BACKGROUND:Obesity is associated with poorly controlled asthma. One reason for this may be the increased mitochondrial oxidative stress in obese asthma. Indeed, targeting oxidative stress reduces airway reactivity in animal models. The purpose of this trial was to investigate the potential efficacy of the mitochondrial-targeted antioxidant MitoQuinone (MitoQ) for the treatment of poorly controlled asthma in people with obesity. METHODS:A twelve week, randomized, double-masked, placebo-controlled trial of 40 mg MitoQ versus placebo in adults with obesity and poorly controlled asthma was performed. The primary outcome was change in airway reactivity, with secondary outcomes of change in asthma control, quality of life, and lung function. RESULTS:Twenty participants were randomized to MitoQ, 18 to placebo. After 12 weeks, there was no difference in change in airway reactivity to methacholine (provocative dose producing a 20% decrease in FEV1) between the group assigned to MitoQ (median change = 0.0 μg [IQR -25.5 to 1.4]) or placebo (change = -0.8 μg [IQR -57 to 0.0]), p = 0.81. There was no difference in the change in Asthma Control Test score (MitoQ 1 [IQR -1 to 2], placebo 1 [-2 to 2]), p = 0.62, or Marks Asthma Quality of Life Score (MitoQ -0.08 [IQR -0.35 to 0.10], placebo -0.05 [-0.30 to 0.15]), p = 0.80. CONCLUSION:MitoQ was well tolerated but did not improve airway reactivity, asthma control, or lung function. Targeting oxidative stress in people with obesity and poorly controlled asthma with MitoQ is unlikely to be efficacious for the treatment of asthma.

Fibrotic lung diseases such as idiopathic pulmonary fibrosis (IPF) are caused by various forms of environmental injury and involve reciprocal interactions between activated (myo)fibroblasts and recruited monocyte-derived macrophages (MoMacs), collectively leading to progressive tissue remodeling. Extending previous findings implicating redox-based mechanisms in IPF pathogenesis, we here highlight the involvement of the NADPH oxidase (NOX) homolog DUOX1 in pulmonary fibrosis, based on observed increases in DUOX1 expression within fibrotic regions of IPF lung tissues or lung tissues from mice with experimentally induced pulmonary fibrosis, localized primarily to (myo)fibroblasts and recruited MoMacs. Building on a previous report implicating DUOX1 in myofibroblast activation, conditional DUOX1 ablation from myeloid cells (including macrophages) using LysM-Cre was found to dramatically attenuate fibrosis, highlighted by impaired MoMac recruitment, reduced collagen production, and improved oxygen saturation. A macrophage-intrinsic role of DUOX1 was further supported by its observed contribution to in vitro migration of bone marrow-derived macrophages (BMDM) and to profibrotic BMDM activation, the latter including production of several epidermal growth factor receptor ligands involved in macrophage-fibroblast cross-talk. Finally, these DUOX1-mediated actions were associated with oxidative activation of Src kinase via cysteine oxidation, and were inhibitable by saracatinib, a clinically used Src inhibitor. Collectively, our findings highlight the involvement of DUOX1 in macrophage-(myo)fibroblast cross-talk in the pathogenesis and/or progression of pulmonary fibrosis, implicating it as a putatively novel therapeutically targetable feature of this devastating disease.

Most people with severe asthma have obesity. Metabolic dysfunction, often associated with obesity, is particularly associated with severe asthma. Mechanisms linking metabolic dysfunction with asthma, and whether improving metabolic function can affect asthma, are not known. The endocannabinoid system plays a significant role in metabolism; inhibition of cannabinoid receptor 1 (CB1R) induces weight loss and improves serum lipid profiles. We used a CB1R inverse agonist, INV-202, in a mouse model of obese asthma and investigated changes in weight, inflammation, airway reactivity, and surfactant lipids. Mice were fed low or high-fat diets (LFD, HFD), and house dust mite (HDM) extract was delivered intranasally to induce allergic airway inflammation. Mice received INV-202 by oral gavage. Airway hyperresponsiveness was measured by FlexiVent, and lung tissue cytokines were measured by ELISA. Leukocytes and lipids in the bronchoalveolar lavage fluid (BALF) were analyzed by flow cytometry and mass spectroscopy, respectively. LFD and HFD mice lost an average of 11% and 27% of their body weight, respectively. LFD mice had a 33% decrease in CCL20 in lung tissue and a 55% decrease in neutrophils in BALF. LFD and HFD mice had improvements in airway hyperresponsiveness, particularly as measured by reduced elastance. Phosphatidylglycerol in BALF increased with INV-202, which significantly correlated with compliance in LFD mice. This study supports a significant contribution of metabolic factors related to the endocannabinoid system in lung compliance and airway reactivity, in part through effects on surfactant lipid composition, and demonstrates the potential of CB1R inverse agonists to treat obese asthma.NEW & NOTEWORTHY Inhibition of the cannabinoid receptor 1, through a pharmacological inverse agonist, not only induces weight loss in a mouse model of obese asthma but also reduces airway hyperresponsiveness, particularly through decreasing elastance/increasing compliance.

