Aged automobile cabin air filters as secondary pollution sources: mechanistic insights into NLRP3 inflammasome activation and discovery of a natural inhibitor

Abstract Automobile cabin air filter-deposited particulate matter represents an underappreciated exposure source, yet the differential toxicity between filter sides and underlying mechanisms remain unclear. Here, we systematically investigated particles extracted from the inside (cabin-facing) and outside (environment-facing) membranes of used automobile air filters. Morphological analysis revealed that inside particles exhibited smaller size and greater dispersion, consistent with ultrafine particle enrichment. In a murine exposure model, inside particles induced more severe pulmonary inflammation, histopathological injury, and elevated interleukin-1β levels compared to outside particles at equivalent doses. Single-cell RNA sequencing identified alveolar macrophages as the predominant responding cell type, with significant enrichment of the NOD-like receptor signaling pathway. Mechanistic investigations demonstrated that particle exposure activated the NLRP3 inflammasome and triggered Gasdermin D-mediated pyroptosis through a reactive oxygen species-independent pathway. Furthermore, we employed structure-based virtual screening of over two thousand natural compounds, from which we identified the cyclic dipeptide Cyclo-(Tyr-Phe) as a novel NLRP3 inhibitor. Molecular dynamics simulations and cellular thermal shift assays confirmed direct NLRP3 binding, while functional validation demonstrated that Cyclo-(Tyr-Phe) effectively attenuated particle-induced pyroptosis and inflammatory responses. These findings illuminate the heightened toxicity of ultrafine-enriched filter particles and provide a promising therapeutic candidate for mitigating particle-induced lung injury. Graphical Abstract