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.

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.

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.

Regulation of dendrite growth and maintenance by exocytosis.

Dendrites lengthen by several orders of magnitude during neuronal development, but how membrane is allocated in dendrites to facilitate this growth remains unclear. Here, we report that Ras opposite (Rop), the Drosophila ortholog of the key exocytosis regulator Munc18-1 (also known as STXBP1), is an essential factor mediating dendrite growth. Neurons with depleted Rop function exhibit reduced terminal dendrite outgrowth followed by primary dendrite degeneration, suggestive of differential requirements for exocytosis in the growth and maintenance of different dendritic compartments. Rop promotes dendrite growth together with the exocyst, an octameric protein complex involved in tethering vesicles to the plasma membrane, with Rop-exocyst complexes and exocytosis predominating in primary dendrites over terminal dendrites. By contrast, membrane-associated proteins readily diffuse from primary dendrites into terminals, but not in the reverse direction, suggesting that diffusion, rather than targeted exocytosis, supplies membranous material for terminal dendritic growth, revealing key differences in the distribution of materials to these expanding dendritic compartments.

Live imaging of multicolor-labeled cells in Drosophila.

We describe LOLLIbow, a Brainbow-based live imaging system with applications in developmental biology and neurobiology. The development of an animal, including the environmentally sensitive adaptation of its brain, is thought to proceed through continual orchestration among diverse cell types as they divide, migrate, transform and interact with one another within the body. To facilitate direct visualization of such dynamic morphogenesis by individual cells in vivo, we have modified the original Brainbow for Drosophila in which live imaging is practical during much of its development. Our system offers permanent fluorescent labels that reveal fine morphological details of individual cells without requiring dissection or fixation of the samples. It also features a non-invasive means to control the timing of stochastic tricolor cell labeling with a light pulse. We demonstrate applicability of the new system in a variety of settings that could benefit from direct imaging of the developing multicellular organism with single-cell resolution.

Part-based motor neuron recognition in the Drosophila ventral nerve cord.

We exploit the morphological stereotypy and relative simplicity of the Drosophila nervous system to model the diverse neuronal morphologies of individual motor neurons and understand underlying principles of synaptic connectivity in a motor circuit. In our analysis, we use images depicting single neurons labeled with green fluorescent protein (GFP) and serially imaged with laser scanning confocal microscopy. We model morphology with a novel formulation of Conditional Random Fields, a hierarchical latent-state CRF, to capture the highly varying compartment-based structure of the neurons (soma-axon-dendrites). In the training phase, we follow two approaches: (i) hierarchical learning, where compartment labels are given, and (ii) latent-state learning, where compartment labels are not given in the samples. We demonstrate the accuracy of our approach using wild-type motor neurons in the larval ventral nerve cord. However, our method can also be used for the identification of motor neuron mutations, as well as the automated annotation of the motor circuitry in wild type and mutant animals. Our method is directly applicable to the recognition of compartment-defined structures.

The role of autophagy in Nmnat-mediated protection against hypoxia-induced dendrite degeneration.

The selective degeneration of dendrites precedes neuronal cell death in hypoxia-ischemia (HI) and is a neuropathological hallmark of stroke. While it is clear that a number of different molecular pathways likely contribute to neuronal cell death in HI, the mechanisms that govern HI-induced dendrite degeneration are largely unknown. Here, we show that the NAD synthase nicotinamide mononucleotide adenylyltransferase (Nmnat) functions endogenously to protect Drosophila class IV dendritic arborization (da) sensory neurons against hypoxia-induced dendritic damage. Whereas dendrites of wild-type class IV neurons are largely resistant to morphological changes during prolonged periods of hypoxia (<1.0% O(2)), class IV neurons of nmnat heterozygous mutants exhibit significant dendrite loss and extensive fragmentation of the dendritic arbor under the same hypoxic conditions. Although basal levels of autophagy are required for neuronal survival, we demonstrate that autophagy is dispensable for maintaining the dendritic integrity of class IV neurons. However, we find that genetically blocking autophagy can suppress hypoxia-induced dendrite degeneration of nmnat heterozygous mutants in a cell-autonomous manner, suggestive of a self-destructive role for autophagy in this context. We further show that inducing autophagy by overexpression of the autophagy-specific kinase Atg1 is sufficient to cause dendrite degeneration of class IV neurons under hypoxia and that overexpression of Nmnat fails to protect class IV dendrites from the effects of Atg1 overexpression. Our studies reveal an essential neuroprotective role for endogenous Nmnat in hypoxia and demonstrate that Nmnat functions upstream of autophagy to mitigate the damage incurred by dendrites in neurons under hypoxic stress.

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.

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.

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.