Adjuvant effect of inhaled particulate matter containing free radicals following house-dust mite induction of asthma in mice.

INTRODUCTION: Exposures to particulate matter (PM) from combustion sources can exacerbate preexisting asthma. However, the cellular and molecular mechanisms by which PM promotes the exacerbation of asthma remain elusive. We used a house dust mite (HDM)-induced mouse model of asthma to test the hypothesis that inhaled DCB230, which are PM containing environmentally persistent free radicals (EPFRs), will aggravate asthmatic responses. METHODS: Groups of 8-10-week-old C57BL/6 male mice were exposed to either air or DCB230 aerosols at a concentration of 1.5 mg/m3 4 h/day for 10 days with or without prior HDM-induction of asthma. RESULTS: Aerosolized DCB230 particles formed small aggregates (30-150 nm). Mice exposed to DCB230 alone showed significantly reduced lung tidal volume, overexpression of the Muc5ac gene, and dysregulation of 4 inflammation related genes, Ccl11, Ccl24, Il-10, and Tpsb2. This suggests DCB230 particles interacted with the lung epithelium inducing mucous hypersecretion and restricting lung volume. In addition to reduced lung tidal volume, compared to respective controls, the HDM + DCB230-exposed group exhibited significantly increased lung tissue damping and up-regulated expression of Muc5ac, indicating that in this model, mucous hypersecretion may be central to pulmonary dysfunction. This group also showed augmented lung eosinophilic inflammation accompanied by an up-regulation of 36 asthma related genes. Twelve of these genes are part of IL-17 signaling, suggesting that this pathway is critical for DCB230 induced toxicity and adjuvant effects in lungs previously exposed to HDM. CONCLUSION: Our data indicate that inhaled DCB230 can act as an adjuvant, exacerbating asthma through IL-17-mediated responses in a HDM mouse model.

Environmentally persistent free radicals: Methods for combustion generation, whole-body inhalation and assessing cardiopulmonary consequences.

Particulate matter (PM) containing environmentally persistent free radicals (EPFRs) results from the incomplete combustion of organic wastes which chemisorb to transition metals. This process generates a particle-pollutant complex that continuously redox cycles to produce reactive oxygen species. EPFRs are well characterized, but their cardiopulmonary effects remain unknown. This publication provides a detailed approach to evaluating these effects and demonstrates the impact that EPFRs have on the lungs and vasculature. Combustion-derived EPFRs were generated (EPFR lo: 2.1e-16 radical/g, EPFR hi: 5.5e-17 radical/g), characterized, and verified as representative of those found in urban areas. Dry particle aerosolization and whole-body inhalation were established for rodent exposures. To verify that these particles and exposures recapitulate findings relevant to known PM-induced cardiopulmonary effects, male C57BL6 mice were exposed to filtered air, ∼280 µg/m3 EPFR lo or EPFR hi for 4 h/d for 5 consecutive days. Compared to filtered air, pulmonary resistance was increased in mice exposed to EPFR hi. Mice exposed to EPFR hi also exhibited increased plasma endothelin-1 (44.6 vs 30.6 pg/mL) and reduced nitric oxide (137 nM vs 236 nM), suggesting vascular dysfunction. Assessment of vascular response demonstrated an impairment in endothelium-dependent vasorelaxation, with maximum relaxation decreased from 80% to 62% in filtered air vs EPFR hi exposed mice. Gene expression analysis highlighted fold changes in aryl hydrocarbon receptor (AhR) and antioxidant response genes including increases in lung Cyp1a1 (8.7 fold), Cyp1b1 (9 fold), Aldh3a1 (1.7 fold) and Nqo1 (2.4 fold) and Gclc (1.3 fold), and in aortic Cyp1a1 (5.3 fold) in mice exposed to EPFR hi vs filtered air. We then determined that lung AT2 cells were the predominate locus for AhR activation. Together, these data suggest the lung and vasculature as particular targets for the health impacts of EPFRs and demonstrate the importance of additional studies investigating the cardiopulmonary effects of EPFRs.

Increased oxidative stress responses in murine macrophages exposed at the air-liquid interface to third- and fourth-generation electronic nicotine delivery system (ENDS) aerosols.

