(2,3) M1-like macrophages play an important role in pathogen clearance, and their sustained activation is linked to postinjury tissue damage. (1) In vitro, classically activated M1 macrophages are generated by polarizing monocyte-derived naïve macrophages (M0) with a mix of cytokines including interferon γ and granulocyte macrophage colony-stimulating factor (GM-CSF). Following infection or tissue damage, macrophages acquire a spectrum of functional phenotypes that are exemplified by pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes at each end of the spectrum, with these subsets expressing unique biomarkers. Macrophages are the sentinels and regulators of the human immune system they display remarkable stimulus-induced functional plasticity, which is key in their ability in responding to a diverse range of pathogens, foreign objects, and dead cells resulting from tissue injury. The first demonstration of this capability illustrates the great potential of direct cell analysis for in situ metabolite profiling with the 3D OrbiSIMS to probe functional phenotype at the single-cell level using molecular signatures and to understand the response of the human body to implanted devices and immune diseases. The diamino acids alanylasparagine and lipid sphingomyelin SM(d18/16:0) are uniquely found in M1 macrophages, while pyridine and pyrimidine are observed at increased intensity in M2 macrophages, findings which link to known biological pathways. Characteristic metabolites in macrophage subsets are identified using a targeted lipid and data-driven multivariate approach highlighting amino acids and other small molecules. This analysis strategy not only requires more than 6 orders of magnitude less sample than traditional mass spectrometry approaches but also allows the study of cell-to-cell variance. Here, the recently developed 3D OrbiSIMS instrument is applied to yield useful insight into the metabolome from individual cells after in vitro differentiation of macrophages into naïve, M1, and M2 phenotypes using different cytokines. Characterizing the metabolic signature (metabolic profiling) of different macrophage subsets is a powerful tool to understand the response of the human immune system to different stimuli. Macrophages are important immune cells that respond to environmental cues acquiring a range of activation statuses represented by pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes at each end of their spectrum.
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