We also discovered that COPD patients and smokers have an increase in apoptosis of airway epithelial cells 25 , 26 and decreased phagocytosis of apoptotic airway cells by alveolar macrophages, a process we termed efferocytosis. 26 , 27 Thus, the present comprehensive in vitro study investigated the effects of E‐cigarette components on both the efferocytic ability of macrophages and the potential cytotoxic/pro‐inflammatory effects on bronchial epithelial cells, with the potential to advise future in vivo studies.

In chronic obstructive pulmonary disease (COPD), non‐typeable Haemophilus influenzae (NTHi) is the most common cause of bacterial airway colonization. 22 We found that alveolar macrophages from smokers/COPD patients have a reduced phagocytosis of NTHi. 23 , 24 We have recently discovered that E‐cigarette vapour extract (EVE) also caused reduced phagocytosis of NTHi by macrophages via reduction of bacterial recognition receptors, 1 and other studies have also shown reduced bacterial phagocytic ability of macrophages exposed to E‐cigarette vapour. 21

There is a growing body of evidence that E‐cigarettes may still be harmful to the lungs. E‐cigarettes were shown to cause toxicity, 14 increased oxidative stress, 15 , 16 reduced proliferation, 14 loss of lung barrier function of endothelial cells 17 and were linked to an asthma‐like response 18 , 19 and increased viral susceptibility of mouse lung cells. 20 , 21 Many of the mouse studies published, however, suffer from varied methodologies or non‐physiological delivery methods, as well as lacking a solid basis in in vitro studies for selection of the E‐liquids given there are thousands of different flavour combinations.

Electronic (E)‐cigarettes are comparatively novel and often advertised as a smoking cessation tool/harm reduction. Recently, there has been a surge in the number of E‐cigarette users/vapers. The most common are individuals who are attempting to quit smoking. 1 However, youth vapers are increasing, 2 - 6 with concerning reports that they were more likely to use tobacco cigarettes. 7 - 10 E‐cigarettes are filled with an E‐liquid, which consists of a carrier base made up of propylene glycol (PG), vegetable glycerine (VG) or a mix, with a flavour added (usually food safe), and often with nicotine (varying concentrations). Fruity flavours, such as apple, are the most popular amongst users. 11 - 13 E‐cigarettes appear on the surface to be a likely safer alternative to tobacco cigarettes. However, the long‐term health risks of E‐cigarettes remain unknown. There is also a need to analyse the effects of the flavouring separate from the debate about the safety of nicotine, especially given some countries, including Australia, ban the sale of nicotine E‐liquids.

Each experiment was performed in duplicate for technical variance and data represent the mean ± SEM of four to six experiments. The Kruskal–Wallis non‐parametric analysis of variance (ANOVA) with Mann–Whitney U ‐test was employed for statistical analysis. SPSS v23 software (IBM, Armonk, NY, USA) was utilized to perform all statistical analyses, and differences between groups of P < 0.05 were considered significant.

THP‐1 macrophages treated with EVE were incubated with apoptotic 16HBE cells. Cytochalasin D was added (20 μM) for 15 min prior for negative controls. Non‐efferocytosed cells were removed, the wells rinsed thoroughly and the THP‐1 cells lifted with ice cold 1× HBSS, 10 mM HEPES (pH 8.2) and 10 000 events were recorded. Macrophages not exposed to apoptotic targets were used as a gating control. Data are expressed as the percentage of efferocytic cells (gating strategy as per Fig. S4 in Supplementary Information).

For all experiments, an EVOD‐2 was used. This device runs at 3.7 V and a resistance of 1.5 Ω. Three apple flavours were tested from two suppliers (Aussie Blue, Windag, Australia and Vape King, Sydney, Australia) in a 70% PG:30% VG base (PG:VG), including one specifically tested to confirm the absence of diacetyl and acetyl propionyl (DA‐AP; Apple 3). Nicotine was tested at 18 mg/mL in 70% PG:30%, and also added to the three flavoured E‐liquids (18 mg/mL). We also tested 100% PG alone, 100% VG alone and self‐mixed 70% PG:30% VG. Based on the average users' puff duration of 2.6 s, 28 50 × 3 s puffs with 5 s in between were bubbled through 10 mL of culture medium (RPMI 1640 medium supplemented with 2 mM l‐glutamine, penicillin (12 μg/mL), and gentamycin (16 μg/mL)) to create E‐cigarette Extract (EVE). A separate EVOD‐2 was used for all nicotine‐containing E‐liquids. The batteries were always fully charged. Control medium was obtained by using the same device to pass air through medium for the same duration as E‐cigarette use. 100% EVE for 24 h was used for all treatments. Cigarette smoke extract (CSE) was prepared as described previously 29 and 10% CSE was used as a positive control. 26 Equal volumes of each treatment were used.