Background: New fourth generation electronic nicotine delivery system (ENDS) devices contain high levels of nicotine salt (up to 60 mg/mL), whose cellular and molecular effects on immune cells are currently unknown. Here, we used a physiologically-relevant in vitro air-liquid interface (ALI) exposure model to assess the toxicity of distinct ENDS, a 3rd-generation electronic-cigarette (e-cig) and two 4th-generation ENDS devices (JUUL and Posh Plus). Methods: Murine macrophages (RAW 264.7) were exposed at the ALI to either air, Menthol or Crème Brûlée-flavored ENDS aerosols generated from those devices for 1-hour per day for 1 or 3 consecutive days. Cellular and molecular toxicity was evaluated 24 h post-exposure. Results: 1-day of Menthol-flavored JUUL aerosol exposure significantly decreased cell viability and significantly increased lactate dehydrogenase (LDH) levels compared to air controls. Further, JUUL Menthol elicited significantly increased reactive oxygen species (ROS) and nitric oxide (NO) production compared to air controls. Posh Crème Brûlée-flavored aerosols displayed significant cytotoxicity - decreased cell viability and increased LDH levels -after 1- and 3-day exposures, while the Crème Brûlée-flavored aerosol produced by the 3rd-generation e-cig device only displayed significant cytotoxicity after 3 days compared to air controls. Further, both Posh and third-generation e-cig Crème Brûlée flavored-aerosols elicited significantly increased ROS plus high levels of 8-isoprostane after 1 and 3 days compared to air controls, indicating increased oxidative stress. Posh and third-generation e-cig Crème Brûlée flavored-aerosols elicited reduction in NO levels after one day, but elicited increase in NO after 3 days. Genes in common dysregulated by both devices after 1 day included α7nAChR, Cyp1a1, Ahr, Mmp12, and iNos. Conclusion: Our results suggest that ENDS Menthol and Crème Brûlée-flavored aerosol exposures from both 3rd- and 4th-generation ENDS devices are cytotoxic to macrophages and cause oxidative stress. This can translate into macrophage dysfunction. Although 4th-generation disposable ENDS devices have no adjustable operational settings and are considered low-powered ENDS devices, their aerosols can induce cellular toxicity compared to air-exposed control cells. This study provides scientific evidence for regulation of nicotine salt-based disposable ENDS products.

The E-cigarette or Vaping Product Use-Associated Lung Injury Epidemic: Pathogenesis, Management, and Future Directions: An Official American Thoracic Society Workshop Report.

E-cigarette or vaping product use-associated lung injury (EVALI) is a severe pulmonary illness associated with the use of e-cigarettes or vaping products that was officially identified and named in 2019. This American Thoracic Society workshop was convened in 2021 to identify and prioritize research and regulatory needs to adequately respond to the EVALI outbreak and to prevent similar instances of disease associated with e-cigarette or vaping product use. An interdisciplinary group of 26 experts in adult and pediatric clinical care, public health, regulatory oversight, and toxicology were convened for the workshop. Four major topics were examined: 1) the public health and regulatory response to EVALI; 2) EVALI clinical care; 3) mechanisms contributing to EVALI; and 4) needed actions to address the health effects of EVALI. Oral presentations and group discussion were the primary modes used to identify top priorities for addressing EVALI. Initiatives including a national EVALI case registry and biorepository, integrated electronic medical record coding system, U.S. Food and Drug Administration regulation and enforcement of nicotine e-cigarette standards, regulatory authority over nontobacco-derived e-cigarettes, training in evaluating exogenous exposures, prospective clinical studies, standardized clinical follow-up assessments, ability to more readily study effects of cannabinoid e-cigarettes, and research to identify biomarkers of exposure and disease were identified as critical needs. These initiatives will require substantial federal investment as well as changes to regulatory policy. Overall, the workshop identified the need to address the root causes of EVALI to prevent future outbreaks. An integrated approach from multiple perspectives is required, including public health; clinical, basic, and translational research; regulators; and users of e-cigarettes. Improving the public health response to reduce the risk of another substantial disease-inducing event depends on coordinated actions to better understand the inhalational toxicity of these products, informing the public of the risks, and developing and enforcing regulatory standards for all e-cigarettes.

Genomic Basis for Individual Differences in Susceptibility to the Neurotoxic Effects of Diesel Exhaust.

Air pollution is a known environmental health hazard. A major source of air pollution includes diesel exhaust (DE). Initially, research on DE focused on respiratory morbidities; however, more recently, exposures to DE have been associated with neurological developmental disorders and neurodegeneration. In this study, we investigated the effects of sub-chronic inhalation exposure to DE on neuroinflammatory markers in two inbred mouse strains and both sexes, including whole transcriptome examination of the medial prefrontal cortex. We exposed aged male and female C57BL/6J (B6) and DBA/2J (D2) mice to DE, which was cooled and diluted with HEPA-filtered compressed air for 2 h per day, 5 days a week, for 4 weeks. Control animals were exposed to HEPA-filtered air on the same schedule as DE-exposed animals. The prefrontal cortex was harvested and analyzed for proinflammatory cytokine gene expression (Il1ß, Il6, Tnfα) and transcriptome-wide response by RNA-seq. We observed differential cytokine gene expression between strains and sexes in the DE-exposed vs. control-exposed groups for Il1ß, Tnfα, and Il6. For RNA-seq, we identified 150 differentially expressed genes between air and DE treatment related to natural killer cell-mediated cytotoxicity per Kyoto Encyclopedia of Genes and Genomes pathways. Overall, our data show differential strain-related effects of DE on neuroinflammation and neurotoxicity and demonstrate that B6 are more susceptible than D2 to gene expression changes due to DE exposures than D2. These results are important because B6 mice are often used as the default mouse model for DE studies and strain-related effects of DE neurotoxicity warrant expanded studies.

