SARS-CoV-2 Mac1 is required for IFN antagonism and efficient virus replication in cell culture and in mice.

Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in these drug targets is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein encoded as a small domain at the N terminus of nonstructural protein 3. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions (ΔMac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV ΔMac1 BACs, SARS-CoV-2 ΔMac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 ΔMac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, ΔMac1 was quickly cleared causing minimal pathology without any morbidity. ΔMac1 SARS-CoV-2 induced increased levels of interferon (IFN) and IFN-stimulated gene expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. ΔMac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target.

E-cigarette aerosols of propylene glycol impair BK channel activity and parameters of mucociliary function.

Propylene glycol (PG) is a common delivery vehicle for nicotine and flavorings in e-cigarette (e-cig) liquids and is largely considered safe for ingestion. However, little is known about its effects as an e-cig aerosol on the airway. Here, we investigated whether pure PG e-cig aerosols in realistic daily amounts impact parameters of mucociliary function and airway inflammation in a large animal model (sheep) in vivo and primary human bronchial epithelial cells (HBECs) in vitro. Five-day exposure of sheep to e-cig aerosols of 100% PG increased mucus concentrations (% mucus solids) of tracheal secretions. PG e-cig aerosols further increased the activity of matrix metalloproteinase-9 (MMP-9) in tracheal secretions. In vitro exposure of HBECs to e-cig aerosols of 100% PG decreased ciliary beating and increased mucus concentrations. PG e-cig aerosols further reduced the activity of large conductance, Ca2+-activated, and voltage-dependent K+ (BK) channels. We show here for the first time that PG can be metabolized to methylglyoxal (MGO) in airway epithelia. PG e-cig aerosols increased levels of MGO and MGO alone reduced BK activity. Patch-clamp experiments suggest that MGO can disrupt the interaction between the major pore-forming BK subunit human Slo1 (hSlo1) and the gamma regulatory subunit LRRC26. PG exposures also caused a significant increase in mRNA expression levels of MMP9 and interleukin 1 beta (IL1B). Taken together, these data show that PG e-cig aerosols cause mucus hyperconcentration in sheep in vivo and HBECs in vitro, likely by disrupting the function of BK channels important for airway hydration.

Nebulized Menthol Impairs Mucociliary Clearance via TRPM8 and MUC5AC/MUC5B in Primary Airway Epithelial Cells.

Flavorings enhance the palatability of e-cigarettes (e-cigs), with menthol remaining a popular choice among e-cig users. Menthol flavor remains one of the only flavors approved by the United States FDA for use in commercially available, pod-based e-cigs. However, the safety of inhaled menthol at the high concentrations used in e-cigs remains unclear. Here, we tested the effects of menthol on parameters of mucociliary clearance (MCC) in air-liquid interface (ALI) cultures of primary airway epithelial cells. ALI cultures treated with basolateral menthol (1 mM) showed a significant decrease in ciliary beat frequency (CBF) and airway surface liquid (ASL) volumes after 24 h. Menthol nebulized onto the surface of ALI cultures similarly reduced CBF and increased mucus concentrations, resulting in decreased rates of mucociliary transport. Nebulized menthol further increased the expression of mucin 5AC (MUC5AC) and mRNA expression of the inflammatory cytokines IL1B and TNFA. Menthol activated TRPM8, and the effects of menthol on MCC and inflammation could be blocked by a specific TRPM8 antagonist. These data provide further evidence that menthol at the concentrations used in e-cigs could cause harm to the airways.

Role of Secondary Organic Matter on Soot Particle Toxicity in Reconstituted Human Bronchial Epithelia Exposed at the Air-Liquid Interface.

