Tobacco and menthol flavored nicotine-free electronic cigarettes induced inflammation and dysregulated repair in lung fibroblast and epithelium.

BACKGROUND: Electronic cigarette (e-cig) vaping has increased in the past decade in the US, and e-cig use is misleadingly marketed as a safe cessation for quitting smoking. The main constituents in e-liquid are humectants, such as propylene glycol (PG) and vegetable glycerine (VG), but different flavoring chemicals are also used. However, the toxicology profile of flavored e-cigs in the pulmonary tract is lacking. We hypothesized that menthol and tobacco-flavored e-cig (nicotine-free) exposure results in inflammatory responses and dysregulated repair in lung fibroblast and epithelium. METHOD: We exposed lung fibroblast (HFL-1) and epithelium (BEAS-2B) to Air, PG/VG, menthol flavored, or tobacco-flavored e-cig, and determined the cytotoxicity, inflammation, and wound healing ability in 2D cells and 3D microtissue chip models. RESULTS: After exposure, HFL-1 showed decreased cell number with increased IL-8 levels in the tobacco flavor group compared to air. BEAS-2B also showed increased IL-8 secretion after PG/VG and tobacco flavor exposure, while menthol flavor exposure showed no change. Both menthol and tobacco-flavored e-cig exposure showed decreased protein abundance of type 1 collagen α 1 (COL1A1), α-smooth-muscle actin (αSMA), and fibronectin as well as decreased gene expression level of αSMA (Acta2) in HFL-1. After tobacco flavor e-cig exposure, HFL-1 mediated wound healing and tissue contractility were inhibited. Furthermore, BEAS-2B exposed to menthol flavor showed significantly decreased tight junction gene expressions, such as CDH1, OCLN, and TJP1. CONCLUSION: Overall, tobacco-flavored e-cig exposure induces inflammation in both epithelium and fibroblasts, and tobacco-flavored e-cig inhibits wound healing ability in fibroblasts.

Prenatal Exposure to Electronic-Cigarette Aerosols Leads to Sex-Dependent Pulmonary Extracellular-Matrix Remodeling and Myogenesis in Offspring Mice.

Electronic-cigarette (e-cig) vaping is a serious concern, as many pregnant women who vape consider it safe. However, little is known about the harmful effects of prenatal e-cig exposure on adult offspring, especially on extracellular-matrix (ECM) deposition and myogenesis in the lungs of offspring. We evaluated the biochemical and molecular implications of maternal exposure during pregnancy to e-cig aerosols on the adult offspring of both sexes, with a particular focus on pulmonary ECM remodeling and myogenesis. Pregnant CD-1 mice were exposed to e-cig aerosols with or without nicotine, throughout gestation, and lungs were collected from adult male and female offspring. Compared with the air-exposed control group, female mice exposed to e-cig aerosols, with or without nicotine, demonstrated increased lung protein abundance of LEF-1 (lymphoid enhancer-binding factor 1), fibronectin, and E-cadherin, whereas altered E-cadherin and PPARγ (peroxisome proliferator-activated receptor γ) levels were observed only in males exposed to e-cig aerosols with nicotine. Moreover, lipogenic and myogenic mRNAs were dysregulated in adult offspring in a sex-dependent manner. PAI-1 (plasminogen activator inhibitor-1), one of the ECM regulators, was significantly increased in females exposed prenatally to e-cig aerosols with nicotine and in males exposed to e-cig aerosols compared with control animals exposed to air. MMP9 (matrix metalloproteinase 9), a downstream target of PAI-1, was downregulated in both sexes exposed to e-cig aerosols with nicotine. No differences in lung histology were observed among any of the treatment groups. Overall, adult mice exposed prenatally to e-cig aerosols could be predisposed to developing pulmonary disease later in life. Thus, these findings suggest that vaping during pregnancy is unsafe and increases the propensity for later-life interstitial lung diseases.

E-cigarette-induced pulmonary inflammation and dysregulated repair are mediated by nAChR α7 receptor: role of nAChR α7 in SARS-CoV-2 Covid-19 ACE2 receptor regulation.