In utero exposures to mint-flavored JUUL aerosol impair lung development and aggravate house dust mite-induced asthma in adult offspring mice.

There are few reports concerning electronic nicotine delivery system (ENDS) use during pregnancy and no studies on asthma in prenatally JUUL-exposed offspring. Here, we tested the hypothesis that in utero JUUL exposure causes unfavorable birth outcomes and lasting pulmonary health effects in adult offspring. BALB/c dams were exposed to either air or mint-flavored JUUL aerosol, 1-hr/d, 20 consecutive days during gestation. Offspring were sacrificed on post-natal day (PND) 0 or at 11-week of age, following house dust mite (HDM) challenge. Gene expression was assessed in the uterine/placental tissue of the dams and lung responses were assessed in offspring at PND0 and at 11 weeks of age. JUUL-exposed offspring exhibited decreased body weights and lengths at PND0. These birth outcomes were accompanied by dysregulation of 54 genes associated with hypoxia and oxidative stress in the uterine/placental tissues of JUUL-exposed dams, as well as 24 genes in the lungs of the offspring related to Wnt signaling, plus 9 genes related to epigenetics, and 7 genes related to inflammation. At 11 weeks of age, JUUL + HDM exposed mice exhibited pulmonary inflammation when compared to their respective air + HDM controls. Additionally, the JUUL + HDM exposure dysregulated several genes associated with allergies and asthma. Further, the JUUL + HDM females showed decreased methylation of the promoter region of the Il10ra gene. Taken together, our mouse model shows that inhalation of JUUL aerosols during pregnancy affects the intrauterine environment, impairs lung development, and heightens the effects of allergic airway responses later in life.

In utero exposure to electronic-cigarette aerosols decreases lung fibrillar collagen content, increases Newtonian resistance and induces sex-specific molecular signatures in neonatal mice.

Approximately 7% of pregnant women in the United States use electronic-cigarette (e-cig) devices during pregnancy. There is, however, no scientific evidence to support e-cig use as being 'safe' during pregnancy. Little is known about the effects of fetal exposures to e-cig aerosols on lung alveologenesis. In the present study, we tested the hypothesis that in utero exposure to e-cig aerosol impairs lung alveologenesis and pulmonary function in neonates. Pregnant BALB/c mice were exposed 2 h a day for 20 consecutive days during gestation to either filtered air or cinnamon-flavored e-cig aerosol (36 mg/mL of nicotine). Lung tissue was collected in offspring during lung alveologenesis on postnatal day (PND) 5 and PND11. Lung function was measured at PND11. Exposure to e-cig aerosol in utero led to a significant decrease in body weights at birth which was sustained through PND5. At PND5, in utero e-cig exposures dysregulated genes related to Wnt signaling and epigenetic modifications in both females (~ 120 genes) and males (40 genes). These alterations were accompanied by reduced lung fibrillar collagen content at PND5-a time point when collagen content is close to its peak to support alveoli formation. In utero exposure to e-cig aerosol also increased the Newtonian resistance of offspring at PND11, suggesting a narrowing of the conducting airways. At PND11, in females, transcriptomic dysregulation associated with epigenetic alterations was sustained (17 genes), while WNT signaling dysregulation was largely resolved (10 genes). In males, at PND11, the expression of only 4 genes associated with epigenetics was dysregulated, while 16 Wnt related-genes were altered. These data demonstrate that in utero exposures to cinnamon-flavored e-cig aerosols alter lung structure and function and induce sex-specific molecular signatures during lung alveologenesis in neonatal mice. This may reflect epigenetic programming affecting lung disease development later in life.

Mmp12 Is Upregulated by in utero Second-Hand Smoke Exposures and Is a Key Factor Contributing to Aggravated Lung Responses in Adult Emphysema, Asthma, and Lung Cancer Mouse Models.

