ER Oxidative Stress Promotes Glutathione-Dependent Oxidation of Collagen-1A1 and Promotes Lung Fibroblast Activation.

Changes in the oxidative (redox) environment accompany idiopathic pulmonary fibrosis (IPF). S-glutathionylation of reactive protein cysteines is a post-translational event that transduces oxidant signals into biological responses. We recently demonstrated that increases in S-glutathionylation promote pulmonary fibrosis, which was mitigated by the deglutathionylating enzyme glutaredoxin (GLRX). However, the protein targets of S-glutathionylation that promote fibrogenesis remain unknown. In the present study we addressed whether the extracellular matrix is a target for S-glutathionylation. We discovered increases in collagen 1A1 S-glutathionylation (COL1A1-SSG) in lung tissues from IPF subjects compared to control subjects in association with increases in ER oxidoreductin 1 (ERO1A) and enhanced oxidation of ER-localized peroxiredoxin 4 (PRDX4) reflecting an increased oxidative environment of the endoplasmic reticulum (ER). Human lung fibroblasts exposed to transforming growth factor beta 1 (TGFB1) show increased secretion of COL1A1-SSG. Pharmacologic inhibition of ERO1A diminished oxidation of PRDX4, attenuated COL1A1-SSG and total COL1A1 levels and dampened fibroblast activation. Absence of Glrx enhanced COL1A1-SSG and overall COL1A1 secretion and promoted activation of mechanosensing pathways. Remarkably, COL1A1-SSG resulted in marked resistance to collagenase degradation. Compared to COL1, lung fibroblasts plated on COL1-SSG proliferated more rapidly, and increased expression of genes encoding extracellular matrix crosslinking enzymes and genes linked to mechanosensing pathways. Overall, these findings suggest that glutathione-dependent oxidation of COL1A1 occurs in settings of IPF in association with enhanced ER oxidative stress and may promote fibrotic remodeling due to increased resistance to collagenase-mediated degradation and fibroblast activation.

Bariatric surgery decreases the capacity of plasma from obese asthmatic subjects to augment airway epithelial cell proinflammatory cytokine production.

Obesity is a risk factor for asthma. Individuals with asthma and obesity often have poor asthma control and do not respond as well to therapies such as inhaled corticosteroids and long-acting bronchodilators. Weight loss improves asthma control, with a 5%-10% loss in body mass necessary and sufficient to lead to clinically relevant improvements. Preclinical studies have demonstrated the pathogenic contribution of adipocytes from obese mice to the augmented production of proinflammatory cytokines from airway epithelial cells and the salutary effects of diet-induced weight loss to decrease these consequences. However, the effects of adipocyte-derived products on airway epithelial function in human obesity remain incompletely understood. We utilized samples collected from a 12-mo longitudinal study of subjects with obesity undergoing weight loss (bariatric) surgery including controls without asthma and subjects with allergic and nonallergic obese asthma. Visceral adipose tissue (VAT) samples were collected during bariatric surgery and from recruited normal weight controls without asthma undergoing elective abdominal surgery. Human bronchial epithelial (HBEC3-KT) cells were exposed to plasma or conditioned media from cultured VAT adipocytes with or without agonists. Human bronchial smooth muscle (HBSM) cells were similarly exposed to adipocyte-conditioned media. Proinflammatory cytokines were augmented in supernatants from HBEC3-KT cells exposed to plasma as compared with subsequent visits. Whereas exposure to obese adipocyte-conditioned media induced proinflammatory responses, there were no differences between groups in both HBEC3-KT and HBSM cells. These data show that bariatric surgery and subsequent weight loss beneficially change the circulating factors that augment human airway epithelial and bronchial smooth muscle cell proinflammatory responses.NEW & NOTEWORTHY This longitudinal study following subjects with asthma and obesity reveals that weight loss following bariatric surgery decreases the capacity for plasma to augment proinflammatory cytokine secretion by human bronchial epithelial cells, implicating that circulating but not adipocyte-derived factors are important modulators in obese asthma.

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.

Mitoquinone mesylate attenuates pathological features of lean and obese allergic asthma in mice.

