The combination of propylene glycol and vegetable glycerin e-cigarette aerosols induces airway inflammation and mucus hyperconcentration.

Despite concerns over their safety, e-cigarettes (e-cigs) remain a popular tobacco product. Although nicotine and flavors found in e-cig liquids (e-liquids) can cause harm in the airways, whether the delivery vehicles propylene glycol (PG) and vegetable glycerin (VG) are innocuous when inhaled remains unclear. Here, we investigated the effects of e-cig aerosols generated from e-liquid containing only PG/VG on airway inflammation and mucociliary function in primary human bronchial epithelial cells (HBEC) and sheep. Primary HBEC were cultured at the air-liquid interface (ALI) and exposed to e-cig aerosols of 50%/50% v/v PG/VG. Ion channel conductance, ciliary beat frequency, and the expression of inflammatory markers, cell type-specific markers, and the major mucins MUC5AC and MUC5B were evaluated after seven days of exposure. Sheep were exposed to e-cig aerosols of PG/VG for five days and mucus concentration and matrix metalloproteinase-9 (MMP-9) activity were measured from airway secretions. Seven-day exposure of HBEC to e-cig aerosols of PG/VG caused a significant reduction in the activities of apical ion channels important for mucus hydration, including the cystic fibrosis transmembrane conductance regulator (CFTR) and large conductance, Ca2+-activated, and voltage-dependent K+ (BK) channels. PG/VG aerosols significantly increased the mRNA expression of the inflammatory markers interleukin-6 (IL6), IL8, and MMP9, as well as MUC5AC. The increase in MUC5AC mRNA expression correlated with increased immunostaining of MUC5AC protein in PG/VG-exposed HBEC. On the other hand, PG/VG aerosols reduced MUC5B expression leading overall to higher MUC5AC/MUC5B ratios in exposed HBEC. Other cell type-specific markers, including forkhead box protein J1 (FOXJ1), keratin 5 (KRT5), and secretoglobin family 1A member 1 (SCGB1A1) mRNAs, as well as overall ciliation, were significantly reduced by PG/VG exposure. Finally, PG/VG aerosols increased MMP-9 activity and caused mucus hyperconcentration in sheep in vivo. E-cig aerosols of PG/VG induce airway inflammation, increase MUC5AC expression, and cause dysfunction of ion channels important for mucus hydration in HBEC in vitro. Furthermore, PG/VG aerosols increase MMP-9 activity and mucus concentration in sheep in vivo. Collectively, these data show that e-cig aerosols containing PG/VG are likely to be harmful in the airways.

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.

Vegetable glycerin e-cigarette aerosols cause airway inflammation and ion channel dysfunction.

Vegetable glycerin (VG) and propylene glycol (PG) serve as delivery vehicles for nicotine and flavorings in most e-cigarette (e-cig) liquids. Here, we investigated whether VG e-cig aerosols, in the absence of nicotine and flavors, impact parameters of mucociliary function in human volunteers, a large animal model (sheep), and air-liquid interface (ALI) cultures of primary human bronchial epithelial cells (HBECs). We found that VG-containing (VG or PG/VG), but not sole PG-containing, e-cig aerosols reduced the activity of nasal cystic fibrosis transmembrane conductance regulator (CFTR) in human volunteers who vaped for seven days. Markers of inflammation, including interleukin-6 (IL6), interleukin-8 (IL8) and matrix metalloproteinase-9 (MMP9) mRNAs, as well as MMP-9 activity and mucin 5AC (MUC5AC) expression levels, were also elevated in nasal samples from volunteers who vaped VG-containing e-liquids. In sheep, exposures to VG e-cig aerosols for five days increased mucus concentrations and MMP-9 activity in tracheal secretions and plasma levels of transforming growth factor-beta 1 (TGF-ß1). In vitro exposure of HBECs to VG e-cig aerosols for five days decreased ciliary beating and increased mucus concentrations. VG e-cig aerosols also reduced CFTR function in HBECs, mechanistically by reducing membrane fluidity. Although VG e-cig aerosols did not increase MMP9 mRNA expression, expression levels of IL6, IL8, TGFB1, and MUC5AC mRNAs were significantly increased in HBECs after seven days of exposure. Thus, VG e-cig aerosols can potentially cause harm in the airway by inducing inflammation and ion channel dysfunction with consequent mucus hyperconcentration.

Persistence of airway inflammation in smokers who switch to electronic cigarettes.

