The current COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has killed over 130,000 Americans in less than 4 months. The majority are over 65 years old, but many younger patients who have underlying medical conditions required admission to the intensive care unit. Current data indicate that patients who have cardiovascular and chronic respiratory conditions, including those caused by tobacco use, are at higher risk of developing severe COVID-19 symptoms and have significantly increased fatality [1
]. There are increasing numbers of reports that smokers have worse clinical outcomes when infected with SARS-CoV-2 [2
]. Information from China shows that people who have cardiovascular and respiratory conditions caused by tobacco use are at higher risk of developing severe COVID-19 symptoms [2
]. Furthermore, tobacco use is the most important risk-factor for chronic obstructive pulmonary disease (COPD) [4
]. Finally, aside from lung damage, it is believed that smokers may be more vulnerable to SARS-CoV-2 because of an altered immune response [5
]. However, many studies reported otherwise. In a preprint, Farsalinos et al. concluded that smoking is not a risk factor for hospitalization due to COVID-19 [6
]. One meta-analysis reported no association between smoking and severity of COVID-19 [7
]. A French study even found that nicotine may protect against COVID-19 infection, prompting interests from the French Health Minister in studying whether nicotine patches could be used for COVID-19 treatment and prevention [8
]. Unfortunately, all reports regarding tobacco and COVID-19 are from epidemiological data and only a few studies reported data. The lack of well-controlled laboratory experiments renders it extremely difficult to determine whether tobacco truly affects COVID-19 outcome and the mechanistic link between them. Furthermore, no peer-reviewed study has reported definitive data demonstrating or rejecting the hypothesis that COVID-19 incidence can be increased by tobacco smoking.
Electronic cigarettes (e-cigs) have dramatically increased in popularity in recent years, especially among the youth [10
], so considerable attention has been directed to any potential link between e-cig and COVID-19 in recent months. E-cigs have been shown to induce inflammation of human airways and may also increase susceptibility to pneumonia by increasing pneumococcal adherence to airway cells [11
]. Notable damage to the lung epithelium could also be done by e-cigs, resulting in increased airway hyperactivity and mucine production [12
]. The detrimental effects of e-cigs thus fueled speculation that e-cigs may increase COVID-19 severity or incidence. However, virtually no study, both on the basic science level and epidemiological level, has linked COVID-19 to e-cigs. Complicating research on e-cigs’ effects on COVID-19 is the fact that e-cigs can be vaped with or without nicotine and with or without flavorings. Flavors and nicotine in e-cigs are frequently associated with more inflammation, epithelial barrier dysregulation, oxidative stress, and DNA damage than e-cigs with only basic components [12
Another possible mechanism linking tobacco smoking to COVID-19 is its propensity to increase lung inflammation. COVID-19 has a strong immunological component, and poor outcomes have recently been associated with cytokine storms and a hyperinflammatory immune system [14
]. Tobacco is known to induce extensive immune dysregulation in smokers, and its influence has been found to lead to airway inflammation [16
]. Although cigarette smoke has been associated with increased susceptibility to COVID-19 [17
], the effect of tobacco on the immune response to SARS-CoV-2 has not been studied. E-cigs are also known to induce significant inflammation in the lungs, but the dysregulated immune landscape is different from that of tobacco [18
]. Therefore, it is plausible that inflammation induced by tobacco or e-cigs could exacerbate inflammation caused by SARS-CoV-2 infection, potentially triggering a lethal cytokine storm.
In this study, we directly investigated the two main hypotheses that might link COVID-19 to e-cig vaping and tobacco smoking, ACE2 upregulation and inflammation, by reanalysis of three independent datasets of gene expression from e-cig vapers and tobacco smokers. We examined whether e-cigs or tobacco upregulate ACE2, dysregulate immune cell population levels, affect cytokine levels, and regulate the activation of inflammasomes. Inflammasomes are critical mediators of inflammation, and their activation would lead to the certain release of cytokines [20
]. Since severe COVID-19 is marked by cytokine storms, we investigated both inflammasome activation and the release of cytokines. Given that nicotine or flavorings present in the e-cig mixture are implicated in promoting lung damage, we examined one e-cig dataset where all participants only vaped e-cigs with neither nicotine nor flavors and another e-cig dataset where participants only vaped e-cigs with nicotine and were free to choose different flavors.