Matrix metalloproteinase-12 (Mmp12) is upregulated by cigarette smoke (CS) and plays a critical role in extracellular matrix remodeling, a key mechanism involved in physiological repair processes, and in the pathogenesis of emphysema, asthma, and lung cancer. While cigarette smoking is associated with the development of chronic obstructive pulmonary diseases (COPD) and lung cancer, in utero exposures to CS and second-hand smoke (SHS) are associated with asthma development in the offspring. SHS is an indoor air pollutant that causes known adverse health effects; however, the mechanisms by which in utero SHS exposures predispose to adult lung diseases, including COPD, asthma, and lung cancer, are poorly understood. In this study, we tested the hypothesis that in utero SHS exposure aggravates adult-induced emphysema, asthma, and lung cancer. Methods: Pregnant BALB/c mice were exposed from gestational days 6-19 to either 3 or 10mg/m3 of SHS or filtered air. At 10, 11, 16, or 17weeks of age, female offspring were treated with either saline for controls, elastase to induce emphysema, house-dust mite (HDM) to initiate asthma, or urethane to promote lung cancer. At sacrifice, specific disease-related lung responses including lung function, inflammation, gene, and protein expression were assessed. Results: In the elastase-induced emphysema model, in utero SHS-exposed mice had significantly enlarged airspaces and up-regulated expression of Mmp12 (10.3-fold compared to air-elastase controls). In the HDM-induced asthma model, in utero exposures to SHS produced eosinophilic lung inflammation and potentiated Mmp12 gene expression (5.7-fold compared to air-HDM controls). In the lung cancer model, in utero exposures to SHS significantly increased the number of intrapulmonary metastases at 58weeks of age and up-regulated Mmp12 (9.3-fold compared to air-urethane controls). In all lung disease models, Mmp12 upregulation was supported at the protein level. Conclusion: Our findings revealed that in utero SHS exposures exacerbate lung responses to adult-induced emphysema, asthma, and lung cancer. Our data show that MMP12 is up-regulated at the gene and protein levels in three distinct adult lung disease models following in utero SHS exposures, suggesting that MMP12 is central to in utero SHS-aggravated lung responses.

Inhalation of particulate matter containing free radicals leads to decreased vascular responsiveness associated with an altered pulmonary function.

Airborne particulate matter (PM) is associated with an increased risk for cardiovascular diseases. Although the goal of thermal remediation is to eliminate organic wastes through combustion, when incomplete combustion occurs, organics chemisorb to transition metals to generate PM-containing environmentally persistent free radicals (EPFRs). Similar EPFR species have been detected in PM found in diesel and gasoline exhaust, woodsmoke, and urban air. Prior in vivo studies demonstrated that EPFRs reduce cardiac function secondary to elevations in pulmonary arterial pressures. In vitro studies showed that EPFRs increase ROS and cytokines in pulmonary epithelial cells. We thus hypothesized that EPFR inhalation would promote lung inflammation and oxidative stress, leading to systemic inflammation, vascular endothelial injury, and a decline in vascular function. Mice were exposed to EPFRs for either 4 h or for 4 h/day for 10 days and lung and vascular function were assessed. After a 4-h exposure, plasma nitric oxide (NO) was reduced while endothelin-1 (ET-1) was increased, however lung function was not altered. After 10 day, plasma NO and ET-1 levels were again altered and lung tidal volume was reduced. These time course studies suggested the vasculature may be an early target of injury. To test this hypothesis, an intermediate time point of 3 days was selected. Though the mice exhibited no marked inflammation in either the lung or the blood, we did note significantly reduced endothelial function concurrent with a reduction in lung tidal volume and an elevation in annexin V protein levels in the lung. Although vascular dysfunction was not dependent upon inflammation, it may be associated with an injury at the air-blood interface. Gene expression analysis suggested roles for oxidative stress and aryl hydrocarbon receptor (Ahr) signaling. Studies probing the relationship between pulmonary oxidative stress and AhR signaling at the air-blood interface with vascular dysfunction seem warranted.NEW & NOTEWORTHY Particulate matter (PM) resulting from the combustion of organic matter is known to contribute to cardiopulmonary disease. Despite hypotheses that cardiovascular dysfunction occurring after PM exposures is secondary to lung or systemic inflammation, these studies investigating exposures to PM-containing environmentally persistent free radicals (EPFRs) demonstrate that cardiovascular dysfunction precedes pulmonary inflammation. The cardiopulmonary health consequences of EPFRs have yet to be thoroughly evaluated, especially in healthy, adult mice. Our data suggest the vasculature as a direct target of PM exposure, and our studies aimed to elucidate the mechanisms contributing to EPFR-induced vascular dysfunction.

Effects of Chronic Secondhand Smoke (SHS) Exposure on Cognitive Performance and Metabolic Pathways in the Hippocampus of Wild-Type and Human Tau Mice.

BACKGROUND: Exposure to secondhand smoke (SHS) is a risk factor for developing sporadic forms of sporadic dementia. A human tau (htau) mouse model is available that exhibits age-dependent tau dysregulation, neurofibrillary tangles, neuronal loss, neuroinflammation, and oxidative stress starting at an early age (3-4 months) and in which tau dysregulation and neuronal loss correlate with synaptic dysfunction and cognitive decline. OBJECTIVE: The goal of this study was to assess the effects of chronic SHS exposure (10 months' exposure to ∼30 mg/m3) on behavioral and cognitive function, metabolism, and neuropathology in mice. METHODS: Wild-type (WT) and htau female and male mice were exposed to SHS (90% side stream, 10% main stream) using the SCIREQ® inExpose™ system or air control for 168 min per day, for 312 d, 7 d per week. The exposures continued during the days of behavioral and cognitive testing. In addition to behavioral and cognitive performance and neuropathology, the lungs of mice were examined for pathology and alterations in gene expression. RESULTS: Mice exposed to chronic SHS exposure showed the following genotype-dependent responses: a) lower body weights in WT, but not htau, mice; b) less spontaneous alternation in WT, but not htau, mice in the Y maze; c) faster swim speeds of WT, but not htau, mice in the water maze; d) lower activity levels of WT and htau mice in the open field; e) lower expression of brain PHF1, TTCM1, IGF1ß, and HSP90 protein levels in WT male, but not female, mice; and f) more profound effects on hippocampal metabolic pathways in WT male than female mice and more profound effects in WT than htau mice. DISCUSSION: The brain of WT mice, in particular WT male mice, might be especially susceptible to the effects of chronic SHS exposure. In WT males, independent pathways involving ascorbate, flavin adenine dinucleotide, or palmitoleic acid might contribute to the hippocampal injury following chronic SHS exposure. https://doi.org/10.1289/EHP8428.

