epithelial cells sense these allergens and here highlight the importance of PAR2 (protease-activated receptor 2) and TRP (transient receptor potential) Ca2+ channels such as TRPV1 (TRP vanilloid 1) in these responses. Combined studies of primary human nasal and mouse tracheal epithelial cells, as well as immortalized human bronchial epithelial cells, indicated the importance of both PAR2 and TRPV1 in IL-33 secretion by both Alternaria alternata and house dust mite, based on both pharmacological and genetic approaches. TRPV1 was also critically involved in allergen-induced ATP release, activation of DUOX1, and redox-dependent activation of EGFR (epidermal growth factor receptor). Moreover, genetic deletion of TRPV1 dramatically attenuated allergen-induced IL-33 secretion and subsequent type 2 responses in mice in vivo. TRPV1 not only contributed to ATP release and P2YR2 signaling but also was critical in downstream innate responses to ATP, indicating potentiating effects of P2YR2 on TRPV1 activation. In aggregate, our studies illustrate a complex relationship between various receptor types, including PAR2 and P2YR2, in epithelial responses to asthma-relevant airborne allergens and highlight the central importance of TRPV1 in such responses.

Proteomic Methods to Evaluate NOX-Mediated Redox Signaling.

The NADPH oxidase (NOX) family of proteins is involved in regulating many diverse cellular processes, which is largely mediated by NOX-mediated reversible oxidation of target proteins in a process known as redox signaling. Protein cysteine residues are the most prominent targets in redox signaling, and to understand the mechanisms by which NOX affect cellular pathways, specific methodology is required to detect specific oxidative cysteine modifications and to identify targeted proteins. Among the many potential redox modifications involving cysteine residues, reversible modifications most relevant to NOX are sulfenylation (P-SOH) and S-glutathionylation (P-SSG), as both can induce structural or functional alterations. Various experimental approaches have been developed to detect these specific modifications, and this chapter will detail state-of-the-art methodology to selectively evaluate these modifications in specific target proteins in relation to NOX activation. We also discuss some of the limitations of these procedures and potential complementary approaches.

Dysregulated Redox Regulation Contributes to Nuclear EGFR Localization and Pathogenicity in Lung Cancer.

Lung cancers are frequently characterized by inappropriate activation of epidermal growth factor receptor (EGFR)-dependent signaling and epigenetic silencing of the NADPH oxidase (NOX) enzyme DUOX1, both potentially contributing to worse prognosis. Based on previous findings linking DUOX1 with redox-dependent EGFR activation, the present studies were designed to evaluate whether DUOX1 silencing in lung cancers may be responsible for altered EGFR regulation. In contrast to normal epithelial cells, EGF stimulation of lung cancer cell lines that lack DUOX1 promotes EGF-induced EGFR internalization and nuclear localization, associated with induction of EGFR-regulated genes and related tumorigenic outcomes. Each of these outcomes could be reversed by overexpression of DUOX1 or enhanced by shRNA-dependent DUOX1 silencing. EGF-induced nuclear EGFR localization in DUOX1-deficient lung cancer cells was associated with altered dynamics of cysteine oxidation of EGFR, and an overall reduction of EGFR cysteines. These various outcomes could also be attenuated by silencing of glutathione S-transferase P1 (GSTP1), a mediator of metabolic alterations and drug resistance in various cancers, and a regulator of cysteine oxidation. Collectively, our findings indicate DUOX1 deficiency in lung cancers promotes dysregulated EGFR signaling and enhanced GSTP1-mediated turnover of EGFR cysteine oxidation, which result in enhanced nuclear EGFR localization and tumorigenic properties.

Direct cysteine sulfenylation drives activation of the Src kinase.