The current COVID-19 pandemic intersects with the ongoing epidemic of tobacco smoking and e-cig vaping in the U.S. and across the globe. Tobacco usage has been declining gradually over the years. In total, 42.3% of U.S. adults smoked cigarettes in 1965, but only 13.7% do so in 2018, according to data from the National Health Interview Survey (NHIS). The smoking rate among young adults have also fallen similarly, with 7% of young adults smoking in 2018 [43
]. Although these trends are encouraging, it does not mask the fact that millions of Americans are still active smokers. The many detrimental effects tobacco can have on the lungs and other organs have made tobacco usage one of the biggest public health crises the world faces. Accumulating evidence suggests that smokers are more susceptible to bacterial or viral infection and exhibit greater severity of these infections [44
]. There have also been limited accounts of cigarette smoking increasing severity of COVID-19 infection [45
]. The millions of smokers worldwide may be especially vulnerable to COVID-19.
However, more concerning than tobacco usage has been the usage of e-cigs. Although not nearly as popular as tobacco, e-cig usage has been gaining traction at alarming rates. Critically, the usage of e-cigs has been increasing dramatically among teenagers. According to CDC data, more than 1.3 million high school students started using e-cigs in one year alone (2017–2018), which is a 78% increase in usage (11.7% to 20.8%) [46
]. From 2011 to 2018, the usage rate among high schoolers increased nearly 14 times (1.5% to 20.8%) [46
]. Even among middle schoolers, usage rate increased over eight times from 2011 to 2018 (0.6% to 4.9%) [46
]. While the health effects of tobacco are well-known, much less is known about e-cigs. In recent days, starting from late June, the surge in COVID-19 across the U.S. has been significantly attributed to increased incidence amongst the younger segment of the population. Given the prevalence of e-cig vaping in the young population, significant attention must be paid to how e-cig use may be related to COVID-19 incidence and severity.
Originally marketed as a safer alternative to traditional cigarettes, alarming health risks of e-cigs have been slowly revealed. E-cigs could be cytotoxic to both endothelial and epithelial cells and may decrease immune functions [47
]. However, study of e-cigs is complicated by the presence of nicotine and flavorings. Not all e-cigs have nicotine and flavoring, but almost all brands have one or the other. These components can be significantly more cytotoxic than the base ingredients of e-cigs, which is propylene glycol (PG) and vegetable glycerin (VG). While nicotine is known to be cytotoxic, we have previously reported that e-cigs without nicotine can also induce DNA double strand breaks in cells [48
A possible molecular mechanism for tobacco or e-cigs to increase COVID-19 susceptibility is the upregulation of ACE2. For SARS-CoV-2, host recognition is carried out by the spike protein on the surface of the viral envelope. The spike protein binds the ACE2 receptor protein in human cells. After this, the spike protein can be cleaved by the serine protease TMPRSS2 [49
]. SARS-CoV uses a similar mechanism for host recognition, after which, fusion of the viral envelope with membranes in the host cell will allow the virus entry into the cell [50
]. This process is vital for the entry of SARS-CoV-2 into human host cells, and therefore plays an integral role in COVID-19 infection and disease progression. Cigarette smoke was found in some studies to increase ACE2 expression in lungs in mammals [17
]. We have previously found that both ACE2 and TMPRSS2 are upregulated in smokers using TCGA samples, although we did not examine e-cig users in that study, and the study focused on the association between ACE2 expression and the androgen pathway [24
]. No study has reported on ACE2 expression’s relationship with e-cig use.
In this study, we compared differences in the activation of key molecular pathways related to COVID-19 in tobacco and e-cig users. Specifically, we examined one cohort of e-cig users who only smoked e-cigs without nicotine and without flavorings and another cohort of e-cig users who smoked e-cigs with nicotine and with any flavorings. We found that tobacco use increases ACE2 expression, which corroborates the results of other studies [52
]. However, e-cig use in any case did not increase ACE2 or TMPRSS2 expression, which has not been reported in the literature. One recent study exposing mice to nicotine-containing e-cigs for 1 month has found induction of ACE2 after exposure to nicotine-containing e-cigs [53
]. In our dataset, humans were also exposed to nicotine-containing e-cigs for at least 1 month, suggesting that the in vivo results may not replicate physiological exposure.