Electronic cigarette (e-cig) vaping is increasing rapidly in the United States, as e-cigs are considered less harmful than combustible cigarettes. However, limited research has been conducted to understand the possible mechanisms that mediate toxicity and pulmonary health effects of e-cigs. We hypothesized that sub-chronic e-cig exposure induces inflammatory response and dysregulated repair/extracellular matrix (ECM) remodeling, which occur through the α7 nicotinic acetylcholine receptor (nAChRα7). Adult wild-type (WT), nAChRα7 knockout (KO), and lung epithelial cell-specific KO (nAChRα7 CreCC10) mice were exposed to e-cig aerosol containing propylene glycol (PG) with or without nicotine. Bronchoalveolar lavage fluids (BALF) and lung tissues were collected to determine e-cig induced inflammatory response and ECM remodeling, respectively. Sub-chronic e-cig exposure with nicotine increased inflammatory cellular influx of macrophages and T-lymphocytes including increased pro-inflammatory cytokines in BALF and increased SARS-Cov-2 Covid-19 ACE2 receptor, whereas nAChRα7 KO mice show reduced inflammatory responses associated with decreased ACE2 receptor. Interestingly, matrix metalloproteinases (MMPs), such as MMP2, MMP8 and MMP9, were altered both at the protein and mRNA transcript levels in female and male KO mice, but WT mice exposed to PG alone showed a sex-dependent phenotype. Moreover, MMP12 was increased significantly in male mice exposed to PG with or without nicotine in a nAChRα7-dependent manner. Additionally, sub-chronic e-cig exposure with or without nicotine altered the abundance of ECM proteins, such as collagen and fibronectin, significantly in a sex-dependent manner, but without the direct role of nAChRα7 gene. Overall, sub-chronic e-cig exposure with or without nicotine affected lung inflammation and repair responses/ECM remodeling, which were mediated by nAChRα7 in a sex-dependent manner.

Developmental perfluorooctane sulfonic acid exposure exacerbates house dust mite induced allergic responses in adult mice.

Perfluorooctane sulfonic acid (PFOS) is a long-chain per- and polyfluoroalkyl substance (PFAS), a persistent organic pollutant, which has been used in aqueous film-forming foams. Emerging epidemiological evidence indicates a significant body burden of PFOS is observed in the lungs. Furthermore, developmental PFOS exposure dysregulates lung development and exacerbates eosinophilic inflammation, which are critical risk factors for asthma. However, it is unknown whether PFOS exerts sex-dependent effects on house dust mite (HDM) induced asthmatic progression and allergic inflammation. In this study, timed pregnant Balb/cJ dams were dosed orally via PFOS (1.0 mg/kg/d) spiked or vehicle control mealworms from gestational day (GD) 0.5 to postnatal day (PND) 21. Subsequently, HDM (30 µg/day) was administered starting at PND 77-82 for 10 days, and the mice were sacrificed 48 h after their final treatment. The serum and lung PFOS concentrations were 3.391 ± 0.189 µg/mL and 3.567 ± 0.1676 µg/g in the offspring, respectively. Male mice exposed to PFOS + HDM showed higher total cell counts in bronchoalveolar lavage fluid (BALF), macrophage counts, and eosinophil counts compared to mice exposed to HDM alone. Female mice exposed to PFOS + HDM had increased BALF eosinophil percentage, mucous production, alternatively activated (M2) macrophage polarization, and M2-associated gene expression compared to female mice exposed to HDM alone. PFOS exposure had no significant effect on HDM-induced IL-4, IL-5, or IL-13, but RANTES was further elevated in female mice. Overall, our data suggest that developmental PFOS exposure increased the risk of exacerbated eosinophilic inflammation and M2 polarization, which were more severe in female mice, suggesting sex-dependent developmental effects of PFOS on allergic airway responses.

Developmental PFOS exposure alters lung inflammation and barrier integrity in juvenile mice.

