Human Bronchial Epithelial Cultures Research Articles

OS01-01 Exploring extracellular and Intracellular interactions and effects of advanced materials on reconstituted primary human bronchial epithelial cultures and connecting their potential impacts on lung health

OS01-01 Exploring extracellular and Intracellular interactions and effects of advanced materials on reconstituted primary human bronchial epithelial cultures and connecting their potential impacts on lung health

Severe Acute Respiratory Syndrome Coronavirus 2 Infection Alters Mediators of Lung Tissue Remodeling In Vitro and In Vivo.

Altered mediators of airway tissue remodeling such as matrix metalloproteinases (MMPs) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may contribute to morbidity in coronavirus disease 2019 (COVID-19); however, the differential impact of SARS-CoV-2 variants of concern (VOCs) on MMPs is unknown. Using both in vitro human airway cell culture model and in vivo transgenic mouse model of SARS-CoV-2 infection, we studied the differential effect of SARS-CoV-2 VOCs on expression of key MMPs and inflammatory mediators in airway cells and tissues. The most consistent findings with all SARS-CoV-2 variants in infected compared to uninfected human bronchial epithelial cell air-liquid interface cultures were the SARS-CoV-2-induced increases in MMP-12 and tissue inhibitor of MMPs. Infection with both SARS-CoV-2 wild type and SARS-CoV-2 Delta variant over 3 days postinfection (dpi) and with Beta variant over 7 dpi increased lung tissue levels of MMP-9 compared to uninfected mice. Overall, SARS-CoV-2 variants had differential dose-dependent impact on secretion of MMP-1, MMP-2, MMP-9, and MMP-12 that varied at the protein versus the gene level and in the early noninflammatory compared to late inflammatory phase of infection. We provide novel mechanistic insight that the differential impact of SARS-CoV-2 variants on severity of COVID-19 may partially be attributed to unique changes in MMPs.

Proximal and Distal Bronchioles Contribute to the Pathogenesis of Non-Cystic Fibrosis Bronchiectasis.

Rationale: Non-cystic fibrosis bronchiectasis (NCFB) may originate in bronchiolar regions of the lung. Accordingly, there is a need to characterize the morphology and molecular characteristics of NCFB bronchioles. Objectives: Test the hypothesis that NCFB exhibits a major component of bronchiolar disease manifest by mucus plugging and ectasia. Methods: Morphologic criteria and region-specific epithelial gene expression, measured histologically and by RNA in situ hybridization and immunohistochemistry, identified proximal and distal bronchioles in excised NCFB lungs. RNA in situ hybridization and immunohistochemistry assessed bronchiolar mucus accumulation and mucin gene expression. CRISPR-Cas9-mediated IL-1R1 knockout in human bronchial epithelial cultures tested IL-1α and IL-1β contributions to mucin production. Spatial transcriptional profiling characterized NCFB distal bronchiolar gene expression. Measurements and Main Results: Bronchiolar perimeters and lumen areas per section area were increased in proximal, but not distal, bronchioles in NCFB versus control lungs, suggesting proximal bronchiolectasis. In NCFB, mucus plugging was observed in ectatic proximal bronchioles and associated nonectatic distal bronchioles in sections with disease. MUC5AC and MUC5B mucins were upregulated in NCFB proximal bronchioles, whereas MUC5B was selectively upregulated in distal bronchioles. Bronchiolar mucus plugs were populated by IL-1β-expressing macrophages. NCFB sterile sputum supernatants induced human bronchial epithelial MUC5B and MUC5AC expression that was >80% blocked by IL-1R1 ablation. Spatial transcriptional profiling identified upregulation of genes associated with secretory cells, hypoxia, interleukin pathways, and IL-1β-producing macrophages in mucus plugs and downregulation of epithelial ciliogenesis genes. Conclusions: NCFB exhibits distinctive proximal and distal bronchiolar disease. Both bronchiolar regions exhibit bronchiolar secretory cell features and mucus plugging but differ in mucin gene regulation and ectasia.

