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Rationale: Nuclear factor (NF)-kappaB is a prominent proinflammatory transcription factor that plays a critical role in allergic airway disease. Previous studies demonstrated that inhibition of NF-kappaB in airway epithelium causes attenuation of allergic inflammation.Objectives: We sought to determine if selective activation of NF-kappaB within the airway epithelium in the absence of other agonists is sufficient to cause allergic airway disease.Methods: A transgenic mouse expressing a doxycycline (Dox)-inducible, constitutively active (CA) version of inhibitor of kappaB (IkappaB) kinase-beta (IKKbeta) under transcriptional control of the rat CC10 promoter, was generated.Measurements and Main Results: After administration of Dox, expression of the CA-IKKbeta transgene induced the nuclear translocation of RelA in airway epithelium. IKKbeta-triggered activation of NF-kappaB led to an increased content of neutrophils and lymphocytes, and concomitant production of proinflammatory mediators, responses that were not observed in transgenic mice not receiving Dox, or in transgene-negative littermate control animals fed Dox. Unexpectedly, expression of the IKKbeta transgene in airway epithelium was sufficient to cause airway hyperresponsiveness and smooth muscle thickening in absence of an antigen sensitization and challenge regimen, the presence of eosinophils, or the induction of mucus metaplasia.Conclusions: These findings demonstrate that selective activation NF-kappaB in airway epithelium is sufficient to induce airway hyperresponsiveness and smooth muscle thickening, which are both critical features of allergic airway disease. [ABSTRACT FROM AUTHOR]

Background: The role of club cells in the pathology of idiopathic pulmonary fibrosis (IPF) is not well understood. Protein disulfide isomerase A3 (PDIA3), an endoplasmic reticulum-based redox chaperone required for the functions of various fibrosis-related proteins; however, the mechanisms of action of PDIA3 in pulmonary fibrosis are not fully elucidated., Objectives: To examine the role of club cells and PDIA3 in the pathology of pulmonary fibrosis and the therapeutic potential of inhibition of PDIA3 in lung fibrosis., Methods: Role of PDIA3 and aberrant club cells in lung fibrosis was studied by analyses of human transcriptome dataset from Lung Genomics Research Consortium, other public resources, the specific deletion or inhibition of PDIA3 in club cells and blocking SPP1 downstream of PDIA3 in mice., Results: PDIA3 and club cell secretory protein ( SCGB1A1 ) signatures are upregulated in IPF compared with control patients. PDIA3 or SCGB1A1 increases also correlate with a decrease in lung function in patients with IPF. The bleomycin (BLM) model of lung fibrosis showed increases in PDIA3 in SCGB1A1 cells in the lung parenchyma. Ablation of Pdia3 , specifically in SCGB1A1 cells, decreases parenchymal SCGB1A1 cells along with fibrosis in mice. The administration of a PDI inhibitor LOC14 reversed the BLM-induced parenchymal SCGB1A1 cells and fibrosis in mice. Evaluation of PDIA3 partners revealed that SPP1 is a major interactor in fibrosis. Blocking SPP1 attenuated the development of lung fibrosis in mice., Conclusions: Our study reveals a new relationship with distally localised club cells, PDIA3 and SPP1 in lung fibrosis and inhibition of PDIA3 or SPP1 attenuates lung fibrosis., Competing Interests: Competing interests: YMWJ-H and VA hold patents: United States Patent No. 8,679,811, 'Treatments Involving Glutaredoxins and Similar Agents', United States Patent No. 8,877,447, 'Detection of Glutathionylated Proteins' (to YMWJ-H), United States Patents, 9,907,828 and 10,688,150 'Treatments of oxidative stress conditions' (YMWJ-H and VA). In the past YMWJ-H and VA have received consulting fees and laboratory contracts from Celdara Medical LLC, NH., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Interleukin-1-dependent increases in glycolysis promote allergic airways disease in mice and disruption of pyruvate kinase M2 (PKM2) activity is critical herein. Glutathione-S-transferase P (GSTP) has been implicated in asthma pathogenesis and regulates the oxidation state of proteins via S-glutathionylation. We addressed whether GSTP-dependent S-glutathionylation promotes allergic airways disease by promoting glycolytic reprogramming and whether it involves the disruption of PKM2., Methods: We used house dust mite (HDM) or interleukin-1β in C57BL6/NJ WT or mice that lack GSTP. Airway basal cells were stimulated with interleukin-1β and the selective GSTP inhibitor, TLK199. GSTP and PKM2 were evaluated in sputum samples of asthmatics and healthy controls and incorporated analysis of the U-BIOPRED severe asthma cohort database., Results: Ablation of Gstp decreased total S-glutathionylation and attenuated HDM-induced allergic airways disease and interleukin-1β-mediated inflammation. Gstp deletion or inhibition by TLK199 decreased the interleukin-1β-stimulated secretion of pro-inflammatory mediators and lactate by epithelial cells. 13 C-glucose metabolomics showed decreased glycolysis flux at the pyruvate kinase step in response to TLK199. GSTP and PKM2 levels were increased in BAL of HDM-exposed mice as well as in sputum of asthmatics compared to controls. Sputum proteomics and transcriptomics revealed strong correlations between GSTP, PKM2, and the glycolysis pathway in asthma., Conclusions: GSTP contributes to the pathogenesis of allergic airways disease in association with enhanced glycolysis and oxidative disruption of PKM2. Our findings also suggest a PKM2-GSTP-glycolysis signature in asthma that is associated with severe disease., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Alcohol use disorders are common both in the United States and globally, and are associated with a variety of co-morbid, inflammation-linked diseases. The pathogenesis of many of these ailments are driven by the activation of the NLRP3 inflammasome, a multi-protein intracellular pattern recognition receptor complex that facilitates the cleavage and secretion of the pro-inflammatory cytokines IL-1β and IL-18. We hypothesized that protracted exposure of leukocytes to ethanol would amplify inflammasome activation, which would help to implicate mechanisms involved in diseases associated with both alcoholism and aberrant NLRP3 inflammasome activation. Here we show that long-term ethanol exposure of human peripheral blood mononuclear cells and a mouse macrophage cell line (J774) amplifies IL-1β secretion following stimulation with NLRP3 agonists, but not with AIM2 or NLRP1b agonists. The augmented NRLP3 activation was mediated by increases in iNOS expression and NO production, in conjunction with increases in mitochondrial membrane depolarization, oxygen consumption rate, and ROS generation in J774 cells chronically exposed to ethanol (CE cells), effects that could be inhibited by the iNOS inhibitor SEITU, the NO scavenger carboxy-PTIO, and the mitochondrial ROS scavenger MitoQ. Chronic ethanol exposure did not alter K + efflux or Zn 2+ homeostasis in CE cells, although it did result in a lower intracellular concentration of NAD + . Prolonged administration of acetaldehyde, the product of alcohol dehydrogenase (ADH) mediated metabolism of ethanol, mimicked chronic ethanol exposure, whereas ADH inhibition prevented ethanol-induced IL-1β hypersecretion. Together, these results indicate that increases in iNOS and mitochondrial ROS production are critical for chronic ethanol-induced IL-1β hypersecretion, and that protracted exposure to the products of ethanol metabolism are probable mediators of NLRP3 inflammasome hyperactivation., (Copyright © 2017. Published by Elsevier B.V.)

