Burn Pit Smoke Condensate-Mediated Toxicity in Human Airway Epithelial Cells.

Burn pits are a method of open-air waste management that was common during military operations in Iraq, Afghanistan, and other regions in Southwest Asia. Veterans returning from deployment have reported respiratory symptoms, potentially from exposure to burn pit smoke, yet comprehensive assessment of such exposure on pulmonary health is lacking. We have previously shown that exposure to condensates from burn pit smoke emissions causes inflammation and cytotoxicity in mice. In this study, we explored the effects of burn pit smoke condensates on human airway epithelial cells (HAECs) to understand their impact on cellular targets in the human lung. HAECs were cultured at the air-liquid interface (ALI) and exposed to burn pit waste smoke condensates (plywood, cardboard, plastic, mixed, and mixed with diesel) generated under smoldering and flaming conditions. Cytotoxicity was evaluated by measuring transepithelial electrical resistance (TEER) and lactate dehydrogenase (LDH) release; toxicity scores (TSs) were quantified for each exposure. Pro-inflammatory cytokine release and modulation of gene expression were examined for cardboard and plastic condensate exposures. Burn pit smoke condensates generated under flaming conditions affected cell viability, with flaming mixed waste and plywood exhibiting the highest toxicity scores. Cardboard and plastic smoke condensates modulated cytokine secretion, with GM-CSF and IL-1ß altered in more than one exposure group. Gene expression of detoxifying enzymes (ALDH1A3, ALDH3A1, CYP1A1, CYP1B1, NQO1, etc.), mucins (MUC5AC, MUC5B), and cytokines was affected by several smoke condensates. Particularly, expression of IL6 was elevated following exposure to all burn pit smoke condensates, and polycyclic aromatic hydrocarbon acenaphthene was positively associated with the IL-6 level in the basolateral media of HAECs. These observations demonstrate that exposure to smoke condensates of materials present in burn pits adversely affects HAECs and that aberrant cytokine secretion and altered gene expression profiles following burn pit material smoke exposure could contribute to the development of airway disease.

Leveraging a Comprehensive Unbiased RNAseq Database Uncovers New Human Monocyte-Derived Macrophage Subtypes Within Commonly Employed In Vitro Polarization Methods.

Macrophages are pivotal innate immune cells which exhibit high phenotypic plasticity and can exist in different polarization states dependent on exposure to external stimuli. Numerous methods have been employed to simulate macrophage polarization states to test their function in vitro. However, limited research has explored whether these polarization methods yield comparable populations beyond key gene, cytokine, and cell surface marker expression. Here, we employ an unbiased comprehensive analysis using data organized through the all RNA-seq and ChIP-seq sample and signature search (ARCHS4) database, which compiles all RNAseq data deposited into the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA). In silico analyses were carried out demonstrating that commonly employed macrophage polarization methods generate distinct macrophage subsets that remained undescribed until now. Our analyses confirm existing knowledge on macrophage polarization, while revealing nuanced differences between M2a and M2c subpopulations, suggesting non-interchangeable stimuli for M2a polarization. Furthermore, we identify divergent gene expression patterns in M1 macrophages following standard polarization protocols, indicating significant subset distinctions. Consequently, equivalence cannot be assumed among polarization regimens for in vitro macrophage studies, particularly in simulating diverse pathogen responses.

Vaping product exposure system (VaPES): a novel in vitro aerosol deposition system.

OBJECTIVE: Due to recent increases in the use of vaping devices, there is a high demand for research addressing the respiratory health effects of vaping products. Given the constantly changing nature of the vaping market with new devices, flavors, metals, and other chemicals rapidly emerging, there is a need for inexpensive and highly adaptable vaping device exposure systems. Here, we describe the design and validation of a novel in vitro aerosol exposure system for toxicity testing of vaping devices. MATERIALS AND METHODS: We developed an inexpensive, open-source in vitro vaping device exposure system that produces even deposition, can be adapted for different vaping devices, and allows for experiments to be performed under physiological conditions. The system was then validated with deposition testing and a representative exposure with human bronchial epithelial cells (hBECs). RESULTS: The Vaping Product Exposure System (VaPES) produced sufficient and uniform deposition for dose-response studies and was precise enough to observe biological responses to vaping exposures. VaPES was adapted to work with both pod and cartridge-based vaping devices. CONCLUSION: We have designed and validated a novel vaping device exposure system that will eliminate the need to use high-cost commercial exposure systems, lowering the barrier to entry of physiologically relevant vaping studies.