Acute and repeated exposures of normal human bronchial epithelial (NHBE) cells culture to particles from a coloured pyrotechnic smoke

Coloured pyrotechnic smokes are frequently used in the military field and occasionally by civilians, but their health hazards have been little studied. The main concern could rise from inhalation of smoke particles. Our previous study showed that acute exposure to particles from a red signalling smoke (RSS) induced an antioxidant and inflammatory responses in small airway epithelial cells. The aim of this study was to further explore the toxicity of RSS particles at a more proximal level of the respiratory tract, using normal human bronchial epithelial cells grown at the Air-Liquid Interface. Acute exposure (24 h) induced an oxidative stress that persisted 24 h post-exposure, associated with particle internalization and epithelium morphological changes (cuboidal appearance and loss of cilia). Repeated exposures (4×16h) to RSS particles did not trigger oxidative stress but cell morphological changes occurred. Overall, this study provides a better overview of the toxic effects of coloured smoke particles.

Vaping-Induced Proteolysis Causes Airway Surface Dehydration.

Proteases such as neutrophil elastase cleave and activate the epithelial sodium channel (ENaC), causing airway dehydration. Our current study explores the impact of increased protease levels in vapers' airways on ENaC activity and airway dehydration. Human bronchial epithelial cultures (HBECs) were exposed to bronchoalveolar lavage fluid (BALF) from non-smokers, smokers and vapers. Airway surface liquid (ASL) height was measured by confocal microscopy as a marker of hydration. ENaC cleavage was measured by Western blotting. Human peripheral blood neutrophils were treated with a menthol-flavored e-liquid (Juul), and the resulting secretions were added to HBECs. BALF from smokers and vapers significantly and equally increased ENaC activity and decreased ASL height. The ASL height decrease was attenuated by protease inhibitors. Non-smokers' BALF had no effect on ENaC or ASL height. BALF from smokers and vapers, but not non-smokers, induced ENaC cleavage. E-liquid-treated neutrophil secretions cleaved ENaC and decreased ASL height. Our study demonstrated that elevated protease levels in vapers' airways have functional significance since they can activate ENaC, resulting in airway dehydration. Lung dehydration contributes to diseases like cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and asthma. Thus, our data predict that vaping, like smoking, will cause airway surface dehydration that likely leads to lung disease.

Acute cigarette smoke exposure leads to higher viral infection in human bronchial epithelial cultures by altering interferon, glycolysis and GDF15-related pathways

BackgroundAcute exacerbations of chronic inflammatory lung diseases, such as chronic obstructive pulmonary disease (COPD), are frequently associated with rhinovirus (RV) infections. Despite these associations, the pathogenesis of virus-induced exacerbations is incompletely understood. We aimed to investigate effects of cigarette smoke (CS), a primary risk factor for COPD, on RV infection in airway epithelium and identify novel mechanisms related to these effects.MethodsPrimary bronchial epithelial cells (PBEC) from COPD patients and controls were differentiated by culture at the air–liquid interface (ALI) and exposed to CS and RV-A16. Bulk RNA sequencing was performed using samples collected at 6 and 24 h post infection (hpi), and viral load, mediator and l-lactate levels were measured at 6, 24 and 48hpi. To further delineate the effect of CS on RV-A16 infection, we performed growth differentiation factor 15 (GDF15) knockdown, l-lactate and interferon pre-treatment in ALI-PBEC. We performed deconvolution analysis to predict changes in the cell composition of ALI-PBEC after the various exposures. Finally, we compared transcriptional responses of ALI-PBEC to those in nasal epithelium after human RV-A16 challenge.ResultsCS exposure impaired antiviral responses at 6hpi and increased viral replication at 24 and 48hpi in ALI-PBEC. At 24hpi, CS exposure enhanced expression of RV-A16-induced epithelial interferons, inflammation-related genes and CXCL8. CS exposure increased expression of oxidative stress-related genes, of GDF15, and decreased mitochondrial membrane potential. GDF15 knockdown experiments suggested involvement of this pathway in the CS-induced increase in viral replication. Expression of glycolysis-related genes and l-lactate production were increased by CS exposure, and was demonstrated to contribute to higher viral replication. No major differences were demonstrated between COPD and non-COPD-derived cultures. However, cellular deconvolution analysis predicted higher secretory cells in COPD-derived cultures at baseline.ConclusionAltogether, our findings demonstrate that CS exposure leads to higher viral infection in human bronchial epithelium by altering not only interferon responses, but likely also through a switch to glycolysis, and via GDF15-related pathways.