Lung tissue remodeling in chronic obstructive pulmonary disease (COPD) is characterized by airway wall thickening and/or emphysema. Although the bronchial and alveolar compartments are functionally independent entities, we recently showed comparable alterations in matrix composition comprised of decreased elastin content and increased collagen and hyaluronan contents of alveolar and small airway walls. Out of several animal models tested, surfactant protein C (SPC)-TNF-α mice showed remodeling in alveolar and airway walls similar to what we observed in patients with COPD. Epithelial cells are able to undergo a phenotypic shift, gaining mesenchymal properties, a process in which c-Jun N-terminal kinase (JNK) signaling is involved. Therefore, we hypothesized that TNF-α induces JNK-dependent epithelial plasticity, which contributes to lung matrix remodeling. To this end, the ability of TNF-α to induce a phenotypic shift was assessed in A549, BEAS2B, and primary bronchial epithelial cells, and phenotypic markers were studied in SPC-TNF-α mice. Phenotypic markers of mesenchymal cells were elevated both in vitro and in vivo, as shown by the expression of vimentin, plasminogen activator inhibitor-1, collagen, and matrix metalloproteinases. Concurrently, the expression of the epithelial markers, E-cadherin and keratin 7 and 18, was attenuated. A pharmacological inhibitor of JNK attenuated this phenotypic shift in vitro, demonstrating involvement of JNK signaling in this process. Interestingly, activation of JNK signaling was also clearly present in lungs of SPC-TNF-α mice and patients with COPD. Together, these data show a role for TNF-α in the induction of a phenotypic shift in vitro, resulting in increased collagen production and the expression of elastin-degrading matrix metalloproteinases, and provide evidence for involvement of the TNF-α-JNK axis in extracellular matrix remodeling.

Background: Lung remodeling and pulmonary fibrosis are serious, life-threatening conditions resulting from diseases such as chronic severe asthma and idiopathic pulmonary fibrosis (IPF). Preclinical evidence suggests that JNK enzyme function is required for key steps in the pulmonary fibrotic process. However, a selective JNK inhibitor has not been investigated in translational models of lung fibrosis with clinically relevant biomarkers, or in IPF patients., Methods: The JNK inhibitor CC-930 was evaluated in the house dust mite-induced fibrotic airway mouse model, in a phase I healthy volunteer pharmacodynamic study, and subsequently in a phase II multicenter study of mild/moderate IPF (n = 28), with a 4-week, placebo-controlled, double-blind, sequential ascending-dose period (50 mg QD, 100 mg QD, 100 mg BID) and a 52-week open-label treatment-extension period., Results: In the preclinical model, CC-930 attenuated collagen 1A1 gene expression, peribronchiolar collagen deposition, airway mucin MUC5B expression in club cells, and MMP-7 expression in lung, bronchoalveolar lavage fluid, and serum. In the phase I study, CC-930 reduced c-Jun phosphorylation induced by UV radiation in skin. In the phase II IPF study, there was a CC-930 dose-dependent trend in reduction of MMP-7 and SP-D plasma protein levels. The most commonly reported adverse events were increased ALT, increased AST, and upper respiratory tract infection (six subjects each, 21.4 %). A total of 13 subjects (46.4 %) experienced adverse events that led to discontinuation of study drug. Nine out of 28 subjects experienced progressive disease in this study. The mean FVC (% predicted) declined after 26-32 weeks at doses of 100 mg QD and 100 mg BID. Changes in MMP-7, SP-D, and tenascin-C significantly correlated with change in FVC (% predicted)., Conclusions: These results illustrate JNK enzymatic activity involvement during pulmonary fibrosis, and support systemic biomarker use for tracking disease progression and the potential clinical benefit of this novel intervention in IPF. Trial registration ClinicalTrials.gov NCT01203943.

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.

Protein S-glutathionylation (PSSG) is an oxidant-induced post-translational modification of protein cysteines that impacts structure and function. The oxidoreductase glutaredoxin-1 (Glrx1) under physiological conditions catalyzes deglutathionylation and restores the protein thiol group. The involvement of Glrx1/PSSG in allergic inflammation induced by asthma-relevant allergens remains unknown. In the present study, we examined the impact of genetic ablation of Glrx1 in the pathogenesis of house dust mite (HDM)-induced allergic airways disease in mice. Wild-type (WT) or Glrx1(-/-) mice were instilled intranasally with HDM on 5 consecutive days for 3 weeks. As expected, overall PSSG was increased in Glrx1(-/-) HDM mice as compared with WT animals. Total cells in bronchoalveolar lavage fluid were similarly increased in HDM-treated WT and Glrx1(-/-) mice. However, in response to HDM, mice lacking Glrx1 demonstrated significantly more neutrophils and macrophages but fewer eosinophils as compared with HDM-exposed WT mice. mRNA expression of the Th2-associated cytokines IL-13 and IL-6, as well as mucin-5AC (Muc5ac), was significantly attenuated in Glrx1(-/-) HDM-treated mice. Conversely, mRNA expression of IFN-γ and IL-17A was increased in Glrx1(-/-) HDM mice compared with WT littermates. Restimulation of single-cell suspensions isolated from lungs or spleens with HDM resulted in enhanced IL-17A and decreased IL-5 production in cells derived from inflamed Glrx1(-/-) mice compared with WT animals. Finally, HDM-induced tissue damping and elastance were significantly attenuated in Glrx1(-/-) mice compared with WT littermates. These results demonstrate that the Glrx1-PSSG axis plays a pivotal role in HDM-induced allergic airways disease in association with enhanced type 2 inflammation and restriction of IFN-γ and IL-17A.

Nuclear Factor kappa B (NF-κB) is a transcription factor family critical in the activation of pro- inflammatory responses. The NF-κB pathway is regulated by oxidant-induced post-translational modifications. Protein S-glutathionylation, or the conjugation of the antioxidant molecule, glutathione to reactive cysteines inhibits the activity of inhibitory kappa B kinase beta (IKKβ), among other NF-κB proteins. Glutathione S-transferase Pi (GSTP) is an enzyme that has been shown to catalyze protein S-glutathionylation (PSSG) under conditions of oxidative stress. The objective of the present study was to determine whether GSTP regulates NF-κB signaling, S-glutathionylation of IKK, and subsequent pro-inflammatory signaling. We demonstrated that, in unstimulated cells, GSTP associated with the inhibitor of NF-κB, IκBα. However, exposure to LPS resulted in a rapid loss of association between IκBα and GSTP, and instead led to a protracted association between IKKβ and GSTP. LPS exposure also led to increases in the S-glutathionylation of IKKβ. SiRNA-mediated knockdown of GSTP decreased IKKβ-SSG, and enhanced NF-κB nuclear translocation, transcriptional activity, and pro-inflammatory cytokine production in response to lipopolysaccharide (LPS). TLK117, an isotype-selective inhibitor of GSTP, also enhanced LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production, suggesting that the catalytic activity of GSTP is important in repressing NF-κB activation. Expression of both wild-type and catalytically-inactive Y7F mutant GSTP significantly attenuated LPS- or IKKβ-induced production of GM-CSF. These studies indicate a complex role for GSTP in modulating NF-κB, which may involve S-glutathionylation of IKK proteins, and interaction with NF-κB family members. Our findings suggest that targeting GSTP is a potential avenue for regulating the activity of this prominent pro-inflammatory and immunomodulatory transcription factor., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is a debilitating lung disease characterized by excessive collagen production and fibrogenesis. Apoptosis in lung epithelial cells is critical in IPF pathogenesis, as heightened loss of these cells promotes fibroblast activation and remodeling. Changes in glutathione redox status have been reported in IPF patients. S-glutathionylation, the conjugation of glutathione to reactive cysteines, is catalyzed in part by glutathione- S -transferase π (GSTP). To date, no published information exists linking GSTP and IPF to our knowledge. We hypothesized that GSTP mediates lung fibrogenesis in part through FAS S-glutathionylation, a critical event in epithelial cell apoptosis. Our results demonstrate that GSTP immunoreactivity is increased in the lungs of IPF patients, notably within type II epithelial cells. The FAS-GSTP interaction was also increased in IPF lungs. Bleomycin- and AdTGFβ-induced increases in collagen content, α-SMA, FAS S-glutathionylation, and total protein S-glutathionylation were strongly attenuated in Gstp -/- mice. Oropharyngeal administration of the GSTP inhibitor, TLK117, at a time when fibrosis was already apparent, attenuated bleomycin- and AdTGFβ-induced remodeling, α-SMA, caspase activation, FAS S-glutathionylation, and total protein S-glutathionylation. GSTP is an important driver of protein S-glutathionylation and lung fibrosis, and GSTP inhibition via the airways may be a novel therapeutic strategy for the treatment of IPF.