Secondary organic matter (SOM) formed from gaseous precursors constitutes a major mass fraction of fine particulate matter. However, there is only limited evidence on its toxicological impact. In this study, air-liquid interface cultures of human bronchial epithelia were exposed to different series of fresh and aged soot particles generated by a miniCAST burner combined with a micro smog chamber (MSC). Soot cores with geometric mean mobility diameters of 30 and 90 nm were coated with increasing amounts of SOM, generated from the photo-oxidation of mesitylene and ozonolysis of α-pinene. At 24 h after exposure, the release of lactate dehydrogenase (LDH), indicating cell membrane damage, was measured and proteome analysis, i.e. the release of 102 cytokines and chemokines to assess the inflammatory response, was performed. The data indicate that the presence of the SOM coating and its bioavailability play an important role in cytotoxicity. In particular, LDH release increased with increasing SOM mass/total particle mass ratio, but only when SOM had condensed on the outer surface of the soot cores. Proteome analysis provided further evidence for substantial interference of coated particles with essential properties of the respiratory epithelium as a barrier as well as affecting cell remodeling and inflammatory activity.

The CFTR Amplifier Nesolicaftor Rescues TGF-ß1 Inhibition of Modulator-Corrected F508del CFTR Function.

Highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulators have led to dramatic improvements in lung function in many people with cystic fibrosis (PwCF). However, the efficacy of CFTR modulators may be hindered by persistent airway inflammation. The cytokine transforming growth factor-beta1 (TGF-ß1) is associated with worse pulmonary disease in PwCF and can diminish modulator efficacy. Thus, strategies to augment the CFTR response to modulators in an inflammatory environment are needed. Here, we tested whether the CFTR amplifier nesolicaftor (or PTI-428) could rescue the effects of TGF-ß1 on CFTR function and ciliary beating in primary human CF bronchial epithelial (CFBE) cells. CFBE cells homozygous for F508del were treated with the combination of elexacaftor/tezacaftor/ivacaftor (ETI) and TGF-ß1 in the presence and absence of nesolicaftor. Nesolicaftor augmented the F508del CFTR response to ETI and reversed TGF-ß1-induced reductions in CFTR conductance by increasing the expression of CFTR mRNA. Nesolicaftor further rescued the reduced ciliary beating and increased expression of the cytokines IL-6 and IL-8 caused by TGF-ß1. Finally, nesolicaftor augmented the F508del CFTR response to ETI in CFBE cells overexpressing miR-145, a negative regulator of CFTR expression. Thus, CFTR amplifiers, but only when used with highly effective modulators, may provide benefit in an inflamed environment.

Hyperglycaemia in cystic fibrosis adversely affects BK channel function critical for mucus clearance.

Large-conductance, Ca2+-activated, voltage-dependent K+ (BK) channel function is critical for adequate airway hydration and mucociliary function. In airway epithelia, BK function is regulated by its γ-subunit, leucine-rich repeat-containing protein 26 (LRRC26). Since patients with cystic fibrosis (CF)-related diabetes mellitus (CFRD) have worse lung function outcomes, this study determined the effects of hyperglycaemia on BK function in CF bronchial epithelial (CFBE) cells in vitro and evaluated the correlation between glycaemic excursions and mRNA expression of LRRC26 in the upper airways of CF and CFRD patients.CFBE cells were redifferentiated at the air-liquid interface (ALI) in media containing either 5.5 mM or 12.5 mM glucose. BK activity was measured in an Ussing chamber. Airway surface liquid (ASL) volume was estimated by meniscus scanning and inflammatory marker expression was measured by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA). CF patients were assessed by 7 days of continuous glucose monitoring (CGM). LRRC26 mRNA expression was measured by quantitative real-time PCR from nasal cells obtained at the end of glucose monitoring.BK currents were significantly decreased in CFBE cells cultured under high glucose. These cells revealed significantly lower ASL volumes and increased inflammation, including the receptor for advanced glycation endproducts (RAGE), compared to cells cultured in normal glucose. In vivo, nasal cell expression of LRRC26 mRNA was inversely correlated with hyperglycaemic excursions, consistent with the in vitro results.Our findings demonstrate that hyperglycaemia induces inflammation and impairs BK channel function in CFBE cells in vitro These data suggest that declining lung function in CFRD patients may be related to BK channel dysfunction.