The Src kinase controls aspects of cell biology and its activity is regulated by intramolecular structural changes induced by protein interactions and tyrosine phosphorylation. Recent studies indicate that Src is additionally regulated by redox-dependent mechanisms, involving oxidative modification(s) of cysteines within the Src protein, although the nature and molecular-level impact of Src cysteine oxidation are unknown. Using a combination of biochemical and cell-based studies, we establish the critical importance of two Src cysteine residues, Cys-185 and Cys-277, as targets for H2O2-mediated sulfenylation (Cys-SOH) in redox-dependent kinase activation in response to NADPH oxidase-dependent signaling. Molecular dynamics and metadynamics simulations reveal the structural impact of sulfenylation of these cysteines, indicating that Cys-277-SOH enables solvent exposure of Tyr-416 to promote its (auto)phosphorylation, and that Cys-185-SOH destabilizes pTyr-527 binding to the SH2 domain. These redox-dependent Src activation mechanisms offer opportunities for development of Src-selective inhibitors in treatment of diseases where Src is aberrantly activated.

Cysteine perthiosulfenic acid (Cys-SSOH): A novel intermediate in thiol-based redox signaling?

The reversible oxidation of protein cysteine residues (Cys-SH) is a key reaction in cellular redox signaling involving initial formation of sulfenic acids (Cys-SOH), which are commonly detected using selective dimedone-based probes. Here, we report that significant portions of dimedone-tagged proteins are susceptible to cleavage by DTT reflecting the presence of perthiosulfenic acid species (Cys-SSOH) due to similar oxidation of hydropersulfides (Cys-SSH), since Cys-S-dimedone adducts are stable toward DTT. Combined studies using molecular modeling, mass spectrometry, and cell-based experiments indicate that Cys-SSH are readily oxidized to Cys-SSOH, which forms stable adducts with dimedone-based probes. We additionally confirm the presence of Cys-SSH within protein tyrosine kinases such as EGFR, and their apparent oxidation to Cys-SSOH in response NADPH oxidase activation, suggesting that such Cys-SSH oxidation may represent a novel, as yet uncharacterized, event in redox-based signaling.

DUOX1 mediates persistent epithelial EGFR activation, mucous cell metaplasia, and airway remodeling during allergic asthma.

Chronic inflammation with mucous metaplasia and airway remodeling are hallmarks of allergic asthma, and these outcomes have been associated with enhanced expression and activation of EGFR signaling. Here, we demonstrate enhanced expression of EGFR ligands such as amphiregulin as well as constitutive EGFR activation in cultured nasal epithelial cells from asthmatic subjects compared with nonasthmatic controls and in lung tissues of mice during house dust mite-induced (HDM-induced) allergic inflammation. EGFR activation was associated with cysteine oxidation within EGFR and the nonreceptor tyrosine kinase Src, and both amphiregulin production and oxidative EGFR activation were diminished by pharmacologic or genetic inhibition of the epithelial NADPH oxidase dual oxidase 1 (DUOX1). DUOX1 deficiency also attenuated several EGFR-dependent features of HDM-induced allergic airway inflammation, including neutrophilic inflammation, type 2 cytokine production (IL-33, IL-13), mucous metaplasia, subepithelial fibrosis, and central airway resistance. Moreover, targeted inhibition of airway DUOX1 in mice with previously established HDM-induced allergic inflammation, by intratracheal administration of DUOX1-targeted siRNA or pharmacological NADPH oxidase inhibitors, reversed most of these outcomes. Our findings indicate an important function for DUOX1 in allergic inflammation related to persistent EGFR activation and suggest that DUOX1 targeting may represent an attractive strategy in asthma management.

The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling.

