Molecular mechanisms underlying NLRP3 inflammasome activation and IL-1β production in air pollution fine particulate matter (PM2.5)-primed macrophages

Abstract

Exposure to air pollution fine particulate matter (PM_2.5) aggravates respiratory and cardiovascular diseases. It has been proposed that PM_2.5 uptake by alveolar macrophages promotes local inflammation that ignites a systemic response, but precise underlying mechanisms remain unclear. Here, we demonstrate that PM_2.5 phagocytosis leads to NLRP3 inflammasome activation and subsequent release of the pro-inflammatory master cytokine IL-1β. Inflammasome priming and assembly was time- and dose-dependent in inflammasome-reporter THP-1-ASC-GFP cells, and consistent across PM_2.5 samples of variable chemical composition. While inflammasome activation was promoted by different PM_2.5 surrogates, significant IL-1β release could only be observed after stimulation with transition-metal rich Residual Oil Fly Ash (ROFA) particles. This effect was confirmed in primary human monocyte-derived macrophages and murine bone marrow-derived macrophages (BMDMs), and by confocal imaging of inflammasome-reporter ASC-Citrine BMDMs. IL-1β release by ROFA was dependent on the NLRP3 inflammasome, as indicated by lack of IL-1β production in ROFA-exposed NLRP3-deficient (Nlrp3^-/-) BMDMs, and by specific NLRP3 inhibition with the pharmacological compound MCC950. In addition, while ROFA promoted the upregulation of pro-inflammatory gene expression and cytokines release, MCC950 reduced TNF-α, IL-6, and CCL2 production. Furthermore, inhibition of TNF-α with a neutralizing antibody decreased IL-1β release in ROFA-exposed BMDMs. Using electron tomography, ROFA particles were observed inside intracellular vesicles and mitochondria, which showed signs of ultrastructural damage. Mechanistically, we identified lysosomal rupture, K⁺ efflux, and impaired mitochondrial function as important prerequisites for ROFA-mediated IL-1β release. Interestingly, specific inhibition of superoxide anion production (O₂^•-) from mitochondrial respiratory Complex I, but not III, blunted IL-1β release in ROFA-exposed BMDMs. Our findings unravel the mechanism by which PM_2.5 promotes IL-1β release in macrophages and provide a novel link between innate immune response and exposure to air pollution PM_2.5.

Keywords: Inflammation; K(+) efflux; Lysosomal disruption; Macrophages; Mitochondria; Particulate matter.