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. 2014 Sep 22;9(9):e108342.
doi: 10.1371/journal.pone.0108342. eCollection 2014.

Electronic cigarette liquid increases inflammation and virus infection in primary human airway epithelial cells

Qun Wu  1 , Di Jiang  1 , Maisha Minor  1 , Hong Wei Chu  1
Affiliations

Electronic cigarette liquid increases inflammation and virus infection in primary human airway epithelial cells

Qun Wu et al. PLoS One. .

Abstract

Background/objective: The use of electronic cigarettes (e-cigarettes) is rapidly increasing in the United States, especially among young people since e-cigarettes have been perceived as a safer alternative to conventional tobacco cigarettes. However, the scientific evidence regarding the human health effects of e-cigarettes on the lung is extremely limited. The major goal of our current study is to determine if e-cigarette use alters human young subject airway epithelial functions such as inflammatory response and innate immune defense against respiratory viral (i.e., human rhinovirus, HRV) infection.

Methodology/main results: We examined the effects of e-cigarette liquid (e-liquid) on pro-inflammatory cytokine (e.g., IL-6) production, HRV infection and host defense molecules (e.g., short palate, lung, and nasal epithelium clone 1, SPLUNC1) in primary human airway epithelial cells from young healthy non-smokers. Additionally, we examined the role of SPLUNC1 in lung defense against HRV infection using a SPLUNC1 knockout mouse model. We found that nicotine-free e-liquid promoted IL-6 production and HRV infection. Addition of nicotine into e-liquid further amplified the effects of nicotine-free e-liquid. Moreover, SPLUNC1 deficiency in mice significantly increased lung HRV loads. E-liquid inhibited SPLUNC1 expression in primary human airway epithelial cells. These findings strongly suggest the deleterious health effects of e-cigarettes in the airways of young people. Our data will guide future studies to evaluate the impact of e-cigarettes on lung health in human populations, and help inform the public about potential health risks of e-cigarettes.

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Conflict of interest statement

Competing Interests: HWC is a PLOS ONE Editorial Board member. This does not alter the authors' adherence to PLOS ONE Editorial policies and criteria.

Figures

Figure 1
Figure 1. The physiological doses of e-liquid do not decrease primary human airway epithelial cell viability.
Normal human tracheobronchial epithelial cells from young healthy non-smokers were treated with medium (control), e-liquid at various concentrations without nicotine (NO-NIC) or with 18 mg/ml of nicotine (NIC-18 mg/ml) for 24 h (A) and 48 h (B). Cell toxicity was assessed by measuring lactate dehydrogenase (LDH) levels in cell culture supernatants. Data (n = 4) are presented as mean ± SEM.
Figure 2
Figure 2. E-liquid induces IL-6 production in primary human airway epithelial cells.
Normal human tracheobronchial epithelial cells from young healthy non-smokers were exposed to medium (control), e-liquid at various concentrations without nicotine (NO-NIC) or with 18 mg/ml of nicotine (NIC-18 mg/ml) for 24 h (A) and 48 h (B). IL-6 protein levels in cell culture supernatants were measured by ELISA. Data (n = 4) are presented as means ± SEM.
Figure 3
Figure 3. E-liquid promotes human rhinovirus (HRV) infection in primary human airway epithelial cells.
Normal human tracheobronchial epithelial cells from young healthy non-smokers were pre-exposed to medium (control), e-liquid at an optimized concentration (0.3% v/v) without nicotine (NO-NIC) or with 18 mg/ml of nicotine (NIC-18 mg/ml) for 24 h and then infected with HRV-16 at 104 TCID50/well or PBS (control) for 6 h (A) and 24 h (B). Viral RNA levels were measured by quantitative real-time RT-PCR. Data (n = 5) are presented as means ± SEM.
Figure 4
Figure 4. E-liquid enhances human rhinovirus-induced IL-6 production in primary human airway epithelial cells.
Normal human tracheobronchial epithelial cells from young healthy non-smokers were pre-exposed to medium (control), e-liquid at an optimized concentration (0.3% v/v) without nicotine (NO-NIC) or with 18 mg/ml of nicotine (NIC-18 mg/ml) for 24 h and then infected with HRV-16 at 104 TCID50/well or PBS (control) for 6 h (A) and 24 h (B). IL-6 protein levels in cell culture supernatants were measured by ELISA. Data (n = 5) are presented as means ± SEM.
Figure 5
Figure 5. E-liquid inhibits SPLUNC1, a host defense molecule against human rhinovirus infection, in primary human airway epithelial cells.
(A) SPLUNC1 knockout (KO, n = 5) mice and littermate controls (WT, n = 5) were intranasally inoculated with HRV-1B at 5×106 pfu/mouse for 24 h to examine lung HRV RNA levels by quantitative real-time RT-PCR. Data are presented as means ± SEM. (B) Normal human tracheobronchial epithelial cells from young healthy non-smokers were pre-exposed to medium (control), e-liquid at an optimized concentration (0.3% v/v) without nicotine (NO-NIC) or with 18 mg/ml of nicotine (NIC-18 mg/ml) for 24 h and then infected with HRV-16 at 104 TCID50/well or PBS (control) for 6 h. SPLUNC1 mRNA levels were measured by quantitative real-time RT-PCR. Data (n = 5) are presented as means ± SEM.
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