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

Lourdes Caceres (Freiburg im Breisgau)1, S. T. Abogunloko (Freiburg im Breisgau)1, X. Li (Freiburg im Breisgau)1, I. Hilgendorf (Freiburg im Breisgau)1, P. Stachon (Freiburg im Breisgau)1, E. Rog-Zielinska (Freiburg im Breisgau)2, O. Groß (Freiburg im Breisgau)3, D. Westermann (Freiburg im Breisgau)1, P. Evelson (Buenos Aires)4, D. Wolf (Freiburg im Breisgau)1, T. Marchini (Freiburg im Breisgau)1

1Universitäts-Herzzentrum Freiburg - Bad Krozingen Cardiology and Angiology Freiburg im Breisgau, Deutschland; 2Universitäts-Herzzentrum Freiburg - Bad Krozingen Institut für Experimentelle Kardiovaskuläre Medizin Freiburg im Breisgau, Deutschland; 3Albert- Ludwigs-Universität Freiburg Freiburg im Breisgau, Deutschland; 4University of Buenos Aires Buenos Aires, Argentinien


Air pollution fine particulate matter (PM2.5) uptake by alveolar macrophages impairs redox metabolism and promotes lung and systemic inflammation, which aggravates cardiorespiratory diseases such as myocardial infarction and stroke by yet unclear mechanisms. Here, we aim to study the molecular pathways leading to NLRP3 inflammasome activation and IL-1β release in human and mice inflammasome-reporter and primary cells incubated with a PM2.5 surrogate (Residual Oil Fly Ash, ROFA) for 6 or 24 hours at 0, 1, 10, or 100 µg/mL. Dose- and time-dependent NLRP3 priming and specks formation was evidenced in THP1-ASC-GFP cells by flow cytometry. This effect was confirmed in primary human monocyte-derived macrophages and by confocal microscopy in ASC-Citrine mice bone marrow derived macrophages (BMDMs) incubated with ROFA particles at 100 µg/mL. While increased IL-1β levels in cell culture supernatants were detected by ELISA following ROFA uptake, lack of IL-1β production was observed in ROFA-exposed NLRP3-deficient (Nlrp3-/-) and Caspase-1-deficient (Casp1-/-) BMDMs, and by specific inhibition of NLRP3 oligomerization with 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 vesicles and mitochondria, which also showed signs of ultrastructural damage. While lysosomal disruption and K+ efflux predominantly drove IL-1β production at the shorter incubation time point (6 hours), impaired mitochondrial function dominated IL-1β release at the longer time point (24 hours). Mechanistically, ROFA-exposed BMDMs showed an up to 1.3-fold increase in MitoSOX+ events by flow cytometry (p<0.001), indicative of enhanced mitochondrial superoxide anion (O2-) production, together with significantly decreased maximal respiration rates and ATP synthesis in the Seahorse MitoStress Test. Interestingly, inhibition of mitochondrial O2- production showed no effect in ROFA-exposed BMDMs after 6 hours. However, after 24 hours, specific inhibition of O2- production from respiratory Complex I by S1QEL, but not from Complex III by S3QEL, significantly decreased IL-1β release by 41% (p<0.01) in ROFA-exposed BMDMs. Our findings unravel the mechanisms by which air pollution PM2.5 promotes IL-1β release in macrophages and provide a novel insight into the innate immune response triggered by PM2.5 exposure that is associated with cardiorespiratory inflammation and disease.

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