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, Ca 2+ -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.

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.

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.

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-β1) 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-β1 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-β1 in the presence and absence of nesolicaftor. Nesolicaftor augmented the F508del CFTR response to ETI and reversed TGF-β1-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-β1. 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.

Historically, airway inflammation has been recognized to play a key role in the pathogenesis of pulmonary disease progression in cystic fibrosis (CF). Inflammatory markers are elevated in the airways of children with CF as well as CF ferret models even before chronic airway infections take hold [ 1 Sly PD Brennan S Gangell C de Klerk N Murray C Mott L et al. Lung disease at diagnosis in infants with cystic fibrosis detected by newborn screening. Am J Respir Crit Care Med. 2009; 180: 146-152 Crossref PubMed Scopus (458) Google Scholar , 2 Sly PD Gangell CL Chen L Ware RS Ranganathan S Mott LS et al. Risk factors for bronchiectasis in children with cystic fibrosis. N Engl J Med. 2013; 368: 1963-1970 Crossref PubMed Scopus (461) Google Scholar , 3 Khan TZ Wagener JS Bost T Martinez J Accurso FJ Riches DW Early pulmonary inflammation in infants with cystic fibrosis. Am J Respir Crit Care Med. 1995; 151: 1075-1082 PubMed Google Scholar , 4 Balough K McCubbin M Weinberger M Smits W Ahrens R Fick R. The relationship between infection and inflammation in the early stages of lung disease from cystic fibrosis. Pediatr Pulmonol. 1995; 20: 63-70 Crossref PubMed Scopus (292) Google Scholar , 5 Keiser NW Birket SE Evans IA Tyler SR Crooke AK Sun X et al. Defective innate immunity and hyperinflammation in newborn cystic fibrosis transmembrane conductance regulator-knockout ferret lungs. Am J Respir Cell Mol Biol. 2015; 52: 683-694 Crossref PubMed Scopus (76) Google Scholar , 6 Rosen BH Evans TIA Moll SR Gray JS Liang B Sun X et al. Infection Is Not Required for Mucoinflammatory Lung Disease in CFTR-Knockout Ferrets. Am J Respir Crit Care Med. 2018; 197: 1308-1318 Crossref PubMed Scopus (96) Google Scholar ], though the lungs of newborn CF pigs show no signs of inflammation [ [7] Stoltz DA Meyerholz DK Pezzulo AA Ramachandran S Rogan MP Davis GJ et al. Cystic fibrosis pigs develop lung disease and exhibit defective bacterial eradication at birth. Sci Transl Med. 2010; 2: 29ra31 Crossref PubMed Scopus (390) Google Scholar ]. Importantly, levels of inflammatory markers are predictive of future pulmonary exacerbations and even structural lung damage [ [1] Sly PD Brennan S Gangell C de Klerk N Murray C Mott L et al. Lung disease at diagnosis in infants with cystic fibrosis detected by newborn screening. Am J Respir Crit Care Med. 2009; 180: 146-152 Crossref PubMed Scopus (458) Google Scholar , [2] Sly PD Gangell CL Chen L Ware RS Ranganathan S Mott LS et al. Risk factors for bronchiectasis in children with cystic fibrosis. N Engl J Med. 2013; 368: 1963-1970 Crossref PubMed Scopus (461) Google Scholar ]. In addition, there is an abnormal immune response, possibly mediated directly by dysfunctional CF transmembrane conductance regulator (CFTR) on inflammatory cells, that hinders bacterial clearance and further propagates the cycle of inflammation [ [8] Lara-Reyna S Holbrook J Jarosz-Griffiths HH Peckham D McDermott MF. Dysregulated signalling pathways in innate immune cells with cystic fibrosis mutations. Cell Mol Life Sci. 2020; 77: 4485-4503 Crossref PubMed Scopus (34) Google Scholar ].

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-β1 in the upper airway. Furthermore, TGF-β1 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-β1 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-β1-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-β1 and improving ASL hydration in the CF airway epithelium in vitro. Importantly, these effects were independent of type 1 angiotensin II receptor inhibition.

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-β1). 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.

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.

"Cystic Fibrosis–related Diabetes Is Associated with Worse Lung Function Trajectory despite Ivacaftor Use." American Journal of Respiratory and Critical Care Medicine, 204(11), pp. 1343–1345

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)-β1 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-β1, 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-β1 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-β signalling. In a prospective clinical study, cigarette smokers showed significantly reduced mRNA expression levels of MUC5AC, TGF-β1, 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.