Obesity is associated with severe, difficult-to-control asthma, and increased airway oxidative stress. Mitochondrial reactive oxygen species (mROS) are an important source of oxidative stress in asthma, leading us to hypothesize that targeting mROS in obese allergic asthma might be an effective treatment. Using a mouse model of house dust mite (HDM)-induced allergic airway disease in mice fed a low- (LFD) or high-fat diet (HFD), and the mitochondrial antioxidant MitoQuinone (MitoQ), we investigated the effects of obesity and ROS on HDM-induced airway inflammation, remodeling, and airway hyperresponsiveness (AHR). Obese allergic mice showed increased lung tissue eotaxin, airway tissue eosinophilia, and AHR compared with lean allergic mice. MitoQ reduced airway inflammation, remodeling, and hyperreactivity in both lean and obese allergic mice, and tissue eosinophilia in obese-allergic mice. Similar effects were observed with decyl triphosphonium (dTPP+), the hydrophobic cationic moiety of MitoQ lacking ubiquinone. HDM-induced oxidative sulfenylation of proteins was increased particularly in HFD mice. Although only MitoQ reduced sulfenylation of proteins involved in protein folding in the endoplasmic reticulum (ER), ER stress was attenuated by both MitoQ and dTPP+ suggesting the anti-allergic effects of MitoQ are mediated in part by effects of its hydrophobic dTPP+ moiety reducing ER stress. In summary, oxidative signaling is an important mediator of allergic airway disease. MitoQ, likely through reducing protein oxidation and affecting the UPR pathway, might be effective for the treatment of asthma and specific features of obese asthma.

Oxidation of peroxiredoxin-4 induces oligomerization and promotes interaction with proteins governing protein folding and endoplasmic reticulum stress.

Peroxiredoxins (PRDXs) catalyze the reduction of hydrogen peroxide (H2O2). PRDX4 is the only peroxiredoxin located within the endoplasmic reticulum (ER) and is the most highly expressed H2O2 scavenger in the ER. PRDX4 has emerged as an important player in numerous diseases, such as fibrosis and metabolic syndromes, and its overoxidation is a potential indicator of ER redox stress. It is unclear how overoxidation of PRDX4 governs its oligomerization state and interacting partners. Herein, we addressed these questions via nonreducing Western blots, mass spectrometry, and site-directed mutagenesis. We report that the oxidation of PRDX4 in lung epithelial cells treated with tertbutyl hydroperoxide caused a shift of PRDX4 from monomer/dimer to high molecular weight (HMW) species, which contain PRDX4 modified with sulfonic acid residues (PRDX4-SO3), as well as of a complement of ER-associated proteins, including protein disulfide isomerases important in protein folding, thioredoxin domain-containing protein 5, and heat shock protein A5, a key regulator of the ER stress response. Mutation of any of the four cysteines in PRDX4 altered the HMW species in response to tertbutyl hydroperoxide as well as the secretion of PRDX4. We also demonstrate that the expression of ER oxidoreductase 1 alpha, which generates H2O2 in the ER, increased PRDX4 HMW formation and secretion. These results suggest a link between SO3 modification in the formation of HMW PRDX4 complexes in cells, whereas the association of key regulators of ER homeostasis with HMW oxidized PRDX4 point to a putative role of PRDX4 in regulating ER stress responses.

Inhibition of PDIA3 in club cells attenuates osteopontin production and lung fibrosis.

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.

Glutathionylation chemistry promotes interleukin-1 beta-mediated glycolytic reprogramming and pro-inflammatory signaling in lung epithelial cells.