Losartan Rescues Inflammation-related Mucociliary Dysfunction in Relevant Models of Cystic Fibrosis.

Rationale: Despite therapeutic progress in treating cystic fibrosis (CF) airway disease, airway inflammation with associated mucociliary dysfunction remains largely unaddressed. Inflammation reduces the activity of apically expressed large-conductance Ca2+-activated and voltage-dependent K+ (BK) channels, critical for mucociliary function in the absence of CFTR (CF transmembrane conductance regulator).Objectives: To test losartan as an antiinflammatory therapy in CF using CF human bronchial epithelial cells and an ovine model of CF-like airway disease.Methods: Losartan's antiinflammatory effectiveness to rescue BK activity and thus mucociliary function was tested in vitro using primary, fully redifferentiated human airway epithelial cells homozygous for F508del and in vivo using a previously validated, now expanded pharmacologic sheep model of CF-like, inflammation-associated mucociliary dysfunction.Measurements and Main Results: Nasal scrapings from patients with CF showed that neutrophilic inflammation correlated with reduced expression of LRRC26 (leucine rich repeat containing 26), the γ subunit mandatory for BK function in the airways. TGF-ß1 (transforming growth factor ß1), downstream of neutrophil elastase, decreased mucociliary parameters in vitro. These were rescued by losartan at concentrations achieved by nebulization in the airway and oral application in the bloodstream: BK dysfunction recovered acutely and over time (the latter via an increase in LRRC26 expression), ciliary beat frequency and airway surface liquid volume improved, and mucus hyperconcentration and cellular inflammation decreased. These effects did not depend on angiotensin receptor blockade. Expanding on a validated and published nongenetic, CF-like sheep model, ewes inhaled CFTRinh172 and neutrophil elastase for 3 days, which resulted in prolonged tracheal mucus velocity reduction, mucus hyperconcentration, and increased TGF-ß1. Nebulized losartan rescued both mucus transport and mucus hyperconcentration and reduced TGF-ß1.Conclusions: Losartan effectively reversed CF- and inflammation-associated mucociliary dysfunction, independent of its angiotensin receptor blockade.

Airway Resistance Caused by Sphingomyelin Synthase 2 Insufficiency in Response to Cigarette Smoke.

Sphingomyelin synthase is responsible for the production of sphingomyelin (SGM), the second most abundant phospholipid in mammalian plasma, from ceramide, a major sphingolipid. Knowledge of the effects of cigarette smoke on SGM production is limited. In the present study, we examined the effect of chronic cigarette smoke on sphingomyelin synthase (SGMS) activity and evaluated how the deficiency of Sgms2, one of the two isoforms of mammalian SGMS, impacts pulmonary function. Sgms2-knockout and wild-type control mice were exposed to cigarette smoke for 6 months, and pulmonary function testing was performed. SGMS2-dependent signaling was investigated in these mice and in human monocyte-derived macrophages of nonsmokers and human bronchial epithelial (HBE) cells isolated from healthy nonsmokers and subjects with chronic obstructive pulmonary disease (COPD). Chronic cigarette smoke reduces SGMS activity and Sgms2 gene expression in mouse lungs. Sgms2-deficient mice exhibited enhanced airway and tissue resistance after chronic cigarette smoke exposure, but had similar degrees of emphysema, compared with smoke-exposed wild-type mice. Sgms2-/- mice had greater AKT phosphorylation, peribronchial collagen deposition, and protease activity in their lungs after smoke inhalation. Similarly, we identified reduced SGMS2 expression and enhanced phosphorylation of AKT and protease production in HBE cells isolated from subjects with COPD. Selective inhibition of AKT activity or overexpression of SGMS2 reduced the production of several matrix metalloproteinases in HBE cells and monocyte-derived macrophages. Our study demonstrates that smoke-regulated Sgms2 gene expression influences key COPD features in mice, including airway resistance, AKT signaling, and protease production.