The epidermal growth factor receptor (EGFR) plays a critical role in regulating airway epithelial homeostasis and responses to injury. Activation of EGFR is regulated by redox-dependent processes involving reversible cysteine oxidation by reactive oxygen species (ROS) and involves both ligand-dependent and -independent mechanisms, but the precise source(s) of ROS and the molecular mechanisms that control tyrosine kinase activity are incompletely understood. Here, we demonstrate that stimulation of EGFR activation by ATP in airway epithelial cells is closely associated with dynamic reversible oxidation of cysteine residues via sequential sulfenylation and S-glutathionylation within EGFR and the non-receptor-tyrosine kinase Src. Moreover, the intrinsic kinase activity of recombinant Src or EGFR was in both cases enhanced by H2O2 but not by GSSG, indicating that the intermediate sulfenylation is the activating modification. H2O2-induced increase in EGFR tyrosine kinase activity was not observed with the C797S variant, confirming Cys-797 as the redox-sensitive cysteine residue that regulates kinase activity. Redox-dependent regulation of EGFR activation in airway epithelial cells was found to strongly depend on activation of either the NADPH oxidase DUOX1 or the homolog NOX2, depending on the activation mechanism. Whereas DUOX1 and Src play a primary role in EGFR transactivation by wound-derived signals such as ATP, direct ligand-dependent EGFR activation primarily involves NOX2 with a secondary role for DUOX1 and Src. Collectively, our findings establish that redox-dependent EGFR kinase activation involves a dynamic and reversible cysteine oxidation mechanism and that this activation mechanism variably involves DUOX1 and NOX2.

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.

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.

Airway epithelial dual oxidase 1 mediates allergen-induced IL-33 secretion and activation of type 2 immune responses.

BACKGROUND: The IL-1 family member IL-33 plays a critical role in type 2 innate immune responses to allergens and is an important mediator of allergic asthma. The mechanisms by which allergens provoke epithelial IL-33 secretion are still poorly understood. OBJECTIVE: Based on previous findings indicating involvement of the NADPH oxidase dual oxidase 1 (DUOX1) in epithelial wound responses, we explored the potential involvement of DUOX1 in allergen-induced IL-33 secretion and potential alterations in airways of asthmatic patients. METHODS: Cultured human or murine airway epithelial cells or mice were subjected to acute challenge with Alternaria alternata or house dust mite, and secretion of IL-33 and activation of subsequent type 2 responses were determined. The role of DUOX1 was explored by using small interfering RNA approaches and DUOX1-deficient mice. Cultured nasal epithelial cells from healthy subjects or asthmatic patients were evaluated for DUOX1 expression and allergen-induced responses. RESULTS: In vitro or in vivo allergen challenge resulted in rapid airway epithelial IL-33 secretion, which depended critically on DUOX1-mediated activation of epithelial epidermal growth factor receptor and the protease calpain-2 through a redox-dependent mechanism involving cysteine oxidation within epidermal growth factor receptor and the tyrosine kinase Src. Primary nasal epithelial cells from patients with allergic asthma were found to express increased DUOX1 and IL-33 levels and demonstrated enhanced IL-33 secretion in response to allergen challenge compared with values seen in nasal epithelial cells from nonasthmatic subjects. CONCLUSION: Our findings implicate epithelial DUOX1 as a pivotal mediator of IL-33-dependent activation of innate airway type 2 immune responses to common airborne allergens and indicate that enhanced DUOX1 expression and IL-33 secretion might present important contributing features of allergic asthma.

Identification of DUOX1-dependent redox signaling through protein S-glutathionylation in airway epithelial cells.

The NADPH oxidase homolog dual oxidase 1 (DUOX1) plays an important role in innate airway epithelial responses to infection or injury, but the precise molecular mechanisms are incompletely understood and the cellular redox-sensitive targets for DUOX1-derived H2O2 have not been identified. The aim of the present study was to survey the involvement of DUOX1 in cellular redox signaling by protein S-glutathionylation, a major mode of reversible redox signaling. Using human airway epithelial H292 cells and stable transfection with DUOX1-targeted shRNA as well as primary tracheal epithelial cells from either wild-type or DUOX1-deficient mice, DUOX1 was found to be critical in ATP-stimulated transient production of H2O2 and increased protein S-glutathionylation. Using cell pre-labeling with biotin-tagged GSH and analysis of avidin-purified proteins by global proteomics, 61 S-glutathionylated proteins were identified in ATP-stimulated cells compared to 19 in untreated cells. Based on a previously established role of DUOX1 in cell migration, various redox-sensitive proteins with established roles in cytoskeletal dynamics and/or cell migration were evaluated for S-glutathionylation, indicating a critical role for DUOX1 in ATP-stimulated S-glutathionylation of ß-actin, peroxiredoxin 1, the non-receptor tyrosine kinase Src, and MAPK phosphatase 1. Overall, our studies demonstrate the importance of DUOX1 in epithelial redox signaling through reversible S-glutathionylation of a range of proteins, including proteins involved in cytoskeletal regulation and MAPK signaling pathways involved in cell migration.