Sub-ohm vaping increases the levels of carbonyls, is cytotoxic, and alters gene expression in human bronchial epithelial cells exposed at the air-liquid interface.

BACKGROUND: Exposure to electronic-cigarette (e-cig) aerosols induces potentially fatal e-cig or vaping-associated lung injury (EVALI). The cellular and molecular mechanisms underlying these effects, however, are unknown. We used an air-liquid interface (ALI) in vitro model to determine the influence of two design characteristics of third-generation tank-style e-cig devices-resistance and voltage-on (1) e-cig aerosol composition and (2) cellular toxicity. METHODS: Human bronchial epithelial cells (H292) were exposed to either butter-flavored or cinnamon-flavored e-cig aerosols at the ALI in a Vitrocell exposure system connected to a third-generation e-cig device. Exposures were conducted following a standard vaping topography profile for 2 h per day, for 1 or 3 consecutive days. 24 h after ALI exposures cellular and molecular outcomes were assessed. RESULTS: We found that butter-flavored e-cig aerosol produced under 'sub-ohm' conditions (< 0.5 Ω) contains high levels of carbonyls (7-15 µg/puff), including formaldehyde, acetaldehyde and acrolein. E-cig aerosol produced under regular vaping conditions (resistance > 1 Ω and voltage > 4.5 V), contains lower carbonyl levels (< 2 µg/puff). We also found that the levels of carbonyls produced in the cinnamon-flavored e-cig aerosols were much lower than that of the butter-flavored aerosols. H292 cells exposed to butter-flavored or cinnamon-flavored e-cig aerosol at the ALI under 'sub-ohm' conditions for 1 or 3 days displayed significant cytotoxicity, decreased tight junction integrity, increased reactive oxygen species production, and dysregulated gene expression related to biotransformation, inflammation and oxidative stress (OS). Additionally, the cinnamon-flavored e-cig aerosol induced pro-oxidant effects as evidenced by increases in 8-hydroxy-2-deoxyguanosine protein levels. Moreover, we confirmed the involvement of OS as a toxicity process for cinnamon-flavored e-cig aerosol by pre-treating the cells with N-acetyl cysteine (NAC), an antioxidant that prevented the cells from the OS-mediated damage induced by the e-cig aerosol. CONCLUSION: The production of high levels of carbonyls may be flavor specific. Overall, inhaling e-cig aerosols produced under 'sub-ohm' conditions is detrimental to lung epithelial cells, potentially via mechanisms associated with OS. This information could help policymakers take the necessary steps to prevent the manufacturing of sub-ohm atomizers for e-cig devices.

Cell-specific toxicity of short-term JUUL aerosol exposure to human bronchial epithelial cells and murine macrophages exposed at the air-liquid interface.

BACKGROUD: JUUL, an electronic nicotine delivery system (ENDS), which first appeared on the US market in 2015, controled more than 75% of the US ENDS sales in 2018. JUUL-type devices are currently the most commonly used form of ENDS among youth in the US. In contrast to free-base nicotine contained in cigarettes and other ENDS, JUUL contains high levels of nicotine salt (35 or 59 mg/mL), whose cellular and molecular effects on lung cells are largely unknown. In the present study, we evaluated the in vitro toxicity of JUUL crème brûlée-flavored aerosols on 2 types of human bronchial epithelial cell lines (BEAS-2B, H292) and a murine macrophage cell line (RAW 264.7). METHODS: Human lung epithelial cells and murine macrophages were exposed to JUUL crème brûlée-flavored aerosols at the air-liquid interface (ALI) for 1-h followed by a 24-h recovery period. Membrane integrity, cytotoxicity, extracellular release of nitrogen species and reactive oxygen species, cellular morphology and gene expression were assessed. RESULTS: Crème brûlée-flavored aerosol contained elevated concentrations of benzoic acid (86.9 µg/puff), a well-established respiratory irritant. In BEAS-2B cells, crème brûlée-flavored aerosol decreased cell viability (≥ 50%) and increased nitric oxide (NO) production (≥ 30%), as well as iNOS gene expression. Crème brûlée-flavored aerosol did not affect the viability of either H292 cells or RAW macrophages, but increased the production of reactive oxygen species (ROS) by ≥ 20% in both cell types. While crème brûlée-flavored aerosol did not alter NO levels in H292 cells, RAW macrophages exposed to crème brûlée-flavored aerosol displayed decreased NO (≥ 50%) and down-regulation of the iNOS gene, possibly due to increased ROS. Additionally, crème brûlée-flavored aerosol dysregulated the expression of several genes related to biotransformation, inflammation and airway remodeling, including CYP1A1, IL-6, and MMP12 in all 3 cell lines. CONCLUSION: Our results indicate that crème brûlée-flavored aerosol causes cell-specific toxicity to lung cells. This study contributes to providing scientific evidence towards regulation of nicotine salt-based products.