Electronic Cigarette Vapor with Nicotine Causes Airway Mucociliary Dysfunction Preferentially via TRPA1 Receptors.

Rationale: Electronic cigarette (e-cig) use has been widely adopted under the perception of safety. However, possibly adverse effects of e-cig vapor in never-smokers are not well understood.Objectives: To test the effects of nicotine-containing e-cig vapors on airway mucociliary function in differentiated human bronchial epithelial cells isolated from never-smokers and in the airways of a novel, ovine large animal model.Methods: Mucociliary parameters were measured in human bronchial epithelial cells and in sheep. Systemic nicotine delivery to sheep was quantified using plasma cotinine levels, measured by ELISA.Measurements and Main Results:In vitro, exposure to e-cig vapor reduced airway surface liquid hydration and increased mucus viscosity of human bronchial epithelial cells in a nicotine-dependent manner. Acute nicotine exposure increased intracellular calcium levels, an effect primarily dependent on TRPA1 (transient receptor potential ankyrin 1). TRPA1 inhibition with A967079 restored nicotine-mediated impairment of mucociliary parameters including mucus transport in vitro. Sheep tracheal mucus velocity, an in vivo measure of mucociliary clearance, was also reduced by e-cig vapor. Nebulized e-cig liquid containing nicotine also reduced tracheal mucus velocity in a dose-dependent manner and elevated plasma cotinine levels. Importantly, nebulized A967079 reversed the effects of e-cig liquid on sheep tracheal mucus velocity.Conclusions: Our findings show that inhalation of e-cig vapor causes airway mucociliary dysfunction in vitro and in vivo. Furthermore, they suggest that the main nicotine effect on mucociliary function is mediated by TRPA1 and not nicotinic acetylcholine receptors.

Protein Phosphatase 2A Reduces Cigarette Smoke-induced Cathepsin S and Loss of Lung Function.

Rationale: CTSS (cathepsin S) is a cysteine protease that is observed at higher concentrations in BAL fluid and plasma of subjects with chronic obstructive pulmonary disease (COPD). Objectives: To investigate whether CTSS is involved in the pathogenesis of cigarette smoke-induced COPD and determine whether targeting upstream signaling could prevent the disease. Methods: CTSS expression was investigated in animal and human tissue and cell models of COPD. Ctss-/- mice were exposed to long-term cigarette smoke and forced oscillation and expiratory measurements were recorded. Animals were administered chemical modulators of PP2A (protein phosphatase 2A) activity. Measurements and Main Results: Here we observed enhanced CTSS expression and activity in mouse lungs after exposure to cigarette smoke. Ctss-/- mice were resistant to cigarette smoke-induced inflammation, airway hyperresponsiveness, airspace enlargements, and loss of lung function. CTSS expression was negatively regulated by PP2A in human bronchial epithelial cells isolated from healthy nonsmokers and COPD donors and in monocyte-derived macrophages. Modulating PP2A expression or activity, with silencer siRNA or a chemical inhibitor or activator, during acute smoke exposure in mice altered inflammatory responses and CTSS expression and activity in the lung. Enhancement of PP2A activity prevented chronic smoke-induced COPD in mice. Conclusions: Our study indicates that the decrease in PP2A activity that occurs in COPD contributes to elevated CTSS expression in the lungs and results in impaired lung function. Enhancing PP2A activity represents a feasible therapeutic approach to reduce CTSS activity and counter smoke-induced lung disease.

Chronic Cigarette Smoke Exposure Subdues PP2A Activity by Enhancing Expression of the Oncogene CIP2A.

