Reply to Sharma and Zeki: Does Vaping Increase Susceptibility to COVID-19?

From the Authors:

We agree with Sharma and Zeki that the increased expression of ACE2 and TMPRSS2 in the small airway epithelium (SAE) in men and cigarette smokers may play a role in the increased susceptibility to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and that our findings are consistent with those of others (1–7). In response to their suggestion that exposure to electronic cigarettes (e-cigs) with or without nicotine may influence ACE2 expression in the airways, we evaluated our prior study assessing the SAE gene expression responses of healthy never-smokers after acute exposure to e-cigs (8). Ten nonsmokers matched for age, sex, and ethnicity with no history of exposure to tobacco products or e-cigs, were assessed at baseline; this included medical examination, routine blood studies, urine analysis, respiratory questionnaires, chest X-ray, and full lung function, followed by bronchoscopy to obtain SAE by brushing. All subjects were documented to be never-smokers on the basis of their self-reported smoking history and by the absence of urine tobacco metabolites. One week later, all subjects inhaled 10 puffs of a “Blu” brand e-cig (first exposure) and 30 minutes later inhaled 10 more puffs (second exposure). Subjects were randomized to inhale e-cigs with (n = 7) or without (n = 3) nicotine. Two hours after the second e-cig exposure, all subjects were reassessed as at baseline, including bronchoscopy to obtain SAE. SAE cell differentials were similar in both exposure groups at both time points and before and after exposure within the same exposure group. Total RNA was extracted, processed, and hybridized on Illumina HiSeq 2500 (Illumina) (8). Genome-wide fragments per kilobase per million expression values are publicly available in the National Center for Biotechnology Information Gene Expression Omnibus (accession number GSE85121).

The SAE transcriptome at baseline was compared with the SAE transcriptome after e-cig exposure within each group using a paired t test using Partek Genomics Suite Software version 6.6 (Partek). A genome-wide analysis of n = 16,044 genes identified 71 genes significantly modified in the SAE after exposure to e-cigs with nicotine and 65 genes significantly modified in the SAE after exposure to e-cigs without nicotine (8). Neither ACE2 nor TMPRSS2 were among the significant genes in either comparison. Gene expression analysis of only ACE2 and TMPRSS2 before and after exposure demonstrated no effect on either ACE2 or TMPRSS2 of e-cigs with or without nicotine (Table 1). Thus, at least for this brand of e-cigs, acute exposure does not affect ACE2 or TMPRSS2 expression, in contrast to cigarette smoke exposure (1). However, only one e-cig brand was evaluated, and e-cig smoke likely varies among brands and added flavorings (9–13). Thus, although our data related to acute e-cig exposure do not show an effect on ACE2 or TMPRSS2 expression, we agree with Sharma and Zeki that further studies of use of e-cigs are warranted because chronic use and different formulations could have an effect on SARS-CoV-2 expression.

Table 1.

Expression of ACE2 and TMPRSS2 in the SAE of Healthy Nonsmokers before and after Acute Exposure to E-Cigarettes^*

SAE Population	E-cig with Nicotine (FPKM)†	E-cig without Nicotine (FPKM)‡
ACE2§
Preexposure	0.9 ± 0.5	0.7 ± 0.4
Postexposure	0.6 ± 0.2	0.7 ± 0.1
P value‖	>0.1	>0.3
TMPRSS2§
Preexposure	22.5 ± 5.0	22.1 ± 2.2
Postexposure	21.9 ± 4.3	23.4 ± 1.9
P value‖	>0.6	>0.05

Definition of abbreviations: E-cig = e-cigarette; FPKM = fragments per kilobase per million; SAE = small airway epithelium.

^*SAE was assessed in never-smokers (n = 10) before and after acute exposure (2 × 10 puffs, 30 min apart) to “Blu” brand e-cigarettes.

^†A random sample (n = 7) of the nonsmokers was exposed to e-cigarettes with nicotine.

^‡A random sample (n = 3) of the nonsmokers was exposed to e-cigarettes without nicotine.

^§The expression of each of the genes was compared before exposure (baseline) and after exposure (Day 7) within each group of exposure, separately.

^‖P values were calculated using a paired t test.