Exposure to electronic cigarettes impairs pulmonary anti-bacterial and anti-viral defenses in a mouse model

© 2015 Sussan et al. Electronic cigarettes (E-cigs) have experienced sharp increases in popularity over the past five years due to many factors, including aggressive marketing, increased restrictions on conventional cigarettes, and a perception that E-cigs are healthy alternatives to cigarettes. Despite this perception, studies on health effects in humans are extremely limited and in vivo animal models have not been generated. Presently, we determined that E-cig vapor contains 7x1011 free radicals per puff. To determine whether E-cig exposure impacts pulmonary responses in mice, we developed an inhalation chamber for E-cig exposure. Mice that were exposed to E-cig vapor contained serum cotinine concentrations that are comparable to human E-cig users. E-cig exposure for 2 weeks produced a significant increase in oxidative stress and moderate macrophage-mediated inflammation. Since, COPD patients are susceptible to bacterial and viral infections, we tested effects of E-cigs on immune response. Mice that were exposed to E-cig vapor showed significantly impaired pulmonary bacterial clearance, compared to air-exposed mice, following an intranasal infection with Streptococcus pneumonia. This defective bacterial clearance was partially due to reduced phagocytosis by alveolar macrophages from E-cig exposed mice. In response to Influenza A virus infection, E-cig exposed mice displayed increased lung viral titers and enhanced virus-induced illness and mortality. In summary, this study reports a murine model of E-cig exposure and demonstrates that E-cig exposure elicits impaired pulmonary anti-microbial defenses. Hence, E-cig exposure as an alternative to cigarette smoking must be rigorously tested in users for their effects on immune response and susceptibility to bacterial and viral infections

Involvement of Noxa in Mediating Cellular ER Stress Responses to Lytic Virus Infection

Noxa is a Bcl-2 homology domain-containing pro-apoptotic mitochondrial protein. Noxa mRNA and protein expression are upregulated by dsRNA or virus, and ectopic Noxa expression enhances cellular sensitivity to virus or dsRNA-induced apoptosis. Here we demonstrate that Noxa null baby mouse kidney (BMK) cells are deficient in normal cytopathic response to lytic viruses, and that reconstitution of the knockout cells with wild-type Noxa restored normal cytopathic responses. Noxa regulation by virus mirrored its regulation by proteasome inhibitors or ER stress inducers and the ER stress response inhibitor salubrinal protected cells against viral cytopathic effects. Noxa mRNA and protein were synergistically upregulated by IFN or dsRNA when combined with ER stress inducers, leading to Noxa/Mcl-1 interaction, activation of Bax and pro-apoptotic caspases, degradation of Mcl-1, loss of mitochondrial membrane potential and initiation of apoptosis. These data highlight the importance of ER stress in augmenting the expression of Noxa following viral infection

Correction: Lack of Effect of Oral Sulforaphane Administration on Nrf2 Expression in COPD: A Randomized, Double-Blind, Placebo Controlled Trial.

[This corrects the article DOI: 10.1371/journal.pone.0163716.]

E-cig exposure impairs viral clearance and causes significant morbidity and mortality in mice following influenza virus infection.

<p>Mice were exposed to air or E-cig for 2 wks, then infected intranasally with either TCID₅₀ 10² (A-B) or TCID₅₀ 10³ (C) of H1N1 virus. (A) Viral titer was determined by TCID₅₀ assay in lung homogenates at 4 days after infection (N = 5 mice per group). (B-C) Mice were weighed daily after infection with either TCID₅₀ 10² (B) or 10³ (C), and values are presented as percent of starting weight (N = 10 mice per group). For mice that died during the experiments, body weights were included in the analysis up until the day of death. (D) Mortality curves in response to intranasal infection with TCID₅₀ 10² or 10³ H1N1 (N = 10 mice per group). *p<0.05 by Student’s two-tailed t-test.</p

E-cig-induced pulmonary response.

<p>(A) EPR spectra of E-cig TPM. The presented spectrum is a result of subtraction of a Cambridge filter pad EPR signal before and after collection of TPM. (B) Lipid peroxidation was measured by thiobarbituric acid reactive substances (TBARS) in lung homogenates from C57BL/6 mice that were exposed to air or E-cig vapor for 1.5 h, twice per day, for 2 wks. (C) Inflammatory cells were quantified in the BAL at 24h after the final exposure. (D) Cytokines were analyzed in cell-free BAL fluid from air and E-cig exposed mice at 24h after the final exposure. N = 10 mice per group. *p<0.05 by Student’s two-tailed t-test.</p

Lack of Effect of Oral Sulforaphane Administration on Nrf2 Expression in COPD: A Randomized, Double-Blind, Placebo Controlled Trial.

COPD patients have high pulmonary and systemic oxidative stress that correlates with severity of disease. Sulforaphane has been shown to induce expression of antioxidant genes via activation of a transcription factor, nuclear factor erythroid-2 related factor 2 (Nrf2).This parallel, placebo-controlled, phase 2, randomized trial was conducted at three US academic medical centers. Patients who met GOLD criteria for COPD and were able to tolerate bronchoscopies were randomly assigned (1:1:1) to receive placebo, 25 μmoles, or 150 μmoles sulforaphane daily by mouth for four weeks. The primary outcomes were changes in Nrf2 target gene expression (NQ01, HO1, AKR1C1 and AKR1C3) in alveolar macrophages and bronchial epithelial cells. Secondary outcomes included measures of oxidative stress and airway inflammation, and pulmonary function tests.Between July 2011 and May 2013, 89 patients were enrolled and randomized. Sulforaphane was absorbed in the patients as evident from their plasma metabolite levels. Changes in Nrf2 target gene expression relative to baseline ranged from 0.79 to 1.45 and there was no consistent pattern among the three groups; the changes were not statistically significantly different from baseline. Changes in measures of inflammation and pulmonary function tests were not different among the groups. Sulforaphane was well tolerated at both dose levels.Sulforaphane administered for four weeks at doses of 25 μmoles and 150 μmoles to patients with COPD did not stimulate the expression of Nrf2 target genes or have an effect on levels of other anti-oxidants or markers of inflammation.Clinicaltrials.gov: NCT01335971

Influenza-induced inflammation is altered by E-cig exposure.

<p>Mice were exposed to air or E-cig for 2 wks, then infected intranasally with TCID₅₀ 10² of H1N1. BAL was collected at day 4 (N = 5) and day 8 (N = 4) after infection, followed by quantification of inflammatory cells (A) and cytokines (B). *p<0.05 by Student’s two-tailed t-test.</p

Schematic of our E-cig exposure model.

<p>Schematic of our E-cig exposure model.</p