Laboratory models reveal how a smoke-damaged epithelial barrier makes airway cells vulnerable to secondary environmental stressors.
Research: Cigarette smoke causes airway epithelial damage and chronic obstructive pulmonary disease-like features associated with polystyrene nanoparticles. Image credit: cerevonstudio / Shutterstock
The recent study was published online as an unedited “published article” in a journal. scientific reportA group of researchers investigated in vitro how tobacco smoke extract (CSE), a laboratory preparation containing smoke-derived substances, affects airway epithelial responses to polystyrene nanoparticles (PS-NPs) and whether continuous exposure to these agents promotes features associated with chronic obstructive pulmonary disease (COPD).
background
People around the world are exposed to tobacco smoke and other air pollutants that can damage the lungs. Smoking is the main cause of COPD, but concerns have been raised about microplastic and nanoplastic particles, which are prevalent in the environment and have even been detected in human tissue. Previous cellular and animal studies suggest that PS-NPs can reach the lungs after inhalation and contribute to inflammation, oxidative stress, and cell damage.
Microplastics and nanoplastics have also been detected in cigarette smoke, so there is a need to understand how cigarette smoke and plastic particles interact. However, direct evidence linking microplastics and nanoplastics to human lung disease remains lacking, and further research is needed to clarify how environmental exposures contribute to chronic lung disease.
About research
Researchers investigated the combined effects of CSE and PS-NPs in two human bronchial epithelial cell lines, immortalized BEAS-2B cells and 16HBE cells. CSE was prepared by bubbling mainstream smoke from 10 standardized 1R6F reference cigarettes into the cell culture medium, resulting in a liquid mixture of smoke-derived components rather than a single isolated chemical.
Cells were first exposed to various dilutions of CSE and subsequently treated with PS-NPs to determine whether smoke-derived mixtures alter cellular responses to plastic nanoparticle exposure.
The researchers used a thiazolyl blue tetrazolium bromide (MTT) assay to assess metabolic activity as an indirect measure of cell viability. They also analyzed membrane damage and cytotoxicity using a lactate dehydrogenase (LDH) assay.
Fluorescently labeled PS-NPs were used to measure nanoparticle-cell association by immunofluorescence microscopy in both cell lines and by flow cytometry in BEAS-2B cells. Additionally, stable isotope analysis was performed on BEAS-2B cells to estimate the proportion of plastic-derived carbon associated with the cell samples.
To determine whether CSE impairs epithelial barrier function, the researchers used quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence to measure transepithelial electrical resistance (TEER), Texas Red dextran permeability, and expression of epithelial binding proteins such as E-cadherin and zonula occludens-1 (ZO-1).
In another series of experiments, we used differentiated 16HBE cells cultured at the air-liquid interface (ALI), providing a differentiated but simplified model that more closely resembles the human airway.
These cell cultures were tested for epithelial integrity, barrier tolerance, periodic acid Schiff (PAS)-positive goblet cell number, ZO-1 protein expression, and CXCL8 secretion.
Statistical analyzes included regular one-way analysis of variance (ANOVA), repeated-measures two-way ANOVA, and Tukey multiple comparisons test, with statistical significance stated as p < 0.05.
Research results
Treatment with CSE and PS-NPs independently reduced the metabolic activity of both BEAS-2B and 16HBE bronchial epithelial cells at specific concentrations. Sequential exposure to CSE and PS-NPs resulted in a significantly greater reduction in metabolic activity than either treatment alone, consistent with potential additive toxicity.
Measurements of LDH release provided more limited evidence for increased cell damage after continuous exposure. In BEAS-2B cells, treatment with 10% CSE followed by 1 μg/mL PS-NPs significantly increased LDH release compared to either exposure alone, while other specific concentration combinations caused increased LDH release in both cell lines. However, no increase was observed in 16HBE cells exposed to CSE treated with the main 1 μg/mL PS-NP dose.
