This is the first study to determine the effects of flavored e-cigarettes with and without nicotine on allergic airways disease. Our findings suggest that flavored e-cigarettes without nicotine can alter allergic airways disease but that the effect is dependent upon the specific flavor. Indeed, the effect of nicotine-free e-cigarette exposure varied greatly between the flavors: Cinnacide suppressed airway inflammation and increased peripheral AHR, Banana Pudding increased soluble collagen content, and there was a trend towards exaggerated airway inflammation in mice exposed to Black Licorice. On the other hand, nicotine universally dampened airway inflammation independent of flavor with no effect on AHR or airway remodeling. These findings suggest that flavored e-cigarettes have the potential to alter the pathophysiology associated with allergic airways disease, but that the direction and extent of the effect is dependent upon exposure to a specific flavor.

Our findings highlight the variable effect of flavored e-cigarettes on airway inflammation and that the suppression caused by nicotine is independent of flavor. In the present study Black Licorice exaggerated airway inflammation whereas Cinnacide caused suppression. Several studies have reported varied in vitro toxicity of flavored e-cigarettes on a variety of respiratory and non-respiratory cell types8,9,10,11. Consistent with our findings regarding Cinnacide, Clapp et al.9 reported suppressed immune responses in human alveolar macrophages and neutrophils exposed to cinnamon flavored e-cigarettes. The authors subsequently replicated these findings using the flavor additive cinnamaldehyde leading them to conclude that cinnamaldehyde was detrimental to immune cell function. This is consistent with the anti-inflammatory effect of cinnamaldehyde in a mouse model of cardiac inflammation22. Although it is likely that Cinnacide used in the present study contained cinnamaldehye, this was not confirmed and therefore we can only speculate on its role in the current findings.

In contrast to nicotine-free e-cigarette exposure, there was a ubiquitous suppression of airway inflammation with e-cigarette exposure containing nicotine. Nicotine is known to have anti-inflammatory properties23,24 and findings with tobacco smoke exposure suggest that reduced airway inflammation is due to abnormal eosinophil migration into the airways25. Reduced eosinophilia in mice with allergic airways disease exposure to e-cigarette containing nicotine is consistent with reduced eosinophilia in asthmatics who smoke tobacco cigarettes compared to asthmatic non-smokers26. Furthermore, a recent study reported that e-cigarette vapor condensate led to alveolar macrophage apoptosis and necrosis, both of which occurred to a greater extent when the exposure contained nicotine27. Alveolar macrophages are crucial to innate immunity28 and suppression of macrophage immune function by e-cigarettes would be expected to compromise the immune response to infection. In addition, wide-spread suppression of immune-related gene expression has been reported in nasal epithelial cells from e-cigarette users29. The airway epithelium is also crucial in coordinating the innate immune response to infection30 with abnormal airway epithelial responses correlated with the severity of viral exacerbations in asthma31. Indeed, there is growing in vitro and mouse model evidence that e-cigarette use is associated with impaired anti-bacterial and anti-viral responses32,33,34. Although exposure to Cinnacide without nicotine suppressed airway inflammation in the present study, it is unclear whether this reflects the potential to contribute to an impaired response to infection. However, recent findings of reduced airway ciliary function in vitro35 strengthen the speculation that cinnamon-flavored e-cigarettes may contribute to immune dysfunction.

There is currently a lack of evidence in both animal models and human data on the effect of e-cigarettes on lung function. In the present study, AHR was increased in mice exposed to Cinnacide without nicotine, despite having reduced airway inflammation. Cinnamaldehye, the characteristic component of cinnamon flavoring, is known to activate transient receptor potential ankyrin 1 (TRPA1) in the upper airways leading to cough36. However, this would be expected to increase AHR measured by central airway narrowing (Rn) and not tissue elastance, as reported in the present study. The reduction in in vitro ciliary function in response to cinnamon flavored e-cigarette exposure37 would be expected to reduce mucus clearance. Mucus production during methacholine challenge is thought to contribute to AHR in allergic airways disease via increased airway closure, as measured by tissue elastance (H)38. Therefore, an impairment in mucus clearance would lead to increased airway closure during methacholine challenge and increased AHR as measured by H, as seen in the present study following exposure to the Cinnacide e-cigarette flavor.