Emerging epidemiological evidence indicates perfluorooctane sulfonic acid (PFOS) is increasingly associated with asthma and respiratory viral infections. Animal studies suggest PFOS disrupts lung development and immuno-inflammatory responses, but little is known about the potential consequences on respiratory health and disease risk. Importantly, PFOS exposure during the critical stages of lung development may increase disease risk later in life. Thus, we hypothesized that developmental PFOS exposure will affect lung inflammation and alveolar/airway development in a sex-dependent manner. To address this knowledge gap, timed pregnant Balb/cJ dams were orally dosed with a PFOS (1.0 or 2.0 mg/kg/d) injected mealworm or a vehicle control daily from gestational day (GD) 0.5 to postnatal day (PND) 21, and offspring were sacrificed at PND 22-23. PFOS-exposed male offspring displayed increased alveolar septa thickness. Occludin was also downregulated in the lungs after PFOS exposure in mice, indicative of barrier dysfunction. BALF macrophages were significantly elevated at 2.0 mg/kg/d PFOS in both sexes compared with vehicles, whereas BALF cytokines (TNF-α, IL-6, KC, MIP-1α, MIP-1ß, and MCP-1) were suppressed in PFOS-exposed male offspring compared with vehicle controls. Multiplex nucleic acid hybridization assay showed male-specific downregulation of cytokine gene expression in PFOS-exposed mice compared with vehicle mice. Overall, these results demonstrate PFOS exposure exhibits male-specific adverse effects on lung development and inflammation in juvenile offspring, possibly predisposing them to later-in-life respiratory disease. Further research is required to elucidate the mechanisms underlying the sex-differentiated pulmonary toxicity of PFOS.

MicroRNA mediated suppression of airway lactoperoxidase by TGF-ß1 and cigarette smoke promotes airway inflammation.

Transforming Growth Factor Beta1 (TGF-ß1) signaling is upregulated in Chronic Obstructive Pulmonary disease (COPD), smokers, and people living with HIV. Cigarette smoking and HIV are also independent risk factors for COPD. Chronic inflammation is a hallmark of COPD. However, the underlying mechanisms remain unknown. Previous research has suggested that TGF-ß1 alters the airway epithelial microRNAome and transcriptome, potentially contributing to lung inflammation. The Lactoperoxidase (LPO) system is an integral component of innate immunity within the airway. LPO plays a crucial role in host defense by catalyzing the oxidation of thiocyanate to hypothiocyanite in the presence of hydrogen peroxide (H2O2), generating a potent antibacterial and antiviral agent. Additionally, the LPO system potentially aids in maintaining cellular redox balance by reducing the levels of H2O2, thus mitigating oxidative stress within the airway epithelium. LPO dysfunction can impair immune responses and exacerbate inflammatory processes in respiratory diseases.In this study, primary bronchial epithelial cells and bronchial cell lines were treated with TGF-ß1 and exposed to cigarette smoke to characterize the effect of these factors on LPO and their downstream effects. RT-qPCR and Western Blot were applied to quantify mRNA and proteins' expression. The levels of H2O2 were detected using the Amplex Red Assay. Magnetofection and transfection were applied to probe the effect of miR-449b-5p. Staining procedures using the MitoTracker Green and C12FDG dyes were used to establish mitochondria mass and senescence. The levels of pro-inflammatory cytokines were measured via Luminex assays.We found that TGF-ß1 and cigarette smoke suppressed airway LPO expression, increasing H2O2 levels. This increase in H2O2 had downstream effects on mitochondrial homeostasis, epithelial cellular senescence, and the pro-inflammatory cytokine response. We demonstrate for the first time that airway LPO is regulated by TGF-ß1-induced miRNA-mediated post-transcriptional silencing through miR-449b-5p in the lungs. Further, we identify and validate miR-449-5p as the candidate miRNA upregulated by TGF-ß1, which is involved in LPO suppression. This paper demonstrates a new mechanism by which TGF-ß1 can lead to altered redox status in the airway.

Tobacco and Menthol flavored electronic cigarettes induced inflammation and dysregulated repair in lung fibroblast and epithelium.

