Background
The contribution of exposure to cigarette smoke (CS) to the development and progression of chronic obstructive pulmonary disease (COPD), cancer, and cardiovascular diseases is widely recognized [
1,
2]. Electronic cigarettes (ECIGs) are commercially available since 2004, but patents for similar devices reach back to 1965 [
3,
4]. ECIGs are devices that produce a vapor by heating a liquid [
3] that usually contains a mixture of glycerol, propylene-glycol, water, flavors, and different concentrations of nicotine. The flavors cover a broad range of tastes from fruit or spices, to different brands of tobacco. An intense scientific and political discussion about the toxicity and potential harm reduction of ECIGs is ongoing.
Glycerol-propylene-glycol-water mixtures have been in use for a long time as artificial fog in aviation emergency training and entertainment business, but only a few studies about possible side effects exist before the emergence of ECIGs [
5,
6]. Several studies have analyzed the effects of ECIGs on lung cells with the goal to evaluate toxic effects on cells and tissues [
7]. A number of negative outcomes on different tissue culture systems (cell death, impaired repair, oxidative stress) have been reported [
7‐
9]. In parallel, several studies compared ECIGs with TCIGs and often found decreased acute toxicity [
10‐
16]. ECIG-vapor induced DNA-strand breaks in vitro [
17] that are known to be induced by oxidative modifications of DNA by free radicals [
18]. Several studies showed increased cytotoxicity and oxidative stress of flavored ECIG-vapor in vitro and in vivo [
8,
19‐
21]. Additionally, it has been shown that ECIG-vapor may damage the pulmonary endothelial barrier and induce pulmonary neutrophilic inflammation [
22,
23]. A recent study showed differences in gene expression in differentiated bronchial epithelial cells between TCIG- and ECIG-exposed cells, with and without nicotine [
24], and showed differences in gene expression signatures in various pathways like phospholipid and fatty acid triacylglycerol, which were significantly enriched after ECIG-exposure.
In the present study we aimed to compare the acute effects of ECIG-vapor and TCIG-smoke on inflammation, host defense and cellular activation of human airway epithelial cells. We used an in vitro TCIG-exposure model [
25] and adopted it to the vaporization of ECIG-liquid. Although we are aware that the composition of TCIG-smoke, besides nictotine and glycerol, is highly different from ECIG-vapor, we normalized the amount of ECIG-vapor to the content of nicotine from our established TCIG-exposure model. This has not been done in previously published studies and was important for direct comparison of the effects of ECIG-vapor and TCIG-smoke exposure. We chose nicotine consumption as a normalization factor to account for the needs of smokers to meet their demands for nicotine, when switching between TCIGs and ECIGs.
Conclusion
The main finding of the present study is that ECIGs impact on the biology of airway epithelial cells with the release of inflammatory mediators but no overt reduction of antibacterial host defense. The acute toxic effect of ECIGs appeared to be less as compared to TCIG-exposure. Based on gene expression analysis, the expression patterns of TCIG-exposed cells were more different from sham exposed cells than ECIG-exposed cells.
Antimicrobial host defense is a fundamental function of epithelial tissues and comprises an active antimicrobial activity mediated by soluble molecules [
41] and the formation of barriers that separate the inside from the outside. Exposure to TCIG is known to cause a breach of epithelial host defense [
27] and impair the barrier function [
42]. In the present study we did not observe a change in antimicrobial activity or barrier integrity after exposure of differentiated airway epithelium to ECIG-vapor. The results from the TCIG-exposed cells are in line with previous reports that TCIG-smoke leads to a defect in host defense and disruption of the epithelial barrier in vivo and in vitro [
27,
34,
43]. We showed earlier that smoking is associated with reduced concentrations of hBD2 in airway secretions of patients with community acquired pneumonia, and that exposure of differentiated pHBE to volatile TCIG-smoke leads to a decreased expression and synthesis of hBD2 after infection with bacteria and an increased inflammatory reaction [
27]. A study by Pace et al. showed that the expression of hBD2 is reduced in central airways of smokers and that the expression hBD2 positively correlates with lung function in COPD [
44]. Exposure of epithelial cell lines with ECIG-vapor caused a decrease of antibacterial host defense and increased biofilm formation in [
18,
45]. In a clinical study, ECIG use was associated with decreased expression of immune-related genes [
46]. The application of ECIG-fluid, not vapor, to primary epithelial cells resulted in increased inflammation and susceptibility to virus infection [
47].
