Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Feb 15;197(4):492-501.
doi: 10.1164/rccm.201708-1590OC.

E-Cigarette Use Causes a Unique Innate Immune Response in the Lung, Involving Increased Neutrophilic Activation and Altered Mucin Secretion

Affiliations
Free PMC article

E-Cigarette Use Causes a Unique Innate Immune Response in the Lung, Involving Increased Neutrophilic Activation and Altered Mucin Secretion

Boris Reidel et al. Am J Respir Crit Care Med. .
Free PMC article

Abstract

Rationale: E-cigarettes have become increasingly popular and little is known about their potential adverse health effects.

Objectives: To determine the effects of e-cigarette use on the airways.

Methods: Induced sputum samples from cigarette smokers, e-cigarette users, and nonsmokers were analyzed by quantitative proteomics, and the total and individual concentrations of mucins MUC5AC and MUC5B were determined by light scattering/refractometry and labeled mass spectrometry, respectively. Neutrophil extracellular trap (NET) formation rates were also determined for the same groups.

Measurements and main results: E-cigarette users exhibited significant increases in aldehyde-detoxification and oxidative stress-related proteins associated with cigarette smoke compared with nonsmokers. The levels of innate defense proteins associated with chronic obstructive pulmonary disease, such as elastase and matrix metalloproteinase-9, were significantly elevated in e-cigarette users as well. E-cigarette users' sputum also uniquely exhibited significant increases in neutrophil granulocyte-related and NET-related proteins, such as myeloperoxidase, azurocidin, and protein-arginine deiminase 4, despite no significant elevation in neutrophil cell counts. Peripheral neutrophils from e-cigarette users showed increased susceptibility to phorbol 12-myristate 13-acetate-induced NETosis. Finally, a compositional change in the gel-forming building blocks of airway mucus (i.e., an elevated concentration of mucin MUC5AC) was observed in both cigarette smokers and e-cigarette users.

Conclusions: Together, our results indicate that e-cigarette use alters the profile of innate defense proteins in airway secretions, inducing similar and unique changes relative to cigarette smoking. These data challenge the concept that e-cigarettes are a healthier alternative to cigarettes.

Keywords: NET; e-cigarette; lung; mucin; neutrophil.