There was no effect of exposure to the vehicle humectant PG/VG or e-cigarettes containing nicotine on AHR. However, we utilized a murine model of allergic airways disease that involves relatively short-term exposure to e-cigarette aerosol. Two previous studies have measured the effect of long-term e-cigarette exposure on airway mechanics and AHR in non-allergic mice. Larcombe39 reported worse baseline mechanics in mice exposed to tobacco flavored e-cig with 100% VG or 100% PG for 8 weeks, with and without nicotine. Furthermore, exposure to 100% VG increased AHR, again regardless of the presence of nicotine, while 100% PG had no effect. In contrast, Garcia-Arcos40 reported increased AHR following 16 weeks of exposure to nebulized e-cigarette liquid containing nicotine (50% PG/VG) but not nicotine-free liquid. Importantly, the two studies differ in respect to heating of the liquid, since Garcia-Arcos et al.40 nebulized the liquid whereas Larcombe et al.39 used a commercial e-cigarette device. Therefore these findings may reveal that long-term nicotine inhalation promotes AHR whereas the development of AHR due to vegetable glycerin requires both long-term exposure and heating of the liquid. This is possibly related to the production of toxic aldehydes and/or acrolein caused by heating of vegetable glycerin41 or metal nanoparticles emitted from device itself when heated42.

There is a similar lack of evidence on the effect of e-cigarettes on lung function in humans. Thirty minutes of nicotine-free e-cigarette use (70% PG/30% VG) was shown to have no effect on spirometric or forced oscillation technique measurements in healthy or asthmatic non-smokers43. Similarly, Flourish et al.44 found no effect of 30 minutes of e-cigarettes containing nicotine and tobacco flavor (>60% PG) on spirometry in healthy tobacco smokers. In contrast, Ferrari et al.16 reported that five minutes of using nicotine-free e-cigarettes containing hazelnut flavor additives reduced PEF and FEV 1 in tobacco smokers but not non-smokers. Vardavas et al.17 also reported that 5 min e-cigarette use using liquid containing nicotine and tobacco flavor (>60% PG) in healthy tobacco smokers increased respiratory system resistance but did not alter reactance. Recently, Lappas et al.18 reported that 10 puffs of an e-cigarette containing nicotine and tobacco flavor worsened respiratory system resistance and reactance in healthy subjects and mild asthmatics. These effects were exaggerated in the asthmatics but had resolved to baseline within 15 min in both groups. Although there are several differences between these human studies, it was the three studies of acute use (5 min or 10 puffs) that reported abnormal lung function whereas the longer studies did not (30 min). It is therefore attractive to speculate that initial inhalation of e-cigarette aerosol induces bronchoconstriction which subsides with continued use. Indeed, one study reported such a time-dependent effect of e-cigarette use on cough reflex sensitivity45. However, the contribution of sustained, long-term use on the development of abnormal lung function and AHR remains to be elucidated.

Exposure to Banana Pudding without nicotine increased soluble lung collagen content in mice with allergic airways disease. In contrast, there was no effect of the other nicotine-free flavored e-cigarettes and all e-cigarette exposures containing nicotine. Similarly, a previous study found no effect of e-cigarette aerosol containing nicotine on lung fibrosis in non-allergic mice46. This may suggest that airway remodeling is linked to specific flavor additives rather than nicotine. For example, diacetyl (2,3-butanedione) is found in numerous common flavors such47 as “buttery/creamy” and fruit flavors, and has been detected in many flavored e-cigarette liquids48. Diacetyl and related chemicals cause lung fibrosis in rodents49 and led to bronchiolitis obliterans in workers at a microwave-popcorn plant50, highlighting the potential for flavored e-cigarettes to contribute to lung fibrosis. Additionally, Crotty-Alexander et al.51 reported renal, hepatic and cardiac fibrosis in mice following long-term (3/6 months) exposure to non-flavored e-cigarette aerosol containing nicotine suggesting the relationship between e-cigarette exposure and fibrosis may differ across different organs.

