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. 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. Methods. MΦ from healthy donors were differentiated into 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), lactate and ammonia release (microfluorimetry) - into stoichiometric equations of metabolism (SAAM II software). Results. 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. Conclusions. 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.