Use of integrated metabolomics, transcriptomics, and signal protein profile to characterize the effector function and associated metabotype of polarized macrophage phenotypes

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 functional phenotype and associated metabotype driving this diverse M phi activation remains. Herein, an ex vivo model was utilized to produce 6 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 4 cell surface markers, similar to 50 secreted proteins, similar to 800 expressed myeloid genes, and similar to 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 that correlate to M phi functional plasticity in these phenotypes.