We also found that tobacco use and use of nicotine/flavor-containing e-cigs led to significant cytokine dysregulation and potential inflammasome activation. However, non-flavored and non-nicotine-containing e-cig use does not lead to either. While it has been reported that there is only limited inflammation in users of e-cigs without nicotine and flavors [21
], no study has compared gene expression changes of this group of e-cig users to users of nicotine/flavor-containing e-cigs. One study also demonstrated inflammasome activation among e-cig users but did not take into account effects of nicotine and flavors [54
Several limitations exist in our study that should be addressed in future studies. First, for the tobacco dataset, current smokers were only compared to former smokers, not never smokers. The tobacco dataset is also derived from adjacent tissue of lung squamous cell tumors. While the adjacent normal tissues are not cancerous, immune dysregulation in the cancer tissue may significantly influence the observed inflammatory response. Second, for the e-cig dataset with flavor/nicotine-containing e-cigs, only former smokers, not never smokers, were participants. However, since both the experimental and control group are composed of former smokers, the effect of smoking should not affect the statistical validity of our comparisons. Third, in the e-cig studies, participants were only exposed to e-cigs for at least 1 month, while certain effects of e-cigs may be expected to develop only after chronic exposure. Nonetheless, given that we observed cytokine/inflammasome dysregulations already given the relative short duration of exposure, we may expect chronic exposure to lead to even more dysregulation.
E-cig manufacturers have proposed that the lack of tobacco in e-cig vapors makes vaping much less deleterious than tobacco smoking. However, chronic vaping is known to correlate to worse outcomes in a variety of diseases. Specifically, in the lungs, vaping has been correlated to toxicity, oxidative stress, and inflammatory response, indicating a positive correlation with general lung damage [55
]. Additionally, short-term e-cig exposure has been shown to induce changes to small airways in the lung and decreased levels of exhaled nitric oxide (NO) [57
]. This latter decrease is associated with changes to inflammation and COPD development, two factors tied to COVID-19-related morbidity [58
]. It is still controversial whether vaping definitively contributes to worse disease states for respiratory diseases [59
]. However, the correlational research between vaping and lung damage indicates that the effects of vaping on COVID-19 infections should be more widely studied before discounting vaping as a significant modulator of severe infections.
Recent studies into the mechanisms behind SARS-CoV-2 infection have gone beyond interactions with the ACE2 receptor. Development of SARS-CoV-2-specific T cells in individuals not previously infected could indicate another variable controlling infection rate [61
]. Epigenetic mechanisms have also been correlated to the ability of the virus to infiltrate the lungs and fuse with epithelial cell membranes. Specifically, syncytium formation by host cells facilitates viral endocytosis but is inhibited via CpG methylation that prevents transcriptional activity of the syncytin-1 protein [62
]. This mechanism has been studied further in SARS-CoV-2. These studies show that syncytium formation is significantly higher in this strain of coronavirus compared to other coronaviridiae, allowing for a high virulence factor due to higher endocytosis rates [64
]. Therefore, despite the lack of a correlation between vaping and ACE2 activity, it is possible that vaping may affect COVID-19 infections via an ACE2-independent mechanism. Indeed, vaping has been correlated to a loss of methylation and immune activation [66
]. We investigated whether the ACE2-independent mechanism is cytokine dysregulation. Although our findings indicate that e-cigs without flavors and nicotine may only modestly dysregulate cytokine expression, we want to emphasize a cautionary note that most vapers use formulations that contain nicotine and flavorings. Our data indicate that flavors and nicotine may significantly dysregulate cytokine levels and promote inflammation. These data should indicate that vaping may still be a potent risk factor for severe COVID-19 infection depending on the flavor and nicotine content.
In conclusion, our study demonstrated that tobacco and flavored or nicotine-containing e-cig use could both lead to increased inflammatory response, but only tobacco upregulates ACE2. Inflammation and ACE2 upregulation may increase susceptibility to COVID-19. While further experiments and epidemiology data are needed to confirm our results, we believe that our study is a critical early step into evaluating the implications of using tobacco and e-cigs during the COVID-19 pandemic.