Vaping-Induced Proteolysis Causes Airway Surface Dehydration

Abstract

Proteases such as neutrophil elastase cleave and activate the epithelial sodium channel (ENaC), causing airway dehydration. Our current study explores the impact of increased protease levels in vapers' airways on ENaC activity and airway dehydration. Human bronchial epithelial cultures (HBECs) were exposed to bronchoalveolar lavage fluid (BALF) from non-smokers, smokers and vapers. Airway surface liquid (ASL) height was measured by confocal microscopy as a marker of hydration. ENaC cleavage was measured by Western blotting. Human peripheral blood neutrophils were treated with a menthol-flavored e-liquid (Juul), and the resulting secretions were added to HBECs. BALF from smokers and vapers significantly and equally increased ENaC activity and decreased ASL height. The ASL height decrease was attenuated by protease inhibitors. Non-smokers' BALF had no effect on ENaC or ASL height. BALF from smokers and vapers, but not non-smokers, induced ENaC cleavage. E-liquid-treated neutrophil secretions cleaved ENaC and decreased ASL height. Our study demonstrated that elevated protease levels in vapers' airways have functional significance since they can activate ENaC, resulting in airway dehydration. Lung dehydration contributes to diseases like cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and asthma. Thus, our data predict that vaping, like smoking, will cause airway surface dehydration that likely leads to lung disease.

Keywords: ENaC; airway surface liquid; bronchoalveolar lavage fluid; e-cigarettes; elastase; menthol.

Figure 1

Vapers’ and smokers’ BALF supernatant alters ENaC subunit expression. HEK293T cells were transfected with α-gfp, β, HA-γ-V5 ENaC subunits and treated with concentrated BALF supernatants from non-smokers, smokers and vapers for 1 h. The impact of BALF supernatant exposure on ENaC subunits is shown for (A) α-, (B) β-, (C) 5′ HA-γ and (D) 3′ V5- γ-ENaC, with bar graphs of corresponding integrated densitometry. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 different to control. All n = 6 per group. All data were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparison post-test.

Figure 2

Vapers’ and smokers’ BALF increase ENaC activity. HBECs were exposed to 15 μL of pooled BALF supernatants from non-smokers, smokers, and vapers for 6 h. (A) Basal, (B) bumetanide- and amiloride-sensitive equivalent currents (I_eq) were determined under thin-film conditions, and (C) macroelectrodes were used to determine transepithelial resistance. ** = p < 0.01, *** = p < 0.001 different as indicated. All n = 18 per group. All data were analyzed by ANOVA followed by Tukey’s post-test.

Figure 3

Vapers’ and smokers’ BALF induce airway dehydration. (A) Representative XZ-confocal micrographs of ASL (red) after 6 h of mucosal exposure to 15 μL PBS or BALF supernatant labeled with 0.1 mg/mL tetramethylrhodamine dextran. (B) Bar graph of 6 h ASL heights following exposure to mucosal BALF ± serosal bumetanide (10 mM) or BALF with mucosal protease inhibitor cocktail. * = p < 0.05 different from control. All n = 9 per group. All data were analyzed using the Kruskal–Wallis Test followed by Dunn’s Multiple Comparison post-test.

Figure 4

Juul e-cigarette condensate induces protease release from neutrophils. Peripheral blood neutrophils were exposed to vehicle (0% e-liquid) vs. 1% and 3% Juul menthol e-liquids diluted in media for 2 h, and the ANS was collected. Neutrophil elastase activity was determined by measuring the cleavage of a fluorogenic substrate by plate reader. The bar graph shows neutrophil elastase activity in ANS treated as indicated. * = p < 0.05, ** = p < 0.01 different as indicated. All n = 6 per group. All data were analyzed using the Kruskal–Wallis Test followed by Dunn’s Multiple Comparison post-test.

Figure 5

ANS from e-liquid-exposed neutrophils decreases total ENaC levels. ANS from control (naïve media), vehicle (ANS, 0% e-liquid) or Juul menthol e-liquid-treated neutrophils (1% and 3%) were added to ENaC-expressing HEK293T cells for 1 h. (A–D) Western blots and bar graphs of integrated densitometry for ENaC subunits as indicated. * = p < 0.05, ** = p < 0.01, different as indicated. All n = 4. All data were analyzed using the Kruskal–Wallis Test followed by Dunn’s Multiple Comparison post-test.

Figure 6

ANS from e-liquid-exposed neutrophils upregulates ENaC activity. HBECs were exposed to 15 μL of PBS or ANS for 6 h. (A) Basal and (B) Bumetanide- and amiloride-sensitive equivalent currents (I_eqs) were determined under thin-film conditions. (C) Macroelectrodes were used to determine transepithelial resistance. * = p < 0.05 different per group as indicated. All n = 12 per group. All data were analyzed with a Student’s t-test.

Figure 7

ANS from Juul-exposed neutrophils induces airway dehydration. HBECs were exposed mucosally to 15 μL of PBS (control), naïve ANS (0% e-liquid), ANS following 1–3% e-liquid exposure or 3% e-liquid in PBS, all labeled with 0.1 mg/mL tetramethylrhodamine-dextran and imaged by XZ-confocal microscopy 6 h later. The bar graph shows 6 h ASL heights. * = p < 0.05, ** = p < 0.01 different as indicated. All n = 6 per group. All data were analyzed using the Kruskal–Wallis Test followed by Dunn’s Multiple Comparison post-test.