A novel cell indirect calorimetry method unveils the metabolic fluxomic signatures of human monocyte-derived M(LPS + IFN-γ) and M(IL-4) macrophages.

Macrophages (MΦ) display distinct immunometabolic phenotypes upon polarization. While transcriptomic analyses have suggested divergent metabolic programs in human M(LPS + IFN-γ) and M(IL-4) MΦ, a comprehensive assessment of their metabolic fluxes is lacking. The aim of this study is to 1) develop and validate a novel indirect microcalorimetry method for quantifying cellular metabolic fluxes, and 2) exploit it to characterize fluxomic signatures of polarized human monocyte-derived macrophages. MΦ from healthy donors were differentiated into unstimulated macrophages (M0), M(LPS + IFN-γ), and M(IL-4) phenotypes and studied in four defined media: substrate-free, glucose, glycyl-glutamine, and glucose + glycyl-glutamine. A steady-state fluxomic model was constructed by integrating four independent measures, oxygen consumption and proton production (Seahorse XFp) and lactate and ammonia release (microfluorimetry), into stoichiometric equations of metabolism (SAAM II software). Fluxes revealed that macrophages rely on glucose to sustain glycolysis, contributing ∼30% of citrate synthase flux, and predominantly on lipids for net citrate synthesis (first step of TCA cycle). Upon polarization, M(LPS + IFN-γ) macrophages showed increased anaerobic glycolysis versus M0 and M(IL-4), with similar TCA fluxes to M0. In contrast, M(IL-4) macrophages exhibited higher TCA and malic enzyme fluxes, especially with glucose and glycyl-glutamine, and a trend toward enhanced lipid oxidation. This novel method enables precise quantification of bioenergetic fluxes. In human MΦ, it reveals that M(LPS + IFN-γ) and M(IL-4) subsets exhibit distinct metabolic phenotypes, consistent with their immunological roles. These results resolve transcriptomic-metabolic discrepancies and provide a robust framework for assessing immunometabolism in primary human cells.NEW & NOTEWORTHY We present a novel microcalorimetry-based approach for quantifying metabolic fluxes in human cells. Applied to primary monocyte-derived human macrophages, this method reveals that M(LPS + IFN-γ) and M(IL-4) subsets exhibit distinct fluxomic profiles: the former favors glycolysis, whereas the latter shows enhanced TCA cycle activity and lipid oxidation. These findings resolve previous discrepancies between transcriptomic and functional data and provide a robust framework for investigating immunometabolism in primary human cells.