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 gamma-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 ASI, 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.

Rationale: Coronavirus disease 2019 (COVID-19) can cause disruption of the renin-angiotensin system in the lungs, possibly contributing to pulmonary capillary leakage. Thus, angiotensin receptor blockers (ARBs) may improve respiratory failure. Objective: Assess safety of losartan for use in respiratory failure related to COVID-19 (NCT04335123). Methods: Single arm, open label trial of losartan in those hospitalized with respiratory failure related to COVID-19. Oral losartan (25 mg daily for 3 days, then 50 mg) was administered from enrollment until day 14 or hospital discharge. A post-hoc external control group with patients who met all inclusion criteria was matched 1:1 to the treatment group using propensity scores for comparison. Measures: Primary outcome was cumulative incidence of any adverse events. Secondary, explorative endpoints included measures of respiratory failure, length of stay and vital status. Results: Of the 34 participants enrolled in the trial, 30 completed the study with a mean age SD of 53.8 ± 17.7 years and 17 males (57%). On losartan, 24/30 (80%) experienced an adverse event as opposed to 29/30 (97%) of controls, with a lower average number of adverse events on losartan relative to control (2.2 vs. 3.3). Using Poisson regression and controlling for age, sex, race, date of enrollment, disease severity at enrollment, and history of high-risk comorbidities, the incidence rate ratio of adverse events on losartan relative to control was 0.69 (95% CI: 0.49–0.97) Conclusions: Losartan appeared safe for COVID-19-related acute respiratory compromise. To assess true efficacy, randomized trials are needed.

The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) caused the devastating ongoing coronavirus disease-2019 (COVID-19) pandemic which poses a great threat to global public health. The spike (S) polypeptide of SARS-CoV-2 consists of the S1 and S2 subunits and is processed by cellular proteases at the S1/S2 boundary. The inclusion of the 4 amino acids (PRRA) at the S1/S2 boundary forms a furin cleavage site (FCS), 682 RRAR↓S 686 , distinguishing SARS-CoV-2 from its closest relative, the SARS-CoV. Various deletions surrounding the FCS have been identified in patients. When SARS-CoV-2 propagated in Vero cells, the virus acquired various deletions surrounding the FCS. In the present study, we studied the viral transcriptome in SARS-CoV-2 infected primary human airway epithelia (HAE) cultured at an air-liquid interface (ALI) with an emphasis on the viral genome stability at the S1/S2 boundary using RNA-seq. 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 S1/S2 boundary of the S gene: one is a deletion of 12 amino acids, 678 TNSP RRAR↓S VAS 689 , which contains the FCS, another is a deletion of 5 amino acids, 675 QTQTN 679 , which is two amino acids upstream of the FCS. Further studies on the dynamics of the FCS deletions in apically released virions revealed that the selective pressure for the FCS maintains the S gene stability in HAE-ALI but with exceptions, in which the FCS deletions are remained at a high rate. Thus, 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 donor-dependent.

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 gamma subunit mandatory for BK function in the airways. TGF-beta 1 (transforming growth factor beta 1), 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 CFTR(inh)172 and neutrophil elastase for 3 days, which resulted in prolonged tracheal mucus velocity reduction, mucus hyperconcentration, and increased TGF-beta 1. Nebulized losartan rescued both mucus transport and mucus hyperconcentration and reduced TGF-beta 1. Conclusions: Losartan effectively reversed CF- and inflammation-associated mucociliary dysfunction, independent of its angiotensin receptor blockade.