Glycolysis is a well-known process by which metabolically active cells, such as tumor or immune cells meet their high metabolic demands. Previously, our laboratory has demonstrated that in airway epithelial cells, the pleiotropic cytokine, interleukin-1 beta (IL1B) induces glycolysis and that this contributes to allergic airway inflammation and remodeling. Activation of glycolysis is known to increase NADPH reducing equivalents generated from the pentose phosphate pathway, linking metabolic reprogramming with redox homeostasis. In addition, numerous glycolytic enzymes are known to be redox regulated. However, whether and how redox chemistry regulates metabolic reprogramming more generally remains unclear. In this study, we employed a multi-omics approach in primary mouse airway basal cells to evaluate the role of protein redox biochemistry, specifically protein glutathionylation, in mediating metabolic reprogramming. Our findings demonstrate that IL1B induces glutathionylation of multiple proteins involved in metabolic regulation, notably in the glycolysis pathway. Cells lacking Glutaredoxin-1 (Glrx), the enzyme responsible for reversing glutathionylation, show modulation of multiple metabolic pathways including an enhanced IL1B-induced glycolytic response. This was accompanied by increased secretion of thymic stromal lymphopoietin (TSLP), a cytokine important in asthma pathogenesis. Targeted inhibition of glycolysis prevented TSLP release, confirming the functional relevance of enhanced glycolysis in cells stimulated with IL1B. Collectively, data herein point to an intriguing link between glutathionylation chemistry and glycolytic reprogramming in epithelial cells and suggest that glutathionylation chemistry may represent a therapeutic target in pulmonary pathologies with perturbations in the glycolysis pathway.

Pyruvate Kinase M2 Promotes Expression of Proinflammatory Mediators in House Dust Mite-Induced Allergic Airways Disease.

Asthma is a chronic disorder characterized by inflammation, mucus metaplasia, airway remodeling, and hyperresponsiveness. We recently showed that IL-1-induced glycolytic reprogramming contributes to allergic airway disease using a murine house dust mite model. Moreover, levels of pyruvate kinase M2 (PKM2) were increased in this model as well as in nasal epithelial cells from asthmatics as compared with healthy controls. Although the tetramer form of PKM2 converts phosphoenolpyruvate to pyruvate, the dimeric form of PKM2 has alternative, nonglycolysis functions as a transcriptional coactivator to enhance the transcription of several proinflammatory cytokines. In the current study, we examined the impact of PKM2 on the pathogenesis of house dust mite-induced allergic airways disease in C57BL/6NJ mice. We report, in this study, that activation of PKM2, using the small molecule activator, TEPP46, augmented PKM activity in lung tissues and attenuated airway eosinophils, mucus metaplasia, and subepithelial collagen. TEPP46 attenuated IL-1ß-mediated airway inflammation and expression of proinflammatory mediators. Exposure to TEPP46 strongly decreased the IL-1ß-mediated increases in thymic stromal lymphopoietin (TSLP) and GM-CSF in primary tracheal epithelial cells isolated from C57BL/6NJ mice. We also demonstrate that IL-1ß-mediated increases in nuclear phospho-STAT3 were decreased by TEPP46. Finally, STAT3 inhibition attenuated the IL-1ß-induced release of TSLP and GM-CSF, suggesting that the ability of PKM2 to phosphorylate STAT3 contributes to its proinflammatory function. Collectively, these results demonstrate that the glycolysis-inactive form of PKM2 plays a crucial role in the pathogenesis of allergic airways disease by increasing IL-1ß-induced proinflammatory signaling, in part, through phosphorylation of STAT3.

Protein Disulfide Isomerase A3 Regulates Influenza Neuraminidase Activity and Influenza Burden in the Lung.

Influenza (IAV) neuraminidase (NA) is a glycoprotein required for the viral exit from the cell. NA requires disulfide bonds for proper function. We have recently demonstrated that protein disulfide isomerase (PDI)A3 is required for oxidative folding of IAV hemagglutinin (HA), and viral propagation. However, it not known whether PDIs are required for NA maturation or if these interactions represent a putative target for the treatment of influenza infection. We sought to determine whether PDIA3 is required for disulfide bonds of NA, its activity, and propagation of the virus. Requirement of disulfides for NA oligomerization and activity were determined using biotin switch and redox assays in WT and PDIA3-/- in A549 cells. A PDI specific inhibitor (LOC14) was utilized to determine the requirement of PDIs in NA activity, IAV burden, and inflammatory response in A549 and primary mouse tracheal epithelial cells. Mice were treated with the inhibitor LOC14 and subsequently examined for IAV burden, NA activity, cytokine, and immune response. IAV-NA interacts with PDIA3 and this interaction is required for NA activity. PDIA3 ablation or inhibition decreased NA activity, viral burden, and inflammatory response in lung epithelial cells. LOC14 treatment significantly attenuated the influenza-induced inflammatory response in mice including the overall viral burden. These results provide evidence for PDIA3 inhibition suppressing NA activity, potentially providing a novel platform for host-targeted antiviral therapies.

