Inflammatory phenotype modulation in the respiratory tract and systemic circulation of e-cigarette users: a pilot study

Abstract

Over 40 million people use e-cigarettes worldwide, but the impact of chronic e-cigarette use on health has not been adequately defined. In particular, effects of e-cigarette aerosol inhalation on inflammation and host defenses across the body are not fully understood. We conducted a longitudinal cohort pilot study to explore changes in the inflammatory state and monocyte function of e-cigarette users (n = 20) versus healthy controls (n = 13) and to evaluate effects of e-cigarette use reduction on the same. Saliva, sputum, and blood were obtained from e-cigarette users at baseline and after a 2-wk intervention of decreased e-cigarette use. Overall, across 38 proteins quantified by multiplex, airway samples from e-cigarette users tended to have decreased levels of immunomodulatory proteins relative to healthy controls, whereas levels of cytokines, chemokines, and growth factors in the circulation tended to be elevated. Specifically, e-cigarette users had lower levels of IL-1 receptor antagonist (IL-1Ra) in saliva (P < 0.0001), with higher IL-1Ra and growth-regulated oncogene (GRO) levels in sputum (P < 0.01 and P < 0.05, respectively), and higher levels of both TNFβ (P < 0.0001) and VEGF (P < 0.0001) in plasma. Circulating monocytes from e-cigarette users had alterations in their inflammatory phenotype in response to reduced e-cigarette use, with blunted IL-8 and IL-6 release upon challenge with bacterial lipopolysaccharide (P < 0.001 and P < 0.05, respectively), suggesting a decreased ability to appropriately respond to bacterial infection. Based on these findings, chronic inhalation of e-cigarette aerosols alters the inflammatory state of the airways and systemic circulation, raising concern for the development of both inflammatory and infectious diseases in chronic users of e-cigarettes.

Keywords: cytokine; e-cigarette; host defense; inflammation; monocyte.

Figure 1.

E-cigarette use associated alterations in immunomodulatory protein levels in saliva, sputum, and plasma. A: inflammatory markers were assessed in the saliva of nonsmoking nonvaping healthy controls (n = 11; blue circles) and e-cigarette users at baseline (n = 17; solid red squares), with lower levels of IL-1RA identified in the oral airways of e-cigarette users. B: IL-1RA remained low in e-cigarette users after 2 wk of reduced e-cigarette usage (n = 14; hollow red squares). C: sputum demonstrated reductions in both GRO and IL-1RA in the lower airways of e-cigarette users vs. healthy controls. D: GRO and IL-1RA remained low in lower airway samples after 2 wk of reduced e-cigarette usage. E: plasma samples demonstrated elevations in TNFβ and VEGF in the circulation of e-cigarette users vs. controls. F: after 2 wk of reduced e-cigarette use, plasma levels of TNFβ and VEGF were persistently elevated. Data are represented as individual data points with the geometric mean. Statistical analysis was conducted with two-way ANOVA with correction for multiple comparisons by controlling the false discovery rate, using the two-stage step-up method of Benjamini, Krieger, and Yekutieli. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Figure 2.

Exploratory assessment of inflammatory modulatory proteins, chemokines, and growth factors in saliva samples. Inflammatory markers were assessed in the saliva of nonsmoking healthy controls (black), e-cigarette users at baseline (red) and after 2 wk of reduced e-cigarette usage (blue). Only the analytes with statistically significant differences by Student’s t test (without controlling for multiple comparisons) were included in this figure and are further separated into cytokines (A–N), chemokines (O–R), and growth factors (S–T). Paired lines between e-cigarette and reduced dose represent the change in the levels in the same individual after reducing the e-cigarette dose. Error bars represent the means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, as determined by Student’s t test.

Figure 3.

Exploratory assessment of inflammatory modulatory proteins, chemokines, and growth factors in sputum samples. Inflammatory markers were assessed in the sputum of nonsmoking healthy controls (black), e-cigarette users at baseline (red) and after 2 wk of reduced e-cigarette usage (blue). Only the analytes with statistically significant differences by Student’s t test (without controlling for multiple comparisons) were included in this figure and are further separated into cytokines (A–J), chemokines (K–L), and growth factors (M–O). Paired lines between e-cigarette and reduced dose represent the change in the levels in the same individual after reducing the e-cigarette dose. Error bars represent the means ± SD. *P < 0.05, and **P < 0.01, as determined by Student’s t test.

Figure 4.

Exploratory assessment of inflammatory modulatory proteins, chemokines, and growth factors in plasma samples. Inflammatory markers were assessed from the plasma in nonsmoking healthy controls (black), e-cigarette users at baseline (red) and after 2 wk of reduced e-cigarette usage (blue). Only the analytes with statistically significant differences by Student’s t test (without controlling for multiple comparisons) were included in this figure and are further separated into cytokines (A–E), chemokines (F–I), and growth factors (J–L). Paired lines between e-cigarette and reduced dose represent the change in the levels in the same individual after reducing the e-cigarette dose. Error bars represent the means ± SD. *P < 0.05, **P < 0.01, and ****P < 0.0001, as determined by Student’s t test.

Figure 5.

Reductions in sputum and plasma immunomodulatory proteins in response to short-term reduction in e-cigarette use. A: IL-1RA in saliva modestly increased after 2 wk reduction in vaping. B: in sputum, four proteins had diminished levels in the setting of short-term reduced vaping, GRO, IL-1RA, IL-8, and IP-10. C: plasma samples demonstrated reduced levels of TNFβ and VEGF in the circulation of e-cigarette users in response to 2 wk of reduced e-cigarette use. Individual data points are shown with the mean and 95% CI. Statistical analysis was conducted with two-way ANOVA with correction for multiple comparisons by controlling the false discovery rate, using the two-stage step-up method of Benjamini, Krieger, and Yekutieli. *P = 0.02, #P = 0.03, **P < 0.01, and ****P < 0.0001. CI, confidence interval.

Figure 6.

E-cigarette alters the inflammatory response of circulating human monocytes. Inflammatory markers TNFα (A–C), IL-6 (D–F), and IL-8 (G–I) were obtained from circulating monocytes in nonsmoking nonvaping healthy controls (black) and from e-cigarette users at baseline (red) and after 2 wk of reduced e-cigarette usage (blue). This figure is further separated into cytokines secreted under unstimulated conditions (A, D, and G) and LPS-stimulated conditions (B, E, and H). Normalized TNFα (C), IL-6 (F), and IL-8 (I) were calculated by dividing cytokine levels from LPS-stimulated monocytes by cytokine levels from nonstimulated monocytes from each subject. Paired lines between e-cigarette and reduced use represent paired changes in cytokine levels from the same subject in the setting of reducing use of e-cigarettes. Error bars represent the means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, as determined by Student’s t test. LPS, lipopolysaccharide.