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Begh and Aveyard (2020) reason that e-cigarettes are valuable smoking cessation tools due to their reduced risk profiles. However, vaping as a smoking cessation tool remains controversial. The largest study in this area (Hajek et al. 2019) seemed flawed and pointed to dangers that traditional smoking cessation programmes do not face. While e-cigarettes appeared associated with higher rates of smoking cessation compared to nicotine replacement therapy (NRT), only 3% in the NRT arm were on varenicline and none on bupropion. If NRT is combined with pharmacological interventions, it achieves quit rates equal to the e-cigarette arm in that study (e.g. Anthenelli et al. 2016). More importantly, an even greater number of participants (25%) became dual users of e-cigarettes and combustible cigarettes than the 18% of participants in the e-cigarette arm who achieved sustained abstinence from tobacco. This is problematic when considering that dual users may be at greater risk of respiratory illness (Bhatta & Glantz, 2020). Furthermore, the NRT arm in the Hajek study showed that only 9% continued NRT with successful cessation at 1 year follow-up, while 80% of the e-cigarette users continued to vape, basically replacing one harmful nicotine delivery device with another. The assertion that vaping is less harmful than smoking is based on lower levels of toxic chemicals in vapour than tobacco smoke. However, chemicals linked to disease are proportionately more toxic at lower levels of exposure (Lanphear, 2017) and long-term studies on vapour constituents are missing. Although the outbreak of vaping-related lung injuries is possibly related to vitamin E acetate and/or tetrahydrocannabinol, other e-cigarette vapour constituents could contribute (Krishnasamy et al. 2020). Furthermore, reduction in toxic exposure is not resulting in linear risk reduction: reducing smoking from 20 cigarettes per day to 1 reduces the risk for developing coronary artery disease only by half (Hackshaw et al. 2018). The same is true for lung function in which a smoking reduction of >85% has to be achieved to change the decline trajectory of the forced expiratory volume in 1 second (FEV1) (Simmons et al. 2005). Vaping is also reaching alarming rates among adolescents (Cullen et al. 2019). The risk of escalating to tobacco (Vogel et al. 2019; Barrington-Trimis et al. 2020) will erode progress in lowering teen smoking over several decades. Amid mounting evidence of possible harm, one must, at the very least, caution on the use of e-cigarettes and require that legislators regulate these products to keep them away from non-smoking adolescents. Readers are invited to give their views on this and the accompanying CrossTalk articles in this issue by submitting a brief (250 word) comment. Comments may be submitted up to 6 weeks after publication of the article, at which point the discussion will close and the CrossTalk authors will be invited to submit a ‘LastWord’. Please email your comment, including a title and a declaration of interest, to jphysiol@physoc.org. Comments will be moderated and accepted comments will be published online only as ‘supporting information’ to the original debate articles once discussion has closed. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. None. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. This work was funded by NIH-NHLBI – F32-HL140729 (S.C.); NCATS – TL1TR002368 (C.D.B.); NIH-NHLBI – R01-HL139365 (M.S.); James and Esther King Florida Biomedical Research Program – no. 5JK02 (M.S.); and FAMRI – CIA no. 130033 (M.S.)

