Chronic E-cigarette use increases neutrophil elastase and matrix metalloprotease levels in the lung

Rationale: Proteolysis is a key aspect of the lung's innate immune system. Proteases, including neutrophil elastase and MMPs (matrix metalloproteases), modulate cell signaling, inflammation, tissue remodeling, and leukocyte recruitment via cleavage of their target proteins. Excessive proteolysis occurs with chronic tobacco use and is causative for bronchiectasis and emphysema. The effect of e-cigarettes (vaping) on proteolysis is unknown. Objectives: We used protease levels as biomarkers of harm to determine the impact of vaping on the lung. Methods: We performed research bronchoscopies on healthy nonsmokers, cigarette smokers, and e-cigarette users (vapers), and determined protease levels in BAL. In parallel, we studied the effects of e-cigarette components on protease secretion in isolated human blood neutrophils and BAL-derived macrophages. We also analyzed the nicotine concentration in induced sputum and BAL. Measurements and Main Results: Neutrophil elastase, MMP-2, and MMP-9 activities and protein levels were equally elevated in both vapers' and smokers' BAL relative to nonsmokers. In contrast, antiprotease levels were unchanged. We also found that exposure of isolated neutrophils and macrophages to nicotine elicited dose-dependent increases in protease release. After vaping, measurable levels of nicotine were detectable in sputum and BAL, which corresponded to the half-maximal effective concentration values for protease release seen in immune cells. Conclusions:We conclude that vaping induces nicotine-dependent protease release from resident pulmonary immune cells. Thus, chronic vaping disrupts the protease-antiprotease balance by increasing proteolysis in lung, which may place vapers at risk of developing chronic lung disease. These data indicate that vaping may not be safer than tobacco smoking

Molecular and functional expression of anion exchangers in cultured normal human nasal epithelial cells

AIMS: Anions have an important role in the regulation of airway surface liquid (ASL) volume, viscosity and pH. However, functional localization and regulation of anion exchangers (AEs) have not been clearly described. The aim of this study was to investigate the regulation of AE mRNA expression level in accordance with mucociliary differentiation and the functional expression of AEs cultured normal human nasal epithelial (NHNE) cells. METHODS: Nasal mucosal specimens from three patients are obtained and serially cultured cells are subjected to morphological examinations, RT-PCR, Western blot analysis and immunocytochemistry. AE activity is assessed by pHi measurements. RESULTS: Expression of ciliated cells on the apical membrane and expression of MUC5AC, a marker of mucous differentiation, increased with time. AE2 and SLC26A4 mRNA expression decreased as mucociliary differentiation progressed, and AE4, SLC26A7 and SLC26A8 mRNA expression increased on the 14th and 28th day after confluence. Accordingly, AE4 protein expression also progressively increased. AE activity in 100 mM K(+) buffer solutions was nearly twofold higher than that in 5 mM K(+) buffer solutions. Moreover, only luminal AE activity increased about fourfold over the control in the presence of 5 microM forskolin. In the presence of 100 microM adenosine-5'-triphosphate (ATP) which evokes intracellular calcium signalling through activation of purinergic receptors, only luminal AE activity was again significantly increased. On the other hand, 500 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of most SLC4 and SLC26AE isoforms, nearly abolished AE activity in both luminal and basolateral membranes. We found that AE activity was affected by intracellular cAMP and calcium signalling in the luminal membrane and was DIDS-sensitive in both membranes of cultured NHNE cells. CONCLUSION: Our findings through molecular and functional studies using cultured NHNE cells suggest that AEs may have an important role in the regulation of ASL.ope

Defective postsecretory maturation of MUC5B mucin in cystic fibrosis airways

In cystic fibrosis (CF), airway mucus becomes thick and viscous, and its clearance from the airways is impaired. The gel-forming mucins undergo an ordered "unpacking/maturation" process after granular release that requires an optimum postsecretory environment, including hydration and pH. We hypothesized that this unpacking process is compromised in the CF lung due to abnormal transepithelial fluid transport that reduces airway surface hydration and alters ionic composition. Using human tracheobronchial epithelial cells derived from non-CF and CF donors and mucus samples from human subjects and domestic pigs, we investigated the process of postsecretory mucin unfolding/maturation, how these processes are defective in CF airways, and the probable mechanism underlying defective unfolding. First, we found that mucins released into a normal lung environment transform from a compact granular form to a linear form. Second, we demonstrated that this maturation process is defective in the CF airway environment. Finally, we demonstrated that independent of HCO3- and pH levels, airway surface dehydration was the major determinant of this abnormal unfolding process. This defective unfolding/maturation process after granular release suggests that the CF extracellular environment is ion/water depleted and likely contributes to abnormal mucus properties in CF airways prior to infection and inflammation

Combustible and Electronic Cigarette Exposures Increase ACE2 Activity and SARS-CoV-2 Spike Binding