Dysregulation of Pyruvate Kinase M2 Promotes Inflammation in a Mouse Model of Obese Allergic Asthma.

Obesity is a risk factor for the development of asthma and represents a difficult-to-treat disease phenotype. Aerobic glycolysis is emerging as a key feature of asthma, and changes in glucose metabolism are linked to leukocyte activation and adaptation to oxidative stress. Dysregulation of PKM2 (pyruvate kinase M2), the enzyme that catalyzes the last step of glycolysis, contributes to house dust mite (HDM)-induced airway inflammation and remodeling in lean mice. It remains unclear whether glycolytic reprogramming and dysregulation of PKM2 also contribute to obese asthma. The goal of the present study was to elucidate the functional role of PKM2 in a murine model of obese allergic asthma. We evaluated the small molecule activator of PKM2, TEPP46, and assessed the role of PKM2 using conditional ablation of the Pkm2 allele from airway epithelial cells. In obese C57BL/6NJ mice, parameters indicative of glycolytic reprogramming remained unchanged in the absence of stimulation with HDM. Obese mice that were subjected to HDM showed evidence of glycolytic reprogramming, and treatment with TEPP46 diminished airway inflammation, whereas parameters of airway remodeling were unaffected. Epithelial ablation of Pkm2 decreased central airway resistance in both lean and obese allergic mice in addition to decreasing inflammatory cytokines in the lung tissue. Lastly, we highlight a novel role for PKM2 in the regulation of glutathione-dependent protein oxidation in the lung tissue of obese allergic mice via a putative IFN-γ-glutaredoxin1 pathway. Overall, targeting metabolism and protein oxidation may be a novel treatment strategy for obese allergic asthma.

Lung epithelial endoplasmic reticulum and mitochondrial 3D ultrastructure: a new frontier in lung diseases.

It has long been appreciated that the endoplasmic reticulum (ER) and mitochondria, organelles important for regular cell function and survival, also play key roles in pathogenesis of various lung diseases, including asthma, fibrosis, and infections. Alterations in processes regulated within these organelles, including but not limited to protein folding in the ER and oxidative phosphorylation in the mitochondria, are important in disease pathogenesis. In recent years it has also become increasingly apparent that organelle structure dictates function. It is now clear that organelles must maintain precise organization and localization for proper function. Newer microscopy capabilities have allowed the scientific community to reveal, via 3D imaging, that the structure of these organelles and their interactions with each other are a main component of regulating function and, therefore, effects on the disease state. In this review, we will examine how 3D imaging through techniques could allow advancements in knowledge of how the ER and mitochondria function and the roles they may play in lung epithelia in progression of lung disease.

DRP1-Mediated Mitochondrial Fission Regulates Lung Epithelial Response to Allergen.

Mitochondria regulate a myriad of cellular functions. Dysregulation of mitochondrial control within airway epithelial cells has been implicated in the pro-inflammatory response to allergens in asthma patients. Because of their multifaceted nature, mitochondrial structure must be tightly regulated through fission and fusion. Dynamin Related Protein 1 (DRP1) is a key driver of mitochondrial fission. During allergic asthma, airway epithelial mitochondria appear smaller and structurally altered. The role of DRP1-mediated mitochondrial fission, however, has not been fully elucidated in epithelial response to allergens. We used a Human Bronchial Epithelial Cell line (HBECs), primary Mouse Tracheal Epithelial Cells (MTECs), and conditional DRP1 ablation in lung epithelial cells to investigate the impact of mitochondrial fission on the pro-inflammatory response to house dust mite (HDM) in vitro and in vivo. Our data suggest that, following HDM challenge, mitochondrial fission is rapidly upregulated in airway epithelial cells and precedes production of pro-inflammatory cytokines and chemokines. Further, deletion of Drp1 in lung epithelial cells leads to decreased fission and enhanced pro-inflammatory signaling in response to HDM in vitro, as well as enhanced airway hyper-responsiveness (AHR), inflammation, differential mucin transcription, and epithelial cell death in vivo. Mitochondrial fission, therefore, regulates the lung epithelial pro-inflammatory response to HDM.