Background: Electronic cigarette (e-cig) vaping has increased in the past decade in the US, and e-cig use is misleadingly marketed as a safe cessation for quitting smoking. The main constituents in e-liquid are humectants, such as propylene glycol (PG) and vegetable glycerine (VG), but different flavoring chemicals are also used. However, the toxicology profile of flavored e-cigs in the pulmonary tract is lacking. We hypothesized that menthol and tobacco-flavored e-cig (nicotine-free) exposure results in inflammatory responses and dysregulated repair in lung fibroblast and epithelium. Method: We exposed lung fibroblast (HFL-1) and epithelium (BEAS-2B) to Air, PG/VG, menthol flavored, or tobacco-flavored e-cig, and determined the cytotoxicity, inflammation, and wound healing ability of the cells in a microtissue chip model. Results: After exposure, HFL-1 showed decreased cell number with increased IL-8 levels in the tobacco flavor group compared to air. BEAS-2B also showed increased IL-8 secretion after PG/VG and tobacco flavor exposure, while menthol flavor exposure showed no change. Both menthol and tobacco-flavored e-cig exposure showed decreased protein abundance of type 1 collagen (COL1A1), α-smooth-muscle actin (αSMA), and fibronectin as well as decreased gene expression level of αSMA (Acta2) in HFL-1. After tobacco flavor e-cig exposure, HFL-1 mediated wound healing and tissue contractility were inhibited. Furthermore, BEAS-2B exposed to menthol flavor showed significantly decreased gene expression of CDH1, OCLN, and TJP1. Conclusion: Overall, tobacco-flavored e-cig exposure induces inflammation in both epithelium and fibroblasts, and tobacco-flavored e-cig inhibits wound healing ability in fibroblast.

Circadian clock molecule REV-ERBα regulates lung fibrotic progression through collagen stabilization.

Molecular clock REV-ERBα is central to regulating lung injuries, and decreased REV-ERBα abundance mediates sensitivity to pro-fibrotic insults and exacerbates fibrotic progression. In this study, we determine the role of REV-ERBα in fibrogenesis induced by bleomycin and Influenza A virus (IAV). Bleomycin exposure decreases the abundance of REV-ERBα, and mice dosed with bleomycin at night display exacerbated lung fibrogenesis. Rev-erbα agonist (SR9009) treatment prevents bleomycin induced collagen overexpression in mice. Rev-erbα global heterozygous (Rev-erbα Het) mice infected with IAV showed augmented levels of collagens and lysyl oxidases compared with WT-infected mice. Furthermore, Rev-erbα agonist (GSK4112) prevents collagen and lysyl oxidase overexpression induced by TGFß in human lung fibroblasts, whereas the Rev-erbα antagonist exacerbates it. Overall, these results indicate that loss of REV-ERBα exacerbates the fibrotic responses by promoting collagen and lysyl oxidase expression, whereas Rev-erbα agonist prevents it. This study provides the potential of Rev-erbα agonists in the treatment of pulmonary fibrosis.

Perfluorooctane Sulfonic Acid Disrupts Protective Tight Junction Proteins via Protein Kinase D in Airway Epithelial Cells.

Perfluorooctane sulfonic acid (PFOS) is a long chain per- and polyfluoroalklyl substance (PFAS) that has been used in aqueous film-forming foams. Emerging epidemiological evidence indicates that PFOS may be associated with chronic lung diseases such as asthma and analysis of human tissues demonstrates that the lungs carry a significant body burden of PFOS. Deficits in barrier function are a major risk factor for asthma. Thus, we hypothesized that PFOS exposure will lead to impaired epithelial barrier function through dysregulated tight junctions. Hence, we assessed the impact of PFOS on epithelial barrier integrity. Bronchial epithelial cells (16HBE) were grown on collagen-coated transwells and treated to 5-25 µM PFOS, and assessed for changes in barrier function and tight junction proteins. Rescue experiments were performed using the protein kinase D (PKD) inhibitor, CID755673. PFOS treatment reduced transepithelial electrical resistance (TEER) and increased 4 kDa FITC-dextran flux. Additionally, PFOS significantly decreased protein levels and the tight junction organization rate of occludin and zonula occludens 1. Increased phosphorylation (Ser744/Ser748) of PKD was observed 3 h following PFOS treatment. Pretreatment with the PKD inhibitor attenuated PFOS-mediated changes in TEER and FITC-dextran flux and restored occludin protein levels. In conclusion, PFOS causes loss of airway barrier integrity and the disruption of tight junctions in bronchial epithelial cells, which was partly attenuated through the inhibition of PKD. These findings demonstrate that PFOS is capable of disrupting airway barrier function, a potentially driving factor underlying associations between PFOS and respiratory diseases such as asthma.

Multi-Walled Carbon Nanotubes (MWCNTs) Cause Cellular Senescence in TGF-ß Stimulated Lung Epithelial Cells.

