Single-cell RNA-sequencing resolves IL-4 and IFN-? into distinct inflammatory trajectories within GM-CSF macrophages

To account for the within-state heterogeneity of GM-CSF compared to M-CSF macrophages (Helft et al. 2015), we performed multiplexed scRNA-seq on 49,441 GM-CSF–derived macrophages stimulated with IL-4, IFN-?, IL-10, or TGF-ß. Cells were visualized by principal component analysis (PC1 = 13.21%, PC2 = 4.65%), which recapitulated patterns previously observed in bulk RNA-seq. IL-4 displaced cells farthest along PC1 towards a cell cluster enriched for NfkB controlled gene expression with high Ccl22 and Ccr7, consistent with a dendritic-cell-like, T-cell-activating phenotype. IFN-? dominated PC2, retaining high MHC-II expression while adding a complement/interferon module (C1qb, C1qc, Gbp2, Irf1), and showed enrichment for IRF1/8 and STAT1 transcription factor signatures. TGF-ß and IL-10 co-clustered in the PC1/PC2 low quadrant and shared expression of reparative markers including Chil3 and Cd24, forming an immunosuppressive, trophic branch distinct from either inflammatory pole. Together, these data show that within the GM-CSF lineage, IL-4 and IFN-? drive orthogonal inflammatory trajectories, whereas IL-10 and TGF-ß consolidate a shared remodeling program. Overall design: GM-CSF–derived macrophages stimulated with IL-4, IFN-?, IL-10, TGF-ß, TNF-a or left untreated were harvested on days 5 and 7 for multiplexed scRNA-seq; only day 7 cells without TNF-a (n = 49,441) were retained for analysis.