As opposed to smoking cessation with nicotine-replacement therapy and/or varenicline, nicotine-containing e-cigarette use does not improve some airway inflammatory markers. https://bit.ly/3FyqIt9.

Losartan ameliorates TGF-ß1-induced CFTR dysfunction and improves correction by cystic fibrosis modulator therapies.

Highly effective modulator therapies dramatically improve the prognosis for those with cystic fibrosis (CF). The triple combination of elexacaftor, tezacaftor, and ivacaftor (ETI) benefits many, but not all, of those with the most common F508del mutation in the CF transmembrane conductance regulator (CFTR). Here, we showed that poor sweat chloride concentration responses and lung function improvements upon initiation of ETI were associated with elevated levels of active TGF-ß1 in the upper airway. Furthermore, TGF-ß1 impaired the function of ETI-corrected F508del-CFTR, thereby increasing airway surface liquid (ASL) absorption rates and inducing mucus hyperconcentration in primary CF bronchial epithelial cells in vitro. TGF-ß1 not only decreased CFTR mRNA, but was also associated with increases in the mRNA expression of TNFA and COX2 and TNF-α protein. Losartan improved TGF-ß1-mediated inhibition of ETI-corrected F508del-CFTR function and reduced TNFA and COX2 mRNA and TNF-α protein expression. This likely occurred by improving correction of mutant CFTR rather than increasing its mRNA (without an effect on potentiation), thereby reversing the negative effects of TGF-ß1 and improving ASL hydration in the CF airway epithelium in vitro. Importantly, these effects were independent of type 1 angiotensin II receptor inhibition.

LRP1 loss in airway epithelium exacerbates smoke-induced oxidative damage and airway remodeling.

The LDL receptor-related protein 1 (LRP1) partakes in metabolic and signaling events regulated in a tissue-specific manner. The function of LRP1 in airways has not been studied. We aimed to study the function of LRP1 in smoke-induced disease. We found that bronchial epithelium of patients with chronic obstructive pulmonary disease and airway epithelium of mice exposed to smoke had increased LRP1 expression. We then knocked out LRP1 in human bronchial epithelial cells in vitro and in airway epithelial club cells in mice. In vitro, LRP1 knockdown decreased cell migration and increased transforming growth factor ß activation. Tamoxifen-inducible airway-specific LRP1 knockout mice (club Lrp1-/-) induced after complete lung development had increased inflammation in the bronchoalveolar space and lung parenchyma at baseline. After 6 months of smoke exposure, club Lrp1-/- mice showed a combined restrictive and obstructive phenotype, with lower compliance, inspiratory capacity, and forced expiratory volume0.05/forced vital capacity than WT smoke-exposed mice. This was associated with increased values of Ashcroft fibrotic index. Proteomic analysis of room air exposed-club Lrp1-/- mice showed significantly decreased levels of proteins involved in cytoskeleton signaling and xenobiotic detoxification as well as decreased levels of glutathione. The proteome fingerprint created by smoke eclipsed many of the original differences, but club Lrp1-/- mice continued to have decreased lung glutathione levels and increased protein oxidative damage and airway cell proliferation. Therefore, LRP1 deficiency leads to greater lung inflammation and damage and exacerbates smoke-induced lung disease.

The SARS-CoV-2 Transcriptome and the Dynamics of the S Gene Furin Cleavage Site in Primary Human Airway Epithelia.

