Cigarette Smoke Drives Polystyrene Nanoparticles-Associated Airway Epithelial Damage and Chronic Obstructive Pulmonary Disease-like features

Introduction: Chronic obstructive pulmonary disease (COPD) is a progressive chronic airway disorder characterized by partially irreversible airflow obstruction, abnormal inflammatory responses, and impaired mucociliary clearance. Cigarette smoke remains the primary risk factor for COPD, contributing to epithelial cell damage, immune cell recruitment, oxidative stress, and cellular senescence. However, a significant number of COPD cases occur in never-smokers, suggesting the involvement of additional environmental contributors. Among these, plastics have raised major environmental concerns due to their widespread presence as micro- and nanoplastics (MNPs), which can accumulate and affect human health. To date, limited data are available on the potential interactions between polystyrene (PS) exposure, cigarette smoke, and COPD onset and progression, and no studies have compared MNPs levels in lung tissue from healthy controls and COPD patients. Therefore, the biochemical interplay between cigarette smoke and MNPs in airway epithelial damage remains unclear. Aim: This PhD project aimed to investigate the relationship between epithelial susceptibility to PS nanoparticles (PS-NPs) and cigarette smoke using in vitro models of human bronchial epithelial cells. Methods: Cigarette smoke extract (CSE)/PS-NPs cytotoxicity was assessed by measuring cell metabolic activity and lactate dehydrogenase (LDH) release in BEAS-2B cells and primary human bronchial epithelial cells (pHBECs). PS-NPs internalization following CSE exposure was evaluated using immunofluorescence (IF) microscopy and flow cytometry. CSE-induced modulation of epithelial integrity was assessed via transepithelial electrical resistance (TEER), a 10 kDa Texas-Red Dextran permeability assay, and Quantitative(q)-PCR/IF analysis of tight junction proteins. To investigate COPD-like features, CXCL8 release was quantified by ELISA in BEAS-2B cells, and neutrophil recruitment and activation were analysed using a transwell migration assay and a myeloperoxidase (MPO) activity assay, respectively. Furthermore, epithelial layer integrity (assessed by Haematoxylin and Eosin staining and TEER), mucus production (via Periodic acid–Schiff [PAS] staining), and CXCL8 release (ELISA) were evaluated in air–liquid interface (ALI) cultured pHBECs. Results and Discussion: Combined exposure to CSE and PS-NPs caused a more pronounced decrease in cell viability compared with individual treatments, indicating enhanced cytotoxicity. CSE exposure significantly increased the number of PS-positive BEAS-2B cells at 1 μg/mL and 100 μg/mL PS, while 5% CSE enhanced PS uptake in pHBECs; conversely, exposure to 10% CSE reduced cell viability, preventing further analysis in pHBECs. Flow cytometry data confirmed IF results in BEAS-2B cells. Both 5% and 10% CSE exposure significantly reduced TEER and E-cadherin levels while increasing dextran permeability, demonstrating epithelial barrier disruption, which likely explains the enhanced PS uptake. In BEAS-2B cells exposed to 5% and 10% CSE, PS treatment further increased CXCL8 protein levels compared to PS treatment alone. This was associated with a significant increase in neutrophil migration after incubation with supernatants from BEAS-2B cells treated with 10% CSE and 1 μg/mL PS, although no additive effect was observed compared to PS treatment alone. Moreover, MPO activity was significantly increased in neutrophils incubated with supernatants from BEAS-2B cells exposed to 10% CSE and then treated with 1 μg/mL PS, indicating enhanced neutrophil activation. In the ALI-pHBEC model, combined exposure to 5% CSE and 1 μg/mL PS significantly decreased epithelial cohesion and increased mucus production, suggesting a crucial role of CSE in PS-induced COPD-like features. Conclusion: Our findings highlight the role of cigarette smoke in enhancing epithelial susceptibility to PS-NPs, promoting inflammation, barrier dysfunction, and neutrophil activation. These results provide new insights into cigarette smoke-driven epithelial vulnerability to environmental MNPs and may inform future studies on their involvement in COPD onset and progression.