Summary Tissue-macrophage populations are constituted by a mosaic of phenotypes, yet new methods are needed to link metabolic status to the range of phenotypes in vivo . We therefore designed a high-dimensional panel for spectral flow cytometry to investigate the heterogeneity of tissue macrophage metabolism at steady-state, and their metabolic adaptation in response to infection. Distinct metabolic profiles were observed between tissue macrophages from different peripheral organs, as well as within populations from a specific site. As such, our data show multiple metabolic states in macrophages corresponding to relative stages of maturity in both the peritoneal cavity and small intestine. Immune perturbation with helminth infection further showed that peritoneal macrophages acquire an overall highly metabolically active profile, whereas responding intestinal macrophages displayed minimal changes in their metabolic phenotype. Thus, we demonstrate that high-dimensional, flow-based analysis is an exciting method to interrogate the metabolic heterogeneity and dynamics of tissue-macrophage populations.

Spectral flow cytometry reveals metabolic heterogeneity in tissue macrophages