Abstract Body: Background:
Residual inflammatory risk persists in atherosclerotic cardiovascular disease despite lipid-lowering therapy. Foamy macrophages accumulate oxidized lipids in the vascular wall and promote vascular inflammation, yet their surface phenotype and functional heterogeneity remain incompletely defined.
Hypothesis:
We hypothesized that surface marker profiling enables functional stratification of human foam-like macrophages under inflammatory conditions and reveals subsets characterized by immune–metabolic reprogramming.
Aims:
To identify surface markers defining human foamy macrophages and characterize their transcriptional programs.
Methods:
Peripheral blood mononuclear cells from three healthy donors were differentiated into macrophages using macrophage colony-stimulating factor with lipopolysaccharide (LPS) and subsequently exposed to oxidized LDL (oxLDL). Surface marker screening was performed using 354 PE-conjugated monoclonal antibodies. Candidate markers were validated with and without LPS. CD14-stratified subsets were isolated for bulk RNA sequencing followed by differential expression and gene set enrichment analyses with FDR correction.
Results:
Surface marker screening identified enrichment of CD14 in oxLDL–treated macrophages. Under LPS-conditioned culture, CD14 expression declined in the absence of oxLDL but was preserved in oxLDL–treated macrophages, whereas CD14 remained detectable under LPS-free conditions regardless of oxLDL exposure. Principal component analysis showed separation of oxLDL+/CD14+, oxLDL+/CD14−, and oxLDL−/CD14− subsets (PC2 26%). Compared with oxLDL+/CD14− cells, oxLDL+/CD14+ macrophages exhibited 256 upregulated and 181 downregulated genes (FDR <0.05). Pro-inflammatory genes including TREM1, IL1B, TLR2, ALOX5, and FCGR3A were increased. Gene set enrichment analysis demonstrated activation of inflammatory response (NES 2.01, FDR 0.014), TNF–NFκB signaling (NES 1.99, FDR 0.024), and IL-6–JAK–STAT3 signaling (NES 1.82, FDR 0.024). Oxidative phosphorylation was suppressed in oxLDL+/CD14+ cells (NES −2.49, FDR 1.6×10^-17), with downregulation of cholesterol homeostasis (NES −1.84, FDR 0.024).
Conclusion(s):
CD14 identifies a distinct pro-inflammatory foamy macrophage subset characterized by inflammatory signaling activation and suppression of mitochondrial oxidative phosphorylation and lipid metabolic programs. These findings reveal heterogeneity in human foamy macrophages and suggest potential therapeutic targets in atherosclerosis.