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., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

S-glutathionylation has emerged as an oxidant-induced post-translational modification of protein cysteines that affects structure and function. The oxidoreductase glutaredoxin-1 (Glrx1), under physiological conditions, catalyzes deglutathionylation and restores the protein thiol group. The involvement of Grx1/S-glutathionylation in allergic inflammation induced by asthma-relevant allergens remains unknown. In the present study we examined the impact of genetic ablation of Glrx1 for the pathogenesis of house dust mite (HDM)-induced allergic airway disease in mice. Wild-type (WT) or Glrx1(-/-) mice in the BALB/c background were instilled intranasally with 50 μg of HDM 5 consecutive days for 3 weeks and killed 72 hours post final exposure. As expected, overall protein S-glutathionylation was increased in Glrx1(-/-) mice exposed to HDM as compared with WT animals. Total cells in the bronchoalveolar lavage fluid were similarly increased in WT and Glrx1(-/-) HDM-treated mice compared with phosphate-buffered saline-treated control mice. However, in response to HDM, mice lacking Glrx1 demonstrated significantly more neutrophils but fewer eosinophils than HDM-exposed WT mice. mRNA expression of the Th2-associated cytokine IL-13, as well as MUC5ac, was significantly attenuated in Glrx1(-/-) HDM-treated mice compared with WT mice. Conversely, expression of IL-17A was increased in Glrx1(-/-) HDM mice compared with WT mice. Last, HDM-induced tissue damping and elastance were significantly attenuated in Glrx1(-/-) mice compared with WT littermates. These results demonstrate that the Grx1/S-glutathionylation redox status plays a pivotal role in HDM-induced allergic inflammation and airway hyperresponsiveness and suggest a potential role of Glrx1/S-glutathionylation in controlling the nature of the HDM-induced adaptive immune responses by promoting Type-2-driven inflammation and restricting IL-17A.

Background: Evidence for association between asthma and the unfolded protein response is emerging. Endoplasmic reticulum resident protein 57 (ERp57) is an endoplasmic reticulum-localized redox chaperone involved in folding and secretion of glycoproteins. We have previously demonstrated that ERp57 is upregulated in allergen-challenged human and murine lung epithelial cells. However, the role of ERp57 in asthma pathophysiology is unknown., Objectives: Here we sought to examine the contribution of airway epithelium-specific ERp57 in the pathogenesis of allergic asthma., Methods: We examined the expression of ERp57 in human asthmatic airway epithelium and used murine models of allergic asthma to evaluate the relevance of epithelium-specific ERp57., Results: Lung biopsy specimens from asthmatic and nonasthmatic patients revealed a predominant increase in ERp57 levels in epithelium of asthmatic patients. Deletion of ERp57 resulted in a significant decrease in inflammatory cell counts and airways resistance in a murine model of allergic asthma. Furthermore, we observed that disulfide bridges in eotaxin, epidermal growth factor, and periostin were also decreased in the lungs of house dust mite-challenged ERp57-deleted mice. Fibrotic markers, such as collagen and α smooth muscle actin, were also significantly decreased in the lungs of ERp57-deleted mice. Furthermore, adaptive immune responses were dispensable for house dust mite-induced endoplasmic reticulum stress and airways fibrosis., Conclusions: Here we show that ERp57 levels are increased in the airway epithelium of asthmatic patients and in mice with allergic airways disease. The ERp57 level increase is associated with redox modification of proinflammatory, apoptotic, and fibrotic mediators and contributes to airways hyperresponsiveness. The strategies to inhibit ERp57 specifically within the airways epithelium might provide an opportunity to alleviate the allergic asthma phenotype., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

In a complex inflammatory airways disease such as asthma, abnormalities in a plethora of molecular and cellular pathways ultimately culminate in characteristic impairments in respiratory function. The ability to study disease pathophysiology in the setting of a functioning immune and respiratory system therefore makes mouse models an invaluable tool in translational research. Despite the vast understanding of inflammatory airways diseases gained from mouse models to date, concern over the validity of mouse models continues to grow. Therefore the aim of this review is twofold; firstly, to evaluate mouse models of asthma in light of current clinical definitions, and secondly, to provide a framework by which mouse models can be continually refined so that they continue to stand at the forefront of translational science. Indeed, it is in viewing mouse models as a continual work in progress that we will be able to target our research to those patient populations in whom current therapies are insufficient., (© 2014 Wiley Periodicals, Inc.)

Interleukin-17A (IL-17A) is a newly emerging player in the pathogenesis of chronic lung diseases that amplifies inflammatory responses and promotes tissue remodeling. Stimulation of lung epithelial cells with IL-17A leads to activation of the transcription factor nuclear factor κB (NF-κB), a key player in the orchestration of lung inflammation. We have previously demonstrated the importance of the redox-dependent posttranslational modification S-glutathionylation in limiting activation of NF-κB and downstream gene induction. Under physiological conditions, the enzyme glutaredoxin 1 (Grx1) acts to deglutathionylate NF-κB proteins, which restores functional activity. In this study, we sought to determine the impact of S-glutathionylation on IL-17A-induced NF-κB activation and expression of proinflammatory mediators. C10 mouse lung alveolar epithelial cells or primary mouse tracheal epithelial cells exposed to IL-17A show rapid activation of NF-κB and the induction of proinflammatory genes. Upon IL-17A exposure, sulfenic acid formation and S-glutathionylated proteins increased. Assessment of S-glutathionylation of NF-κB pathway components revealed S-glutathionylation of RelA (RelA-SSG) and inhibitory κB kinase α (IKKα-SSG) after stimulation with IL-17A. SiRNA-mediated ablation of Grx1 increased both RelA-SSG and IKKα-SSG and acutely increased nuclear content of RelA and tended to decrease nuclear RelB. SiRNA-mediated ablation or genetic ablation of Glrx1 decreased the expression of the NF-κB-regulated genes KC and CCL20 in response to IL-17A, but conversely increased the expression of IL-6. Last, siRNA-mediated ablation of IKKα attenuated nuclear RelA and RelB content and decreased expression of KC and CCL20 in response to IL-17A. Together, these data demonstrate a critical role for the S-glutathionylation/Grx1 redox axis in regulating IKKα and RelA S-glutathionylation and the responsiveness of epithelial cells to IL-17A., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Chronic allergic asthma leads to airway remodeling and subepithelial fibrosis via mechanisms not fully understood. Airway remodeling is amplified by profibrotic mediators, such as transforming growth factor-β1 (TGF-β1), which plays a cardinal role in various models of fibrosis. We recently have identified a critical role for c-Jun-NH2-terminal-kinase (JNK) 1 in augmenting the profibrotic effects of TGF-β1, linked to epithelial-to-mesenchymal transition of airway epithelial cells. To examine the role of JNK1 in house dust mite (HDM)-induced airway remodeling, we induced allergic airway inflammation in wild-type (WT) and JNK1-/- mice by intranasal administration of HDM extract. WT and JNK1-/- mice were sensitized with intranasal aspirations of HDM extract for 15 days over 3 wk. HDM caused similar increases in airway hyperresponsiveness, mucus metaplasia, and airway inflammation in WT and JNK1-/- mice. In addition, the profibrotic cytokine TGF-β1 and phosphorylation of Smad3 were equally increased in WT and JNK1-/- mice. In contrast, increases in collagen content in lung tissue induced by HDM were significantly attenuated in JNK1-/- mice compared with WT controls. Furthermore HDM-induced increases of α-smooth muscle actin (α-SMA) protein and mRNA expression as well as the mesenchymal markers high-mobility group AT-hook 2 and collagen1A1 in WT mice were attenuated in JNK1-/- mice. The let-7 family of microRNAs has previously been linked to fibrosis. HDM exposure in WT mice and primary lung epithelial cells resulted in striking decreases in let-7g miRNA that were not observed in mice or primary lung epithelial cells lacking JNK1-/- mice. Overexpression of let-7g in lung epithelial cells reversed the HDM-induced increases in α-SMA. Collectively, these findings demonstrate an important requirement for JNK1 in promoting HDM-induced fibrotic airway remodeling.

