117 Winter emissions triggered stress and antiviral cellular responses in the human airway model

Abstract Vehicle exhaust is a major source of urban particulate matter, yet the toxicological effects of subfreezing emissions remain insufficiently understood. We investigated how emissions from light-duty (LD) and heavy-duty (HD) vehicles affect human airway epithelial cells in subfreezing temperatures. Five LD vehicles across different Euro classes were tested under a Real Driving Emissions cycle at +23°C and −9°C at BOSMAL (Poland) and two HD trucks using standard diesel (EN590) and sustainable fuel (HVO) were driven in winter conditions in Tampere (Finland). Complete exhaust was delivered directly to MucilAir™ cultures using a portable toxicity incubator, with clean-air and blank controls included. Toxicity was evaluated through cytotoxicity (adenylate kinase, LDH), barrier integrity (TEER), oxidative stress (isoprostanes), and genotoxicity (Comet assay). Transcriptomic responses were analysed by RNA sequencing. Cytotoxicity remained low (viability 80%) under all conditions. In the LD campaign: diesel vehicles at −9°C caused a small but consistent TEER decrease, indicating mild barrier disturbance, and transcriptomics clearly reflected the temperature effect. Across LD vehicles, 1,563 genes were upregulated and 916 downregulated at −9°C. The Euro6 hybrid and Euro4 diesel shared 72 differentially expressed genes enriched in interferon signalling and antiviral defence, suggesting a viral-like stress response. In the HD campaign: overall toxicity was low; EN590 caused slightly stronger gene expression changes than HVO, triggering stress-adaptive and epithelial defence responses without direct damage. These findings show that subfreezing operation affects the biological impact of vehicle emissions and support including low-temperature conditions in regulatory assessments