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

Abstract

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.

Keywords: Airway epithelium; CFTR; Mucus; e-cigarette; vegetable glycerin.

FIGURE 1

VG and PG/VG e-cig aerosols impair nasal CFTR function in vivo. (A) Consolidated Standards of Reporting Trials diagram demonstrating the flow of participants and their samples used in this study. Compliance was defined as initiating at least 40 puffs per day with ≥ 200 s daily puff time. Nasal potential difference (NPD) recordings and nasal brushings were collected at baseline and Day 8. (B) Example NPD recordings from a volunteer who vaped VG e-liquid performed at initial (baseline) and final (Day 8) visits. (C) Before-after plots of nasal CFTR function (measured as change following “Zero Cl⁻” and “Isoproterenol” treatments) from all volunteers at baseline (BL) and at Day 8 after one week of vaping. n = 7. The reduction in nasal CFTR activity is seen in all volunteers who inhaled aerosol generated from e-liquids containing VG (PG/VG and VG only; n = 3).

FIGURE 2

Inflammatory markers in nasal cells from volunteers who vaped VG and PG/VG e-liquids. (A,B) Before-after plots of mRNA expressions of IL6, IL8, MMP2, MMP9, and TGFB1 measured in HNECs at baseline (BL) and day 8 from volunteers who vaped VG (A) or PG/VG (B). n = 4 for VG, n = 3 for PG/VG.

FIGURE 3

Levels of MMP-9 activity and MUC5AC protein in nasal epithelial lining fluid (ELF) from volunteers who vaped VG e-liquid. (A,B) Before-after plots of levels of MMP-9 activity (A) and MUC5AC protein (B) measured from nasal ELF at baseline (BL) and day 8 from volunteers who vaped VG. n = 3.

FIGURE 4

Effects of VG e-cig aerosols in a large animal model (sheep). (A) Five-day exposure of sheep to 100% VG e-cig aerosols causes a significant increase in mucus concentrations (measured as % mucus solids from tracheal secretions) after five days. n = 6 from 2 sheep for baseline, n = 9 from 3 sheep for Day 5. (B) Five-day exposure of sheep to VG aerosols causes a significant increase in MMP-9 activity measured from tracheal secretions. n = 6 from 2 sheep for baseline, n = 9 from 3 sheep for Day 5. (C–E) Five-day exposure of sheep to VG e-cig aerosols does not cause a significant change in plasma levels of IL-6 (C) or IL-8 (D) proteins but causes a significant increase in the expression of TGF-β1 protein (E) in plasma. n = 6 from 2 sheep (C) or n = 9 from 3 sheep (D,E). Statistics: Data are presented as mean ± SEM. *p < 0.05, ns = not significant. Data were analyzed using a mixed-effects model.

FIGURE 5

VG e-cig aerosols induce mucociliary dysfunction in primary HBECs in vitro. (A) Mucus concentrations are significantly increased in HBECs exposed to VG e-cig aerosols after five days compared to air-exposed controls. n = 6 from 5 lungs. (B) Ciliary beat frequency (CBF) of HBECs exposed to VG e-cig aerosols is significantly reduced after five days compared to air-exposed controls. n ≥ 23 from 5 lungs. (C) Five-day exposure of HBECs to VG e-cig aerosols does not affect cytotoxicity as assessed by lactate dehydrogenase (LDH) release into basolateral media. n = 6 lungs. Statistics: Data are presented as mean ± SEM. *p < 0.05, ns = not significant. Data were analyzed by two-tailed t-test (A,C) or mixed-effects model (B) after assessing normality by Shapiro-Wilk.

FIGURE 6

VG aerosols impair CFTR function in primary HBECs in vitro by reducing membrane fluidity. (A) VG only aerosols cause a significant reduction in CFTR function compared to air-exposed HBECs after seven days. n = 10 lungs. (B) HBECs exposed to VG aerosols for two days have significantly reduced CFTR function compared to air only controls. n = 8 lungs. (C) VG exposure reduces total MC540 emissions, indicative of reduced membrane fluidity, while air only controls do not cause a change in MC540 emissions. n = 8 lungs. (D) Schematic illustrating the activities of cholesterol oxidase and cholesterol esterase in cholesterol metabolism pathway. (E) To reduce membrane fluidity, HBECs were acutely treated with cholesterol oxidase (COase, 1 U/ml, 30 min). COase-treated HBECs have significantly reduced CFTR function compared to buffer only controls. n = 6 from 4 lungs. (F) To increase membrane fluidity, VG-exposed HBECs were treated with CEase (0.2 U/ml, 30 min). CEase significantly improves CFTR function. n = 7 lungs. Statistics: Data are presented as mean ± SEM. *p < 0.05, ns = not significant. Data were analyzed by two-tailed paired t-test (A,B), one-tailed Wilcoxon test (C), or one-tailed paired t-test (E,F) depending on normality assessment by Shapiro-Wilk.

FIGURE 7

Inflammatory markers in primary HBECs exposed to VG e-cig aerosols. (A–E) VG aerosol exposure significantly increases IL6 (B), IL8 (C), and TGFB1 (E), but not IL1B (A) and MMP9 (D), mRNA expressions in HBECs after seven days. (F) Seven-day exposure of HBECs to VG aerosols causes a significant increase in MUC5AC mRNA expression. n ≥ 12 lungs. Data are shown as relative to GAPDH and air control. Statistics: Data are presented as mean ± SEM. *p < 0.05, ns = not significant. Data were analyzed by two-tailed Wilcoxon test or two-tailed paired t-test depending on normality assessment by Shapiro-Wilk.