Mass spectrometry revealed that there were differences in the flavouring chemicals used to create the three apple flavours with only menthol being found in common between any two flavours. Nicotine was confirmed to be absent from all three E‐liquids (Table S1 in Supplementary Information).

Only Apple 2 and 3 EVE exposure, with or without nicotine, caused a release of DNA (up to 422% of control; Fig. 6 ). Nicotine alone and Apple 1 EVE only showed a trend towards increased DNA release whilst the bases had no effects. Doses of Apple 3 EVE showed that this effect was only significant at 100% EVE (Fig. S5 D in Supplementary Information).

We previously showed that EVE altered cytokine secretion by macrophages. 1 Bronchial epithelial cells are also an important source of inflammatory cytokines. Thus, we assessed whether EVE exposure altered their cytokine secretion. TNF‐α was significantly reduced following EVE treatment (0.4–11.9 pg/mL) versus control (66.8 pg/mL) and nicotine alone also caused a reduction (6.1 pg/mL). Interestingly, the glycol bases also reduced TNF‐α secretion (5.82 pg/mL). IL‐6 was significantly reduced by EVE exposure (264–948 pg/mL) versus control (1985 pg/mL; Fig. 5 A). IP‐10 (also known as CXCL10) secretion by bronchial epithelial cells was also significantly reduced by EVE exposure (3.2–7.2 pg/mL) versus control (21.9 pg/mL). Nicotine alone also reduced IP‐10 secretion (8.1 pg/mL) (Fig. 5 C). MIP‐1α was reduced by Apple 3 EVE ± nicotine (4.3–5.7 pg/mL) versus control (17.7 pg/mL) (Fig. 5 D). MIP‐1β secretion was reduced by all three flavours EVE (6.4–35.1 pg/mL) versus control (68.9 pg/mL) and nicotine alone (21.6 pg/mL) (Fig. 5 E). IL‐8 was unchanged for all treatments (Fig. 5 F). IFN‐γ, IL‐1β, IL‐10, IL‐12p70 and MCP‐1 were below detection for this cell line.

We show that exposure to apple EVE also reduces expression of the constitutively expressed efferocytic receptor, CD36 (1063–1174 MFI) versus control (1415 MFI; Fig. 4 B). Nicotine alone also reduced CD36 (1067 MFI). Interestingly, expression of CD36 was also reduced by exposure to the glycol bases alone or in combination (1077–1121 MFI). Expression of constitutively expressed hyaluronan receptor CD44, which is also an apoptotic cell recognition receptor, was reduced for Apple 3 + nicotine EVE only (1756 MFI) versus control (2332 MFU). Ten percent CSE treatment caused a decrease in CD44 in line with previous findings (1716 MFI; Fig. 4 D). 30

Effect of E‐cigarette vapour on efferocytosis of apoptotic airway cells by macrophages. THP‐1 macrophages were treated with control media, 10% CSE or 100% EVE for 24 h. Fivefold pHrodo‐labelled apoptotic 16HBE targets were added to the macrophages for 1.5 h. Non‐efferocytosed targets were removed and the macrophages washed. THP‐1 macrophages containing apoptotic targets were assessed by flow cytometry. Data are mean ± SEM. n ≥ 4. *Significance from control. **Significant from CSE P < 0.05. 16HBE, CSE, cigarette smoke extract; CyD, Cytochalasin D; EVE, E‐cigarette vapour extract; PG, propylene glycol; VG, vegetable glycerine.

Exposure to EVE of all three apple flavours significantly decreased efferocytosis of apoptotic airway cells (10.4–13%) versus control (21.3%), as did nicotine (12.9%). The reduction in efferocytosis with treatment of 10% CSE (3.7%) was consistent with our previous publications 26 , 27 (Fig. 3 ). Testing of more diluted Apple 3 EVE showed that even dilution to 75% led to significant decreases but not at 50% or below (Fig. S5 C in Supplementary Information).