Tissue injury and inflammation are associated with increased production of reactive oxygen species (ROS), which have the ability to induce oxidative injury to various biomolecules resulting in protein dysfunction, genetic instability, or cell death. However, recent observations indicate that formation of hydrogen peroxide (H2 O2 ) during tissue injury is also an essential feature of the ensuing wound healing response, and functions as an early damage signal to control several critical aspects of the wound healing process. Because innate oxidative wound responses must be tightly coordinated to avoid chronic inflammation or tissue injury, a more complete understanding is needed regarding the origins and dynamics of ROS production, and their critical biological targets. This prospect highlights the current experimental evidence implicating H2 O2 in early epithelial wound responses, and summarizes technical advances and approaches that may help distinguish its beneficial actions from its more deleterious actions in conditions of chronic tissue injury or inflammation., (© 2013 Wiley Periodicals, Inc.)

Rationale: The death receptor Fas is critical for bacterial clearance and survival of mice after Pseudomonas aeruginosa infection., Objectives: Fas ligand (FasL)-induced apoptosis is augmented by S-glutathionylation of Fas (Fas-SSG), which can be reversed by glutaredoxin-1 (Grx1). Therefore, the objective of this study was to investigate the interplay between Grx1 and Fas in regulating the clearance of P. aeruginosa infection., Methods: Lung samples from patients with bronchopneumonia were analyzed by immunofluorescence. Primary tracheal epithelial cells, mice lacking the gene for Grx1 (Glrx1(-/-)), Glrx1(-/-) mice treated with caspase inhibitor, or transgenic mice overexpressing Grx1 in the airway epithelium were analyzed after infection with P. aeruginosa., Measurements and Main Results: Patient lung samples positive for P. aeruginosa infection demonstrated increased Fas-SSG compared with normal lung samples. Compared with wild-type primary lung epithelial cells, infection of Glrx1(-/-) cells with P. aeruginosa showed enhanced caspase 8 and 3 activities and cell death in association with increases in Fas-SSG. Infection of Glrx1(-/-) mice with P. aeruginosa resulted in enhanced caspase activity and increased Fas-SSG as compared with wild-type littermates. Absence of Glrx1 significantly enhanced bacterial clearance, and decreased mortality postinfection with P. aeruginosa. Inhibition of caspases significantly decreased bacterial clearance postinfection with P. aeruginosa, in association with decreased Fas-SSG. In contrast, transgenic mice that overexpress Grx1 in lung epithelial cells had significantly higher lung bacterial loads, enhanced mortality, decreased caspase activation, and Fas-SSG in the lung after infection with P. aeruginosa, compared with wild-type control animals., Conclusions: These results suggest that S-glutathionylation of Fas within the lung epithelium enhances epithelial apoptosis and promotes clearance of P. aeruginosa and that glutaredoxin-1 impairs bacterial clearance and increases the severity of pneumonia in association with deglutathionylation of Fas.

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 β-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.

NF-κB activation within the epithelium has been implicated in the pathogenesis of asthma, yet the exact role of epithelial NF-κB in allergen-induced inflammation and airway remodeling remains unclear. In the current study, we used an intranasal house dust mite (HDM) extract exposure regimen time course in BALB/c mice to evaluate inflammation, NF-κB activation, airway hyperresponsiveness (AHR), and airway remodeling. We used CC10-IκBαSR transgenic mice to evaluate the functional importance of epithelial NF-κB in response to HDM. After a single exposure of HDM, mRNA expression of proinflammatory mediators was significantly elevated in lung tissue of wild-type (WT) mice, in association with increases in nuclear RelA and RelB, components of the classical and alternative NF-κB pathway, respectively, in the bronchiolar epithelium. In contrast, CC10-IκBαSR mice displayed marked decreases in nuclear RelA and RelB and mRNA expression of proinflammatory mediators compared with WT mice. After 15 challenges with HDM, WT mice exhibited increases in inflammation, AHR, mucus metaplasia, and peribronchiolar fibrosis. CC10-IκBαSR transgenic mice displayed marked decreases in neutrophilic infiltration, tissue damping, and elastance parameters, in association will less peribronchiolar fibrosis and decreases in nuclear RelB in lung tissue. However, central airway resistance and mucus metaplasia remained elevated in CC10-IκBαSR transgenic mice, in association with the continued presence of lymphocytes, and partial decreases in eosinophils and IL-13. The current study demonstrates that following airway exposure with an asthma-relevant allergen, activation of classical and alternative NF-κB pathways occurs within the airway epithelium and may coordinately contribute to allergic inflammation, AHR, and fibrotic airway remodeling.

Glutathione has traditionally been considered as an antioxidant that protects cells against oxidative stress. Hence, the loss of reduced glutathione and formation of glutathione disulfide is considered a classical parameter of oxidative stress that is increased in diseases. Recent studies have emerged that demonstrate that glutathione plays a more direct role in biological and pathophysiological processes through covalent modification to reactive cysteines within proteins, a process known as S-glutathionylation. The formation of an S-glutathionylated moiety within the protein can lead to structural and functional modifications. Activation, inactivation, loss of function, and gain of function have all been attributed to S-glutathionylation. In pathophysiological settings, S-glutathionylation is tightly regulated. This perspective offers a concise overview of the emerging field of protein thiol redox modifications. We will also cover newly developed methodology to detect S-glutathionylation in situ, which will enable further discovery into the role of S-glutathionylation in biology and disease., (Copyright © 2013 Wiley Periodicals, Inc.)