Understanding the Relationship Between Neutrophil Function and Demographic Variables.

Neutrophils play a crucial role in the body's defense against respiratory pathogens, and dysregulation is linked to airway diseases. The study presented here explores the association between demographic factors (age, BMI, and sex) and functional phenotypes (oxidative burst and bioenergetics) of neutrophils. We measured PMA-stimulated oxidative burst (Seahorse XF) and phagocytosis (pHrodo red S. aureus) of human peripheral blood neutrophils and determined whether there were significant demographic associations with cellular function. There were no significant associations between neutrophil oxidative burst bioenergetic parameters or phagocytosis and BMI or age. However, our data revealed sexual dimorphism in neutrophil phagocytosis, with males exhibiting significantly higher phagocytic capacity than females. Additionally, phagocytic capacity and bioenergetic parameters were correlated in males but not in females. The study indicates potential variations in neutrophil activation pathways between males and female and emphasizes the importance of considering sex as a biological variable in respiratory host defense research.

Expanded characterization of in vitro polarized M0, M1, and M2 human monocyte-derived macrophages: Bioenergetic and secreted mediator profiles.

Respiratory macrophage subpopulations exhibit unique phenotypes depending on their location within the respiratory tract, posing a challenge to in vitro macrophage model systems. Soluble mediator secretion, surface marker expression, gene signatures, and phagocytosis are among the characteristics that are typically independently measured to phenotype these cells. Bioenergetics is emerging as a key central regulator of macrophage function and phenotype but is often not included in the characterization of human monocyte-derived macrophage (hMDM) models. The objective of this study was to expand the phenotype characterization of naïve hMDMs, and their M1 and M2 subsets by measuring cellular bioenergetic outcomes and including an expanded cytokine profile. Known markers of M0, M1 and M2 phenotypes were also measured and integrated into the phenotype characterization. Peripheral blood monocytes from healthy volunteers were differentiated into hMDM and polarized with either IFN-γ + LPS (M1) or IL-4 (M2). As expected, our M0, M1, and M2 hMDMs exhibited cell surface marker, phagocytosis, and gene expression profiles indicative of their different phenotypes. M2 hMDMs however were uniquely characterized and different from M1 hMDMs by being preferentially dependent on oxidativte phosphorylation for their ATP generation and by secreting a distinct cluster of soluble mediators (MCP4, MDC, and TARC). In contrast, M1 hMDMs secreted prototypic pro-inflammatory cytokines (MCP1, eotaxin, eotaxin-3, IL12p70, IL-1α, IL15, TNF-ß, IL-6, TNF-α, IL12p40, IL-13, and IL-2), but demonstrated a relatively constitutively heightened bioenergetic state, and relied on glycolysis for ATP generation. These data are similar to the bioenergetic profiles we previously observed in vivo in sputum (M1) and BAL (M2)-derived macrophages in healthy volunteers, supporting the notion that polarized hMDMs can provide an acceptable in vitro model to study specific human respiratory macrophage subtypes.

Biomarkers of Airway Immune Homeostasis Differ Significantly with Generation of E-Cigarettes.

