Pro-inflammatory effects of e-cigarette vapour condensate on human alveolar macrophages

Abstract

Objective: Vaping may increase the cytotoxic effects of e-cigarette liquid (ECL). We compared the effect of unvaped ECL to e-cigarette vapour condensate (ECVC) on alveolar macrophage (AM) function.

Methods: AMs were treated with ECVC and nicotine-free ECVC (nfECVC). AM viability, apoptosis, necrosis, cytokine, chemokine and protease release, reactive oxygen species (ROS) release and bacterial phagocytosis were assessed.

Results: Macrophage culture with ECL or ECVC resulted in a dose-dependent reduction in cell viability. ECVC was cytotoxic at lower concentrations than ECL and resulted in increased apoptosis and necrosis. nfECVC resulted in less cytotoxicity and apoptosis. Exposure of AMs to a sub-lethal 0.5% ECVC/nfECVC increased ROS production approximately 50-fold and significantly inhibited phagocytosis. Pan and class one isoform phosphoinositide 3 kinase inhibitors partially inhibited the effects of ECVC/nfECVC on macrophage viability and apoptosis. Secretion of interleukin 6, tumour necrosis factor α, CXCL-8, monocyte chemoattractant protein 1 and matrix metalloproteinase 9 was significantly increased following ECVC challenge. Treatment with the anti-oxidant N-acetyl-cysteine (NAC) ameliorated the cytotoxic effects of ECVC/nfECVC to levels not significantly different from baseline and restored phagocytic function.

Conclusions: ECVC is significantly more toxic to AMs than non-vaped ECL. Excessive production of ROS, inflammatory cytokines and chemokines induced by e-cigarette vapour may induce an inflammatory state in AMs within the lung that is partly dependent on nicotine. Inhibition of phagocytosis also suggests users may suffer from impaired bacterial clearance. While further research is needed to fully understand the effects of e-cigarette exposure in humans in vivo, we caution against the widely held opinion that e-cigarettes are safe.

Keywords: macrophage biology; oxidative stress; respiratory infection.

Figure 1

Effect of e-cigarette vapour condensate (ECVC) and -cigarette liquid (ECL) on alveolar macrophage viability. Viability was assessed by 4 hour incubation with cell titre aqueous assay following 24 hour exposure to a range of doses with (A) ECL, (B) nicotine-free ECL (nfECL), (C) ECVC, (D) nicotine-free ECVC (nfECVC). Graphs presented as median with IQR of eight independent experiments. The central horizontal line on each box plot represents the median, the upper and lower horizontal lines represent the first (Q1) and third (Q3) quartiles, respectively, and the vertical lines represent the range of values within the limits Q1–1.5 (Q3–Q1) and Q3–1.5 (Q3–Q1). n=8, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 2

Effect of e-cigarette vapour condensate (ECVC)/nicotine-free ECVC (nfECVC) on alveolar macrophage (AM) apoptosis and necrosis. Induction of apoptosis and necrosis in AM following a 24 hour exposure to 0.8% ECVC/nfECVC. Graphs presented as median with IQR of eight independent experiments. The central horizontal line on each box plot represents the median, the upper and lower horizontal lines represent the first (Q1) and third (Q3) quartiles, respectively, and the vertical lines represent the range of values within the limits Q1–1.5 (Q3–Q1) and Q3–1.5 (Q3–Q1). n=8, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. UTC, untreated control.

Figure 3

Functional effects of e-cigarette vapour condensate (ECVC)/nicotine-free ECVC (nfECVC) exposure to alveloar macrophages (AMs) on reactive oxygen species (ROS). AMs were exposed to 0.5% ECVC/nfECVC for 4 hours. Following this, production of ROS was assessed by DCFDA assay. Graphs presented as median with IQR of eight independent experiments. The central horizontal line on each box plot represents the median, the upper and lower horizontal lines represent the first (Q1) and third (Q3) quartiles, respectively, and the vertical lines represent the range of values within the limits Q1–1.5 (Q3–Q1) and Q3–1.5 (Q3–Q1). n=8, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 4

Functional effects of e-cigarette vapour conensate (ECVC)/nicotine-free ECVC (nfECVC) exposure to alveolar macrophages (AMs). Production of inflammatory cytokines (A, B), chemokines (C, D) and (E) matrix metalloproteinase (MMP-9). AMs following 24 hour exposure to ECVC (0.5%) as assessed by ELISA. Data are presented as pg/106 live cells at the end of the experiment to account for cell loss. n=8, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 5

Functional effects of e-cigarette vapour condensate (ECVC)/nicotine-free ECVC (nfECVC) exposure to (A) alveolar macrophage (AM) and (B) THP-1 macrophage phagocytosis. Cells were exposed to 0.5% ECVC/nfECVC for 6 hours, following which uptake of pHrodo bioparticles was assessed. Graphs presented as median with IQR of eight independent experiments. The central horizontal line on each box plot represents the median, the upper and lower horizontal lines represent the first (Q1) and third (Q3) quartiles, respectively, and the vertical lines represent the range of values within the limits Q1–1.5 (Q3–Q1) and Q3–1.5 (Q3–Q1). n=8, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. UTC, untreated control.

Figure 6

Effect of antioxidant treatment on macrophages. (A) Viability following 24 hour exposure to e-cigarette vapour condensate (ECVC)/nicotine-free vapour condensate (nfECVC) in the presence or absence of N-acetyl cysteine (NAC). Graphs presented as median with IQR of eight independent experiments. (B) Apoptosis following 24 hour exposure to ECVC/nfECVC, in the presence or absence of NAC. Graphs presented as median with IQR of six independent experiments. The central horizontal line on each box plot represents the median, the upper and lower horizontal lines represent the first (Q1) and third (Q3) quartiles, respectively, and the vertical lines represent the range of values within the limits Q1–1.5 (Q3–Q1) and Q3–1.5 (Q3–Q1). n=8, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. UTC, untreated control.

Figure 7

Functional effects of e-cigarette vapour condensate (ECVC)/nicotine-free vapour condensate (nfECVC) phagocytosis. (A) Alveolar macrophages (AMs) exposed to 0.5% ECVC/nfECVC for 6 hours in the presence or absence of 1 mM NAC treatment, following which uptake of pHrodo bioparticles was assessed. (B) THP-1 macrophages were exposed to 0.5% ECVC/nfECVC for 6 hours, in the presence or absence of 5 mM NAC treatment, following which uptake of pHrodo bioparticles was assessed. Graphs presented as median with IQR of eight independent experiments. The central horizontal line on each box plot represents the median, the upper and lower horizontal lines represent the first (Q1) and third (Q3) quartiles, respectively, and the vertical lines represent the range of values within the limits Q1–1.5 (Q3–Q1) and Q3–1.5 (Q3–Q1). n=8, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. UTC, untreated control.

Figure 8

Effect of e-cigarette vapour condensate (ECVC)/nicotine-free ECVC (nfECVC) and phosphopinositol 3 kinase (PI3K) inhibitor PIK-75 on alveolar macrophage viability. Viability was assessed by 4 hour incubation with cell titre aqueous assay following 24 hour exposure to class one specific PI3K inhibitor PIK-75 and challenged with ECVC or nfECVC (0.8%). Graphs presented as median with IQR of six independent experiments. The central horizontal line on each box plot represents the median, the upper and lower horizontal lines represent the first (Q1) and third (Q3) quartiles, respectively, and the vertical lines represent the range of values within the limits Q1–1.5 (Q3–Q1) and Q3–1.5 (Q3–Q1). n=8, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. UTC, untreated control.