Rationale: Pro-inflammatory cytokines released during influenza infection is one of the major causes of lung injury. Detection of virus by pattern recognition receptors (PRRs) like toll like receptors (TLRs), and endoplasmic reticulum (ER) stress contributes to exacerbated release of cytokines following infection. Influenza A virus (IAV) – induced ER stress occurs due to rapid replication of viral RNPs and increased translational burden on the ER causing accumulation of unfolded proteins and activation of an adaptive response called the unfolded protein response (UPR). PKR-like kinase (PERK), one of the members of the UPR pathway plays a direct role in decreasing the translational burden. Phosphorylation of PERK (P-PERK) during UPR activation and its involvement in pro-inflammatory cytokine release is well established, but only in various models of sterile infection. Although a decrease in cytokine production was observed when P-PERK was inhibited in the above-mentioned studies, it is not clear if it was under transcriptional or translational regulation by P-PERK. Our objective is to determine the role of P-PERK in pro-inflammatory cytokine release during IAV infection. In addition, we will also determine the mode of regulation by which these cytokines are controlled and whether P-PERK can be targeted to prevent cytokine storm during IAV infection. Methods: Cell culture experiments to study ER-stress and cytokine release were studied in human bronchial epithelial cells (HBEC3KT) infected with IAV. Activation of UPR and PERK in IAV infected cells was confirmed by Western blots. Pro-inflammatory cytokines released under the control of P-PERK were identified using an inhibitor, GSK2606414 (PERKi) that inhibits PERK activation. Transcriptional and translational regulation was studied using qPCR and polysome profiling respectively. C57BL/6 mice were infected with 2000 EIU of IAV/PR8 intranasally and treated with varying concentrations of PERKi. Results: We found that PERK was phosphorylated 24 hours post infection and sustained until 48 hours post infection in HBEC3KT cells. These cells were treated with 5 M PERKi during infection showed significant decrease in PERK phosphorylation. Pro-inflammatory cytokines like IL6, IL8, CCL4 and CCL20 were significantly decreased in the supernatants following PERKi treatment. Both IL6 and CCL4 exhibited a decrease in translational efficiency following PERKi treatment. Conclusion: These results suggest PERK's involvement in overt cytokine release during IAV infection.

RATIONALE: During periods of stress or high protein synthesis, unfolded and misfolded proteins accumulate in the lumen of the endoplasmic reticulum (ER). In response, a series of pathways called the unfolded protein response (UPR) are activated to process unfolded proteins. Elevated ER stress and UPR signaling has been implicated in allergic asthma and is regulated by chaperone protein BiP, which, under homeostatic conditions, is bound to three UPR transducers: IRE1, PERK, and ATF6. UPR activation occurs when BiP dissociates from these transducers, however, the mechanism for dissociation in allergic airway disease is unknown. Mass spectrometry data from our allergic airway disease mouse model revealed a novel sulfenylation modification of BiP which was associated with enhanced UPR activation, and attenuation of mitochondrial ROS decreased BiP sulfenylation in allergen challenged mice, including decrease in inflammation and AHR. Yeast studies have shown that sulfenylation of the lone cysteine residue results in increased chaperone capability of BiP, however, the effect sulfenylation on BiP dissociation from the UPR transducers in mammalian system is unknown. Additionally, mammalian BiP has two cysteines and the effect of sulfenyation remains unknown. We hypothesize that sulfenylation of BiP results in BiP/IRE1 dissociation and elevated IRE1 activity which could provide a potential target for mitigating ER stress in allergic asthma. METHODS: HBEC3KT cells were dosed with 25ug/mL of HDM. Protein and RNA lysates were collected 6, 24, and 48hrs after HDM dose. Cells were transfected with 750ng of mutant BiP plasmids 24hrs prior to HDM treatment. UPR expression was measured using western blot and RT-qPCR. BiP sulfenylation was measured using DCP-Bio1 and neutravidin pulldown. Interaction between BiP and IRE1 was evaluated through immunoprecipitation. Mice were sensitized intranasally with 25ug HDM on days 1 and 7 then challenged on days 14-18. MitoQ was administered via intraperitoneal injection on days 17-19. Cytokine analysis was evaluated via ELISA.RESULTS: BiP sulfenylation is associated with increased activity of IRE1 and decreased association of BiP with IRE1 in response to HDM. BiP C41a mutant overexpression resulted in increased association with IRE1 and decreased IRE1 signaling. BiP C420a mutant overexpression resulted in the formation of a truncated product implying that C420 is crucial for BiP structure. In mice, MitoQ treatment decreased BiP sulfenylation, IRE1 activation, inflammation, and AHR. CONCLUSIONS: These findings provide evidence for a novel oxidative post translational modification involved in activation of the IRE1 pathway and can provide potential targets for treatment of allergic airway disease.