Rationale: Numerous studies have demonstrated that e-cigarettes can impact respiratory immune homeostasis; however, the extent of these effects remains an active area of investigation, and most previous studies were conducted with model systems or subjects exposed to third-generation e-cigarettes, such as vape pens and box mods. Objectives: Given the rise in popularity of nicotine-salt-containing pods and disposable e-cigarettes (fourth generation), we set out to better understand the respiratory effects of these newer e-cigarettes and compare their effects to early-generation devices. Methods: We collected induced sputum samples from a cohort of nonsmokers, smokers, third-generation e-cigarette users, and fourth-generation e-cigarette users (n = 20-30 per group) and evaluated the cellular and fluid-phase composition for markers of inflammation, host defense, and lung injury. Measurements and Main Results: Fourth-generation e-cigarette users had significantly more bronchial epithelial cells in the sputum, suggestive of airway injury. Concentrations of soluble intercellular adhesion molecule 1 (sICAM1) and soluble vascular cell adhesion molecule 1 (sVCAM1) were significantly lower in fourth-generation e-cigarette users in comparison with all other groups, and CRP (C-reactive protein), IFN-γ, MCP-1 (monocyte chemoattractant protein-1), MMP-2 (matrix metalloproteinase 2), uteroglobin, and VEGF (vascular endothelial growth factor) were significantly lower in fourth- versus third-generation e-cigarette users, suggestive of overall immune suppression in fourth-generation e-cigarette users. Predictive modeling also demonstrated clear separation between exposure groups, indicating that the overall mediator milieu is different between groups, particularly fourth-generation e-cigarette users. Conclusions: Our results indicate disrupted immune homeostasis in fourth-generation e-cigarette users and demonstrate that the biological effects of fourth-generation e-cigarette use are unique compared with those associated with previous-generation e-cigarettes.

A Mixed Methods Approach to Improving Provider Counseling of Patients Who Vape.

Significance: Vaping is an epidemic among young people, but there is little guidance on how medical providers should counsel young adults about vaping. To address this gap, we examined how electronic health record (EHR) systems prompt providers to collect vaping data and interviewed young adults about vaping communications with providers and preferred information sources. Methods: In this mixed methods study, we used survey research methods to explore if prompts exist in EHR systems to guide discussions about vaping with youth seen in primary care. We collected primary care practice information about EHR prompts regarding e-cigarette use from 10 rural North Carolina practices from August 2020 through November 2020 and interviewed 17 young adults (age 18-21 years) who reviewed resources and provided their opinion on the resource's relevance for their age group. Interviews were stratified by vaping status, transcribed, coded, and thematically analyzed. Results: Only 5 of 10 EHR systems included prompts to capture information about vaping and data capture was optional in all 5 cases. Of the 17 interviewees, 10 were female, 14 were White, 3 were non-White and the mean age was 19.6 years. Two central themes emerged. Young adults: 1) were open to confidential, non-confrontational interactions with trusted providers and supported the use of a 2-page resource/discussion guide, questionnaires about vaping, and other waiting room resources, and 2) wanted prevention and cessation resources to be age-appropriate, including medical facts from a trusted source, and to be disseminated via social media platforms used by young adults. Conclusions: We found a lack of EHR functionalities in screening for vaping status hindered patients from receiving counseling on use. Young adults report a willingness to communicate with and learn from trusted providers and to gain understanding from information accessed via social media.

Differential responses to e-cig generated aerosols from humectants and different forms of nicotine in epithelial cells from nonsmokers and smokers.