Multi-walled carbon nanotubes are engineered nanomaterials (ENMs) that have a fiber-like structure which may be a concern for the development of cellular senescence. Premature senescence, a state of irreversible cell cycle arrest, is implicated in the pathogenesis of chronic lung diseases such as pulmonary fibrosis (PF). However, the crosstalk between downstream pathways mediating fibrotic and senescent responses of MWCNTs is not well-defined. Here, we exposed human bronchial epithelial cells (BEAS-2B) to MWCNTs for up to 72 h and demonstrate that MWCNTs increase reactive oxygen species (ROS) production accompanied by inhibition of cell proliferation. In addition, MWCNT exposure resulted in the increase of p21 protein abundance and senescence associated ß-galactosidase (SA ß-gal) activity. We also determined that co-exposure with the cytokine, transforming growth factor-ß (TGF-ß) exacerbated cellular senescence indicated by increased protein levels of p21, p16, and γH2A.X. Furthermore, the production of fibronectin and plasminogen activator inhibitor (PAI-1) was significantly elevated with the co-exposure compared to MWCNT or TGF-ß alone. Together, our study suggests that the cellular senescence potential of MWCNTs may be enhanced by pro-fibrotic mediators, such as TGF-ß in the surrounding microenvironment.

Molecular clock REV-ERBα regulates cigarette smoke-induced pulmonary inflammation and epithelial-mesenchymal transition.

Cigarette smoke (CS) is the main etiological factor in the pathogenesis of emphysema/chronic obstructive pulmonary disease (COPD), which is associated with abnormal epithelial-mesenchymal transition (EMT). Previously, we have shown an association among circadian rhythms, CS-induced lung inflammation, and nuclear heme receptor α (REV-ERBα), acting as an antiinflammatory target in both pulmonary epithelial cells and fibroblasts. We hypothesized that molecular clock REV-ERBα plays an important role in CS-induced circadian dysfunction and EMT alteration. C57BL/6J WT and REV-ERBα heterozygous (Het) and -KO mice were exposed to CS for 30 days (subchronic) and 4 months (chronic), and WT mice were exposed to CS for 10 days with or without REV-ERBα agonist (SR9009) administration. Subchronic/chronic CS exposure caused circadian disruption and dysregulated EMT in the lungs of WT and REV-ERBα-KO mice; both circadian and EMT dysregulation were exaggerated in the REV-ERBα-KO condition. REV-ERBα agonist, SR9009 treatment reduced acute CS-induced inflammatory response and abnormal EMT in the lungs. Moreover, REV-ERBα agonist (GSK4112) inhibited TGF-ß/CS-induced fibroblast differentiation in human fetal lung fibroblast 1 (HFL-1). Thus, CS-induced circadian gene alterations and EMT activation are mediated through a Rev-erbα-dependent mechanism, which suggests activation of REV-ERBα as a novel therapeutic approach for smoking-induced chronic inflammatory lung diseases.

Pulmonary Toxicity and Inflammatory Response of E-Cigarette Vape Cartridges Containing Medium-Chain Triglycerides Oil and Vitamin E Acetate: Implications in the Pathogenesis of EVALI.

Recently, there has been an outbreak associated with the use of e-cigarette or vaping products, associated lung injury (EVALI). The primary components of vaping products, vitamin E acetate (VEA) and medium-chain triglycerides (MCT), may be responsible for acute lung toxicity. Currently, little information is available on the physiological and biological effects of exposure to these products. We hypothesized that these e-cig vape cartridges and their constituents (VEA and MCT) induce pulmonary toxicity, mediated by oxidative damage and inflammatory responses, leading to acute lung injury. We studied the potential mechanisms of e-cig vape cartridge aerosol induced inflammatory response by evaluating the generation of reactive oxygen species by MCT, VEA, and cartridges and their effects on the inflammatory state of pulmonary epithelium and immune cells both in vitro and in vivo. Cells exposed to these aerosols generated reactive oxygen species, caused cytotoxicity, induced epithelial barrier dysfunction, and elicited an inflammatory response. Using a murine model, the parameters of acute toxicity to aerosol inhalation were assessed. Infiltration of neutrophils and lymphocytes was accompanied by significant increases in IL-6, eotaxin, and G-CSF in the bronchoalveolar lavage fluid (BALF). In mouse plasma, eicosanoid inflammatory mediators, leukotrienes, were significantly increased. Plasma from e-cig users also showed increased levels of hydroxyeicosatetraenoic acid (HETEs) and various eicosanoids. Exposure to e-cig vape cartridge aerosols showed the most significant effects and toxicity compared to MCT and VEA. In addition, we determined SARS-CoV-2 related proteins and found no impact associated with aerosol exposures from these tested cartridges. Overall, this study demonstrates acute exposure to specific e-cig vape cartridges induces in vitro cytotoxicity, barrier dysfunction, and inflammation and in vivo mouse exposure induces acute inflammation with elevated proinflammatory markers in the pathogenesis of EVALI.

