E-cigarette vapour enhances pneumococcal adherence to airway epithelial cells

Abstract

E-cigarette vapour contains free radicals with the potential to induce oxidative stress. Since oxidative stress in airway cells increases platelet-activating factor receptor (PAFR) expression, and PAFR is co-opted by pneumococci to adhere to host cells, we hypothesised that E-cigarette vapour increases pneumococcal adhesion to airway cells.Nasal epithelial PAFR was assessed in non-vaping controls, and in adults before and after 5 min of vaping. We determined the effect of vapour on oxidative stress-induced, PAFR-dependent pneumococcal adhesion to airway epithelial cells in vitro, and on pneumococcal colonisation in the mouse nasopharynx. Elemental analysis of vapour was done by mass spectrometry, and oxidative potential of vapour assessed by antioxidant depletion in vitroThere was no difference in baseline nasal epithelial PAFR expression between vapers (n=11) and controls (n=6). Vaping increased nasal PAFR expression. Nicotine-containing and nicotine-free E-cigarette vapour increased pneumococcal adhesion to airway cells in vitro Vapour-stimulated adhesion in vitro was attenuated by the PAFR blocker CV3988. Nicotine-containing E-cigarette vapour increased mouse nasal PAFR expression, and nasopharyngeal pneumococcal colonisation. Vapour contained redox-active metals, had considerable oxidative activity, and adhesion was attenuated by the antioxidant N-acetyl cysteine.This study suggests that E-cigarette vapour has the potential to increase susceptibility to pneumococcal infection.

Figure 1

(a) Platelet-activating factor receptor (PAFR) expression in nasal epithelial cells from vaping adults (n=11) prior to experimental E-cigarette vapour exposure (baseline) and in non-vaping controls (n=6). There is no difference between the two groups (p =0.18). Data are expressed as mean (SEM) and analysed by t test, (b) change in PAFR expression (expressed as median fluorescence intensity (MFI) adjusted for isotopic control) in nasal epithelial cells from adult vapers (n=11), before, and 1 h after, 5 min vaping (*p <0.05, paired t-test).

Figure 2

(a) Dose-dependent effect of nicotine-free (N-) and nicotine-containing (N+) E-cigarette vapour extract (ECV). Increased pneumococcal adhesion, reflected by increased colony forming unit count (CFU), is significant at 5% for N- ECV and 2% for N+ ECV, (b) effect of 5% N- ECV and N+ ECV on pneumococcal entry into A549 cells. Intracellular bacteria were assessed after treatment with antibiotics to kill cell surface bacteria, (c) effect of ECV on platelet-activating factor receptor (PAFR) expression in A549 cells. Expression was determined by flow cytometry and expressed as median fluorescence intensity, (d) effect of the PAFR blocker CV3988 on pneumococcal adhesion to A549 cells after exposure to either 5% N-, or 5% N+ ECV. Data, from either 6 or 7 separate experiments, are expressed as median (IQR; interquartile range), and analysed by Kruskal-Wallis with post-hoc multiple comparison testing. *p <0.05, **p <0.01, ***p <0.001.

Figure 3

Effect of the antioxidant N-acetyl cysteine (NAC) on pneumococcal adhesion to; (a) A549, and (b) BEAS-2B cells cultured with either 5% nicotine-free (N-) E-cigarette vapour extract (ECV), or 5% nicotine-containing (N+) ECV. Data, from either 4 or 5 separate experiments, are expressed as median (IQR; interquartile range) and analysed by Kruskal-Wallis with post-hoc multiple comparison testing. *p <0.05.

Figure 4

Adhesion of S. pneumoniae D39 to; (a) primary nasal HPNEpC cells, (b) primary bronchial HBEpC cells, and (c) bronchial cell line BEAS-2B, after exposure to either 5% nicotine-free (N-) E-cigarette vapour extract (ECV), 5% nicotine-containing (N+) ECV, or DPBS control, with and without incubation with PAFR blocker CV3988. Data from 6 separate experiments are expressed as median (IQR; interquartile range) and analysed by Kruskal-Wallis with post-hoc multiple comparison testing. *p <0.05, and **p <0.01.

Figure 5

Effect of nicotine-free (N-) E-cigarette vapour extract (ECV), and nicotine-containing (N+) ECV on platelet-activating factor receptor (PAFR) median fluorescence intensity (MRI) determined by flow cytometry in; (a) primary nasal HPNEpC cells, (b) primary bronchial HBEpC cells, and (c) BEAS-2B cells. Data, from 6 to 10 separate experiments, are expressed as median (IQR; interquartile range), and analysed by Kruskal-Wallis with post-hoc multiple comparison testing. *p <0.05, **p <0.01, and ***p <0.001, (d) PAFR MFI in nasal epithelial cells obtained from vapers (n=8) before vaping (baseline), and then cultured in vitro with 5% nicotine containing (N+) ECV extract for 2.5 h. Data are analysed by paired t-test. **p <0.01.

Figure 6

(a) Nasopharyngeal carriage of S. pneumoniae in mice exposed to either nicotine-free (N-) E-cigarette vapour extract (ECV), or nicotine-containing (N+) ECV, or DPBS control. Mice (DPBS controls; n= 17 controls; N- and N+ ECV; n=18) were dosed intranasally twice daily with 100% N+ ECV, 100% N- ECV, or DPBS. On the 4th day of dosing, mice were infected with 1 x 10⁵ CFU S. pneumoniae. Mice were culled on the 4^th day post-infection and nasopharyngeal tissue collected. Colony forming unit count (CFU) values of S. pneumoniae are given as count per mL, (b) PAFR expression determined by flow cytometry (median fluorescence intensity; MFI) in nasopharyngeal epithelial cells of mice after exposure to 100% N+ ECV. Expression was determined on the 4^th day of intranasal ECV dosing. Data are expressed as mean (SEM) and analysed by one-way ANOVA with post hoc comparison testing; *p <0.05, ***p <0.001.

Figure 7

The percentage loss of (a) ascorbate, and (b) glutathione from a synthetic respiratory tract lining fluid (RTLF) following a 4 h incubation with 5% nicotine-free (N-) E cigarette vapour extract (ECV), and 5% nicotine-containing (N+) ECV. Included in the assay is a control particle with low oxidative potential (M120), and a particle with higher oxidative potential (SRM-1648). The percentage loss is expressed relative to a particle/ECV-free control. Data are expressed as mean (SEM), and analysed by ANOVA with post-hoc multiple comparison testing (n=9). **p <0.01, and ***p <0.001.