Beyond M1/M2 dichotomy: Toward an operationally defined, modular framework for macrophage functional states

Macrophages are versatile immune cells involved in tissue homeostasis, host defense, inflammation, and disease progression. Their biology has traditionally been interpreted through the M1/M2 paradigm, but single-cell and spatial profiling, lineage tracing, and multi-omics reveal substantially greater heterogeneity. This review organizes macrophage diversity around recurring functional programs: immunosuppressive/regulatory, matrix-remodeling, hypoxia-associated metabolic, IFN-responsive/antigen-presenting, and proliferative. Angiogenic/perivascular activity is treated as a putative program because its status as a distinct transcriptional entity remains uncertain. We introduce an operational framework based on three criteria-co-expression coherence, combinatorial independence, and microenvironmental inducibility-to distinguish candidate functional modules from broader programs. We also conduct an evidence-weighted assessment of these programs, examining their independent variation, contextual coupling, and co-occurrence. Furthermore, we propose a Macrophage Dominance Index (MDI) as a hypothesis-generating numerical framework, with provisional marker sets, weighting, and formula requiring independent validation. Finally, we derive falsifiable predictions and outline experimental, single-cell, spatial, and clinical strategies to test relationships among programs. Overall, available evidence suggests substantial coupling among macrophage functional programs rather than uniformly independent functional axes. Macrophage heterogeneity is therefore better understood as context-dependent combinations of programs, some of which remain unconfirmed as distinct transcriptional modules. Although the framework may inform macrophage biology broadly, most supporting evidence, particularly concerning program relationships, biomarkers, prognosis, and therapeutic implications, derives from tumor-associated macrophages (TAMs). Its applicability to non-cancer settings remains to be established. The MDI and proposed predictions are intended to facilitate testable research, not to serve as validated diagnostic, prognostic, or therapeutic tools.