Exposure of the lung to inhaled smoke or other noxious components is often associated with inflammation, which is involved in host defense, systemic reaction, and repair. There is a large body of data that exposure of airway epithelial cells to TCIG-exposure results in the release of a complex mixture of inflammatory mediators [
42,
48‐
50] albeit the compound being responsible has not been identified so far. In the present study, TCIG-smoke leads to an increased synthesis and expression of pro-onflammatory mediators. In contrast, ECIG-treatment induced a lower stimulation of IL-8 sysnthesis or transcription of other inflammatory markers like S100A7 and S100A12. However, Calu-3 cells produced significantly elevated concentrations of IL-8 after ECIG-treatment compared to NCI-H292 or pHBE. Our results are partly in line with other studies published so far. It has been shown that the exposure of different bronchial epithelial cell lines to ECIG-vapor induced the release of IL-8 (NCI-H292-cells) [
8,
51], increased IL-1β release and reduced cell proliferation (A549 cells) [
52], and impaired barrier function of BEAS-2B cells (flavoring chemicals) [
51].
To get a detailed view on the differentially regulated genes after TCIG- and ECIG-exposure, we analyzed the gene expression of differentiated airway epithelium after exposure. The changes of the transcriptome of the ECIG-exposed cells was much less as compared to that of TCIG-exposed cells. Glutathione peroxidase-2 (GPX2) is one example of a gene, whose expression is significantly deregulated in the group comparison of the top 100 most differentially expressed genes after ECIG-exposure. GPX2 reduces H
2O
2 to H
2O and O
2−, thereby oxidizing glutathione, which is reduced by glutathione reductase, that reduces NADPH to NADP [
53]. The expression of GPX2 is slightly increased by ECIG-vapor and significantly more after TCIG-treatment. In contrast to GPX2, the expression of the S100-proteins S100A7 and S100A12 was significantly upregulated 24 h after ECIG-exposure. S100 proteins comprise a family of calcium binding proteins involved in inflammation, host defense, and carcinogenesis [
40,
54]. S100A7 and S100A12 belong to a group of danger-associated proteins [
55], which bind to cell surface receptors like RAGE and induce inflammation [
56]. Additionally, S100A12 has been shown to induce the secretion of MUC5AC from airway epithelial cells [
57]. Among the genes with the highest variance in gene expressen (Fig.
3) and the significantly upgregualted genes (Fig.
4) in the TCIG-group we also found IL1-β, another prominent pro-inflammatory mediator. A recent study applied RNA-seq analysis of differentiated airway epithelial cells and found similar impact of ECIG-vapor: The effects were detectable but less as compared to conventional TCIG. Various pathways such as the phospholipid and fatty acid triacylglycerol metabolisms were significantly enriched after ECIG-exposure [
24]. Another study investigated the micro RNA (miRNA) response and the exposure of differentiated airway epithelial cells with ECIG-vapor resulted in the upregulation of oxaidative stress genes [
58]. ECIGs also modify the metabolome of epithelial cells and showing significant changes partially overlapping with the effect of TCIG [
59].
Our results indicate that ECIGs impact on epithelial biology with an effect on inflammation and metabolism. As compared to the exposure with TCIG, there was less impact on host defense, inflammation and gene expression. Other studies have shown that ECIGs induce antioxidant defenses and oxidative DNA damage in primary epithelial cells [
60‐
63]. We used two different cell lines and pHBEs to account for different reactivity to TCIG and ECIG vapor. While NCI-H292 cells retain their original mucoepidermoid characteristics with nearly diploid chromosome counts, Calu-3 cells are highly transformed adenocarcinoma cells with hypotriploid chromosome counts [
64]. The finding that ECIG-vapor induced a significant release of IL-8 only from the tumor cell line Calu-3 that was comparable with TCIG-exposed cells indicates that the vapor of ECIGs may induce inflammation in certain lung tumors, while it may not be pro-inflammatory for non-transformed bronchial epithelial cells (Fig.