Phosphatase activity of the major serine threonine phosphatase, protein phosphatase 2A (PP2A), is blunted in the airways of individuals with chronic obstructive pulmonary disease (COPD), which results in heightened inflammation and proteolytic responses. The objective of this study was to investigate how PP2A activity is modulated in COPD airways. PP2A activity and endogenous inhibitors of PP2A were investigated in animal and cell models of COPD. In primary human bronchial epithelial (HBE) cells isolated from smokers and donors with COPD, we observed enhanced expression of cancerous inhibitor of PP2A (CIP2A), an oncoprotein encoded by the KIAA1524 gene, compared with cells from nonsmokers. CIP2A expression was induced by chronic cigarette smoke exposure in mice that coincided with a reduction in PP2A activity, airspace enlargements, and loss of lung function, as determined by PP2A phosphatase activity, mean linear intercept analysis, and forced expiratory volume in 0.05 second/forced vital capacity. Modulating CIP2A expression in HBE cells by silencing RNA or chemically with erlotinib enhanced PP2A activity, reduced extracellular-signal-regulated kinase phosphorylation, and reduced the responses of matrix metalloproteinases 1 and 9 in HBE cells isolated from subjects with COPD. Enhanced epithelial growth factor receptor responses in cells from subjects with COPD were observed to modulate CIP2A expression levels. Our study indicates that chronic cigarette smoke induction of epithelial growth factor receptor signaling and CIP2A expression can impair PP2A responses that are associated with loss of lung function and enhancement of proteolytic responses. Augmenting PP2A activity by manipulating CIP2A expression may represent a feasible therapeutic approach to counter smoke-induced lung disease.

Fibroblast growth factor 23 and Klotho contribute to airway inflammation.

Circulating levels of fibroblast growth factor (FGF)23 are associated with systemic inflammation and increased mortality in chronic kidney disease. α-Klotho, a co-receptor for FGF23, is downregulated in chronic obstructive pulmonary disease (COPD). However, whether FGF23 and Klotho-mediated FGF receptor (FGFR) activation delineates a pathophysiological mechanism in COPD remains unclear. We hypothesised that FGF23 can potentiate airway inflammation via Klotho-independent FGFR4 activation.FGF23 and its effect were studied using plasma and transbronchial biopsies from COPD and control patients, and primary human bronchial epithelial cells isolated from COPD patients as well as a murine COPD model.Plasma FGF23 levels were significantly elevated in COPD patients. Exposure of airway epithelial cells to cigarette smoke and FGF23 led to a significant increase in interleukin-1ß release via Klotho-independent FGFR4-mediated activation of phospholipase Cγ/nuclear factor of activated T-cells signalling. In addition, Klotho knockout mice developed COPD and showed airway inflammation and elevated FGFR4 expression in their lungs, whereas overexpression of Klotho led to an attenuation of airway inflammation.Cigarette smoke induces airway inflammation by downregulation of Klotho and activation of FGFR4 in the airway epithelium in COPD. Inhibition of FGF23 or FGFR4 might serve as a novel anti-inflammatory strategy in COPD.

Airway Surface Dehydration by Transforming Growth Factor ß (TGF-ß) in Cystic Fibrosis Is Due to Decreased Function of a Voltage-dependent Potassium Channel and Can Be Rescued by the Drug Pirfenidone.