Pulmonary toxicity and inflammatory response of e-cigarettes containing medium-chain triglyceride oil and vitamin E acetate: Implications in the pathogenesis of EVALI but independent of SARS-COV-2 COVID-19 related proteins.

Recently, there has been an outbreak associated with the use of e-cigarette or vaping products, associated lung injury (EVALI). The primary components of vaping products, vitamin E acetate (VEA) and medium-chain triglycerides (MCT) may be responsible for acute lung toxicity. Currently, little information is available on the physiological and biological effects of exposure to these products. We hypothesized that these e-cig cartridges and their constituents (VEA and MCT) induce pulmonary toxicity, mediated by oxidative damage and inflammatory responses, leading to acute lung injury. We studied the potential mechanisms of cartridge aerosol induced inflammatory response by evaluating the generation of reactive oxygen species by MCT, VEA, and cartridges, and their effects on the inflammatory state of pulmonary epithelium and immune cells both in vitro and in vivo. Cells exposed to these aerosols generated reactive oxygen species, caused cytotoxicity, induced epithelial barrier dysfunction, and elicited an inflammatory response. Using a murine model, the parameters of acute toxicity to aerosol inhalation were assessed. Infiltration of neutrophils and lymphocytes was accompanied by significant increases in IL-6, eotaxin, and G-CSF in the bronchoalveolar lavage fluid (BALF). In mouse plasma, eicosanoid inflammatory mediators, leukotrienes, were significantly increased. Plasma from e-cig users also showed increased levels of hydroxyeicosatetraenoic acid (HETEs) and various eicosanoids. Exposure to e-cig cartridge aerosols showed the most significant effects and toxicity compared to MCT and VEA. In addition, we determined at SARS-COV-2 related proteins and found no impact associated with aerosol exposures from these tested cartridges. Overall, this study demonstrates acute exposure to specific e-cig cartridges induces in vitro cytotoxicity, barrier dysfunction, and inflammation and in vivo mouse exposure induces acute inflammation with elevated pro-inflammatory markers in the pathogenesis of EVALI.

E-Liquid Containing a Mixture of Coconut, Vanilla, and Cookie Flavors Causes Cellular Senescence and Dysregulated Repair in Pulmonary Fibroblasts: Implications on Premature Aging.

Electronic cigarette (e-cig) usage has risen dramatically worldwide over the past decade. While they are touted as a safe alternative to cigarettes, recent studies indicate that high levels of nicotine and flavoring chemicals present in e-cigs may still cause adverse health effects. We hypothesized that an e-liquid containing a mixture of tobacco, coconut, vanilla, and cookie flavors would induce senescence and disrupt wound healing processes in pulmonary fibroblasts. To test this hypothesis, we exposed pulmonary fibroblasts (HFL-1) to e-liquid at varying doses and assessed cytotoxicity, inflammation, senescence, and myofibroblast differentiation. We found that e-liquid exposure caused cytotoxicity, which was accompanied by an increase in IL-8 release in the conditioned media. E-liquid exposure resulted in elevated senescence-associated beta-galactosidase (SA-ß-gal) activity. Transforming growth factor-ß1 (TGF-ß1) induced myofibroblast differentiation was inhibited by e-liquid exposure, resulting in decreased α-smooth muscle actin and fibronectin protein levels. Together, our data suggest that an e-liquid containing a mixture of flavors induces inflammation, senescence and dysregulated wound healing responses.