2). We may speculate that this finding implicates that ECIG vapor could be more pathologic for individuals with pre-existing, yet silent cancerous lesions, but yet have to investigate this in a more detailed study.
The present study and other studies have limitations. In the first instance, all in vitro experiments provide only data on short term outcomes and do not allow to make predictions about the long-term effects of ECIG use. Therefore, it is also not possible to draw conclusions about the long-term safety or harm reduction potential of ECIGs. This study focus on specific cell lines and primary cells, while other studies focus on diverse other cellular systems.
The availability of flavors as additives for ECIGs and different types of vaporizing devices is growing and not regulated, and is not addressed in the present study. It has already been shown that falvours, especially cinnamon containing liquids, induce toxicity in vitro and in vivo [
8,
19]. The variability in taste and nicotine strength is one of the features of ECIGs that surely attracts many people but on the other hand makes standardisation in research more complicated. In addition, the various exposure systems vaporize at different temperatures. This will lead to a different taste but also different chemical decomposition of ingredients. The boiling point of most of the ingredients found in flavored liquids is below 300 °C [
20]. It has been shown, that the amount of formaldehyde, acetaldehyde and formalin is proportional to the output voltage of the device [
65]. The devices offered currently range from 1 to 75 W (Joytech eGo AIO ECO 6–8 W, iStick Pico Melo 4 D22 1–75 W), producing temperatures of up to more than 300 °C.
The aim of the present study was to compare the impact of ECIG-vapor and TCIG on airway-epithelial cell biology. Comparing two physically different agents and trying to normalize is not straight forward. ECIGs produce a fine mist, containing the vaporized ingredients of the ECIG-fluid and trace amounts of emissions from the heating element. TCIG-smoke in contrast is a mixture of combustion-degraded ingredients from tobacco containing small particles, gas, and small amounts of humidity. Nicotine is one of the few ingredients that is common to TCIG-smoke and ECIG-vapor that is absorbed, and, what is even more important, with known toxicity and addictive properties. Normalizing on nicotine consumption therefore seems reasonable, since the user will be in need for certain amounts of nicotine, which will make the overall uptake between TCIG and ECIG comparable [
66].
Only a few studies directly compare TCIG and ECIG in airway epithelial cells. One of the publications that closely reflects our setup and normalization procedures [
67] used several dilutions of ECIG and TCIG and determined the concentration of nicotine after the exposure in the chambers. Their data show a significantly different gene expression pattern between ECIG- and TCIG-exposed samples when comparing conditions with similar nicotine concentration. In addition, they also showed an upregulation of many genes after TCIG-exposure, that were similar to our experiments (i.e. IL1A, IL1B, GPX2, CYP1A1, CYP1B1, S100A12). Although a slightly different setup was used [
68], another publication showed that in comparison to TCIGs the vapor of ECIGs induced only minor changes in gene expression, although their smoking protocol used with ECIGs was more intense than the corresponding TCIG-exposure. In their 6 × 8 min TCIG-exposure protocol similar genes were upregulated like in our protocol (i.e. IL1A, IL1B, GPX2, CYP1A1, CYP1B1, S100A12) [
68]. Although different protocols for ECIG-exposure and different cells were used, both studies agree with our findings, that TCIG-exposure induces a transcriptomic profile, which is different from ECIG-exposed cells and that the majority of differentially expressed genes can be found in TCIG-exposed samples.
In conclusion, ECIG-vapor has an acute effect on the biology of AECs. ECIGs had no significant effects on the secretion of chemokines or antimicrobial peptides after bacterial stimulation but induced the expression of S100A7 and S100A12. While the effects of ECIGs on epithelial cells appears to be less toxic as compared to TCIGs, in vitro results do not permit to draw conclusions about the long-term safety.
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