Acrolein exposure suppresses antigen-induced pulmonary inflammation.

BACKGROUND: Adverse health effects of tobacco smoke arise partly from its influence on innate and adaptive immune responses, leading to impaired innate immunity and host defense. The impact of smoking on allergic asthma remains unclear, with various reports demonstrating that cigarette smoke enhances asthma development but can also suppress allergic airway inflammation. Based on our previous findings that immunosuppressive effects of smoking may be largely attributed to one of its main reactive electrophiles, acrolein, we explored the impact of acrolein exposure in a mouse model of ovalbumin (OVA)-induced allergic asthma. METHODS: C57BL/6 mice were sensitized to ovalbumin (OVA) by intraperitoneal injection with the adjuvant aluminum hydroxide on days 0 and 7, and challenged with aerosolized OVA on days 14-16. In some cases, mice were also exposed to 5 ppm acrolein vapor for 6 hrs/day on days 14-17. Lung tissues or brochoalveolar lavage fluids (BALF) were collected either 6 hrs after a single initial OVA challenge and/or acrolein exposure on day 14 or 48 hrs after the last OVA challenge, on day 18. Inflammatory cells and Th1/Th2 cytokine levels were measured in BALF, and lung tissue samples were collected for analysis of mucus and Th1/Th2 cytokine expression, determination of protein alkylation, cellular thiol status and transcription factor activity. RESULTS: Exposure to acrolein following OVA challenge of OVA-sensitized mice resulted in markedly attenuated allergic airway inflammation, demonstrated by decreased inflammatory cell infiltrates, mucus hyperplasia and Th2 cytokines. Acrolein exposure rapidly depleted lung tissue glutathione (GSH) levels, and induced activation of the Nrf2 pathway, indicated by accumulation of Nrf2, increased alkylation of Keap1, and induction of Nrf2-target genes such as HO-1. Additionally, analysis of inflammatory signaling pathways showed suppressed activation of NF-κB and marginally reduced activation of JNK in acrolein-exposed lungs, associated with increased carbonylation of RelA and JNK. CONCLUSION: Acrolein inhalation suppresses Th2-driven allergic inflammation in sensitized animals, due to direct protein alkylation resulting in activation of Nrf2 and anti-inflammatory gene expression, and inhibition of NF-κB or JNK signaling. Our findings help explain the paradoxical anti-inflammatory effects of cigarette smoke exposure in allergic airways disease.

Protein alkylation by the α,ß-unsaturated aldehyde acrolein. A reversible mechanism of electrophile signaling?

Acrolein, a reactive aldehyde found in cigarette smoke, is thought to induce its biological effects primarily by irreversible adduction to cellular nucleophiles such as cysteine thiols. Here, we demonstrate that acrolein rapidly inactivates the seleno-enzyme thioredoxin reductase (TrxR) in human bronchiolar epithelial HBE1 cells, which recovered over 4-8h by a mechanism depending on the presence of cellular GSH and thioredoxin 1 (Trx1), and corresponding with reversal of protein-acrolein adduction. Our findings indicate that acrolein-induced protein alkylation is not necessarily a feature of irreversible protein damage, but may reflect a reversible signaling mechanism that is regulated by GSH and Trx1.

Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1.

Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1-30 µM) resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases such as JNK, and may therefore be important in acrolein-induced alterations in airway epithelial function, as a contributing mechanism in tobacco-related respiratory disease.