The respiratory epithelium plays a critical role in innate defenses against airborne pathogens and pollutants, and alterations in epithelial homeostasis and repair mechanisms are thought to contribute to chronic lung diseases associated with airway remodeling. Previous studies implicated the nicotinamide adenine dinucleotide phosphate-reduced oxidase dual oxidase-1 (DUOX1) in redox signaling pathways involved in in vitro epithelial wound responses to infection and injury. However, the importance of epithelial DUOX1 in in vivo epithelial repair pathways has not been established. Using small interfering (si)RNA silencing of DUOX1 expression, we show the critical importance of DUOX1 in wound responses in murine tracheal epithelial (MTE) cells in vitro, as well as its contribution to epithelial regeneration in vivo in a murine model of epithelial injury induced by naphthalene, a selective toxicant of nonciliated respiratory epithelial cells (club cells [Clara]). Whereas naphthalene-induced club-cell injury is normally followed by epithelial regeneration after 7 and 14 days, such airway reepithelialization was significantly delayed after the silencing of airway DUOX1 by oropharyngeal administration of DUOX1-targeted siRNA. Wound closure in MTE cells was related to DUOX1-dependent activation of the epidermal growth factor receptor (EGFR) and the transcription factor signal transducer and activator of transcription-3 (STAT3), known mediators of epithelial cell migration and wound responses. Moreover, in vivo DUOX1 silencing significantly suppressed naphthalene-induced activation of STAT3 and EGFR during early stages of epithelial repair. In conclusion, these experiments demonstrate for the first time an important function for epithelial DUOX1 in lung epithelial regeneration in vivo, by promoting EGFR-STAT3 signaling and cell migration as critical events in initial repair.

The transcription factor NF-κB has been causally linked to inflammatory lung diseases. Recent studies have unraveled the complexity of NF-κB activation by identifying two parallel activation pathways: the classical NF-κB pathway, which is controlled by IκB kinase complex-β (IKKβ) and RelA/p50, and the alternative pathway, which is controlled by IKKα and RelB/p52. The alternative pathway regulates adaptive immune responses and lymphoid development, yet its role in the regulation of innate immune responses remains largely unknown. In this study, we determined the relevance of the alternative NF-κB pathway in proinflammatory responses in lung epithelial cells. The exposure of C10 murine alveolar lung epithelial cells to diverse stimuli, or primary murine tracheal epithelial cells to LPS, resulted in the activation of both NF-κB pathways, based on the nuclear translocation of RelA, p50, RelB, and p52. Increases in the nuclear content of RelA occurred rapidly, but transiently, whereas increases in nuclear RelB content were protracted. The small interfering (si) RNA-mediated knockdown of IKKα, RelA, or RelB resulted in decreases of multiple LPS-induced proinflammatory cytokines. Surprisingly, the siRNA ablation of IKKα or RelB led to marked increases in the production of IL-6 in response to LPS. The simultaneous expression of constitutively active (CA)-IKKα and CA-IKKβ caused synergistic increases in proinflammatory mediators. Lastly, the disruption of the IKK signalsome inhibited the activation of both NF-κB pathways. These results demonstrate that the coordinated activation of both NF-κB pathways regulates the magnitude and nature of proinflammatory responses in lung epithelial cells.

Protein-S-glutathionylation (PSSG) is an oxidative modification of reactive cysteines that has emerged as an important player in pathophysiological processes. Under physiological conditions, the thiol transferase, glutaredoxin-1 (Glrx1) catalyses deglutathionylation. Although we previously demonstrated that Glrx1 expression is increased in mice with allergic inflammation, the impact of Glrx1/PSSG in the development of allergic airways disease remains unknown. In the present study we examined the impact of genetic ablation of Glrx1 in the pathogenesis of allergic inflammation and airway hyperresponsiveness (AHR) in mice. Glrx1(-/-) or WT mice were subjected to the antigen, ovalbumin (OVA), and parameters of allergic airways disease were evaluated 48 h after three challenges, and 48 h or 7 days after six challenges with aerosolized antigen. Although no clear increases in PSSG were observed in WT mice in response to OVA, marked increases were detected in lung tissue of mice lacking Glrx1 48 h following six antigen challenges. Inflammation and expression of proinflammatory mediators were decreased in Glrx1(-/-) mice, dependent on the time of analysis. WT and Glrx1(-/-) mice demonstrated comparable increases in AHR 48 h after three or six challenges with OVA. However, 7 days postcessation of six challenges, parameters of AHR in Glrx1(-/-) mice were resolved to control levels, accompanied by marked decreases in mucus metaplasia and expression of Muc5AC and GOB5. These results demonstrate that the Glrx1/S-glutathionylation redox status in mice is a critical regulator of AHR, suggesting that avenues to increase S-glutathionylation of specific target proteins may be beneficial to attenuate AHR.

Recent studies suggest the importance of the transition of airway epithelial cells (EMT) in pulmonary fibrosis, and also indicate a role for Wingless protein (Wnt)/β-catenin signaling in idiopathic pulmonary fibrosis. We investigated the possible role of the Wnt signaling pathway in inducing EMT in lung epithelial cells, and sought to unravel the role of c-Jun-N-terminal-kinase-1 (JNK1). The exposure of C10 lung epithelial cells or primary mouse tracheal epithelial cells (MTECs) to Wnt3a resulted in increases in JNK phosphorylation and nuclear β-catenin content. Because the role of β-catenin as a transcriptional coactivator is well established, we investigated T-cell factor/lymphocyte-enhancement factor (TCF/LEF) transcriptional activity in C10 lung epithelial cells after the activation of Wnt. TCF/LEF transcriptional activity was enhanced after the activation of Wnt, and this increase in TCF/LEF transcriptional activity was diminished after the small interfering (si)RNA-mediated ablation of JNK. The activation of the Wnt pathway by Wnt3a, or the expression of either wild-type or constitutively active β-catenin (S37A), led to the activation of an EMT transcriptome, manifested by the increased mRNA expression of CArG box-binding factor-A, fibroblast-specific protein (FSP)-1, α-smooth muscle actin (α-SMA), and vimentin, increases in the content of α-SMA and FSP1, and the concomitant loss of zona occludens-1. The siRNA-mediated ablation of β-catenin substantially decreased Wnt3a-induced EMT. The siRNA ablation of JNK1 largely abolished Wnt3a, β-catenin, and β-catenin S37a-induced EMT. In MTECs lacking Jnk1, Wnt3a-induced increases in nuclear β-catenin, EMT transcriptome, and the content of α-SMA or FSP1 were substantially diminished. These data show that the activation of the Wnt signaling pathway is capable of inducing an EMT program in lung epithelial cells through β-catenin, and that this process is controlled by JNK1.

We recently demonstrated that S-glutathionylation of the death receptor Fas (Fas-SSG) amplifies apoptosis (V. Anathy et al., J. Cell Biol. 184:241-252, 2009). In the present study, we demonstrate that distinct pools of Fas exist in cells. Upon ligation of surface Fas, a separate pool of latent Fas in the endoplasmic reticulum (ER) underwent rapid oxidative processing characterized by the loss of free sulfhydryl content (Fas-SH) and resultant increases in S-glutathionylation of Cys294, leading to increases of surface Fas. Stimulation with FasL rapidly induced associations of Fas with ERp57 and glutathione S-transferase π (GSTP), a protein disulfide isomerase and catalyst of S-glutathionylation, respectively, in the ER. Knockdown or inhibition of ERp57 and GSTP1 substantially decreased FasL-induced oxidative processing and S-glutathionylation of Fas, resulting in decreased death-inducing signaling complex formation and caspase activity and enhanced survival. Bleomycin-induced pulmonary fibrosis was accompanied by increased interactions between Fas-ERp57-GSTP1 and S-glutathionylation of Fas. Importantly, fibrosis was largely prevented following short interfering RNA-mediated ablation of ERp57 and GSTP. Collectively, these findings illuminate a regulatory switch, a ligand-initiated oxidative processing of latent Fas, that controls the strength of apoptosis.