In the United States, millions of adults use electronic cigarettes (e-cigs), and a majority of these users are former or current cigarette smokers. It is unclear, whether prior smoking status affects biological responses induced by e-cigs. In this study, differentiated human nasal epithelial cells (hNECs) from nonsmokers and smokers at air-liquid interface were acutely exposed to the e-cig generated aerosols of humectants, propylene glycol (PG), and glycerol (GLY). Mucin levels were examined in the apical washes, and cytokine levels were assessed in the basolateral supernatants 24 h postexposure. The aerosol from the GLY exposure increased mucin 5, subtype AC (MUC5AC) levels in the apical wash of hNECs from nonsmokers, but not smokers. However, the aerosol from GLY induced pro-inflammatory responses in hNECs from smokers. We also exposed hNECs from nonsmokers and smokers to e-cig generated aerosol from PG:GLY with freebase nicotine or nicotine salt. The PG:GLY with freebase nicotine exposure increased MUC5AC and mucin 5, subtype B (MUC5B) levels in hNECs from nonsmokers, but the nicotine salt exposure did not. The PG:GLY with nicotine salt exposure increased pro-inflammatory cytokines in hNECs from smokers, which was not seen with the freebase nicotine exposure. Taken together, these data indicate that the e-cig generated aerosols from the humectants, mostly GLY, and the type of nicotine used cause differential effects in airway epithelial cells from nonsmokers and smokers. As e-cig use is increasing, it is important to understand that the biological effects of e-cig use are likely dependent on prior cigarette smoke exposure.

Electronic Cigarettes and Their Impact on Allergic Respiratory Diseases: A Work Group Report of the AAAAI Environmental Exposures and Respiratory Health Committee.

The explosive rise in popularity of electronic cigarette (e-cig) devices over the past decade has led to controversies over the role of these devices in smoking cessation and harm reduction from combustible cigarette smoking. Increased recognition of potential direct harms of e-cigs, including life-threatening and fatal cases of e-cig and vaping product use-associated lung injury, has emphasized the need to curb use until safety can be established. Of particular concern is the steep rise in e-cig use among teenagers and young adults who have never smoked and among individuals with underlying lung disease, such as asthma. In this report, we describe the different types of e-cig devices available, summarize the available data on the potential health benefits and detriments of e-cig use, and highlight the findings of studies examining e-cigs as smoking cessation tools. Because e-cigs have only gained popularity in the last few years, very few studies have been able to demonstrate an impact of e-cig use on harm reduction related to combustible cigarettes. Moreover, the health effects of e-cigs at a population level must be balanced against the harms of e-cig use, which include nicotine dependence and promoting initiation of cigarette use amongst "never smokers." With respect to smoking cessation, e-cigs appear to serve as switching products that may help individuals reduce or quit cigarette use, but do not address nicotine addiction. Finally, we discuss our recommendations for ways that health care providers can screen and counsel patients on e-cig use. The goal of this report is to provide health care providers with the most recent information on this topic so that they can educate patients on the potential pros and cons of e-cig use.

Current E-Cigarette Research in the Context of Asthma.

PURPOSE OF REVIEW: The purpose of this review is to integrate recent research on the respiratory immune effects of e-cigarettes with the pathogenesis of asthma to better understand how e-cigarettes may affect asthmatics and to note critical knowledge gaps regarding the effects of e-cigarettes on asthmatics. RECENT FINDINGS: Human, rodent, and cell culture studies show that key cellular functions of epithelial cells, macrophages, and neutrophils are altered by e-cigarette exposure. Because respiratory immunity is already dysregulated in asthma, further alteration of cellular function by e-cigarettes could impact asthma development, severity, and/or exacerbations. Future research is needed to more directly investigate this relationship using controlled human exposure studies and exposure of cell culture or animal models of asthma to e-cigarettes.

Common E-Cigarette Flavoring Chemicals Impair Neutrophil Phagocytosis and Oxidative Burst.

E-cigarette flavorings have not been thoroughly evaluated for inhalational toxicity. We have shown that the flavoring chemical cinnamaldehyde impairs human neutrophils, macrophages, and natural killer cells. Here we investigated the effects of other common e-liquid flavoring chemicals on phagocytosis and oxidative burst in neutrophils. We demonstrate that cinnamaldehyde and ethyl vanillin dose-dependently decrease oxidative burst and that benzaldehyde and benzaldehyde propylene glycol acetal dose-dependently impair phagocytosis. Isoamyl acetate did not affect either measure of neutrophil function. These data suggest that inhaling aromatic aldehydic flavoring chemicals, such as cinnamaldehyde, benzaldehyde, benzaldehyde propylene glycol acetal, or ethyl vanillin, could impair neutrophil function.