Effect of E‐cigarette vapour on primary bronchial epithelial cell necrosis, apoptosis and cell membrane integrity. Primary bronchial cells from healthy non‐smoking deceased donors were treated with control media, 10% CSE or 100% EVE for 24 h. Cells were assessed for (A) necrosis by Sytox Green, (B) apoptosis by Annexin V labelling and (C) cell membrane integrity by comparing LDH release compared with control media treated cells fully lysed with Tween20 (LDH max). Data are mean ± SEM. n = 6 (three donors, two passages, performed in duplicate). *Significance from control P < 0.05, ** P < 0.01. CSE, cigarette smoke extract; EVE, E‐cigarette vapour extract; LDH, lactate dehydrogenase; PG, propylene glycol; VG, vegetable glycerine.

We assessed toxicity of EVE on human bronchial epithelial cells, via release of LDH (Fig. 1 A), and found that two of the three apple flavours induced significant toxicity (36.2–40.1%) versus control (11.6%). EVE caused an increase in necrosis (17.7–30.2%) versus control (9.6%) (Fig. 1 B) and apoptosis (16.7–20.1%) versus control (8.7%) (Fig. 1 B) without monolayer disruption (Fig. S1 in Supplementary Information). Testing of more diluted Apple 3 EVE showed that even dilution to 75% led to insignificant increases (Fig. S5 A,B in Supplementary Information). These results were validated in primary bronchial epithelial cells (Fig. 2 ).

DISCUSSION

Many studies now show that E‐cigarettes have toxic effects on a range of cells31-36 including airway epithelial cells16, 17; however, many have been limited to looking at cell viability and looking at a range of flavours without investigating the variants in the same flavour. In this study, we assessed the EVE from three apple E‐liquids ± nicotine (18 mg/mL) from two retailers including one free of DA‐AP, which has been associated with popcorn lung.37, 38 We observed increased cytotoxicity with EVE exposure consistent with findings from other studies looking at the toxicity of EVE,15, 39-41 many of which also show the effect is dependent on the flavour.14, 42-46

Many studies have looked at cell viability with EVE exposure; however, few have specifically investigated whether it is via apoptosis. Gingival cells,47 head and neck squamous cancer cells41 and murine fibroblasts40 also showed increased apoptosis with EVE exposure. This is the first study showing EVE can cause apoptosis in epithelial lung cells. In healthy lungs, there is a basal turnover of airway cells via apoptosis and these would normally be cleared away by alveolar macrophages to prevent build‐up of apoptotic debris which can cause inflammation. We and others have previously found a higher rate of apoptosis of airway cells in COPD and smokers,25, 48, 49 and reduced ability of alveolar macrophages to efferocytose these cells.23, 30, 49-53 We and others previously showed that EVE exposure of THP‐1 PMA‐differentiated macrophages caused reduced bacterial phagocytosis.1, 21, 54, 55 We show that EVE caused a significant reduction in efferocytosis. This effect was nicotine independent/flavour dependent and nicotine alone also significantly reduced efferocytosis compared with control which is an interesting finding as nicotine is often thought not to be of concern beyond addiction. The dose of EVE used in this study does not increase cell death of the THP‐1 cells.1

Previous studies have shown cigarette smoke‐exposed and COPD alveolar macrophages had reduced efferocytic ability via reduced apoptotic cell recognition receptors including SR‐A156 and SR‐B1 (CD36),52, 57 CD44,30, 58 CD31,30 and LRP‐1/CD91.30 We recently published reduced macrophage bacterial recognition receptors post EVE treatment including SR‐A1 and TLR‐2.1 This study showed that apoptotic cell recognition receptor CD44 was only significantly reduced by one flavour EVE in the presence of nicotine whilst CD36 was significantly reduced for all treatments, including PG and VG, providing evidence that the bases themselves are not inert. The fact that there was no reduction in efferocytosis seen for the bases suggests that the effect on one single receptor is not enough to significantly affect total efferocytosis. We previously reported that SR‐A1, which recognizes apoptotic cells as well as bacteria, was also reduced,1 suggesting that this receptor may play a bigger role in efferocytosis than CD36 or CD44, or other receptors may also be involved. This is the first study that shows that E‐cigarettes may alter expression of apoptotic cell recognition receptors on the surface of macrophages resulting in reduced efferocytic ability, which in addition to increased apoptosis of bronchial epithelial, may lead in to increased inflammation in the airways.

