A laboratory model reveals how smoke-damaged epithelial barriers may leave airway cells more vulnerable to a second environmental stressor.
Study: Cigarette smoke drives polystyrene nanoparticles-associated airway epithelial damage and chronic obstructive pulmonary disease-like features. Image Credit: cerevonstudio / Shutterstock
In a recent study published online as an unedited 'Article in Press' in the journal Scientific Reports, a group of researchers investigated in vitro how cigarette smoke extract (CSE), a laboratory preparation containing smoke-derived substances, influences airway epithelial responses to polystyrene nanoparticles (PS-NPs) and whether sequential exposure to these agents promotes features associated with chronic obstructive pulmonary disease (COPD).
Background
People worldwide are exposed to cigarette smoke and airborne pollutants that may damage the lungs. Although smoking is the primary cause of COPD, concerns have emerged about microplastic and nanoplastic particles, which are widespread in the environment and have been detected in human tissues. Previous cell and animal studies suggest that PS-NPs can reach the lungs following inhalation and may contribute to inflammation, oxidative stress, and cellular injury.
Since microplastics and nanoplastics have also been detected in cigarette smoke, it is necessary to understand how cigarette smoke and plastic particles may interact. However, direct evidence linking microplastics and nanoplastics to human lung disease remains lacking, and further research is needed to clarify how environmental exposures may contribute to chronic lung diseases.
About the study
The researchers investigated the combined effects of CSE and PS-NPs in two human bronchial epithelial cell lines: the immortalized BEAS-2B cells and the 16HBE cells. CSE was prepared by bubbling the mainstream smoke from 10 standardized 1R6F reference cigarettes through cell culture medium, yielding a liquid mixture of smoke-derived constituents rather than a single isolated chemical.
Cells were first exposed to various dilutions of CSE and subsequently treated with PS-NPs to determine whether the smoke-derived mixture altered cellular responses to plastic nanoparticle exposure.
The researchers used the thiazolyl blue tetrazolium bromide (MTT) assay to evaluate metabolic activity as an indirect measure of cell viability. They also used a lactate dehydrogenase (LDH) assay to analyze membrane damage and cytotoxicity.
Fluorescently labeled PS-NPs were used to measure cellular association with the nanoparticles by immunofluorescence microscopy in both cell lines and by flow cytometry in BEAS-2B cells. In addition, stable isotope analysis was performed in BEAS-2B cells to estimate the fraction of plastic-derived carbon associated with the cellular samples.
To determine whether CSE impaired epithelial barrier function, the investigators measured transepithelial electrical resistance (TEER), Texas Red-dextran permeability, and the expression of epithelial junction proteins, including E-cadherin and zonula occludens-1 (ZO-1), using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence.
A separate series of experiments used differentiated 16HBE cells cultured at an air-liquid interface (ALI), providing a differentiated yet simplified model that more closely resembles the human airway.
These cell cultures were tested for epithelial integrity, barrier resistance, periodic acid-Schiff (PAS)-positive goblet cell numbers, ZO-1 protein expression, and CXCL8 secretion.
Statistical analysis included ordinary one-way analysis of variance (ANOVA), repeated-measures two-way ANOVA, and Tukey multiple comparisons tests, with statistical significance described as p < 0.05.
Study 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 decrease in metabolic activity than either treatment alone, consistent with potential additive toxicity.
Measurements of LDH release provided more limited evidence of increased cellular injury following sequential exposure. In BEAS-2B cells, treatment with 10% CSE followed by 1 µg/mL PS-NPs significantly increased LDH release compared with either exposure alone, whereas increased LDH release occurred under certain other concentration combinations in both cell lines. However, no increase was observed in CSE-exposed 16HBE cells treated with the principal 1 µg/mL PS-NP dose.
The researchers explored whether CSE affected interactions 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 proportion of PS-NP-positive cells.
More specifically, exposure of BEAS-2B cells to 5% and 10% CSE increased the proportion of PS-NP-positive cells, with 10% CSE also increasing median fluorescence intensity, indicating higher levels of cell-associated nanoparticles.
Immunofluorescence showed a comparable increase in PS-NP-positive 16HBE cells after exposure to 5% CSE. Measurements of cytokeratin-8 expression and cell area showed no significant differences, suggesting that the increased nanoparticle signal was not attributable to changes in these measures.
To investigate a possible explanation for why CSE increased cellular susceptibility to PS-NPs, the researchers evaluated epithelial barrier function. CSE significantly increased intracellular Texas Red-dextran uptake, indicating greater membrane permeability.
In Transwell cultures, CSE reduced TEER after six to eight days and increased epithelial permeability in a dose-dependent manner after eight days. These functional changes were associated with changes in E-cadherin levels, an epithelial junction protein measured at the messenger ribonucleic acid (mRNA) and protein levels.
Although ZO-1 mRNA expression remained unchanged, ZO-1 protein expression decreased following treatment with 10% CSE, supporting evidence of disrupted junctional integrity and impaired epithelial barrier function.
Studies using differentiated 16HBE cells grown under ALI conditions supported and extended these barrier findings in a model that more closely resembles the human airway.
Treatment with either CSE or PS-NPs significantly reduced TEER and was accompanied by disorganized epithelial architecture. CSE markedly decreased ZO-1 expression, whereas PS-NPs alone produced little effect on this protein.
Combined exposure did not further reduce ZO-1 expression or TEER compared with PS-NP treatment alone, but significantly increased the number of periodic acid-Schiff-positive goblet cells compared with PS-NPs alone or untreated controls, indicating an increase in cells with a mucus-producing phenotype.
Although PS-NPs alone significantly increased CXCL8 levels compared with untreated cultures, the combined treatment did not significantly alter CXCL8 levels relative to controls.
Overall, the findings showed that CSE impaired airway epithelial barrier function, increased the proportion of PS-NP-positive cells, and enhanced selected responses to subsequent PS-NP exposure, including changes resembling some COPD-like epithelial features.
Conclusions
The results indicated that CSE increased selected harmful responses to subsequent PS-NP exposure in these human bronchial epithelial cell models. Their sequential exposure led to decreased metabolic activity, enhanced LDH release under selected conditions, a higher proportion of PS-NP-positive cells, and increased PAS-positive goblet cell numbers.
CSE itself compromised epithelial barrier integrity and reduced junctional protein expression, whereas PS-NPs independently reduced TEER in the ALI model. These changes collectively produced several epithelial features associated with COPD but did not demonstrate COPD development.
The study suggested that constituents captured in CSE may enhance the susceptibility of airway epithelial cells to subsequent exposure to plastic nanoparticles, raising questions about the potential respiratory effects of exposure to both tobacco smoke and environmental plastic pollution and emphasizing the need for continued investigation into their combined respiratory effects.
Because the study used immortalized cell lines, commercial 100-nm polystyrene particles, a liquid CSE preparation that could not reproduce inhalation of the complete smoke mixture, and sequential liquid exposures, the findings require validation using primary airway cells, more physiologically relevant exposure systems, and a broader range of lower CSE and PS-NP concentrations, as well as animal models and human studies.
Journal reference:
- 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). Cigarette smoke drives polystyrene nanoparticles-associated airway epithelial damage and chronic obstructive pulmonary disease-like features. Scientific Reports. DOI: 10.1038/s41598-026-61077-6, https://www.nature.com/articles/s41598-026-61077-6