Electronic-Cigarette Vehicles and Flavoring Affect Lung Function and Immune Responses in a Murine Model.

The use of electronic nicotine delivery systems (ENDS), also known as electronic-cigarettes (e-cigs), has raised serious public health concerns, especially in light of the 2019 outbreak of e-cig or vaping product use-associated acute lung injury (EVALI). While these cases have mostly been linked to ENDS that contain vitamin E acetate, there is limited research that has focused on the chronic pulmonary effects of the delivery vehicles (i.e., without nicotine and flavoring). Thus, we investigated lung function and immune responses in a mouse model following exposure to the nearly ubiquitous e-cig delivery vehicles, vegetable glycerin (VG) and propylene glycol (PG), used with a specific 70%/30% ratio, with or without vanilla flavoring. We hypothesized that mice exposed sub-acutely to these e-cig aerosols would exhibit lung inflammation and altered lung function. Adult female C57BL/6 mice (n = 11-12 per group) were exposed to filtered air, 70%/30% VG/PG, or 70%/30% VG/PG with a French vanilla flavoring for 2 h a day for 6 weeks. Prior to sacrifice, lung function was assessed. At sacrifice, broncho-alveolar lavage fluid and lung tissue were collected for lipid mediator analysis, flow cytometry, histopathology, and gene expression analyses. Exposures to VG/PG + vanilla e-cig aerosol increased lung tidal and minute volumes and tissue damping. Immunophenotyping of lung immune cells revealed an increased number of dendritic cells, CD4+ T cells, and CD19+ B cells in the VG/PG-exposed group compared to air, irrespective of the presence of vanilla flavoring. Quantification of bioactive lung lipids demonstrated a >3-fold increase of 2-arachidonoylglycerol (2-AG), an anti-inflammatory mediator, and a 2-fold increase of 12-hydroxyeicosatetraenoic acid (12-HETE), another inflammatory mediator, following VG/PG exposure, with or without vanilla flavoring. This suggests that e-cig aerosol vehicles may affect immunoregulatory molecules. We also found that the two e-cig aerosols dysregulated the expression of lung genes. Ingenuity Pathway Analysis revealed that the gene networks that are dysregulated by the VG/PG e-cig aerosol are associated with metabolism of cellular proteins and lipids. Overall, our findings demonstrate that VG and PG, the main constituents of e-liquid formulations, when aerosolized through an e-cig device, are not harmless to the lungs, since they disrupt immune homeostasis.

In utero exposures to electronic-cigarette aerosols impair the Wnt signaling during mouse lung development.

Currently, more than 9 million American adults, including women of childbearing age, use electronic-cigarettes (e-cigs). Further, the prevalence of maternal vaping now approaching 10% is similar to that of maternal smoking. Little, however, is known about the effects of fetal exposures to nicotine-rich e-cig aerosols on lung development. In this study, we assessed whether in utero exposures to e-cig aerosols compromised lung development in mice. A third-generation e-cig device was used to expose pregnant BALB/c mice by inhalation to 36 mg/mL of nicotine cinnamon-flavored e-cig aerosols for 14-31 days. This included exposures for either 12 days before mating plus during gestation (preconception groups) or only during gestation (prenatal groups). Respective control mice were exposed to filtered air. Subgroups of offspring were euthanized at birth or at 4 wk of age. Compared with respective air-exposed controls, both preconception and prenatal exposures to e-cig aerosols significantly decreased the offspring birth weight and body length. In the preconception group, 7 inflammation-related genes were downregulated, including 4 genes common to both dams and fetuses, denoting an e-cig immunosuppressive effect. Lung morphometry assessments of preconception e-cig-exposed offspring showed a significantly increased tissue fraction at birth. This result was supported by the downregulation of 75 lung genes involved in the Wnt signaling, which is essential to lung organogenesis. Thus, our data indicate that maternal vaping impairs pregnancy outcomes, alters fetal lung structure, and dysregulates the Wnt signaling. This study provides experimental evidence for future regulations of e-cig products for pregnant women and developmentally vulnerable populations.

Mitochondrial - nuclear genetic interaction modulates whole body metabolism, adiposity and gene expression in vivo.

