A transcriptomic atlas of macrophage polarization induced by inflammatory and reparative cytokines reveals cytokine-specific and synergistic polarization programs.

Macrophages are highly plastic immune cells derived from bone marrow progenitors that respond dynamically to environmental cues and coordinate inflammation, debris clearance, tissue remodeling, wound repair, and regeneration. Although traditionally classified into pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes, macrophage activation is now recognized as a continuum of overlapping states shaped by complex signaling environments. However, how diverse microenvironmental cues shape macrophage states and functions remains incompletely defined. Here, we performed a systematic transcriptomic analysis of murine bone marrow-derived macrophages stimulated with a broad panel of inflammatory, regulatory, and reparative cytokines, both individually and in selected combinations. Our analysis identified distinct and overlapping transcriptional programs associated with classical, alternative, and hybrid activation states, and showed that combinatorial cytokine signals modulate these programs beyond the effects of individual stimuli. Notably, combining IL-4 or IL-13 with IL-6 or IL-33 enhanced gene-expression programs associated with tissue repair, angiogenesis, and metabolic reprogramming. Cytokine array analysis further supported distinct, combination-specific secretory responses. Addition of IL-33 to the IL-4 + IL-13 backbone increased secretion of the alternative activation-associated chemokines CCL17 and CCL22, whereas addition of IL-6 uniquely increased secretion of the immunoregulatory cytokine IL-10. The combinations also showed condition-dependent increases in inflammatory mediators, including TNF-α, IL-18, CXCL1, and, to a lesser extent, IL-1β. Together, these findings support distinct mixed secretory programs rather than a uniform macrophage activation state. Overall, this study provides a systematic framework for understanding how individual and combinatorial cytokine cues shape macrophage transcriptional programs. This comprehensive dataset serves as a resource for interpreting macrophage heterogeneity in complex inflammatory and tissue-repair microenvironments and for prioritizing candidate regulatory pathways for future mechanistic investigation.