Background and PurposeFibrotic lung remodelling after a respiratory viral infection represents a debilitating clinical sequela. Studying or managing viral-fibrotic sequela remains challenging, due to limited therapeutic options and lack of understanding of mechanisms. This study determined whether protein disulfide isomerase A3 (PDIA3) and secreted phosphoprotein 1 (SPP1), which are associated with pulmonary fibrosis, can promote influenza-induced lung fibrotic remodelling and whether inhibition of PDIA3 or SPP1 can resolve viral-mediated fibrotic remodelling.Experimental ApproachA retrospective analysis of TriNetX data sets was conducted. Serum from healthy controls and influenza A virus (IAV)-infected patients was analysed. An inhibitor of PDIA3, punicalagin, and a neutralizing antibody for SPP1 were administered in mice. Macrophage cells treated with macrophage colony-stimulating factor (M-CSF) were used as a cell culture model.Key ResultsThe TriNetX data set showed an increase in lung fibrosis and decline in lung function in flu-infected acute respiratory distress syndrome (ARDS) patients compared with non-ARDS patients. Serum samples revealed a significant increase in SPP1 and PDIA3 in influenza-infected patients. Lung PDIA3 and SPP1 expression increased following viral infection in mouse models. Punicalagin administration 2 weeks after IAV infection in mice caused a significant decrease in lung fibrosis and improved oxygen saturation. Administration of neutralizing SPP1 antibody decreased lung fibrosis. Inhibition of PDIA3 decreased SPP1secretion from macrophages, in association with diminished disulfide bonds in SPP1.Conclusion and ImplicationsThe PDIA3-SPP1 axis promotes post-influenza lung fibrosis in mice and that pharmacological inhibition of PDIA3 or SPP1 can treat virus-induced lung fibrotic sequela. image

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 COL1A1 (collagen 1A1) S-glutathionylation (COL1A1-SSG) in lung tissues from subjects with IPF compared with control subjects in association with increases in ERO1A (endoplasmic reticulum [ER] oxidoreductin 1) and enhanced oxidation of ER-localized PRDX4 (peroxiredoxin 4), reflecting an increased oxidative environment of the ER. Human lung fibroblasts exposed to TGFB1 (transforming growth factor-β1) show increased secretion of COL1A1-SSG. Pharmacologic inhibition of ERO1A diminished the oxidation of PRDX4, attenuated COL1A1-SSG and total COL1A1 concentrations, and dampened fibroblast activation. Absence of Glrx enhanced COL1A1-SSG and overall COL1A1 secretion and promoted the activation of mechanosensing pathways. Remarkably, COL1A1-SSG resulted in marked resistance to collagenase degradation. Compared with COL1, lung fibroblasts plated on COL1-SSG proliferated more rapidly and increased the 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 because of increased resistance to collagenase-mediated degradation and fibroblast activation.

Obesity is a risk factor for severe influenza, and asthma exacerbations caused by respiratory viral infections. We investigated mechanisms that increase the severity of airway disease related to influenza in obesity using cells derived from obese and lean individuals, and in vitro and in vivo models. Primary human nasal epithelial cells (pHNECs) derived from obese compared with lean individuals developed increased inflammation and injury in response to influenza A virus (IAV). Obese mice infected with influenza developed increased airway inflammation, lung injury and elastance, but had a decreased interferon response, compared with lean mice. Lung arachidonic acid (AA) levels increased in obese mice infected with IAV; arachidonic acid increased inflammatory cytokines and injury markers in response to IAV in human bronchial epithelial (HBE) cells. Obesity in mice, and AA in HBE cells, increased activation of p38 MAPK signaling following IAV infection; inhibiting this pathway attenuated inflammation, injury and tissue elastance responses, and improved survival. In summary, obesity increases disease severity in response to influenza infection through activation of the p38 MAPK pathway in response to altered arachidonic acid signaling.

Protein-S-glutathionylation is a post-translational modification involving the conjugation of glutathione to protein thiols, which can modulate the activity and structure of key cellular proteins. Glutaredoxins (GLRX) are oxidoreductases that regulate this process by performing deglutathionylation. However, GLRX has five cysteines that are potentially vulnerable to oxidative modification, which is associated with GLRX aggregation and loss of activity. To date, GLRX cysteines that are oxidatively modified and their relative susceptibilities remain unknown. We utilized molecular modeling approaches, activity assays using recombinant GLRX, coupled with site-directed mutagenesis of each cysteine both individually and in combination to address the oxidizibility of GLRX cysteines. These approaches reveal that C8 and C83 are targets for S-glutathionylation and oxidation by hydrogen peroxide in vitro. In silico modeling and experimental validation confirm a prominent role of C8 for dimer formation and aggregation. Lastly, combinatorial mutation of C8, C26, and C83 results in increased activity of GLRX and resistance to oxidative inactivation and aggregation. Results from these integrated computational and experimental studies provide insights into the relative oxidizability of GLRX's cysteines and have implications for the use of GLRX as a therapeutic in settings of dysregulated protein glutathionylation.

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.

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.