We hypothesized that changes in the mitochondrial DNA (mtDNA) would significantly influence whole body metabolism, adiposity and gene expression in response to diet. Because it is not feasible to directly test these predictions in humans we used Mitochondrial-Nuclear eXchange mice, which have reciprocally exchanged nuclear and mitochondrial genomes between different Mus musculus strains. Results demonstrate that nuclear-mitochondrial genetic background combination significantly alters metabolic efficiency and body composition. Comparative RNA sequencing analysis in adipose tissues also showed a clear influence of the mtDNA on regulating nuclear gene expression on the same nuclear background (up to a 10-fold change in the number of differentially expressed genes), revealing that neither Mendelian nor mitochondrial genetics unilaterally control gene expression. Additional analyses indicate that nuclear-mitochondrial genome combination modulates gene expression in a manner heretofore not described. These findings provide a new framework for understanding complex genetic disease susceptibility.

Generation of Electronic Cigarette Aerosol by a Third-Generation Machine-Vaping Device: Application to Toxicological Studies.

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and flavoring chemicals. The aerosol produced includes fine and ultrafine particles, and potentially nicotine and aldehydes, which can be harmful to human health. E-cig users inhale these aerosols and, with the third-generation of e-cig devices, control design features (resistance and voltage) in addition to the choice of e-liquids, and the puffing profile. These are key factors that can significantly impact the toxicity of the inhaled aerosols. E-cig research, however, is challenging and complex mostly due to the absence of standardized assessments and to the numerous varieties of e-cig models and brands, as well as e-liquid flavors and solvents that are available on the market. These considerations highlight the urgent need to harmonize e-cig research protocols, starting with e-cig aerosol generation and characterization techniques. The current study focuses on this challenge by describing a detailed step-by-step e-cig aerosol generation technique with specific experimental parameters that are thought to be realistic and representative of real-life exposure scenarios. The methodology is divided into four sections: preparation, exposure, post-exposure analysis, plus cleaning and maintenance of the device. Representative results from using two types of e-liquid and various voltages are presented in terms of mass concentration, particle size distribution, chemical composition and cotinine levels in mice. These data demonstrate the versatility of the e-cig exposure system used, aside from its value for toxicological studies, as it allows for a broad range of computer-controlled exposure scenarios, including automated representative vaping topography profiles.

Early Postnatal Secondhand Smoke Exposure Disrupts Bacterial Clearance and Abolishes Immune Responses in Muco-Obstructive Lung Disease.

Secondhand smoke (SHS) exposure has been linked to the worsening of ongoing lung diseases. However, whether SHS exposure affects the manifestation and natural history of imminent pediatric muco-obstructive airway diseases such as cystic fibrosis remains unclear. To address these questions, we exposed Scnn1b transgenic (Scnn1b-Tg+) mice to SHS from postnatal day (PND) 3-21 and lung phenotypes were examined at PND22. Although a majority of filtered air (FA)-exposed Scnn1b-Tg+ (FA-Tg+) mice successfully cleared spontaneous bacterial infections by PND22, the SHS-exposed Scnn1b-Tg+ (SHS-Tg+) mice failed to resolve these infections. This defect was associated with suppressed antibacterial defenses, i.e., phagocyte recruitment, IgA secretion, and Muc5b expression. Whereas the FA-Tg+ mice exhibited marked mucus obstruction and Th2 responses, SHS-Tg+ mice displayed a dramatic suppression of these responses. Mechanistically, downregulated expression of IL-33, a stimulator of type II innate lymphoid cells, in lung epithelial cells was associated with suppression of neutrophil recruitment, IgA secretions, Th2 responses, and delayed bacterial clearance in SHS-Tg+ mice. Cessation of SHS exposure for 21 d restored previously suppressed responses, including phagocyte recruitment, IgA secretion, and mucous cell metaplasia. However, in contrast with FA-Tg+ mice, the SHS-Tg+ mice had pronounced epithelial necrosis, alveolar space consolidation, and lymphoid hyperplasia; indicating lagged unfavorable effects of early postnatal SHS exposure in later life. Collectively, our data show that early postnatal SHS exposure reversibly suppresses IL-33 levels in airspaces which, in turn, results in reduced neutrophil recruitment and diminished Th2 response. Our data indicate that household smoking may predispose neonates with muco-obstructive lung disease to bacterial exacerbations.

Sex-specific lung functional changes in adult mice exposed only to second-hand smoke in utero.