Figures

Figure 1.
Figure 1.
Analysis of induced samples of tobacco product users’ sputum indicates a uniquely altered airway secretome for e-cigarette users in comparison with cigarette smokers and nonsmokers. (A) Venn diagram showing the number of proteins with significantly higher levels than the means in the sputum of cigarette smokers (CS), e-cigarette users (ECU), and nonsmokers (NS). (B) Heatmap of proteins displaying significantly (ANOVA P ≤ 0.05) changed levels with respect to nonsmokers based on the total precursor intensity. The protein order in the heatmap is based on descending fold change in the e-cigarette user group. (C) Interactome map showing proteins with significant increases in cigarette smokers, e-cigarette users, and both with respect to nonsmokers. Quantified protein hits were based on at least two assigned peptides per protein.
Figure 2.
Figure 2.
Levels of proteins known to be affected by cigarette smoke exposure are also altered in e-cigarette users. The total precursor intensity of each sample was plotted for comparison among the groups. (A) Aldehyde dehydrogenase 3A1, (B) nucleobindin-1, (C) thioredoxin, (D) glutathione S-transferase, and (E) microseminoprotein β. Mean and SEM values are indicated by major and minor horizontal bars, respectively. Statistical significance was determined by one-way ANOVA. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005, and ****P ≤ 0.001. CS = cigarette smokers; ECU = e-cigarette users; NS = nonsmokers.
Figure 3.
Figure 3.
Airway epithelial defense protein levels are significantly altered in tobacco product users. The total precursor intensity of each sample was plotted for comparison among the groups. (A) Deleted in malignant brain tumors 1, (B) lactotransferrin, (C) trefoil factor 3, and (D) lysozyme C. Mean and SEM values are indicated by major and minor horizontal bars, respectively. Statistical significance was determined by one-way ANOVA. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005, ****P ≤ 0.001. CS = cigarette smokers; DMBT1 = deleted in malignant brain tumors 1; ECU = e-cigarette users; NS = nonsmokers.
Figure 4.
Figure 4.
Neutrophilic granule enzyme levels are significantly increased in e-cigarette users’ airways, despite no increase in neutrophil cell counts. The total precursor intensity of each sample was plotted for comparison among the groups. (A) Neutrophil elastase, (B) myeloperoxidase, (C) proteinase 3, (D) matrix metalloproteinase 9, and (E) azurocidin. (F) Neutrophil cell counts in sputum samples from e-cigarette users and cigarette smokers and nonsmokers. Mean and SEM values are indicated by major and minor horizontal bars, respectively. Statistical significance was determined by one-way ANOVA. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005. CS = cigarette smokers; ECU = e-cigarette users; MMP = matrix metalloproteinase; NS = nonsmokers; PMN = polymorphonuclear neutrophil.
Figure 5.
Figure 5.
Evidence for increased neutrophil extracellular trap formation in current e-cigarette users. The levels of neutrophil extracellular trapmarker proteins, such as S100A8 (A) and S100A9 (B), which form a heterodimer called calprotectin, (C) coronin-1 (CORO1A), and (D) peptidyl arginine deiminase, type IV (PADI4) were significantly increased. Statistical significance was determined by one-way ANOVA. *P ≤ 0.05, ***P ≤ 0.005. CS = cigarette smokers; ECU = e-cigarette users; NS = nonsmokers.
Figure 6.
Figure 6.
Isolated peripheral (blood) neutrophils from e-cigarette users are more susceptible to phorbol ester stimulation-induced NETosis. Quantitation of phorbol 12-myristate 13-acetate–induced neutrophil extracellular traps (NETs) formed by neutrophils from nonsmokers, cigarette smokers, and e-cigarette users. Neutrophils isolated from the venous blood of nonsmokers (solid squares), cigarette smokers (solid triangles), and e-cigarette users (solid circles) were challenged with 25 nM phorbol 12-myristate 13-acetate, a protein kinase C activator, and potent NET agonist, and assayed for nucleic acid release over time. Mean and SEM values are indicated by major and minor horizontal bars, respectively. Statistical significance was determined by one-way ANOVA. *P ≤ 0.05. CS = cigarette smokers; ECU = e-cigarette users; NS = nonsmokers.
Figure 7.
Figure 7.
The ratio between the major airway mucins MUC5AC and MUC5B is significantly shifted toward MUC5AC in cigarette smokers and follows the same trend in e-cigarette users. Comparison of total and individual mucin concentrations and their ratios in sputum samples from nonsmokers, cigarette smokers, and e-cigarette users. Individual mucin concentrations of the dominant airway mucin MUC5B (A) increased slightly but not significantly in cigarette smokers, and the concentrations of MUC5AC (B) and the MUC5AC/MUC5B ratio (C) significantly increased in both cigarette and e-cigarette users. Total mucin concentrations were increased in cigarette smokers but not in e-cigarette users (D). Mean and SEM values are indicated by major and minor horizontal bars, respectively. *P < 0.05. CS = cigarette smokers; ECU = e-cigarette users; NS = nonsmokers.
Figure 8.
Figure 8.
Schematic depiction of the impact of e-cigarette smoking on the airways. Vaping uniquely alters the airway innate immune response by causing an increase in the release of neutrophil extracellular trap–associated proteins; proteins involved in maintaining the redox balance of the airways; and the ratio of the building blocks of airway mucus, namely, the mucins MUC5AC and MUC5B. e-cig = e-cigarette.

Comment in

  • E-Cigarettes: Mucus Measurements Make Marks.
    Evans CM, Dickey BF, Schwartz DA. Evans CM, et al. Am J Respir Crit Care Med. 2018 Feb 15;197(4):420-422. doi: 10.1164/rccm.201711-2157ED. Am J Respir Crit Care Med. 2018. PMID: 29161057 Free PMC article. No abstract available.
  • Schlechte Nachricht für E-Zigaretten-Fans.
    Bargon J. Bargon J. MMW Fortschr Med. 2018 Apr;160(7):37. doi: 10.1007/s15006-018-0428-x. MMW Fortschr Med. 2018. PMID: 29663211 German. No abstract available.

Similar articles

See all similar articles

Cited by 52 articles

See all "Cited by" articles

Publication types

Feedback