73 Evaluating the Risks of Prolonged Exposure to Graphene Oxide on Healthy and Streptococcus Pneumoniae Infected 3D Reconstituted Human Lung Cultures

Abstract The growing production of graphene oxide (GO) and GO-based products has led to an increased risk of human exposure and caused scientific and public concern about human health and safety. Even though human exposure to GO can occur via different pathways, the main exposure route for airborn GO is through the lungs, thus placing the respiratory tract at particular risk. Occupational exposure to GO might occur repeatedly over a prolonged period of time but insights on lung toxicity of long-term repeated GO administration is largely lacking. Moreover, there is a risk that GO co-exposure with prevalent lung pathogens (e.g. Streptococcus pneumoniae (SP)) could increase the susceptibility to lung infections. In this study, we aimed to investigate the impact of repeated and prolonged GO exposure in healthy and SP-infected lungs using well-characterized materials with distinct properties and reconstituted primary human bronchial epithelial cultures. Our results revealed that GO had no major toxicity effects on healthy airway epithelium even upon prolonged exposure, but alterations in inflammatory/immune signaling was apparent and showed a correlation with the lateral particle size. GO pre-exposure slightly altered the response of the airway epithelium towards subsequent SP infection as evidenced by a decreased mitochondrial activity, increased bacterial translocation and altered inflammatory/immune responses. Whole genome transcriptomic profiling is ongoing to gain insights in deregulated genes and pathways following chronic GO administration. Our findings on the health risks of GO inhalation in healthy and infected human lung is important to risk assessment and management of this promising material.

Passaging Primary Human Bronchial Epithelia Reduces CFTR-Mediated Fluid Transport and Alters mRNA Expression.

Primary human bronchial epithelial cultures (HBECs) are used to study airway physiology, disease, and drug development. HBECs often replicate human airway physiology/pathophysiology. Indeed, in the search for cystic fibrosis (CF) transmembrane conductance regulator (CFTR) therapies, HBECs were seen as the "gold standard" in preclinical studies. However, HBECs are not without their limitations: they are non-immortalized and the requirement for human donors, especially those with rare genetic mutations, can make HBECs expensive and/or difficult to source. For these reasons, researchers may opt to expand HBECs by passaging. This practice is common, but to date, there has not been a robust analysis of the impact of expanding HBECs on their phenotype. Here, we used functional studies of airway surface liquid (ASL) homeostasis, epithelial barrier properties, and RNA-seq and Western blotting to investigate HBEC changes over two passage cycles. We found that passaging impaired CFTR-mediated ASL secretion and led to a reduction in the plasma membrane expression of the epithelial sodium channel (ENaC) and CFTR. Passaging also resulted in an increase in transepithelial resistance and a decrease in epithelial water permeability. We then looked for changes at the mRNA level and found that passaging significantly affected 323 genes, including genes involved in inflammation, cell growth, and extracellular matrix remodeling. Collectively, these data highlight the potential for HBEC expansion to impact research findings.

Evaluation of chronic cigarette smoke exposure in human bronchial epithelial cultures.

Cigarette smoke (CS) exposure induces both cytotoxicity and inflammation, and often causes COPD, a growing cause of morbidity and mortality. CS also inhibits the CFTR Cl- channel, leading to airway surface liquid dehydration, which is predicated to impair clearance of inhaled pathogens and toxicants. Numerous in vitro studies have been performed that utilize acute (≤24 h) CS exposures. However, CS exposure is typically chronic. We evaluated the feasibility of using British-American Tobacco (BAT)-designed CS exposure chambers for chronically exposing human bronchial epithelial cultures (HBECs) to CS. HBECs are polarized and contain mucosal and serosal sides. In vivo, inhaled CS interacts with mucosal membranes, and BAT chambers are designed to direct CS to HBEC mucosal surfaces while keeping CS away from serosal surfaces via a perfusion system. We found that serosal perfusion was absolutely required to maintain HBEC viability over time following chronic CS exposure. Indeed, with this system, we found that CS increased inflammation and mucin levels, while decreasing CFTR function. Without this serosal perfusion, CS was extremely toxic within 24 h. We therefore propose that 5- and 10-day CS exposures with serosal perfusion are suitable for measuring chronic CS exposure and can be used for monitoring new and emerging tobacco products.

Vaping additives cannabinoid oil and vitamin E acetate adhere to and damage the human airway epithelium.