The use of e-cigarettes has risen in popularity over the past decade, especially among adolescents. E-cigarette is a generic term that covers a variety of nicotine delivery systems using vapour. These devices were invented and initially marketed as a safer alternative to tobacco cigarettes. However, vast advertising campaigns on social media highlighting pleasant flavours made them appealing to adolescents. The dearth of evidence regarding their safety compelled research efforts to understand how e-cigarette use, or ‘vaping’, affects human physiology. Current literature highlights their potential for adverse effects on human health and nicotine addiction. E-cigarette vapour is typically generated from a mixture of propylene glycol and vegetable glycerine, serving as a vehicle for nicotine plus countless flavouring agents. Heating e-cigarette liquid (e-liquid) generates aerosols that contain reactive oxygen species (ROS) and other volatile chemicals (e.g. acrolein, formaldehyde). The important question is whether levels of these agents generated in e-cigarettes are sufficient to cause harm. Increasing applied wattage to the heating coil generally reduces particle size, allowing greater lung penetration (Son et al. 2020), but also increases ROS generation and toxic metal concentrations (Olmedo et al. 2018). Other diluents are also used, including vitamin E acetate (VEA) and medium-chain triglycerides, but typically with tetrahydrocannabinol-containing liquids (Blount et al. 2019). Vaping has been touted as a safer form of nicotine delivery, given the reduction in measured chemicals in vapour compared to cigarette smoke (Goniewicz et al. 2014). However, reduced number or concentration of chemicals does not linearly relate to risk reduction. A Public Health England report advocates e-cigarettes to quit smoking (McNeill et al. 2018), but longitudinal information on safety is sparse. Moreover, FDA approval for oral ingestion of common e-liquid constituents cannot equate to or predict safety for inhalation. For example, inhalation of diacetyl, a butter-like flavouring added to popcorn, also found in some e-liquids, can cause bronchiolitis obliterans. New vaping-related case reports, including cases of bronchiolitis obliterans (Landman et al. 2019) and hard metal pneumoconiosis (Fels Elliott et al. 2019), underscore adverse effects unique to e-cigarette vapour exposure. Studies in cell culture models, animals and humans have begun to reveal adverse respiratory effects of vaping (Gotts et al. 2019). Acute exposure of human bronchial epithelial cells (HBECs) to e-cigarette vapour impaired apical ion channel function (Garcia-Arcos et al. 2016), which was consistent with another study that implicated elevated acrolein levels causing this effect (Lin et al. 2019). Acute exposure of HBECs to nicotine or nicotine-containing vapour impaired mucociliary clearance by increasing mucus viscosity and reducing airway surface liquid volume that was dependent on the function of the transient receptor potential ankyrin 1 (TRPA1), a novel nicotine receptor (Chung et al. 2019). Nicotine-containing vapour exposure of sheep increased their airway mucus concentrations and reduced tracheal mucus velocity in a TRPA1-dependent manner (Chung et al. 2019). These data seem to be supported by human studies: preliminary data show serious peripheral and central dysfunctions of the mucociliary apparatus associated with vaping, compared to those who had not smoked or vaped (Dr W. Bennett, University of North Carolina, Chapel Hill, personal communication). Historically, Dr Bennett's lab did not find any difference in clearance between young ‘healthy’ smokers and non-smokers (both non-vapers). Consistent with these data on mucociliary dysfunction, adolescent e-cigarette users were found to have an almost twofold increase in risk of chronic bronchitis compared to those who never vaped (McConnell et al. 2017). Moreover, e-cigarette users were 1.81 times as likely to report asthma-like symptoms than non-users (Perez et al. 2019). Animal models further reveal inflammation and tissue damage caused by e-cigarette vapour. Chronic exposure of mice to nicotine-containing vapour increased airway protease activity, caused emphysematous changes, and also increased lung cancer susceptibility (Garcia-Arcos et al. 2016; Tang et al. 2019). Again, data from humans confirm inflammatory changes: chronic vaping increased bronchoalveolar lavage (BAL) protease activity equal to that of smoking, even though many in this group were ex-smokers (Ghosh et al. 2019). Furthermore, proteomic approaches showed that unique changes in BAL fluid can be detected with vaping (Ghosh et al. 2018). These in vitro and in vivo studies suggest that vaping leads to dysfunction of airway ion channels such as TRPA1 and the epithelial sodium channel (ENaC), which has been shown to be activated by airway proteases (Donaldson et al. 2002). These channels are critical for mucociliary clearance and their activation potentially leads to chronic airway disease similar to smoking. Less known are possible cardiovascular effects of e-cigarette use (Buchanan et al. 2020). Nicotine-containing e-cigarettes caused acute increases in arterial stiffness and blood pressure in young smokers (Vlachopoulos et al. 2016), which was seen by others. Even nicotine- and flavouring-free e-cigarettes increase blood pressure and systemic inflammation (Caporale et al. 2019), suggesting that e-liquid vehicles alone may not be benign. Adolescent e-cigarette use is a significant risk factor for future smoking (Miech et al. 2017). A meta-analysis showed that current e-cigarette users had 3.5 times the odds of smoking compared to never-smokers and never-vapers (Soneji et al. 2017). This may be due to the increased nicotine craving that is no longer satiated even with nicotine salt e-liquids containing >50 mg ml−1 nicotine (Gotts et al. 2019). The α4β2-nicotinic acetylcholine receptor is highly expressed in the brain and mediates nicotine's neurophysiological outcomes such as increased dopamine levels, eliciting reward effects (Benowitz, 2010). Chronic nicotine exposure causes desensitization and requires higher nicotine concentrations to be effective. Thus, vaping nicotine is expected to lead to addiction in never-smokers. Indeed, recent studies demonstrate that cotinine levels and vaping frequency increased in adolescent e-cigarette users who continued to vape over 12 months and that 28.8% of previous sole e-cigarette users started using traditional cigarettes to satisfy their craving (Vogel et al. 2019). Most e-liquids contain flavours that increase their appeal to users who normally would not smoke. A survey of US adolescents found that wide availability of flavours is the most frequent factor for decision to vape (Ambrose et al. 2015). Users who vape non-menthol flavours have greater odds of continuing to vape and more frequent vape use after 6 months (Leventhal et al. 2019). However, flavouring compounds themselves can have adverse effects on airway epithelial cells (Sassano et al. 2018). For instance, direct exposure to cinnamaldehyde has irreversible effects on ciliary beating of airway cultures (Clapp et al. 2019) and impairs immune responses (Clapp et al. 2017). A recent surge in vaping-related acute lung injury in the USA, termed EVALI, highlights the unique risks associated with unregulated modification of e-liquid with VEA and other harmful substances. As of 14 January 2020, 2668 hospitalized EVALI cases have been reported (Krishnasamy et al. 2020). Though the main culprit for EVALI is unknown, a candidate is VEA found in all BAL samples from EVALI patients who underwent bronchoscopy (Blount et al. 2019). While differing degrees of respiratory disease are observed in EVALI patients, the potential for recovery from EVALI and long-term health implications are currently unknown. Accumulating novel evidence indicates that vaping may not be as benign as perceived. In addition, e-cigarettes may not necessarily be a ‘harm reduced’ tobacco alternative (Hiemstra & Bals, 2018), especially since quitting rates with vaping seem equal to smoking cessation attempts with nicotine replacement and medication therapy, but with a high continued vaping rate at 1 year (Hajek et al. 2019). Worse, widespread e-cigarette use is exposing a new generation of non-smokers to possible nicotine addiction and adverse health effects. Therefore, we should advocate against vaping in non-smokers and possibly pause with recommending vaping for smoking cessation. Readers are invited to give their views on this and the accompanying CrossTalk articles in this issue by submitting a brief (250 word) comment. Comments may be submitted up to 6 weeks after publication of the article, at which point the discussion will close and the CrossTalk authors will be invited to submit a ‘LastWord’. Please email your comment, including a title and a declaration of interest, to jphysiol@physoc.org. Comments will be moderated and accepted comments will be published online only as ‘supporting information’ to the original debate articles once discussion has closed. Samuel Chung is a Postdoctoral Scholar in the Salathe Lab. His work focuses on how airway nicotine receptors mediate e-cigarette vapour constituents’ effects on mucociliary clearance. Charles D. Bengtson is an Assistant Professor of Medicine at the University of Kansas Medical Centre (KUMC). His clinical and research interests include the acute health effects of e-cigarette use and the role of hyperglycaemia on pulmonary outcomes in cystic fibrosis. Michael D. Kim is a Research Assistant Professor of Medicine at KUMC. His interests include the role of glucose metabolism on ion channel function in the airway epithelium, with particular focus on cystic fibrosis. Matthias Salathe is the Peter T. Bohan Professor of Medicine and Chair of the Department of Internal Medicine at KUMC. His research focuses on mechanisms regulating homeostasis of the airway epithelium and impaired mucociliary clearance observed in smoking-related chronic bronchitis and cystic fibrosis. His group specializes in basic and translational studies on the acute and chronic health effects of vaping amongst nonsmokers and current smokers. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. None. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. This work was funded by NIH-NHLBI – F32-HL140729 (S.C.); NCATS – TL1TR002368 (C.D.B.); NIH-NHLBI – R01-HL139365 (M.S.); James and Esther King Florida Biomedical Research Program – no. 5JK02 (M.S.); and FAMRI – CIA no. 130033 (M.S.)

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.

Protein interaction network (PIN) or interactome has been mapped vigorously for the entire genome. We recognize, nonetheless, that such a map could illuminate profound insights had its context been revealed. We describe a scalable protein lableling method that could re-supply natural context back to the map of protein interactome. Genetically encoded fluorescent proteins, position-specific genomic integration and GAL4-responsive expression control enable labeling proteinsA, BandCeach with a either an eGFP, mCherry or NirFP in specified cells of optically transparent animals such asDrosophilaembryos. While following multiple proteins through development and behavior, these labels offer separable pairs of Förster resonance energy transfer between proteinsAandBand proteinsBandC. We test and observe FRET interactions between specific protein pairs controlling cytoskeleton, nuclear signaling and cell polarity. By using our protein labeling method, it will be possible to map protein interaction networkin situ— isPIN.

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.