Bi-phasic Metabolic Responses During Macrophage Activation

The role of cell metabolism is an emerging element regulating macrophage-inflammatory response. Inflammatory macro-phages with highly glycolytic phenotype are also known to elevate their glycolytic activity upon pathogenic stimulation, such as lipopolysaccharide (LPS) injection, through toll-like receptors (TRLs). The dynamic changes in macrophage metabolism were traced in a real-time manner by measuring extracellular acidification (as proton efflux rates, PER) and oxygen consumption rates (OCR) after injection of LPS using Seahorse XFe96 analyzer. The PER of human peripheral blood monocytes (PBMC) derived M1 macrophages increases within an hour after stimulation, corresponding with the release of key cytokines as tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β). In contrast to PBMC-derived M1 macrophages, full activation of murine macrophage cell lines RAW264.7 and J774.A1 required co-stimulation with interferon gamma (IFNγ). Interestingly, LPS and IFNγ co-stimulation modulates glycolytic rates in a bi-phasic manner which was identified only in long-term (> 6 hr) monitoring. A series of long-term real-time XF analysis after activation revealed that the immediate early glycolytic response totally relies on LPS stimulation while the secondary elevation in PER depends on IFNγ stimulus and is abolished in the presence of inducible nitric oxide synthase (iNOS) inhibitors. These results strongly support the role of iNOS-derived nitric oxide in stimulating late glycolytic activation through inhibition of mitochondrial respiration. The data presented imply a temporal orchestration mechanism of TLR and IFNγ-receptor signaling in the metabolic regulation and activation of inflammatory macrophages.