BACKGROUND: An increasing number of epidemiological and experimental studies have associated exposure to second-hand smoke (SHS) during pregnancy with adverse outcomes in newborns. As we have previously shown in mice, in utero exposure to SHS at critical stages of fetal development, results in altered lung responses and increased disease susceptibility upon re-exposure to irritants (SHS or ovalbumin) in adulthood. In this study, we asked whether the in utero SHS exposure alone is sufficient to alter lung structure and function in adult mice. METHODS: Pregnant BALB/c mice were exposed from days 6 to 19 of pregnancy to 10 mg/m3 of SHS or HEPA-filtered air. Male and female offspring (n = 13-15/group) were sacrificed at 15 weeks of age. We measured lung function with non-invasive and invasive methods, performed lung morphometric analysis on trichrome-stained lung tissue samples, and assessed lung gene expression via RNA sequencing and protein assays. RESULTS: In utero SHS exposure significantly increased mean linear intercept and decreased the surface area per unit volume of the lungs in both males and females, indicating perturbation in alveolar developmental processes. Tidal volume, minute volume and inspiratory capacity were significantly decreased compared with the controls only in male mice exposed in utero to SHS, suggesting that males are more sensitive than females to an SHS insult during lung development. This also suggests that in our model, lung structure changes may be necessary but are not sufficient to impair lung function. SERPINA1A, the mouse ortholog of human α1-antitrypsin, deficiency of which is a known genetic risk factor for emphysema, was down-regulated at the protein level in the in utero SHS-exposed mice. Additionally, DNMT3A protein expression was dysregulated, indicating that DNA methylation occurred in the lungs. CONCLUSIONS: Our results indicate that in utero SHS exposure alone alters both lung function and structure well into adulthood (15 weeks) in male mice. Furthermore, lung function alterations in this model are sex-specific, with males being more susceptible to in utero SHS effects. Overall, our data suggest that in utero SHS exposure alone can predispose to adult lung diseases.

In utero exposure to second-hand smoke activates pro-asthmatic and oncogenic miRNAs in adult asthmatic mice.

Exposures to environmental pollutants contribute to dysregulated microRNA (miRNA) expression profiles, which have been implicated in various diseases. Previously, we reported aggravated asthmatic responses in ovalbumin (OVA)-challenged adult mice that had been exposed in utero to second-hand smoke (SHS). Whether in utero SHS exposure dysregulates miRNA expression patterns in the adult asthma model has not been investigated. Pregnant BALB/c mice were exposed (days 6-19 of pregnancy) to SHS (10 mg/m(3)) or HEPA-filtered air. All offspring were sensitized and challenged with OVA (19-23 weeks) before sacrifice. RNA samples extracted from lung homogenates, were subjected to RNA sequencing (RNA-seq). RNA-seq identified nine miRNAs that were most significantly up-regulated by in utero SHS exposure. Among these nine, miR-155-5p, miR-21-3p, and miR-18a-5p were also highly correlated with pro-asthmatic Th2 cytokine levels in bronchoalveolar lavage fluid. Further analysis indicated that these up-regulated miRNAs shared common chromosome locations, particularly Chr 11C, with pro-asthmatic genes. These three miRNAs have also been characterized as oncogenic miRNAs (oncomirs). We cross-referenced miRNA-mRNA expression profiles and identified 16 tumor suppressor genes that were down-regulated in the in utero-exposed offspring and that are predicted targets of the up-regulated oncomirs. In conclusion, in utero SHS exposure activates pro-asthmatic genes and miRNAs, which colocalize at specific chromosome locations, in OVA-challenged adult mice. The oncogenic characteristics of the miRNAs and putative miRNA-mRNA regulatory networks suggest that the synergistic effect of in utero SHS exposure and certain adult irritants may promote an oncogenic milieu in mouse lungs via inhibition of miRNA-regulated tumor suppressor genes.

In utero exposure to second-hand smoke aggravates the response to ovalbumin in adult mice.

Second-hand smoke (SHS) exposure in utero exacerbates adult responses to environmental irritants. We tested the hypothesis that effects of in utero SHS exposure on modulating physiological and transcriptome responses in BALB/c mouse lungs after ovalbumin (OVA) challenge extend well into adulthood, and that the responses show a sex bias. We exposed BALB/c mice in utero to SHS or filtered air (AIR), then sensitized and challenged all offspring with OVA from 19 to 23 weeks of age. At the end of the adult OVA challenge, we evaluated pulmonary function, examined histopathology, analyzed bronchoalveolar lavage fluid (BALF), and assessed gene expression changes in the lung samples. All groups exhibited lung inflammation and inflammatory cell infiltration. Pulmonary function testing (airway hyperresponsiveness [AHR], breathing frequency [f]) and BALF (cell differentials, Th1/Th2 cytokines) assessments showed significantly more pronounced lung responses in the SHS-OVA groups than in AIR-OVA groups (AHR, f; eosinophils, neutrophils; IFN-γ, IL-1b, IL-4, IL-5, IL-10, IL-13, KC/CXCL1, TNF-α), with the majority of responses being more pronounced in males than in females. SHS exposure in utero also significantly altered lung gene expression profiles, primarily of genes associated with inflammatory responses and respiratory diseases, including lung cancer and lung fibrosis. Altered expression profiles of chemokines (Cxcl2, Cxcl5, Ccl8, Ccl24), cytokines (Il1b, Il6, Il13) and acute phase response genes (Saa1, Saa3) were confirmed by qRT-PCR. In conclusion, in utero exposure to SHS exacerbates adult lung responses to OVA challenge and promotes a pro-asthmatic milieu in adult lungs; further, males are generally more affected by SHS-OVA than are females.