Influenza A virus (IAV) infection is known to induce endoplasmic reticulum (ER) stress, Fas-dependent apoptosis, and TGF-β production in a variety of cells. However, the relationship between these events in murine primary tracheal epithelial cells (MTECS), which are considered one of the primary sites of IAV infection and replication, is unclear. We show that IAV infection induced ER stress marker activating transcription factor-6 and endoplasmic reticulum protein 57-kD (ERp57), but not C/EBP homologous protein (CHOP). In contrast, the ER stress inducer thapsigargin (THP) increased CHOP. IAV infection activated caspases and apoptosis, independently of Fas and caspase-8, in MTECs. Instead, apoptosis was mediated by caspase-12. A decrease in ERp57 attenuated the IAV burden and decreased caspase-12 activation and apoptosis in epithelial cells. TGF-β production was enhanced in IAV-infected MTECs, compared with THP or staurosporine. IAV infection caused the activation of c-Jun N-terminal kinase (JNK). Furthermore, IAV-induced TGF-β production required the presence of JNK1, a finding that suggests a role for JNK1 in IAV-induced epithelial injury and subsequent TGF-β production. These novel findings suggest a potential mechanistic role for a distinct ER stress response induced by IAV, and a profibrogenic/repair response in contrast to other pharmacological inducers of ER stress. These responses may also have a potential role in acute lung injury, fibroproliferative acute respiratory distress syndrome, and the recently identified H1N1 influenza-induced exacerbations of chronic obstructive pulmonary disease (Wedzicha JA. Proc Am Thorac Soc 2004;1:115-120) and idiopathic pulmonary fibrosis (Umeda Y, et al. Int Med 2010;49:2333-2336).

Significance: Redox-based signaling governs a number of important pathways in tissue homeostasis. Consequently, deregulation of redox-controlled processes has been linked to a number of human diseases. Among the biological processes regulated by redox signaling, apoptosis or programmed cell death is a highly conserved process important for tissue homeostasis. Apoptosis can be triggered by a wide variety of stimuli, including death receptor ligands, environmental agents, and cytotoxic drugs. Apoptosis has also been implicated in the etiology of many human diseases., Recent Advances: Recent discoveries demonstrate that redox-based changes are required for efficient activation of apoptosis. Among these redox changes, alterations in the abundant thiol, glutathione (GSH), and the oxidative post-translational modification, protein S-glutathionylation (PSSG) have come to the forefront as critical regulators of apoptosis., Critical Issues: Although redox-based changes have been documented in apoptosis and disease pathogenesis, the mechanistic details, whereby redox perturbations intersect with pathogenic processes, remain obscure., Future Directions: Further research will be needed to understand the context in which of the members of the death receptor pathways undergo ligand dependent oxidative modifications. Additional investigation into the interplay between oxidative modifications, redox enzymes, and apoptosis pathway members are also critically needed to improve our understanding how redox-based control is achieved. Such analyses will be important in understanding the diverse chronic diseases. In this review we will discuss the emerging paradigms in our current understanding of redox-based regulation of apoptosis with an emphasis on S-glutathionylation of proteins and the enzymes involved in this important post-translational modification.

Asthma represents a growing problem in the developing world, affecting millions of children and adults. Features of the disease are reversible airflow obstruction, airway hyperresponsiveness and airway inflammation leading to tissue damage and remodeling. Many studies have attempted to address whether inflammation and airway hyperresponsiveness are mechanistically linked. In this study, data are presented from several mouse models that illustrate that a clear link between these features of asthma remains elusive. The impact of altering inflammatory signaling (NF-κB or JNK1) on inflammation and airway hyperresponsiveness was examined. In addition, the effect of antigen sensitization and the route of antigen delivery were investigated. The data herein show that in many cases, inflammation and airway hyperresponsiveness do not directly correlate. In conclusion, the need for mechanistic studies in mouse models is highlighted to address the interplay between these components thought to be critical to asthma pathogenesis.

The c-Jun N-terminal kinase (JNK) - 1 pathway has been implicated in the cellular response to stress in many tissues and models. JNK1 is known to play a role in a variety of signaling cascades, including those involved in lung disease pathogenesis. Recently, a role for JNK1 signaling in immune cell function has emerged. The goal of the present study was to determine the role of JNK1 in host defense against both bacterial and viral pneumonia, as well as the impact of JNK1 signaling on IL-17 mediated immunity. Wild type (WT) and JNK1 -/- mice were challenged with Escherichia coli, Staphylococcus aureus, or Influenza A. In addition, WT and JNK1 -/- mice and epithelial cells were stimulated with IL-17A. The impact of JNK1 deletion on pathogen clearance, inflammation, and histopathology was assessed. JNK1 was required for clearance of E. coli, inflammatory cell recruitment, and cytokine production. Interestingly, JNK1 deletion had only a small impact on the host response to S. aureus. JNK1 -/- mice had decreased Influenza A burden in viral pneumonia, yet displayed worsened morbidity. Finally, JNK1 was required for IL-17A mediated induction of inflammatory cytokines and antimicrobial peptides both in epithelial cells and the lung. These data identify JNK1 as an important signaling molecule in host defense and demonstrate a pathogen specific role in disease. Manipulation of the JNK1 pathway may represent a novel therapeutic target in pneumonia.

It is established that cigarette smoke (CS) causes irreversible oxidations in lung epithelial cells, and can lead to their death. However, its impact on reversible and physiologically relevant redox-dependent protein modifications remains to be investigated. Glutathione is an important antioxidant against inhaled reactive oxygen species as a direct scavenger, but it can also covalently bind protein thiols upon mild oxidative stress to protect them against irreversible oxidation. This posttranslational modification, known as S-glutathionylation, can be reversed under physiological conditions by the enzyme, glutaredoxin 1 (Grx1). The aim of this study was to investigate if CS modifies Grx1, and if this impacts on protein S-glutathionylation and epithelial cell death. Upon exposure of alveolar epithelial cells to CS extract (CSE), a decrease in Grx1 mRNA and protein expression was observed, in conjunction with decreased activity and increased protein S-glutathionylation. Using mass spectrometry, irreversible oxidation of recombinant Grx1 by CSE and acrolein was demonstrated, which was associated with attenuated enzyme activity. Furthermore, carbonylation of Grx1 in epithelial cells after exposure to CSE was shown. Overexpression of Grx1 attenuated CSE-induced increases in protein S-glutathionylation and increased survival. Conversely, primary tracheal epithelial cells of mice lacking Grx1 were more sensitive to CS-induced cell death, with corresponding increases in protein S-glutathionylation. These results show that CS can modulate Grx1, not only at the expression level, but can also directly modify Grx1 itself, decreasing its activity. These findings demonstrate a role for the Grx1/S-glutathionylation redox system in CS-induced lung epithelial cell death.