E-cigarette, or vaping product use-associated lung injury (EVALI), is a severe respiratory disorder that caused a sudden outbreak of hospitalized young people in 2019. Using cannabis oil containing vaping products, including vitamin E acetate contaminants, was found to be strongly associated with EVALI. However, the underlying tissue impacts of the condition are still largely unknown. Here, we focused on the vehicle cannabinoid oil (CBD oil) and contaminant vitamin E acetate (VEA) effects on airway epithelial cells. Primary human bronchial epithelial (HBE) cultures were exposed to e-liquid aerosols that contained CBD oil and VEA in combination or the common e-liquid components PG/VG with and without nicotine. Cell viability analysis indicated dramatically increased cell death counts after 3 days of CBD exposure, and this effect was even higher after CBD + VEA exposure. Microscopic examination of the cultures revealed cannabinoid and VEA depositions on the epithelial surfaces and cannabinoid accumulation in exposed cells, followed by cell death. These observations were supported by proteomic analysis of the cell secretions that exhibited increases in known markers of airway epithelial toxicity, such as xenobiotic enzymes, factors related to oxidative stress response, and cell death indicators. Overall, our study provides insights into the association between cannabinoid oil and vitamin E acetate vaping and lung injury. Collectively, our results suggest that the adherent accumulation of CBD oil on airway surfaces and the cellular uptake of both CBD oil- and VEA-containing condensates cause elevated metabolic stress, leading to increased cell death rates in human airway epithelial cultures.

Mucus from human bronchial epithelial cultures: rheology and adhesion across length scales.

Mucus is a viscoelastic aqueous fluid that participates in the protective barrier of many mammals' epithelia. In the airways, together with cilia beating, mucus rheological properties are crucial for lung mucociliary function, and, when impaired, potentially participate in the onset and progression of chronic obstructive pulmonary disease (COPD). Samples of human mucus collected in vivo are inherently contaminated and are thus poorly characterized. Human bronchial epithelium (HBE) cultures, differentiated from primary cells at an air-liquid interface, are highly reliable models to assess non-contaminated mucus. In this paper, the viscoelastic properties of HBE mucus derived from healthy subjects, patients with COPD and from smokers are measured. Hallmarks of shear-thinning and elasticity are obtained at the macroscale, whereas at the microscale mucus appears as a heterogeneous medium showing an almost Newtonian behaviour in some extended regions and an elastic behaviour close to boundaries. In addition, we developed an original method to probe mucus adhesion at the microscopic scale using optical tweezers. The measured adhesion forces and the comparison with mucus-simulants rheology as well as mucus imaging collectively support a structure composed of a network of elastic adhesive filaments with a large mesh size, embedded in a very soft gel.

Editorial: theme issue on complex rheology in biological systems

Editorial: theme issue on complex rheology in biological systems

631 Comparing F508del cystic fibrosis transmembrane conductance regulator modulator responses in human primary enteric monolayer and human bronchial epithelial cultures

631 Comparing F508del cystic fibrosis transmembrane conductance regulator modulator responses in human primary enteric monolayer and human bronchial epithelial cultures

Prevalence and Mechanisms of Mucus Accumulation in COVID-19 Lung Disease.

Rationale: The incidence and sites of mucus accumulation and molecular regulation of mucin gene expression in coronavirus (COVID-19) lung disease have not been reported. Objectives: To characterize the incidence of mucus accumulation and the mechanisms mediating mucin hypersecretion in COVID-19 lung disease. Methods: Airway mucus and mucins were evaluated in COVID-19 autopsy lungs by Alcian blue and periodic acid-Schiff staining, immunohistochemical staining, RNA in situ hybridization, and spatial transcriptional profiling. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected human bronchial epithelial (HBE) cultures were used to investigate mechanisms of SARS-CoV-2-induced mucin expression and synthesis and test candidate countermeasures. Measurements and Main Results: MUC5B and variably MUC5AC RNA concentrations were increased throughout all airway regions of COVID-19 autopsy lungs, notably in the subacute/chronic disease phase after SARS-CoV-2 clearance. In the distal lung, MUC5B-dominated mucus plugging was observed in 90% of subjects with COVID-19 in both morphologically identified bronchioles and microcysts, and MUC5B accumulated in damaged alveolar spaces. SARS-CoV-2-infected HBE cultures exhibited peak titers 3 days after inoculation, whereas induction of MUC5B/MUC5AC peaked 7-14 days after inoculation. SARS-CoV-2 infection of HBE cultures induced expression of epidermal growth factor receptor (EGFR) ligands and inflammatory cytokines (e.g., IL-1α/β) associated with mucin gene regulation. Inhibiting EGFR/IL-1R pathways or administration of dexamethasone reduced SARS-CoV-2-induced mucin expression. Conclusions: SARS-CoV-2 infection is associated with a high prevalence of distal airspace mucus accumulation and increased MUC5B expression in COVID-19 autopsy lungs. HBE culture studies identified roles for EGFR and IL-1R signaling in mucin gene regulation after SARS-CoV-2 infection. These data suggest that time-sensitive mucolytic agents, specific pathway inhibitors, or corticosteroid administration may be therapeutic for COVID-19 lung disease.