Researchers investigated whether CSE affects the interaction between airway epithelial cells and PS-NPs. Immunofluorescence analysis in both cell lines and flow cytometry in BEAS-2B cells revealed that pretreatment with CSE increased the percentage of PS-NP-positive cells.
More specifically, when BEAS-2B cells were exposed to 5% and 10% CSE, the percentage of PS-NP-positive cells increased, and 10% CSE also increased the median fluorescence intensity. This indicates high levels of cell-associated nanoparticles.
Immunofluorescence showed a comparable increase in PS-NP-positive 16HBE cells after exposure to 5% CSE. Cytokeratin-8 expression and cell area measurements showed no significant differences, suggesting that the increase in nanoparticle signal was not due to changes in these measurements.
To investigate possible explanations for why CSE increases cellular sensitivity to PS-NPs, the researchers evaluated epithelial barrier function. CSE significantly increased the uptake of Texas Red dextran into cells and showed higher membrane permeability.
In transwell cultures, CSE decreased TEER after 6–8 days and increased epithelial permeability after 8 days in a dose-dependent manner. These functional changes were associated with changes in E-cadherin levels, an epithelial binding protein, measured at the messenger ribonucleic acid (mRNA) and protein levels.
Although ZO-1 mRNA expression was unchanged, ZO-1 protein expression decreased after treatment with 10% CSE, supporting evidence of disruption of junctional integrity and impaired epithelial barrier function.
Studies using differentiated 16HBE cells grown under ALI conditions confirmed and extended these barrier findings in a model that more closely resembles the human airway.
Treatment with CSE or PS-NPs significantly reduced TEER and was accompanied by disturbance of epithelial structure. CSE significantly decreased ZO-1 expression, whereas PS-NPs alone had little effect on this protein.
Combined exposure did not further reduce ZO-1 expression or TEER compared to PS-NP treatment alone, but significantly increased the number of periodic acid Schiff-positive goblet cells compared to PS-NP alone or untreated controls, indicating an increase in cells with a mucus-producing phenotype.
PS-NPs alone significantly increased CXCL8 levels compared to untreated cultures, whereas the combination treatment did not significantly change CXCL8 levels compared to the control.
Overall, the findings showed that CSE impairs airway epithelial barrier function, increases the proportion of PS-NP-positive cells, and enhances selected responses to subsequent PS-NP exposure, including changes resembling some COPD-like epithelial features.
conclusion
Results showed that in these human bronchial epithelial cell models, CSE increased selected adverse responses to subsequent PS-NP exposure. Their continuous exposure resulted in decreased metabolic activity, enhanced LDH release under selected conditions, increased proportion of PS-NP-positive cells, and increased number of PAS-positive goblet cells.
CSE itself impaired epithelial barrier integrity and reduced binding protein expression, whereas PS-NPs independently reduced TEER in the ALI model. Collectively, these changes produced several epithelial features associated with COPD but were not indicative of COPD development.
This study suggests that components entrapped in CSE may increase the susceptibility of airway epithelial cells to subsequent exposure to plastic nanoparticles, raising questions about the potential respiratory effects of exposure to both cigarette smoke and environmental plastic pollution, and highlighting the need for continued investigation of their combined respiratory effects.
Because this study used an immortalized cell line, commercially available 100 nm polystyrene particles, a liquid CSE preparation that cannot reproduce the inhalation of a complete smoke mixture, and continuous liquid exposure, this result requires validation using primary airway cells, a more physiologically relevant exposure system, a broader range of lower CSE and PS-NP concentrations, as well as studies in animal models and humans.
Reference magazines:
- Mirra, D., Panico, F., De Meersman, Y., Spaziano, G., Altieri, S., Caiazzo, E., Bracke, K. R., Iovino, P., Marfella, R., Lubritto, C., Paolisso, G., Maes, T. (2026). Tobacco smoke causes airway epithelial damage and chronic obstructive pulmonary disease-like symptoms associated with polystyrene nanoparticles. Scientific report. Doi: 10.1038/s41598-026-61077-6, https://www.nature.com/articles/s41598-026-61077-6