Transforming growth factor ß1 (TGF-ß1) is not only elevated in airways of cystic fibrosis (CF) patients, whose airways are characterized by abnormal ion transport and mucociliary clearance, but TGF-ß1 is also associated with worse clinical outcomes. Effective mucociliary clearance depends on adequate airway hydration, governed by ion transport. Apically expressed, large-conductance, Ca(2+)- and voltage-dependent K(+) (BK) channels play an important role in this process. In this study, TGF-ß1 decreased airway surface liquid volume, ciliary beat frequency, and BK activity in fully differentiated CF bronchial epithelial cells by reducing mRNA expression of the BK γ subunit leucine-rich repeat-containing protein 26 (LRRC26) and its function. Although LRRC26 knockdown itself reduced BK activity, LRRC26 overexpression partially reversed TGF-ß1-induced BK dysfunction. TGF-ß1-induced airway surface liquid volume hyper-absorption was reversed by the BK opener mallotoxin and the clinically useful TGF-ß signaling inhibitor pirfenidone. The latter increased BK activity via rescue of LRRC26. Therefore, we propose that TGF-ß1-induced mucociliary dysfunction in CF airways is associated with BK inactivation related to a LRRC26 decrease and is amenable to treatment with clinically useful TGF-ß1 inhibitors.

TLR9 expression is required for the development of cigarette smoke-induced emphysema in mice.

The expression of Toll-like receptor (TLR)-9, a pathogen recognition receptor that recognizes unmethylated CpG sequences in microbial DNA molecules, is linked to the pathogenesis of several lung diseases. TLR9 expression and signaling was investigated in animal and cell models of chronic obstructive pulmonary disease (COPD). We observed enhanced TLR9 expression in mouse lungs following exposure to cigarette smoke. Tlr9(-/-) mice were resistant to cigarette smoke-induced loss of lung function as determined by mean linear intercept, total lung capacity, lung compliance, and tissue elastance analysis. Tlr9 expression also regulated smoke-mediated immune cell recruitment to the lung; apoptosis; expression of granulocyte-colony stimulating factor (G-CSF), the CXCL5 protein, and matrix metalloproteinase-2 (MMP-2); and protein tyrosine phosphatase 1B (PTP1B) activity in the lung. PTP1B, a phosphatase with anti-inflammatory abilities, was identified as binding to TLR9. In vivo delivery of a TLR9 agonist enhanced TLR9 binding to PTP1B, which inactivated PTP1B. Ptp1b(-/-) mice had elevated lung concentrations of G-CSF, CXCL5, and MMP-2, and tissue expression of type-1 interferon following TLR9 agonist administration, compared with wild-type mice. TLR9 responses were further determined in fully differentiated normal human bronchial epithelial (NHBE) cells isolated from nonsmoker, smoker, and COPD donors, and then cultured at air liquid interface. NHBE cells from smokers and patients with COPD expressed more TLR9 and secreted greater levels of G-CSF, IL-6, CXCL5, IL-1ß, and MMP-2 upon TLR9 ligand stimulation compared with cells from nonsmoker donors. Although TLR9 combats infection, our results indicate that TLR9 induction can affect lung function by inactivating PTP1B and upregulating expression of proinflammatory cytokines.

Chronic electronic cigarette exposure in mice induces features of COPD in a nicotine-dependent manner.

BACKGROUND: The use of electronic (e)-cigarettes is increasing rapidly, but their lung health effects are not established. Clinical studies examining the potential long-term impact of e-cigarette use on lung health will take decades. To address this gap in knowledge, this study investigated the effects of exposure to aerosolised nicotine-free and nicotine-containing e-cigarette fluid on mouse lungs and normal human airway epithelial cells. METHODS: Mice were exposed to aerosolised phosphate-buffered saline, nicotine-free or nicotine-containing e-cigarette solution, 1-hour daily for 4 months. Normal human bronchial epithelial (NHBE) cells cultured at an air-liquid interface were exposed to e-cigarette vapours or nicotine solutions using a Vitrocell smoke exposure robot. RESULTS: Inhalation of nicotine-containing e-cigarettes increased airway hyper-reactivity, distal airspace enlargement, mucin production, cytokine and protease expression. Exposure to nicotine-free e-cigarettes did not affect these lung parameters. NHBE cells exposed to nicotine-containing e-cigarette vapour showed impaired ciliary beat frequency, airway surface liquid volume, cystic fibrosis transmembrane regulator and ATP-stimulated K+ ion conductance and decreased expression of FOXJ1 and KCNMA1. Exposure of NHBE cells to nicotine for 5 days increased interleukin (IL)-6 and IL-8 secretion. CONCLUSIONS: Exposure to inhaled nicotine-containing e-cigarette fluids triggered effects normally associated with the development of COPD including cytokine expression, airway hyper-reactivity and lung tissue destruction. These effects were nicotine-dependent both in the mouse lung and in human airway cells, suggesting that inhaled nicotine contributes to airway and lung disease in addition to its addictive properties. Thus, these findings highlight the potential dangers of nicotine inhalation during e-cigarette use.