Differential gene expression of 3D primary human airway cultures exposed to cigarette smoke and electronic nicotine delivery system (ENDS) preparations

BackgroundAcute exposure to cigarette smoke alters gene expression in several biological pathways such as apoptosis, immune response, tumorigenesis and stress response, among others. However, the effects of electronic nicotine delivery systems (ENDS) on early changes in gene expression is relatively unknown. The objective of this study was to evaluate the early toxicogenomic changes using a fully-differentiated primary normal human bronchial epithelial (NHBE) culture model after an acute exposure to cigarette and ENDS preparations.ResultsRNA sequencing and pathway enrichment analysis identified time and dose dependent changes in gene expression and several canonical pathways when exposed to cigarette preparations compared to vehicle control, including oxidative stress, xenobiotic metabolism, SPINK1 general cancer pathways and mucociliary clearance. No changes were observed with ENDS preparations containing up to 28 µg/mL nicotine. Full model hierarchical clustering revealed that ENDS preparations were similar to vehicle control.ConclusionThis study revealed that while an acute exposure to cigarette preparations significantly and differentially regulated many genes and canonical pathways, ENDS preparations containing the same concentration of nicotine had very little effect on gene expression in fully-differentiated primary NHBE cultures.

Assessment of in vitro kinetics and biological impact of nebulized trehalose on human bronchial epithelium

Trehalose is added in drug formulations to act as fillers or improve aerosolization performance. Its characteristics as a carrier molecule have been explored; however, the fate of trehalose in human airway tissues has not been thoroughly investigated. Here, we investigated the fate of nebulized trehalose using in vitro human air-liquid bronchial epithelial cultures. First, a tracing experiment was conducted using 13C12-trehalose; we measured trehalose distribution in different culture compartments (apical surface liquid, epithelial culture, and basal side medium) at various time points following acute exposure to 13C12-labeled trehalose. We found that 13C12-trehalose was metabolized into 13C6-glucose. The data was then used to model the kinetics of trehalose disappearance from the apical surface of bronchial cultures. Secondly, we evaluated the potential adverse effects of nebulized trehalose on the bronchial cultures after they were acutely exposed to nebulized trehalose up to a level just below its solubility limit (50 g/100 g water). We assessed the ciliary beating frequency and histological characteristics. We found that nebulized trehalose did not lead to marked alteration in ciliary beating frequency and morphology of the epithelial cultures. The in vitro testing approach used here may enable the early selection of excipients for future development of inhalation products.

The larger attachment glycoprotein of respiratory syncytial virus produced in primary human bronchial epithelial cultures reduces infectivity for cell lines.

Respiratory syncytial virus (RSV) infects the upper and lower respiratory tracts and can cause lower respiratory tract infections in children and elders. RSV has traditionally been isolated, grown, studied and quantified in immortalized cell lines, most frequently HEp-2 cells. However, in vivo RSV infection is modeled more accurately in primary well differentiated human bronchial epithelial (HBE) cultures where RSV targets the ciliated cells and where the putative RSV receptor differs from the receptor on HEp-2 cells. The RSV attachment (G) glycoprotein in virions produced by HEp-2 cells is a highly glycosylated 95 kDa protein with a 32 kDa peptide core. However, virions produced in HBE cultures, RSV (HBE), contain an even larger, 170 kDa, G protein (LgG). Here we show that LgG is found in virions from both subgroups A and B lab-adapted and clinical isolates. Unexpectedly, RSV (HBE) virions were approximately 100-fold more infectious for HBE cultures than for HEp-2 cells. Surprisingly, the cause of this differential infectivity, was reduced infectivity of RSV (HBE) on HEp-2 cells rather than enhanced infectivity on HBE cultures. The lower infectivity of RSV(HBE) for HEp-2 cells is caused by the reduced ability of LgG to interact with heparan sulfate proteoglycans (HSPG), the RSV receptor on HEp-2 cells. The discovery of different infectivity corresponding with the larger form of the RSV attachment protein when produced by HBE cultures highlights the importance of studying a virus produced by its native host cell and the potential impact on quantifying virus infectivity on cell lines where the virus entry mechanisms differ from their natural target cell.