Although the present study does not directly compare to tobacco exposure, findings from several tobacco exposure models allow indirect comparison. In the present study, the effect of e-cigarettes with nicotine was confined to suppression of allergic airway inflammation, with no effect on AHR or features of airway remodeling. Lancaster et al.52 induced allergic airways disease over 2.5 weeks with tobacco cigarette smoke exposure throughout. Cigarette smoke exaggerated allergic airways inflammation, due to effects on eosinophils and neutrophils, increased AHR and increased mucous hyperplasia. Using an identical study design, Kumar et al.53 reported that tobacco cigarette smoke increased HDM-induced airway eosinophils, neutrophils and mucous metaplasia, although effects on AHR did not reach significance. Similarly, concurrent exposure to ovalbumin and tobacco cigarette smoke contributes to more severe allergic airways inflammation, due to effects on eosinophils and neutrophils, and AHR54. These findings of increased airway inflammation, AHR and features of airway remodeling in tobacco models similar to the present study may suggest that e-cigarettes are less likely to worsen asthma pathophysiology than tobacco cigarette smoking. However, the present study used a mild exposure regime when compared to those used for tobacco exposure. In our study, mice were exposed to e-cigarettes for 30 min twice/day whereas Lanckacker et al.52 and Moerloose et al.54 used four exposures/day, and Kumar et al.53 used three exposures/day. Therefore, it is possible that the lack of effect of e-cigarettes with nicotine on AHR and airway remodeling in the present study is due to reduced exposure compared to these tobacco mouse models. Nonetheless, a potential reduction in harm with e-cigarettes compared to tobacco cigarettes is consistent with the reduction in the extent and number of toxic particles in e-cigarette aerosol compared to tobacco smoke55. This speculation of reduced harm with e-cigarettes is strengthened by the single report of improved lung function, AHR and symptoms in asthmatic tobacco smokers who transition to e-cigarettes15.

The present study evaluated several different flavored e-cigarettes in a well-described animal model of allergic disease; however, there are a few limitations that should be mentioned. Firstly, liquids were purchased from different companies who are likely to have different manufacturing practices. The effect of e-cigarettes containing nicotine on airway inflammation was the same for all liquids suggesting minimal effects of any differences in manufacturing procedures. However, there was a small difference in the percentage of PG and VG between the liquids, with control and Black Licorice used at 55%/45% while the other liquids were 50%/50%. The combination of flavor aldehydes and PG produces PG acetals, with the amount measured dependent upon PG content i.e. 70% PG > 50% PG > 30% PG56. However, it is unknown whether PG acetal formation is greater with 55% PG compared to 50% PG, and whether this could lead to the increased airway inflammation measured in mice exposed to Black Licorice. Lastly, while we endeavored to use the same commercial liquid with and without nicotine, we were unable to obtain the Atomic Cinnacide flavor without nicotine due to discontinuation by the manufacturer. We therefore utilized a similarly strong cinnamon flavor based on consumer reviews (Cinnacide). Given that both e-cigarettes liquids dampened airway inflammation, the difference in manufacturer did not appear to have a significant effect on the overall findings.

To assess the potential toxicity of flavored e-cigarette to patients with asthma the present study investigated the effects of various flavored e-cigarettes with and without nicotine on a mouse model of allergic airways disease. In this model of mild exposure, e-cigarettes with and without nicotine had substantially different effects. Depending upon the specific flavor, e-cigarettes without nicotine enhanced or suppressed airway inflammation, increased airway hyperresponsiveness and increased features of airway remodeling. In contrast, there was a consistent suppression of airway inflammation without any effect on AHR or airway remodelling due to e-cigarettes with nicotine, regardless of the flavor. These findings highlight the potential for flavored e-cigarettes, in the absence of nicotine, to negatively contribute to asthma outcomes and warrant caution in promoting their use to patients with asthma. Future investigation into the individual chemical constituents of e-cigarettes driving these toxic effects may provide important insight for the regulation of e-cigarettes.