High Salt (NaCl) Induces Metabolic Modifications and Affects Classic and Alternative Macrophage Activation.

High intake of dietary sodium is a risk factor for cardiovascular disease. Previously, we have shown that an increase in extracellular hypertonic sodium (+40 mM Na + ; HS) inhibits alternatively-activated M2 macrophage (M(IL4+IL13)) gene signature and function, while boosting classic M1 macrophage polarization (M(LPS)) and bacterial killing capacity. M1 macrophages predominantly rely on glycolysis, whereas M2 macrophages use oxidative phosphorylation, fueled by the oxidation of fatty acids, at a late phase of activation (24 h). The aim of this study was to elucidate the role of HS on the metabolic signature of M1 and M2 macrophages. Therefore, we quantified 13 C incorporation into intermediates of central carbon metabolism via GC-MS based pulsed stable isotope resolved metabolomics (pSIRM), and performed Seahorse analyses at varying time points during M1/M2-activation. After 24 h, LPS-induced increase in basal ECAR (extracellular acidification as surrogate parameter for glycolysis) and lactate production were further boosted under HS compared to isotonic salt conditions (NS), whereas mitochondrial respiration (OCR) was not affected. On the other hand, IL4+IL13-induced increase in OCR was decreased under HS compared to NS, while ECAR was not affected at this stage. Regarding TCA-cycle, we have found an early (1 h and 3 h post-stimulation) increase in citrate levels in M1 and M2 macrophages independent of HS, which further increased after 24 h in M1 and M1+HS. Interestingly, both in early and late phase of activation, glucose-derived 13 C incorporation into citrate was lower in HS-treated compared to NS-treated M0, M1 and M2 macrophages, suggesting an alternative replenishing source. Consistently, other TCA-cycle intermediates increased after 24h (especially in M1 and even further in M1+HS), whereas glucose-derived 13 C incorporation was minimal. Our data suggests that under hypertonic sodium macrophages re-direct TCA-cycle intermediates into the production of pro-inflammatory mediators (as described for late-phase M1 macrophages), whereas TCA-cycle itself is replenished by other alternative metabolic substrates. These HS-induced metabolic modifications might play an important role for macrophage activation and function.