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- FIGURE 1.
Characterization of polarized peritoneal macrophages. qRT-PCR confirms distinctive polarization states of macrophages in our ex vivo system. M1 and MLPS polarization phenotypes were verified by overexpression of Nos-2 and Tnf-α. M2 polarization phenotype was characterized by increased expression of Arg-1 and Fizz-1. LPS treatment induced expression of Arg-1, consistent with the development of an endotoxin-tolerance phenotype. Data are expressed relative to expression of housekeeping gene Hprt (n = 3–4). mRNA = messenger RNA.
- FIGURE 2.
Macrophage polarization and glucose utilization. Glucose uptake and RNA expression of genes involved in intracellular trapping of 3H-2-deoxyglucose is enhanced in MLPS macrophages. Inflammatory polarization of macrophages by LPS (MLPS), unlike IFN-γ plus TNF-α (M1), markedly enhances 3H-2-deoxyglucose uptake (A), which is associated with overexpression of Glut-1 (B), Hk-1 (C), and Hk-2 (D). M2 polarization has no effect on glucose uptake or expression of Glut-1. Modest increase is present in Hk-1 and Hk-2 expression in M2 and Hk-2 expression in M1 polarized macrophages without concomitant increase in Glut-1 expression or 3H-2-deoxyglucose uptake (n = 5 for deoxyglucose uptake and n = 3 for gene expression experiments). mRNA = messenger RNA.
- FIGURE 3.
Mitochondrial bioenergetics and macrophage polarization. Real-time analysis of OCR demonstrates markedly different respiratory profiles among polarized murine peritoneal macrophages at baseline and after incubation with specific mitochondrial inhibitors. Basal mitochondrial respiration is significantly lower in MLPS than in nonpolarized macrophages. Spare respiratory capacity is negligible in nonpolarized and classically polarized (M1 and MLPS) macrophages. On the other hand, OCR reaches to about 2.2-fold of basal level in M2 polarized macrophages in presence of FCCP, indicating significant spare respiratory capacity of these cells. Consistently, maximal respiratory capacity of M2 polarized macrophages is also significantly higher in M2 than in classically (M1 and MLPS) polarized macrophages (n = 7).
- FIGURE 4.
Glycolysis vs. oxidative phosphorylation in polarized macrophages. Basal ECAR-to-OCR ratio is significantly higher in MLPS macrophages than in M0, M1, and M2 polarized macrophages, demonstrating that LPS induced shift away from oxidative phosphorylation toward glycolysis (n = 7).
- FIGURE 5.
Mitochondrial biogenesis and macrophage polarization. Mitochondrial content and RNA expression of genes involved in mitochondrial biogenesis and oxidative phosphorylation is significantly enhanced in M2 polarized murine peritoneal macrophages. M2 polarization promotes mitochondrial biogenesis, as shown by increased mitochondrial DNA copy number (mtDNA-to-nDNA ratio) (A) and expression of Tfam (B), and Cox-1 (C). No significant difference exists in expression of Tfam and Cox-1 or mitochondrial content of M0 and M1 polarized macrophages. LPS treatment induced expression of both Tfam and Cox-1, but this was not associated with significant increase in mitochondrial content of MLPS macrophages (n = 5 for mtDNA copy number and n = 3 for gene expression experiments). mRNA = messenger RNA.
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