Glutathione Peroxidase-1 Suppresses the Unfolded Protein Response upon Cigarette Smoke Exposure.

Oxidative stress provokes endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) in the lungs of chronic obstructive pulmonary (COPD) subjects. The antioxidant, glutathione peroxidase-1 (GPx-1), counters oxidative stress induced by cigarette smoke exposure. Here, we investigate whether GPx-1 expression deters the UPR following exposure to cigarette smoke. Expression of ER stress markers was investigated in fully differentiated normal human bronchial epithelial (NHBE) cells isolated from nonsmoking, smoking, and COPD donors and redifferentiated at the air liquid interface. NHBE cells from COPD donors expressed heightened ATF4, XBP1, GRP78, GRP94, EDEM1, and CHOP compared to cells from nonsmoking donors. These changes coincided with reduced GPx-1 expression. Reintroduction of GPx-1 into NHBE cells isolated from COPD donors reduced the UPR. To determine whether the loss of GPx-1 expression has a direct impact on these ER stress markers during smoke exposure, Gpx-1-/- mice were exposed to cigarette smoke for 1 year. Loss of Gpx-1 expression enhanced cigarette smoke-induced ER stress and apoptosis. Equally, induction of ER stress with tunicamycin enhanced antioxidant expression in mouse precision-cut lung slices. Smoke inhalation also exacerbated the UPR response during respiratory syncytial virus infection. Therefore, ER stress may be an antioxidant-related pathophysiological event in COPD.

The Endoplasmic Reticulum Resident Protein AGR3. Required for Regulation of Ciliary Beat Frequency in the Airway.

Protein disulfide isomerase (PDI) family members regulate protein folding and calcium homeostasis in the endoplasmic reticulum (ER). The PDI family member anterior gradient (AGR) 3 is expressed in the airway, but the localization, regulation, and function of AGR3 are poorly understood. Here we report that AGR3, unlike its closest homolog AGR2, is restricted to ciliated cells in the airway epithelium and is not induced by ER stress. Mice lacking AGR3 are viable and develop ciliated cells with normal-appearing cilia. However, ciliary beat frequency was lower in airways from AGR3-deficient mice compared with control mice (20% lower in the absence of stimulation and 35% lower after ATP stimulation). AGR3 deficiency had no detectable effects on ciliary beat frequency (CBF) when airways were perfused with a calcium-free solution, suggesting that AGR3 is required for calcium-mediated regulation of ciliary function. Decreased CBF was associated with impaired mucociliary clearance in AGR3-deficient airways. We conclude that AGR3 is a specialized member of the PDI family that plays an unexpected role in the regulation of CBF and mucociliary clearance in the airway.

Roflumilast partially reverses smoke-induced mucociliary dysfunction.