The transcription factor nuclear factor κB (NF-κB) is a critical regulator of inflammation and immunity and is negatively regulated via S-glutathionylation. The inhibitory effect of S-glutathionylation is overcome by glutaredoxin-1 (Grx1), which under physiological conditions catalyzes deglutathionylation and enhances NF-κB activation. The mechanisms whereby expression of the Glrx1 gene is regulated remain unknown. Here we examined the role of NF-κB in regulating activation of Glrx1. Transgenic mice that express a doxycycline-inducible constitutively active version of inhibitory κB kinase-β (CA-IKKβ) demonstrate elevated expression of Grx1. Transient transfection of CA-IKKβ also resulted in significant induction of Grx1. A 2-kb region of the Glrx1 promoter that contains two putative NF-κB binding sites was activated by CA-IKKβ, RelA/p50, and lipopolysaccharide (LPS). Chromatin immunoprecipitation experiments confirmed binding of RelA to the promoter of Glrx1 in response to LPS. Stimulation of C10 lung epithelial cells with LPS caused transient increases in Grx1 mRNA expression and time-dependent increases in S-glutathionylation of IKKβ. Overexpression of Grx1 decreased S-glutathionylation of IKKβ, prolonged NF-κB activation, and increased levels of proinflammatory mediators. Collectively, this study demonstrates that the Glrx1 gene is positively regulated by NF-κB and suggests a feed-forward mechanism to promote NF-κB signaling by decreasing S-glutathionylation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Activation of NF-κB in airway epithelium is observed in allergic asthma and is induced by inhalation of numerous infectious and reactive substances. Many of the substances that activate NF-κB in the airway epithelium are also capable of acting as adjuvants to elicit antigen-specific sensitization to concomitantly inhaled protein, thereby circumventing the inherent bias of the lung to promote tolerance to innocuous antigens. We have used a transgenic mouse inducibly expressing a constitutively active mutant of the inhibitor of nuclear factor κB (IκB) kinase β ((CA)IKKβ) that activates NF-κB only in nonciliated airway epithelial cells to test whether activation of this intracellular signaling pathway in this specific cell type is sufficient to establish a pulmonary environment permissive to the development of allergic sensitization to inhaled protein. When airway epithelial (CA)IKKβ was transiently expressed in antigen-naive mice only during initial inhalation of ovalbumin, the mice became allergically sensitized to the antigen. As a consequence, subsequent inhalation of ovalbumin alone led to an allergic asthma-like response that included airway hyperresponsiveness to methacholine, eosinophilia, mucus expression, elevated serum levels of antigen-specific IgE and IgG1, and splenic CD4(+) T cells that secreted T helper type 2 and type 17 cytokines in response to in vitro antigen restimulation. Furthermore, CD11c(+) cells in the mediastinal lymph nodes (MLN) of (CA)IKKβ-expressing mice displayed significantly elevated levels of activation markers. These data implicate airway epithelial NF-κB activation as a critical modulator of the adaptive immune response to inhaled antigens via the secretion of soluble mediators that affect the capacity of CD11c(+) cells to undergo maturation and promote antigen-specific allergic responses.

Transforming growth factor (TGF)-β1 is a key mediator of lung remodeling and fibrosis. Epithelial cells are both a source of and can respond to TGF-β1 with epithelial-to-mesenchymal transition (EMT). We recently determined that TGF-β1-induced EMT in lung epithelial cells requires the presence of c-Jun N-terminal kinase (JNK) 1. Because TGF-β1 signals via Smad complexes, the goal of the present study was to determine the impact of JNK1 on phosphorylation of Smad3 and Smad3-dependent transcriptional responses in lung epithelial cells. Evaluation of JNK1-deficient lung epithelial cells demonstrated that TGF-β1-induced terminal phosphorylation of Smad3 was similar, whereas phosphorylation of mitogen-activated protein kinase sites in the linker regions of Smad3 was diminished, in JNK1-deficient cells compared with wild-type cells. In comparison to wild-type Smad3, expression of a mutant Smad3 in which linker mitogen-activated protein kinase sites were ablated caused a marked attenuation in JNK1 or TGF-β1-induced Smad-binding element transcriptional activity, and expression of plasminogen activator inhibitor-1, fibronectin-1, high-mobility group A2, CArG box-binding factor-A, and fibroblast-specific protein-1, genes critical in the process of EMT. JNK1 enhanced the interaction between Smad3 and Smad4, which depended on linker phosphorylation of Smad3. Conversely, Smad3 with phosphomimetic mutations in the linker domain further enhanced EMT-related genes and proteins, even in the absence of JNK1. Finally, we demonstrated a TGF-β1-induced interaction between Smad3 and JNK1. Collectively, these results demonstrate that Smad3 phosphorylation in the linker region and Smad transcriptional activity are directly or indirectly controlled by JNK1, and provide a putative mechanism whereby JNK1 promotes TGF-β1-induced EMT.

Protein S-glutathionylation (PSSG), a reversible posttranslational modification of reactive cysteines, recently emerged as a regulatory mechanism that affects diverse cell-signaling cascades. The extent of cellular PSSG is controlled by the oxidoreductase glutaredoxin-1 (Grx1), a cytosolic enzyme that specifically de-glutathionylates proteins. Here, we sought to evaluate the impact of the genetic ablation of Grx1 on PSSG and on LPS-induced lung inflammation. In response to LPS, Grx1 activity increased in lung tissue and bronchoalveolar lavage (BAL) fluid in WT (WT) mice compared with PBS control mice. Glrx1(-/-) mice consistently showed slight but statistically insignificant decreases in total numbers of inflammatory cells recovered by BAL. However, LPS-induced concentrations of IL-1β, TNF-α, IL-6, and Granulocyte/Monocyte Colony-Stimulating Factor (GM-CSF) in BAL were significantly decreased in Glrx1(-/-) mice compared with WT mice. An in situ assessment of PSSG reactivity and a biochemical evaluation of PSSG content demonstrated increases in the lung tissue of Glrx1(-/-) animals in response to LPS, compared with WT mice or PBS control mice. We also demonstrated that PSSG reactivity was prominent in alveolar macrophages (AMs). Comparative BAL analyses from WT and Glrx1(-/-) mice revealed fewer and smaller AMs in Glrx1(-/-) mice, which showed a significantly decreased expression of NF-κB family members, impaired nuclear translocation of RelA, and lower levels of NF-κB-dependent cytokines after exposure to LPS, compared with WT cells. Taken together, these results indicate that Grx1 regulates the production of inflammatory mediators through control of S-glutathionylation-sensitive signaling pathways such as NF-κB, and that Grx1 expression is critical to the activation of AMs.

Background: Respiratory infections including Mycoplasma pneumoniae (Mp) contribute to various chronic lung diseases. We have shown that mouse short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein was able to inhibit Mp growth. Further, airway epithelial cells increased SPLUNC1 expression upon Mp infection. However, the mechanisms underlying SPLUNC1 regulation remain unknown. In the current study, we investigated if SPLUNC1 production following Mp infection is regulated through Toll-like receptor 2 (TLR2) signaling., Methods: Airway epithelial cell cultures were utilized to reveal the contribution of TLR2 signaling including NF-κB to SPLUNC1 production upon bacterial infection and TLR2 agonist stimulation., Results: Mp and TLR2 agonist Pam3CSK4 increased SPLUNC1 expression in tracheal epithelial cells from wild type, but not TLR2(-/-) BALB/c mice. RNA interference (short-hairpin RNA) of TLR2 in normal human bronchial epithelial cells under air-liquid interface cultures significantly reduced SPLUNC1 levels in Mp-infected or Pam3CSK4-treated cells. Inhibition and activation of NF-κB pathway decreased and increased SPLUNC1 production in airway epithelial cells, respectively., Conclusions: Our data for the first time suggest that airway epithelial TLR2 signaling is pivotal in mycoplasma-induced SPLUNC1 production, thus improving our understanding of the aberrant SPLUNC1 expression in airways of patients suffering from chronic lung diseases with bacterial infections.