SARS-CoV-2 spike D614G change enhances replication and transmission

During the evolution of SARS-CoV-2 in humans, a D614G substitution in the spike glycoprotein (S) has emerged; virus containing this substitution has become the predominant circulating variant in the COVID-19 pandemic1. However, whether the increasing prevalence of this variant reflects a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains unknown. Here we use isogenic SARS-CoV-2 variants to demonstrate that the variant that contains S(D614G) has enhanced binding to the human cell-surface receptor angiotensin-converting enzyme2 (ACE2), increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a human ACE2 knock-in mouse model, and markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Our data show that the D614G substitution in S results in subtle increases in binding and replication in vitro, and provides a real competitive advantage in vivo-particularly during the transmission bottleneck. Our data therefore provide an explanation for the global predominance of the variant that contains S(D614G) among the SARS-CoV-2 viruses that are currently circulating.

Clinical and molecular characterization of the R751L-CFTR mutation.

Cystic fibrosis (CF) arises from mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in progressive and life-limiting respiratory disease. R751L is a rare CFTR mutation that is poorly characterized. Our aims were to describe the clinical and molecular phenotypes associated with R751L. Relevant clinical data were collected from three heterozygote individuals harboring R751L (2 patients with G551D/R751L and 1 with F508del/R751L). Assessment of R751L-CFTR function was made in primary human bronchial epithelial cultures (HBEs) and Xenopus oocytes. Molecular properties of R751L-CFTR were investigated in the presence of known CFTR modulators. Although sweat chloride was elevated in all three patients, the clinical phenotype associated with R751L was mild. Chloride secretion in F508del/R751L HBEs was reduced compared with non-CF HBEs and associated with a reduction in sodium absorption by the epithelial sodium channel (ENaC). However, R751L-CFTR function in Xenopus oocytes, together with folding and cell surface transport of R751L-CFTR, was not different from wild-type CFTR. Overall, R751L-CFTR was associated with reduced sodium chloride absorption but had functional properties similar to wild-type CFTR. This is the first report of R751L-CFTR that combines clinical phenotype with characterization of functional and biological properties of the mutant channel. Our work will build upon existing knowledge of mutations within this region of CFTR and, importantly, inform approaches for clinical management. Elevated sweat chloride and reduced chloride secretion in HBEs may be due to alternative non-CFTR factors, which require further investigation.

Comparison of the biological impact of aerosol of e-vapor device with MESH® technology and cigarette smoke on human bronchial and alveolar cultures

Exposure to aerosol from electronic vapor (e-vapor) products has been suggested to result in less risk of harm to smokers than cigarette smoke (CS) exposure. Although many studies on e-vapor products have tested the effects of liquid formulations on cell cultures, few have evaluated the effects of aerosolized formulations. We examined the effects of acute exposure to the aerosol of an e-vapor device that uses the MESH® technology (IQOS® MESH, Philip Morris International) and to CS from the 3R4F reference cigarette on human organotypic bronchial epithelial culture and alveolar triculture models. In contrast to 3R4F CS exposure, exposure to the IQOS MESH aerosol (Classic Tobacco flavor) did not cause cytotoxicity in bronchial epithelial cultures or alveolar tricultures despite its greater concentrations of deposited nicotine (3- and 4-fold, respectively). CS exposure caused a marked decrease in the frequency and active area of ciliary beating in bronchial cultures, whereas IQOS MESH aerosol exposure did not. Global mRNA expression and secreted protein profiles revealed a significantly lower impact of IQOS MESH aerosol exposure than 3R4F CS exposure. Overall, our whole aerosol exposure study shows a clearly reduced impact of IQOS MESH aerosol relative to CS in bronchial and alveolar cultures, even at greater nicotine doses.