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

Additional file 1: Figure S1. of PARP inhibition in leukocytes diminishes inflammation via effects on integrins/cytoskeleton and protects the blood-brain barrier

PARPi decrease PARP activity in a dose-dependent manner. PARP activity of primary monocytes treated with different concentrations of PARPi AIQ (A) and olaparib (B). Results are presented as the mean ± SEM (*P < 0.05, **P < 0.01 vs. untreated control) from two independent experiments for at least three replicates. Figure S2. PARP activity significantly diminished in ex vivo treated mouse leukocytes. (A) PARP activity was measured in freshly isolated and ex vivo PARPi-treated mouse leukocytes. Data are presented as mean ± SEM for at least three replicates from three/four donor mice. ***P < 0.005 indicate significance vs. non-treated. (B) Flow cytometry data presenting leukocyte profile of isolated leukocytes with or without PARPi treatment

Secoisolariciresinol diglucoside is a blood-brain barrier protective and anti-inflammatory agent: implications for neuroinflammation

Abstract Background Secoisolariciresinol diglucoside (SDG), the main lignan in flaxseed, is known for its beneficial effects in inflammation, oxidative stress, heart disease, tumor progression, atherosclerosis, and diabetes. SDG might be an attractive natural compound that protects against neuroinflammation. Yet, there are no comprehensive studies to date investigating the effects of SDG on brain endothelium using relevant in vivo and in vitro models. Methods We evaluated the effects of orally administered SDG on neuroinflammatory responses using in vivo imaging of the brain microvasculature during systemic inflammation and aseptic encephalitis. In parallel, the anti-inflammatory actions of SDG on brain endothelium and monocytes were evaluated in vitro blood-brain barrier (BBB) model. Multiple group comparisons were performed by one-way analysis of variance with Dunnet’s post hoc tests. Results We found that SDG diminished leukocyte adhesion to and migration across the BBB in vivo in the setting of aseptic encephalitis (intracerebral TNFα injection) and prevented enhanced BBB permeability during systemic inflammatory response (LPS injection). In vitro SDG pretreatment of primary human brain microvascular endothelial cells (BMVEC) or human monocytes diminished adhesion and migration of monocytes across brain endothelial monolayers in conditions mimicking CNS inflammatory responses. Consistent with our in vivo observations, SDG decreased expression of the adhesion molecule, VCAM1, induced by TNFα, or IL-1β in BMVEC. SDG diminished expression of the active form of VLA-4 integrin (promoting leukocyte adhesion and migration) and prevented the cytoskeleton changes in primary human monocytes activated by relevant inflammatory stimuli. Conclusion This study indicates that SDG directly inhibits BBB interactions with inflammatory cells and reduces the inflammatory state of leukocytes. Though more work is needed to determine the mechanism by which SDG mediates these effects, the ability of SDG to exert a multi-functional response reducing oxidative stress, inflammation, and BBB permeability makes it an exciting potential therapeutic for neuroinflammatory diseases. SDG can serve as an anti-inflammatory and barrier-protective agent in neuroinflammation

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

Activation of TonEBP by Calcium Controls β1,3-Glucuronosyltransferase-I Expression, a Key Regulator of Glycosaminoglycan Synthesis in Cells of the Intervertebral Disc*

The goal of this investigation was to study the expression and regulation of β1,3-Glucuronosyltransferase-I (GlcAT-I), a key enzyme regulating GAG synthesis in cells of the intervertebral disc. There was a robust expression of GlcAT-I in the nucleus pulposus in vivo. Treatment with the calcium ionophore ionomycin resulted in increased GlcAT-I expression, whereas GlcAT-I promoter constructs lacking TonE site or a mutant TonE were unresponsive to the ionophore. Experiments using TonEBP and DN-TonEBP constructs showed that TonEBP positively regulated GlcAT-I promoter activity. ChIP analysis confirmed binding of TonEBP to the promoter. We further validated the role of TonEBP in controlling GlcAT-I expression using mouse embryo fibroblasts from TonEBP null mice. GlcAT-I promoter activity in null cells was significantly lower than the wild type cells. In contrast to wild type cells, treatment with ionomycin failed to increase GlcAT-I promoter activity in null cells. We then investigated if calcineurin (Cn)-NFAT signaling played a regulatory role in GlcAT-I expression. Inhibition of Cn following ionomycin treatment did not block GlcAT-I and tauT, a TonEBP-responsive reporter activity. GlcAT-I promoter activity was suppressed by co-expression of Cn, NFAT2, NFAT3, and NFAT4. Moreover, following ionomycin treatment, fibroblasts from CnAα and CnAβ null mice exhibited robust induction in GlcAT-I promoter activity compared with wild type cells. Results of these studies demonstrate that calcium regulates GlcAT-I expression in cells of the nucleus pulposus through a signaling network comprising both activator and suppressor molecules. The results suggest that by controlling both GAG and aggrecan synthesis, disc cells can autoregulate their osmotic environment and accommodate mechanical loading

Schematic of our E-cig exposure model.

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

E-cig exposure reduces pulmonary bacterial clearance in mice infected with <i>S</i>. <i>pneumoniae</i>.

<p>Mice were exposed to air or E-cig for 2 wks, then infected intranasally with 1x10⁵ colony forming units (CFU) of <i>S</i>. <i>pneumoniae</i>. Inflammation (A) and bacterial CFUs (B) were determined in BAL at 24h after infection (N = 10 mice per group). (C) In a separate group of mice, bacterial CFUs were quantified in lung homogenates (N = 10 mice per group). (D) Alveolar macrophages from air or E-cig exposed mice were harvested and infected with <i>S</i>. <i>pneumoniae</i> at multiplicities of infection of 10 and 20. Bacterial CFUs were quantified in cell-free culture media at 4 h after infection. (E) Intracellular (internalized) and extracellular bacteria (cell-free culture media) were quantified at 1 h after <i>ex vivo</i> infection of alveolar macrophages with an MOI of 20. (F) Alveolar macrophages were harvested at 2h after final exposure, stained with antibodies against CD36 and MARCO, and analyzed by flow cytometry. (G) Bacterial clearance was measured in alveolar macrophages from mice exposed to air or traditional E-cig vapor. *p<0.05 by Student’s two-tailed t-test.</p

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