Reactive oxidants such as nitrogen dioxide (NO(2)) injure the pulmonary epithelium, causing airway damage and inflammation. We previously demonstrated that nuclear factor-κ B (NF-κB) activation within airway epithelial cells occurs in response to NO(2) inhalation, and is critical for lipopolysaccharide-induced or antigen-induced inflammatory responses. Here, we investigated whether manipulation of NF-κB activity in lung epithelium affected severe lung injuries induced by NO(2) inhalation. Wild-type C57BL/6J, CC10-IκBα(SR) transgenic mice with repressed airway epithelial NF-κB function, or transgenic mice expressing a doxycycline-inducible, constitutively active I κ B kinase β (CC10-rTet-(CA)IKKβ) with augmented NF-κB function in airway epithelium, were exposed to toxic levels of 25 ppm or 50 ppm NO(2) for 6 hours a day for 1 or 3 days. In wild-type mice, NO(2) caused the activation of NF-κB in airway epithelium after 6 hours, and after 3 days resulted in severe acute lung injury, characterized by neutrophilia, peribronchiolar lesions, and increased protein, lactate dehydrogenase, and inflammatory cytokines. Compared with wild-type mice, neutrophilic inflammation and elastase activity, lung injury, and several proinflammatory cytokines were significantly suppressed in CC10-IκBα(SR) mice exposed to 25 or 50 ppm NO(2). Paradoxically, CC10-rTet-(CA)IKKβ mice that received doxycycline showed no further increase in NO(2)-induced lung injury compared with wild-type mice exposed to NO(2), instead displaying significant reductions in histologic parameters of lung injury, despite elevations in several proinflammatory cytokines. These intriguing findings demonstrate distinct functions of airway epithelial NF-κB activities in oxidant-induced severe acute lung injury, and suggest that although airway epithelial NF-κB activities modulate NO(2)-induced pulmonary inflammation, additional NF-κB-regulated functions confer partial protection from lung injury.

Tissue fibrosis is believed to be a manifestation of dysregulated repair following injury, in association with impaired reepithelialization, and aberrant myofibroblast activation and proliferation. Numerous pathways have been linked to the pathogenesis of fibrotic lung disease, including the death receptor Fas, which contributes to apoptosis of lung epithelial cells. A redox imbalance also has been implicated in disease pathogenesis, although mechanistic details whereby oxidative changes intersect with profibrotic signaling pathways remain elusive. Oxidation of cysteines in proteins, such as S-glutathionylation (PSSG), is known to act as a regulatory event that affects protein function. This manuscript will discuss evidence that S-glutathionylation regulates death receptor induced apoptosis, and the potential implications for cysteine oxidations in the pathogenesis of in fibrotic lung disease.

Fifteen years have passed since we published findings in the AJRCMB demonstrating that induction of early response fos/jun proto-oncogenes in rodent tracheal and mesothelial cells correlates with fibrous geometry and pathogenicity of asbestos. Our study was the first to suggest that the aberrant induction of signaling responses by crocidolite asbestos and erionite, a fibrous zeolite mineral associated with the development of malignant mesotheliomas (MMs) in areas of Turkey, led to altered gene expression. New data questioned the widely held belief at that time that the carcinogenic effects of asbestos in the development of lung cancer and MM were due to genotoxic or mutagenic effects. Later studies by our group revealed that proto-oncogene expression and several of the signaling pathways activated by asbestos were redox dependent, explaining why antioxidants and antioxidant enzymes were elevated in lung and pleura after exposure to asbestos and how they alleviated many of the phenotypic and functional effects of asbestos in vitro or after inhalation. Since these original studies, our efforts have expanded to understand the interface between asbestos-induced redox-dependent signal transduction cascades, the relationship between these pathways and cell fate, and the role of asbestos and cell interactions in development of asbestos-associated diseases. Of considerable significance is the fact that the signal transduction pathways activated by asbestos are also important in survival and chemoresistance of MMs and lung cancers. An understanding of the pathogenic features of asbestos fibers and dysregulation of signaling pathways allows strategies for the prevention and therapy of asbestos-related diseases.

The oxidation of protein cysteine residues represents significant posttranslational modifications that impact a wide variety of signal transduction cascades and diverse biological processes. Oxidation of cysteines occurs through reactions with reactive oxygen as well as nitrogen species. These oxidative events can lead to irreversible modifications, such as the formation of sulfonic acids, or manifest as reversible modifications such as the conjugation of glutathione with the cysteine moiety, a process termed S-glutathionylation (also referred to as S-glutathiolation, or protein mixed disulfides). Similarly, S-nitrosothiols can also react with the thiol group in a process known as S-nitrosylation (or S-nitrosation). It is the latter two events that have recently come to the forefront of cellular biology through their ability to reversibly impact numerous cellular processes. Herein we describe two protocols for the detection of S-glutathionylated or S-nitrosylated proteins in situ. The protocol for the detection of S-glutathionylated proteins relies on the catalytic specificity of glutaredoxin-1 for the reduction of S-glutathionylated proteins. The protocol for the detection of S-nitrosylated proteins represents a modification of the previously described biotin switch protocol, which relies on ascorbate in the presence of chelators to decompose S-nitrosylated proteins. These techniques can be applied in situ to elucidate which compartments in tissues are affected in diseased states whose underlying pathologies are thought to represent a redox imbalance., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

NF-kappaB activation in the airway epithelium has been established as a critical pathway in ovalbumin (Ova)-induced airway inflammation in BALB/c mice (Poynter ME, Cloots R, van Woerkom T, Butnor KJ, Vacek P, Taatjes DJ, Irvin CG, Janssen-Heininger YM. J Immunol 173: 7003-7009, 2004). BALB/c mice are susceptible to the development of allergic airway disease, whereas other strains of mice, such as C57BL/6, are considered more resistant. The goal of the present study was to determine the proximal signals required for NF-kappaB activation in the airway epithelium in allergic airway disease and to unravel whether these signals are strain-dependent. Our previous studies, conducted in the BALB/c mouse background, demonstrated that transgenic mice expressing a dominant-negative version of IkappaBalpha in the airway epithelium (CC10-IkappaBalpha(SR)) were protected from Ova-induced inflammation. In contrast to these earlier observations, we demonstrate here that CC10-IkappaBalpha(SR) transgenic mice on the C57BL/6 background were not protected from Ova-induced allergic airway inflammation. Consistent with this finding, Ova-induced nuclear localization of the RelA subunit of NF-kappaB was not observed in C57BL/6 mice, in contrast to the marked nuclear presence of RelA in BALB/c mice. Evaluation of cytokine profiles in bronchoalveolar lavage demonstrated elevated expression of TNF-alpha in BALB/c mice compared with C57BL/6 mice after an acute challenge with Ova. Finally, neutralization of TNF-alpha by a blocking antibody prevented nuclear localization of RelA in BALB/c mice after Ova challenge. These data suggest that the mechanism of response of the airway epithelium of immunized C57BL/6 mice to antigen challenge is fundamentally different from that of immunized BALB/c mice and highlight the potential importance of TNF-alpha in regulating epithelial NF-kappaB activation in allergic airway disease.

Protein S-glutathionylation (PSSG) is a posttranslational modification that involves the conjugation of the small antioxidant molecule glutathione to cysteine residues and is emerging as a critical mechanism of redox-based signaling. PSSG levels increase under conditions of oxidative stress and are controlled by glutaredoxins (Grx) that, under physiological conditions, preferentially deglutathionylate cysteines and restore sulfhydryls. Both the occurrence and distribution of PSSG in tissues is unknown because of the labile nature of this oxidative event and the lack of specific reagents. The goal of this study was to establish and validate a protocol that enables detection of PSSG in situ, using the property of Grx to deglutathionylate cysteines. Using Grx1-catalyzed cysteine derivatization, we evaluated PSSG content in mice subjected to various models of lung injury and fibrosis. In control mice, PSSG was detectable primarily in the airway epithelium and alveolar macrophages. Exposure of mice to NO(2) resulted in enhanced PSSG levels in parenchymal regions, while exposure to O(2) resulted in minor detectable changes. Finally, bleomycin exposure resulted in marked increases in PSSG reactivity both in the bronchial epithelium as well as in parenchymal regions. Taken together, these findings demonstrate that Grx1-based cysteine derivatization is a powerful technique to specifically detect patterns of PSSG expression in lungs, and will enable investigations into regional changes in PSSG content in a variety of diseases.