Dysregulation of the glutaredoxin/S-glutathionylation redox axis in lung diseases.

Glutathione is a major redox buffer, reaching millimolar concentrations within cells and high micromolar concentrations in airways. While glutathione has been traditionally known as an antioxidant defense mechanism that protects the lung tissue from oxidative stress, glutathione more recently has become recognized for its ability to become covalently conjugated to reactive cysteines within proteins, a modification known as S-glutathionylation (or S-glutathiolation or protein mixed disulfide). S-glutathionylation has the potential to change the structure and function of the target protein, owing to its size (the addition of three amino acids) and charge (glutamic acid). S-glutathionylation also protects proteins from irreversible oxidation, allowing them to be enzymatically regenerated. Numerous enzymes have been identified to catalyze the glutathionylation/deglutathionylation reactions, including glutathione S-transferases and glutaredoxins. Although protein S-glutathionylation has been implicated in numerous biological processes, S-glutathionylated proteomes have largely remained unknown. In this paper, we focus on the pathways that regulate GSH homeostasis, S-glutathionylated proteins, and glutaredoxins, and we review methods required toward identification of glutathionylated proteomes. Finally, we present the latest findings on the role of glutathionylation/glutaredoxins in various lung diseases: idiopathic pulmonary fibrosis, asthma, and chronic obstructive pulmonary disease.

IL-1/inhibitory κB kinase ε-induced glycolysis augment epithelial effector function and promote allergic airways disease.

BACKGROUND: Emerging studies suggest that enhanced glycolysis accompanies inflammatory responses. Virtually nothing is known about the relevance of glycolysis in patients with allergic asthma. OBJECTIVES: We sought to determine whether glycolysis is altered in patients with allergic asthma and to address its importance in the pathogenesis of allergic asthma. METHODS: We examined alterations in glycolysis in sputum samples from asthmatic patients and primary human nasal cells and used murine models of allergic asthma, as well as primary mouse tracheal epithelial cells, to evaluate the relevance of glycolysis. RESULTS: In a murine model of allergic asthma, glycolysis was induced in the lungs in an IL-1-dependent manner. Furthermore, administration of IL-1ß into the airways stimulated lactate production and expression of glycolytic enzymes, with notable expression of lactate dehydrogenase A occurring in the airway epithelium. Indeed, exposure of mouse tracheal epithelial cells to IL-1ß or IL-1α resulted in increased glycolytic flux, glucose use, expression of glycolysis genes, and lactate production. Enhanced glycolysis was required for IL-1ß- or IL-1α-mediated proinflammatory responses and the stimulatory effects of IL-1ß on house dust mite (HDM)-induced release of thymic stromal lymphopoietin and GM-CSF from tracheal epithelial cells. Inhibitor of κB kinase ε was downstream of HDM or IL-1ß and required for HDM-induced glycolysis and pathogenesis of allergic airways disease. Small interfering RNA ablation of lactate dehydrogenase A attenuated HDM-induced increases in lactate levels and attenuated HDM-induced disease. Primary nasal epithelial cells from asthmatic patients intrinsically produced more lactate compared with cells from healthy subjects. Lactate content was significantly higher in sputum supernatants from asthmatic patients, notably those with greater than 61% neutrophils. A positive correlation was observed between sputum lactate and IL-1ß levels, and lactate content correlated negatively with lung function. CONCLUSIONS: Collectively, these findings demonstrate that IL-1ß/inhibitory κB kinase ε signaling plays an important role in HDM-induced glycolysis and pathogenesis of allergic airways disease.

Skeletal muscle atrophy and dysfunction in breast cancer patients: role for chemotherapy-derived oxidant stress.