The spike (S) polypeptide of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consists of the S1 and S2 subunits and is processed by cellular proteases at the S1/S2 boundary that contains a furin cleavage site (FCS), 682RRAR↓S686 Various deletions surrounding the FCS have been identified in patients. When SARS-CoV-2 propagated in Vero cells, it acquired deletions surrounding the FCS. We studied the viral transcriptome in Vero cell-derived SARS-CoV-2-infected primary human airway epithelia (HAE) cultured at an air-liquid interface (ALI) with an emphasis on the viral genome stability of the FCS. While we found overall the viral transcriptome is similar to that generated from infected Vero cells, we identified a high percentage of mutated viral genome and transcripts in HAE-ALI. Two highly frequent deletions were found at the FCS region: a 12 amino acid deletion (678TNSPRRAR↓SVAS689) that contains the underlined FCS and a 5 amino acid deletion (675QTQTN679) that is two amino acids upstream of the FCS. Further studies on the dynamics of the FCS deletions in apically released virions from 11 infected HAE-ALI cultures of both healthy and lung disease donors revealed that the selective pressure for the FCS maintains the FCS stably in 9 HAE-ALI cultures but with 2 exceptions, in which the FCS deletions are retained at a high rate of >40% after infection of ≥13 days. Our study presents evidence for the role of unique properties of human airway epithelia in the dynamics of the FCS region during infection of human airways, which is likely donor dependent.IMPORTANCE Polarized human airway epithelia at an air-liquid interface (HAE-ALI) are an in vitro model that supports efficient infection of SARS-CoV-2. The spike (S) protein of SARS-CoV-2 contains a furin cleavage site (FCS) at the boundary of the S1 and S2 domains which distinguishes it from SARS-CoV. However, FCS deletion mutants have been identified in patients and in vitro cell cultures, and how the airway epithelial cells maintain the unique FCS remains unknown. We found that HAE-ALI cultures were capable of suppressing two prevalent FCS deletion mutants (Δ678TNSPRRAR↓SVAS689 and Δ675QTQTN679) that were selected during propagation in Vero cells. While such suppression was observed in 9 out of 11 of the tested HAE-ALI cultures derived from independent donors, 2 exceptions that retained a high rate of FCS deletions were also found. Our results present evidence of the donor-dependent properties of human airway epithelia in the evolution of the FCS during infection.

Losartan reduces cigarette smoke-induced airway inflammation and mucus hypersecretion.

The aim was to determine whether losartan reduces cigarette smoke (CS)-induced airway inflammation and mucus hypersecretion in an in vitro model and a small clinical trial. Primary human bronchial epithelial cells (HBECs) were differentiated at the air-liquid interface (ALI) and exposed to CS. Expression of transforming growth factor (TGF)-ß1 and the mucin MUC5AC, and expression or activity of matrix metalloproteinase (MMP)-9 were measured after CS exposure. Parameters of mucociliary clearance were evaluated by measuring airway surface liquid volumes, mucus concentrations, and conductance of cystic fibrosis transmembrane conductance regulator (CFTR) and large conductance, Ca2+-activated and voltage-dependent potassium (BK) channels. Nasal cells were collected from study participants and expression of MUC5AC, TGF-ß1, and MMP-9 mRNAs was measured before and after losartan treatment. In vitro, CS exposure of HBECs caused a significant increase in mRNA expression of MUC5AC and TGF-ß1 and MMP-9 activity and decreased CFTR and BK channel activities, thereby reducing airway surface liquid volumes and increasing mucus concentrations. Treatment of HBECs with losartan rescued CS-induced CFTR and BK dysfunction and caused a significant decrease in MUC5AC expression and mucus concentrations, partially by inhibiting TGF-ß signalling. In a prospective clinical study, cigarette smokers showed significantly reduced mRNA expression levels of MUC5AC, TGF-ß1, and MMP-9 in the upper airways after 2 months of losartan treatment. Our findings suggest that losartan may be an effective therapy to reduce inflammation and mucus hypersecretion in CS-induced chronic airway diseases.

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.

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.

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.

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.

Nmnat exerts neuroprotective effects in dendrites and axons.

Dendrites can be maintained for extended periods of time after they initially establish coverage of their receptive field. The long-term maintenance of dendrites underlies synaptic connectivity, but how neurons establish and then maintain their dendritic arborization patterns throughout development is not well understood. Here, we show that the NAD synthase Nicotinamide mononucleotide adenylyltransferase (Nmnat) is cell-autonomously required for maintaining type-specific dendritic coverage of Drosophila dendritic arborization (da) sensory neurons. In nmnat heterozygous mutants, dendritic arborization patterns of class IV da neurons are properly established before increased retraction and decreased growth of terminal branches lead to progressive defects in dendritic coverage during later stages of development. Although sensory axons are largely intact in nmnat heterozygotes, complete loss of nmnat function causes severe axonal degeneration, demonstrating differential requirements for nmnat dosage in the maintenance of dendritic arborization patterns and axonal integrity. Overexpression of Nmnat suppresses dendrite maintenance defects associated with loss of the tumor suppressor kinase Warts (Wts), providing evidence that Nmnat, in addition to its neuroprotective role in axons, can function as a protective factor against progressive dendritic loss. Moreover, motor neurons deficient for nmnat show progressive defects in both dendrites and axons. Our studies reveal an essential role for endogenous Nmnat function in the maintenance of both axonal and dendritic integrity and present evidence of a broad neuroprotective role for Nmnat in the central nervous system.