We recently published that EVE exposure caused altered cytokine secretion by macrophages.1 Our current study assessed TNF‐α, IP‐10, MIP‐1α, MIP‐1β and IL‐6. Some showed reduction with all three apple EVE whilst others were flavour specific. Our study showed nicotine alone reduced TNF‐α, IP‐10 and MIP‐1β, adding to the data that suggests nicotine can cause harm beyond addiction. Interestingly, TNF‐α was also reduced by PG and VG, again showing that the bases may not be as inert as thought. A few studies have investigated cytokine secretion by lung cells; however, none of these were performed using the same normal bronchial epithelial cell line and exposure, E‐liquid and method/time, making comparison with our novel data difficult. One study testing A549 and BEAS‐2B cells found no change in IL‐8 or IL‐6 with EVE but a decrease with CSE.59 Another showed that E‐cigarette exposure decreased IL‐6 release by A549 lung cancer cells, but increased other cytokines including IP‐10 and MIP‐1β.15 Other studies showed that IL‐6 secretion was increased in lung cells after E‐cigarette exposure.20, 43 Mice exposed to E‐cigarettes showed increased IL‐6 in the bronchoalveolar lavage fluid (BALF).43 IL‐8 was found to be increased by human foetal lung fibroblasts exposed to only some flavours EVE for 24h.43 Normal epithelial lung cells exposed to pure flavouring chemicals showed increases in IL‐8 only with specific flavours whilst mucoepidermoid lung cells exposed to the flavouring chemicals showed no change in IL‐8.60 Mucoepidermoid lung cells,43 human foetal lung fibroblasts61 and cancerous lung epithelial cells15 exposed for a short time to one flavour of vapour directly at the air liquid interface (<1 h) had increased IL‐8 secretion. The latter study found in general that different cell lines had very different cytokine responses overall.15 Other cytokines have also been shown to have altered secretion from airway epithelial cells by E‐cigarette exposure.15, 20, 43, 60, 62 Reduction in inflammatory cytokines in vivo could lead to poorer response to foreign agents and more severe or longer infections.

However, a previous study showed that the length of exposure may be a factor in deciding whether an upregulation or downregulation is observed with CSE exposure,63 and a mouse study showed that the signs of inflammation in vaped mice varied depending on the exposure time.64 This combined with the fact that the purpose of this study was to investigate variants of apple flavourings, make it very important to consider that it is likely that flavour, exposure route and length of exposure could affect cytokine release, and that some flavours may cause inflammation and others could dampen the immune response.

As it is well known that cigarette smoke exposure can lead to release of damage‐associated molecular pattern (DAMP)/‘find me’ signals,65 we tested for the release of dsDNA as we could find no evidence of this being investigated in the literature for this cell type. We found that the release of DNA was flavour specific and independent of nicotine. This suggests that despite the right chemoattractant being released, the phagocytes will be unlikely to phagocytose their targets even if drawn to them. We look forward to reading future studies investigating DAMP release.

There was little in common between the three apple E‐liquid flavour profiles (Table S1 in Supplementary Information), and none were positive for the well‐known apple flavourings ethyl valerate or ethyl‐2‐methyl butanoate. Most studies assessing E‐liquids by mass spectrometry have focused on the presences of diacetyl, nicotine or cancerous compounds. Very few previous studies have assessed which chemicals are responsible for the flavour. Two studies simply looked for a particular library of flavouring chemicals66, 67and another assessed the flavour profile of two apple E‐liquids, there was no overlap with our flavour profiles.68 This helps emphasize our results further that no two E‐liquids labelled as the same flavour are going to have the same chemical composition, especially as each flavour can have many flavouring chemicals present.

There are limitations to our study, in that this is an in vitro/ex vivo culture system, and bronchial epithelial cells do not exist in isolation and this model removes intercellular cross‐talk leading to cytokine secretion. Human studies would also likely suffer from the wide range of E‐liquids used by the participants, which could mask flavour‐specific results that more and more studies are suggesting may be a key factor.14, 42-46, 69 As more studies, especially animal studies, using one flavoured E‐liquid per group are published, we will begin to see a clearer picture of the effects of E‐cigarettes on the lungs.

We conclude that E‐cigarettes can cause airway epithelial cell death and apoptosis and efferocytic dysfunction of macrophages via alteration of apoptotic cell recognition receptors, and can alter bronchial epithelial cell cytokine secretion pathways in a flavour‐dependent manner with some variation between different apple E‐liquids observed. Nicotine alone was also shown to affect efferocytosis and some cytokines. As such, E‐cigarettes should be treated with caution by users, especially those who are non‐smokers.