To the Editor: The outbreak of coronavirus disease (COVID-19) has extensively impacted global health. The spike protein on the surface of the causative pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to the angiotensin-converting enzyme 2 (ACE2) receptor, a metallocarboxypeptidase, which is expressed in both mACE2 (membrane-anchored ACE2) and sACE2 (soluble ACE2) forms in the lung. Although mACE2 is responsible for viral entry, recent observations also suggest that sACE2 is involved, by its interaction with the spike protein, followed by receptor-mediated endocytosis of the viral particles (1). Tobacco use has been speculated as a risk factor for contracting SARS-CoV-2 infection and subsequent disease severity (2, 3), and electronic cigarettes (e-cigarettes) have been shown to induce harmful proteomic and immune changes in the lungs of vapers (4). In addition, the effect of vaping, and the role of nicotine, in the regulation of ACE2 expression has been demonstrated in animal models and cell culture systems (5–8). We therefore tested the hypothesis that combustible tobacco (e.g., cigarettes) and noncombustible e-cigarettes could affect ACE2 activity and subsequent SARS-CoV-2 infection. Some of our data have been reported previously at an international conference (9) and deposited in a preprint repository (https://doi.org/10.1101/2021.06.04.447156)

Mucus Hydration in Subjects with Stable Chronic Bronchitis: A Comparison of Spontaneous and Induced Sputum

Mucus hydration is important in mucus clearance and lung health. This study sought to test the relative utility of spontaneous sputum (SS) versus the reasonably noninvasive induced sputum (IS) samples for measurement of mucus hydration. SS and IS samples were collected over a 2-day study interval. Sputum was induced with escalating inhaled nebulized 3–5% hypertonic saline. Viscous portions of the samples (“plugs”) were utilized for percent solids and total mucin analyses. Cytokines, nucleotides/nucleosides and cell differentials were measured in plugs diluted into 0.1% Sputolysin. Overall, 61.5% of chronic bronchitis (CB) subjects produced a SS sample and 95.2% an IS sample. Total expectorate sample weights were less for the SS (0.94 ± 0.98 g) than the IS (2.67 ± 2.33 g) samples. Percent solids for the SS samples (3.56% ± 1.95; n = 162) were significantly greater than the IS samples (3.08% ± 1.81; n = 121), p = 0.133. Total mucin concentrations also exhibited a dilution of the IS samples: SS = 4.15 ± 3.23 mg/ml (n = 62) versus IS= 3.34 ± 2.55 mg/ml (n = 71) (p = 0.371). Total mucins (combined SS and IS) but not percent solids, were inversely associated with FEV 1 percent predicted (p = 0.052) and FEV 1 ,/FVC % (p = 0.035). There were no significant differences between sample types in cytokine or differential cell counts. The probability of sample collections was less for SS than IS samples. Measurements of hydration revealed modest dilution of the IS samples compared to SS. Thus for measurements of mucus hydration, both SS and IS samples appear to be largely interchangeable

A daily snack containing green leafy vegetables, fruit and milk before and during pregnancy prevented gestational diabetes in a randomized controlled trial in Mumbai

Background: Prospective observational studies suggest that maternal diets rich in leafy green vegetables and fruit may help prevent gestational diabetes mellitus (GDM).Objective: Our objective was to test whether increasing women's dietary intake of leafy green vegetables, fruit, and milk before conception and throughout pregnancy reduced their risk of GDM.Methods: Project SARAS (''excellent'') (2006-2012) was a nonblinded, individually randomized, controlled trial in women living in slums in the city of Mumbai, India. The interventions included a daily snack made from leafy green vegetables, fruit, and milk for the treatment group or low-micronutrient vegetables (e.g., potato and onion) for the control group, in addition to the usual diet. Results for the primary outcome, birth weight, have been reported. Women were invited to take an oral-glucose-tolerance test (OGTT) at 28-32 wk gestation to screen for GDM (WHO 1999 criteria). The prevalence of GDM was compared between the intervention and control groups, and Kernel density analysis was used to compare distributions of 120-min plasma glucose concentrations between groups.Results: Of 6513 women randomly assigned, 2291 became pregnant; of these, 2028 reached a gestation of 28 wk, 1008 (50%) attended for an OGTT, and 100 (9.9%) had GDM. In an intention-to-treat analysis, the prevalence of GDM was reduced in the treatment group (7.3% compared with 12.4% in controls; OR: 0.56; 95% CI: 0.36, 0.86; P = 0.008). The reduction in GDM remained significant after adjusting for prepregnancy adiposity and fat or weight gain during pregnancy. Kernel density analysis showed that this was explained by the fact that fewer women in the treatment group had a 2-h glucose concentration in the range 7.5-10.0 mmol/L.Conclusions: In low-income settings, in which women have a low intake of micronutrient-rich foods, improving dietary micronutrient quality by increasing intake of leafy green vegetables, fruit, and/or milk may have an important protective effect against the development of GDM