Diesel particulate matter induces dose-dependent cytotoxicity, multi-faceted inflammation, MAPK activation, and oxidative stress in RAW 264.7 macrophages: an integrated transcriptomic and protein–protein interaction network analysis
TL;DR
Beyond classical inflammation, these integrated findings reveal coordinated network-level perturbations consistent with ferroptosis vulnerability, genome instability, paradoxical immune dysregulation, and potential macrophage identity loss consistent with ferroptosis vulnerability, genome instability, paradoxical immune dysregulation, and potential macrophage identity loss.
Abstract
Diesel particulate matter (DPM), a Group 1 carcinogen classified by the International Agency for Research on Cancer, is a major component of traffic-related air pollution. Although macrophages are central to the response to inhaled particles, the integrated network-level cellular response to DPM remains incompletely characterized. A combined three analytical methods was applied to RAW 264.7 macrophages exposed to DPM (NIST SRM2975) for 24 h: (i) dose–response phenotyping across twelve CCK-8 concentrations (0–750 μg/mL) and multi-modal characterization (Western blot, RT-qPCR, Griess assay, DCFH-DA ROS imaging, TNF-α ELISA) at sub-cytotoxic to near-IC50 doses; (ii) 3′ mRNA-sequencing at 100 μg/mL with edgeR-based differential expression (|fold change|≥ 1.5) and Gene Ontology, KEGG, and Reactome enrichment; and (iii) STRING-based protein–protein interaction (PPI) network analysis with Markov Cluster (MCL) modular clustering in Cytoscape. DPM reduced viability dose-dependently (IC50 ≈ 135–140 μg/mL) and induced concerted activation of iNOS, COX-2, MAPK phosphorylation, pro-inflammatory cytokines (TNF-α, IL-1β), antioxidant enzymes (SOD2, HO-1, Catalase), nitrite, ROS, and secreted TNF-α. Transcriptomics identified 750 differentially expressed genes (331 up; 419 down), with enrichment in cytokine production, NF-κB/MAPK signaling, inflammasome, and oxidative stress. PPI analysis resolved ten up- and ten down-regulated functional modules, with key hubs Ccl2, Gpx4, Ifit3/Stat1, Dna2/Rad52, and Csf1r. Beyond classical inflammation, these integrated findings reveal coordinated network-level perturbations consistent with ferroptosis vulnerability, genome instability, paradoxical immune dysregulation, and potential macrophage identity loss — hypothesis-generating axes for future mechanistic validation.