How breast cancer and its treatments affect skeletal muscle is not well defined. To address this question, we assessed skeletal muscle structure and protein expression in 13 women who were diagnosed with breast cancer and receiving adjuvant chemotherapy following tumor resection and 12 nondiseased controls. Breast cancer patients showed reduced single-muscle fiber cross-sectional area and fractional content of subsarcolemmal and intermyofibrillar mitochondria. Drugs commonly used in breast cancer patients (doxorubicin and paclitaxel) caused reductions in myosin expression, mitochondrial loss, and increased reactive oxygen species (ROS) production in C2C12 murine myotube cell cultures, supporting a role for chemotherapeutics in the atrophic and mitochondrial phenotypes. Additionally, concurrent treatment of myotubes with the mitochondrial-targeted antioxidant MitoQ prevented chemotherapy-induced myosin depletion, mitochondrial loss, and ROS production. In patients, reduced mitochondrial content and size and increased expression and oxidation of peroxiredoxin 3, a mitochondrial peroxidase, were associated with reduced muscle fiber cross-sectional area. Our results suggest that chemotherapeutics may adversely affect skeletal muscle in patients and that these effects may be driven through effects of these drugs on mitochondrial content and/or ROS production.

Epigenetic and Transcriptomic Regulation of Lung Repair during Recovery from Influenza Infection.

Seasonal and pandemic influenza is a cause of morbidity and mortality worldwide. Most people infected with influenza virus display mild-to-moderate disease phenotypes and recover within a few weeks. Influenza is known to cause persistent alveolitis in animal models; however, little is known about the molecular pathways involved in this phenotype. We challenged C57BL/6 mice with influenza A/PR/8/34 and examined lung pathologic processes and inflammation, as well as transcriptomic and epigenetic changes at 21 to 60 days after infection. Influenza induced persistent parenchymal lung inflammation, alveolar epithelial metaplasia, and epithelial endoplasmic reticulum stress that were evident after the clearance of virus and resolution of morbidity. Influenza infection induced robust changes in the lung transcriptome, including a significant impact on inflammatory and extracellular matrix protein expression. Despite the robust changes in lung gene expression, preceding influenza (21 days) did not exacerbate secondary Staphylococcus aureus infection. Finally, we examined the impact of influenza on miRNA expression in the lung and found an increase in miR-155. miR-155 knockout mice recovered from influenza infection faster than controls and had decreased lung inflammation and endoplasmic reticulum stress. These data illuminate the dynamic molecular changes in the lung in the weeks after influenza infection and characterize the repair process, identifying a novel role for miR-155.

Protein disulfide isomerase-endoplasmic reticulum resident protein 57 regulates allergen-induced airways inflammation, fibrosis, and hyperresponsiveness.

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.

Ablation of Glutaredoxin-1 Modulates House Dust Mite-Induced Allergic Airways Disease in Mice.

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.

Effect of a chemical chaperone, tauroursodeoxycholic acid, on HDM-induced allergic airway disease.

Endoplasmic reticulum (ER) stress-induced unfolded protein response plays a critical role in inflammatory diseases, including allergic airway disease. However, the benefits of inhibiting ER stress in the treatment of allergic airway disease are not well known. Herein, we tested the therapeutic potential of a chemical chaperone, tauroursodeoxycholic acid (TUDCA), in combating allergic asthma, using a mouse model of house dust mite (HDM)-induced allergic airway disease. TUDCA was administered during the HDM-challenge phase (preventive regimen), after the HDM-challenge phase (therapeutic regimen), or therapeutically during a subsequent HDM rechallenge (rechallenge regimen). In the preventive regimen, TUDCA significantly decreased HDM-induced inflammation, markers of ER stress, airway hyperresponsiveness (AHR), and fibrosis. Similarly, in the therapeutic regimen, TUDCA administration efficiently decreased HDM-induced airway inflammation, mucus metaplasia, ER stress markers, and AHR, but not airway remodeling. Interestingly, TUDCA administered therapeutically in the HDM rechallenge regimen markedly attenuated HDM-induced airway inflammation, mucus metaplasia, ER stress markers, methacholine-induced AHR, and airway fibrotic remodeling. These results indicate that the inhibition of ER stress in the lungs through the administration of chemical chaperones could be a valuable strategy in the treatment of allergic airway diseases.

Epithelial NF-κB orchestrates house dust mite-induced airway inflammation, hyperresponsiveness, and fibrotic remodeling.

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.