Metabolic Immunomodulation, Transcriptional Regulation, and Signal Protein Expression Define the Metabotype and Effector Functions of Five Polarized Macrophage Phenotypes

Macrophages (M{Phi}s) display remarkable plasticity and the ability to activate diverse responses to a host of intracellular and external stimuli. Despite extensive characterization of M1 M{Phi}s and a broad set of M2 M{Phi}s, comprehensive characterization of metabolic shifts driving M{Phi} activation remains. Herein, we utilized an ex vivo model to produce six M{Phi} functional phenotypes. Isolated CD14+ PBMCs were differentiated into resting M0 M{Phi}s, and then polarized into M1 (IFN-{gamma}/LPS), M2a (IL-4/IL-13), M2b (IC/LPS), M2c (IL-10), and M2d (IL-6/LIF) M{Phi}s. The M{Phi}s were profiled using a bioanalyte matrix of four cell surface markers, [~]50 secreted proteins, [~]800 expressed myeloid genes, and [~]450 identified metabolites relative to M0 M{Phi}s. Signal protein and expressed gene profiles grouped the M{Phi}s into inflammatory (M1 and M2b) and wound resolution (M2a, M2c, and M2d) phenotypes; however, each had a unique metabolic profile. While both M1 and M2b M{Phi}s shared metabotype profiles consistent with an inflammatory signature; key differences were observed in the TCA cycle, FAO and OXPHOS. Additionally, M2a, M2c and M2d M{Phi}s all profiled as tissue repair M{Phi}s; however, metabotype differences were observed in multiple pathways including hexosamine, polyamine, and fatty acid metabolism. These metabolic and other key functional distinctions suggest phagocytic and proliferative functions for M2a M{Phi}s, and angiogenesis and ECM assembly capabilities for M2b, M2c and M2d M{Phi}s. By integrating metabolomics into a systems analysis of M{Phi} phenotypes, we provide the most comprehensive map of M{Phi} diversity to date, along with the global metabolic shifts driving M{Phi} functional plasticity in these phenotypes. Summary SentenceMacrophage functional plasticity of six macrophage phenotypes correlates with unique distinctions in cell-surface marker expression, signal protein secretion, transcriptomics profiles, and metabolic processes.