Application Note
Real-time discrimination of inflammatory macrophage activation using Agilent Seahorse XF Technology
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mmunometabolism research has dra- matically expanded our understanding of immune cell function in recent years. There is a contemporary paradigm shift that is moving beyond characterisation of cell phenotype to controlling cell fate by modulating metabolism1. This Application Note demonstrates how XF technology can be used in routine analyses to detect and monitor M1 macrophage activation in a live-cell, real-time activation assay. The model can be adapted for various routine studies on pathogenic and phagocytic acti- vation of macrophages, and for kinetic analysis of dendritic cells and other mononuclear phagocytotic cell types. In this study, XF technology was used to simultaneously measure both pathways that regulate immune cell function for human M1 macrophages derived from peripheral blood mononuclear cells (PBMC). Notably, glycolytic flux was accu- rately quantified by real-time proton efflux rates (PER), which corrects for acidifica- tion from the TCA cycle. Simultaneously with PER measurements, mitochondrial function was monitored via oxygen con- sumption rate (OCR).
I) Early and rapid elevation of glycolysis with activation, followed by sustained kinetic response Real-time detection of the immediate gly- colytic response can be used as a rapid and reliable method for macrophage activation detection. Simultaneous measurements of PER and OCR during inflammatory M1 PBMC-derived macrophage activation show elevated glycolysis, indicating activa- tion (Figure 1). Glycolytic rates (ie PER) of macrophages increased rapidly upon co- injection of lipopolysaccharide (LPS) and interferon gamma (IFN) and elevated rates were sustained for hours (Figure 1A). The injection slightly decreased the OCR,
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but the effect was small compared to the changes in PER (Figure 1B). Both measure- ments work together to show the metabolic switch towards glycolysis as an indicator of M1 macrophage activation.
II) Functional relevance: validation with cytokine accumulation Real-time activation with XF technology is a simple and direct way to examine macrophage reaction to internal (ie cancer cells) or external (ie pathogens) cues. To demonstrate this capability, fully-differenti- ated, PBMC-derived M1 macrophages were stimulated by injection of LPS, IFN, or both. Figure 2B shows the rapid increase in glycolytic rates (PER) induced by LPS with or without IFN, but not by IFN alone, indicating LPS stimulated-activation, while IFN alone does not stimulate activation.
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The non-destructive nature of the XF activation assay enables correlation with other downstream biological measure- ments, as exemplified in Figure 2. The immediate increase in glycolytic rates cor- responded to the production of M1 activa- tion-associated cytokines, TNF and IL-1 (Figures 2C and 2D), as functional confir- mation of the XF real-time activation assay. Interestingly, the duration of cytokine pro- duction and accumulation differed between TNF (declining < 26h) and IL-1 (contin- uing > 26h). Differing cytokine expression kinetics highlights the added value of con- tinuous kinetic XF measurements, since endpoint assays would need to be designed appropriately to identify proper cytokine sets and timing. The real-time activation assay eliminates this optimisation issue and provides time-resolved information which enables a better understanding of the dynamic activation mechanism and contin- ued response. By correlating real-time func- tional information to orthogonal biological data, greater insight may be gained with respect to the causes and metabolic impli- cations of immune cell activation. DDW
Reference 1 O’Neill et al. (2016) Nat. Rev. Immunol. 16:553-565.
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