In order to fulfill their function immune cells tightly regulate their central carbon metabolism depending on the microenvironment they encounter. Fully activated classic M1 macrophages are known to increase glycolysis and pentose phosphate pathway, while repurposing their tricarboxylic acid (TCA) cycle and electron transport chain (ETC), away from energy production towards the generation of pro-inflammatory mediators. On the other hand, fully-activated alternative M2 macrophages highly depend on intact TCA cycling and oxidative phosphorylation (OXPHOS). One important factor of local microenvironment is sodium. Dietary salt, local infection and aging can induce accumulation of high amounts of sodium (without concomitant water retention) in tissues. Immune cells residing or invading such hypertonic saline compartments are differentially regulated and exhibit an altered function. Interestingly, it has been shown that HS boosts macrophage bacterial killing capacity after only 4h, a time point rarely studied in regards to immune cell metabolism. Based on these data, we hypothesized that high extracellular sodium affects the central carbon metabolism of murine and human mononuclear phagocyte cells. Here we show that upon stimulation, M(LPS) and M(IL4+IL13) macrophages very quickly show an induction of respective pro-inflammatory and anti-inflammatory marker genes, which only partially further increase over time. Furthermore, activation under hypertonic saline (+40mM NaCl, HS) conditions had immediate effects on marker gene expression. Surprisingly, anaerobic glycolysis, the main M1-associated energy source, was not affected by HS at this early stage of activation. We therefore analyzed TCA cycle and OXPHOS by pulsed stable isotope resolved metabolomics (pSIRM) and Seahorse technology. At 3h of activation, glucose- and glutamine-derived label incorporation into the TCA cycle were not affected by HS. Only the conversion of succinate into fumarate was inhibited upon HS in both M(LPS) and M(IL4+IL13). By contrast, Seahorse analysis revealed a significant decrease in basal and maximal oxygen consumption rate (OCR) under HS in both M(LPS) and M(IL4+IL13). This reduction was accompanied by a decrease in ATP production and mitochondrial membrane potential after only 3h of activation. These data suggest a mitochondrial dysfunction and metabolic uncoupling of TCA cycle and mitochondrial respiration under HS. We could show that HS inhibited ETC complex III and that pharmacologic inhibition of complex III as well as pharmacologic uncoupling mimicked the HS effect on macrophage phenotype. In a translational approach in healthy individuals, we demonstrated that dietary salt intake resulted in a significant increase in plasma sodium. Strikingly, this increase correlated with a decrease in mitochondrial function in peripheral blood-derived monocytes. Taken together, we suggest that HS induces a mitochondrial dysfunction in murine macrophages and human monocytes. This could constitute a novel mechanism by which HS modulates murine and human immune cell function.