BACKGROUND: Phosphodiesterases (PDEs) break down cAMP, thereby regulating intracellular cAMP concentrations and diffusion. Since PDE4 predominates in airway epithelial cells, PDE4 inhibitors can stimulate Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by increasing cAMP. Tobacco smoking and COPD are associated with decreased CFTR function and impaired mucociliary clearance (MCC). However, the effects of the PDE4 inhibitor roflumilast on smoke-induced mucociliary dysfunction have not been fully explored. METHODS: Primary normal human bronchial epithelial cells (NHBE) from non-smokers, cultured at the air-liquid interface (ALI) were used for most experiments. Cultures were exposed to cigarette smoke in a Vitrocell VC-10 smoking robot. To evaluate the effect of roflumilast on intracellular cAMP concentrations, fluorescence resonance energy transfer (FRET) between CFP- and YFP-tagged protein kinase A (PKA) subunits was recorded. Airway surface liquid (ASL) was measured using light refraction scanning and ciliary beat frequency (CBF) employing infrared differential interference contrast microscopy. Chloride conductance was measured in Ussing chambers and CFTR expression was quantified with qPCR. RESULTS: While treatment with 100 nM roflumilast had little effect alone, it increased intracellular cAMP upon stimulation with forskolin and albuterol in cultures exposed to cigarette smoke and in control conditions. cAMP baselines were lower in smoke-exposed cells. Roflumilast prolonged cAMP increases in smoke-exposed and control cultures. Smoke-induced reduction in functional, albuterol-mediated chloride conductance through CFTR was improved by roflumilast. ASL volumes also increased in smoke-exposed cultures in the presence of roflumilast while it did not in its absence. Cigarette smoke exposure decreased CBF, an effect rescued with roflumilast, particularly when used together with the long-acting ß-mimetic formoterol. Roflumilast also enhanced forskolin-induced CBF stimulation in ASL volume supplemented smoked and control cells, confirming the direct stimulatory effect of rising cAMP on ciliary function. In active smokers, CFTR mRNA expression was increased compared to non-smokers and ex-smokers. Roflumilast also increased CFTR mRNA levels in cigarette-smoke exposed cell cultures. CONCLUSIONS: Our results show that roflumilast can rescue smoke-induced mucociliary dysfunction by reversing decreased CFTR activity, augmenting ASL volume, and stimulating CBF, the latter particularly in combination with formoterol. As expected, CFTR mRNA expression was not indicative of apical CFTR function.

A soluble adenylyl cyclase form targets to axonemes and rescues beat regulation in soluble adenylyl cyclase knockout mice.

Ciliary beating is important for effective mucociliary clearance. Soluble adenylyl cyclase (sAC) regulates ciliary beating, and a roughly 50-kD sAC variant is expressed in axonemes. Normal human bronchial epithelial (NHBE) cells express multiple sAC splice variants: full-length sAC; variants with catalytic domain 1 (C1) deletions; and variants with partial C1. One variant, sACex5v2-ex12v2, contains two alternative splices creating new exons 5 (ex5v2) and 12 (ex12v2), encoding a roughly 45-kD protein. It is therefore similar in size to ciliary sAC. The variant increases in expression upon ciliogenesis during differentiation at the air-liquid interface. When expressed in NHBE cells, this variant was targeted to cilia. Exons 5v2-7 were important for ciliary targeting, whereas exons 2-4 prevented it. In vitro, cytoplasmic sACex2-ex12v2 (containing C1 and C2) was the only variant producing cAMP. Ciliary sACex5v2-ex12v2 was not catalytically active. Airway epithelial cells isolated from wild-type mice revealed sAC-dependent ciliary beat frequency (CBF) regulation, analogous to NHBE cells: CBF rescue from HCO3(-)/CO2-mediated intracellular acidification was sensitive to the sAC inhibitor, KH7. Compared with wild type, sAC C2 knockout (KO) mice revealed lower CBF baseline, and the HCO3(-)/CO2-mediated CBF decrease was not inhibited by KH7, confirming lack of functional sAC. Human sACex5v2-ex12v2 was targeted to cilia and sACex2-ex12v2 to the cytoplasm in these KO mice. Introduction of the ciliary sACex5v2-ex12v2 variant, but not the cytoplasmic sACex2-ex12v2, restored functional sAC activity in C2 KO mice. Thus, we show, for the first time, a mammalian axonemal targeting sequence that localizes a sAC variant to cilia to regulate CBF.