New Insights into How JUUL™ Electronic Cigarette Aerosols and Aerosol Constituents Affect SARS-CoV-2 Infection of Human Bronchial Epithelial Cells

Abstract

Background: The relationship between the use of tobacco products and SARS-CoV-2 infection is poorly understood and controversial. Most studies have been done with tobacco cigarettes, while few have examined the effect of electronic cigarettes (ECs) on SARS-CoV-2 infection. We tested the hypothesis that EC fluids and aerosols with high concentrations of nicotine promote SARS-COV-2 infection by increasing viral entry into human respiratory epithelial cells. Methods: Responses of BEAS-2B cells to authentic JUUL aerosols or their individual constituents (propylene glycol (PG)/vegetable glycerin (VG) and nicotine) were compared using three exposure platforms: submerged culture, air-liquid-interface (ALI) exposure in a cloud chamber, and ALI exposure in a Cultex system, which produces authentic heated EC aerosols. SARS-CoV-2 infection machinery was assessed using immunohistochemistry and Western blotting. Specifically, the levels of the SARS-CoV-2 receptor ACE2 (angiotensin converting enzyme 2) and a spike modifying enzyme, TMPRSS2 (transmembrane serine protease 2), were evaluated. Following each exposure, lentivirus pseudoparticles with spike protein and a green-fluorescent reporter were used to test viral penetration and the susceptibility of BEAS-2B cells to infection. Results: Nicotine, EC fluids, and authentic JUUL aerosols increased both ACE2 levels and TMPRSS2 activity, which in turn increased viral particle entry into cells. While most data were in good agreement across the three exposure platforms, cells were more responsive to treatments when exposed at the ALI in the Cultex system, even though the exposures were brief and intermittent. In the Cultex system, PG/VG, PG/VG/nicotine, and JUUL aerosols significantly increased infection above clean air controls. However, both the PG/VG and JUUL treatments were significantly lower than nicotine/PG/VG. PG/VG increased infection only in the Cultex system, which produces heated aerosol. Conclusion: Our data are consistent with the conclusion that authentic JUUL aerosols or their individual constituents (nicotine or PG/VG) increase SARS-CoV-2 infection. The strong effect produced by nicotine was modulated in authentic JUUL aerosols, demonstrating the importance of studying mixtures and aerosols from actual EC products. These data support the idea that vaping increases the likelihood of contracting COVID-19.

Does Vaping Increase the Likelihood of SARS-CoV-2 Infection? Paradoxically Yes and No

“…Our finding that nicotine increased ACE2 agrees with prior studies [15][16][17][18][19][20][21][22][23][24]. To the best of our knowledge, our data are the first to show that PG/VG aerosol can increase ACE2, an important finding since these solvents are the dominant chemicals in e-liquids [2,48].…”

“…Similarly, there is some discrepancy in the EC literature regarding the influence of EC use to SARS-CoV-2 susceptibility [15][16][17][18][19][20][21][22][23]. Our study clearly shows that the presence of benzoic acid in e-liquids mitigates the enhanced infection caused by nicotine.…”

“…In our previous study, we used monolayers of BEAS-2B cells in submerged cultures and ALI exposures [21]. Treatments that increased ACE2 (nicotine, nicotine plus PG/VG, and authentic JUUL™ aerosol) also increased viral pseudoparticle infection; however, the increase in the JUUL™ group was significantly less than in the nicotine only group, even though both groups were exposed to a similar concentration of nicotine [21]. It is likely that benzoic acid in JUUL™ aerosol suppressed nicotine-enhanced infection in our prior study, but the effect was not as great as in the current study.…”

“…Although human data are limited, Ghosh et al, 2022 [19] showed that EC aerosol increased ACE2 activity and soluble ACE2 levels in EC users’ bronchoalveolar lavage fluid; these changes were correlated with infection of human bronchial epithelial cells by SARS-CoV-2 pseudoparticles following exposure to JUUL™ “Virginia Tobacco” e-liquid in submerged cultures. Additionally, e-liquid with and without nicotine increased ACE2 expression in BEAS-2B cells after submerged treatment [20], an effect subsequently linked to nicotine concentration in air-liquid interface (ALI) exposures of BEAS-2B cells [21]. Using BEAS-2B monolayer cultures, we further showed that submerged and ALI exposures with nicotine and e-liquid with nicotine increased ACE2 levels, TMPRSS2 activity, and SARS-CoV-2 pseudoparticle infection [21].…”

“…Additionally, e-liquid with and without nicotine increased ACE2 expression in BEAS-2B cells after submerged treatment [20], an effect subsequently linked to nicotine concentration in air-liquid interface (ALI) exposures of BEAS-2B cells [21]. Using BEAS-2B monolayer cultures, we further showed that submerged and ALI exposures with nicotine and e-liquid with nicotine increased ACE2 levels, TMPRSS2 activity, and SARS-CoV-2 pseudoparticle infection [21]. While preliminary, these studies suggest that EC use may increase the likelihood of contracting COVID- 19.…”