NOTCH reprograms mitochondrial metabolism for proinflammatory macrophage activation
Jun Xu, … , Samuel W. French, Hidekazu Tsukamoto
Jun Xu, … , Samuel W. French, Hidekazu Tsukamoto
Published March 23, 2015
Citation Information: J Clin Invest. 2015;125(4):1579-1590. https://doi.org/10.1172/JCI76468.
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Research Article Immunology

NOTCH reprograms mitochondrial metabolism for proinflammatory macrophage activation

Abstract

Metabolic reprogramming is implicated in macrophage activation, but the underlying mechanisms are poorly understood. Here, we demonstrate that the NOTCH1 pathway dictates activation of M1 phenotypes in isolated mouse hepatic macrophages (HMacs) and in a murine macrophage cell line by coupling transcriptional upregulation of M1 genes with metabolic upregulation of mitochondrial oxidative phosphorylation and ROS (mtROS) to augment induction of M1 genes. Enhanced mitochondrial glucose oxidation was achieved by increased recruitment of the NOTCH1 intracellular domain (NICD1) to nuclear and mitochondrial genes that encode respiratory chain components and by NOTCH-dependent induction of pyruvate dehydrogenase phosphatase 1 (Pdp1) expression, pyruvate dehydrogenase activity, and glucose flux to the TCA cycle. As such, inhibition of the NOTCH pathway or Pdp1 knockdown abrogated glucose oxidation, mtROS, and M1 gene expression. Conditional NOTCH1 deficiency in the myeloid lineage attenuated HMac M1 activation and inflammation in a murine model of alcoholic steatohepatitis and markedly reduced lethality following endotoxin-mediated fulminant hepatitis in mice. In vivo monocyte tracking further demonstrated the requirement of NOTCH1 for the migration of blood monocytes into the liver and subsequent M1 differentiation. Together, these results reveal that NOTCH1 promotes reprogramming of mitochondrial metabolism for M1 macrophage activation.

Authors

Jun Xu, Feng Chi, Tongsheng Guo, Vasu Punj, W.N. Paul Lee, Samuel W. French, Hidekazu Tsukamoto

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Figure 2

NOTCH directly activates Nos2 transcription.

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NOTCH directly activates Nos2 transcription. (A) Schematic response elem...
(A) Schematic response elements for HIF-1α (HRE), NF-κB (κB), and NOTCH partner CSL in murine Nos2 promoter. ChIP-qPCR on these sites in cultured HMacs under either 21% (normoxia) or 2% (hypoxia) oxygen for 16 hours. Values are fold enrichment relative to control after normalization with IgG (n = 3). *P < 0.05 vs. control under normoxia, t test. (B) ChIP-qPCR for NICD1 and CSL (RBP-Jκ) in Raw 264.7 cells treated with or without LI. Results are representative of 2 separate experiments. (C) Luciferase Nos2 promoter activity in Raw 264.7 cells treated with vehicle, DAPT, or LPS plus DAPT under 21% (NOMO) or 2% (HYPO) O2. Values are percentage change of Firefly over Renilla luciferase activity as compared with control under NOMO (n = 4). *P < 0.05 vs. normoxic control, **P < 0.05 vs. hypoxic control, #P < 0.05 with LPS treatment under respective conditions, 2-way ANOVA. (D) Nos2 promoter activity in Raw 264.7 cells transduced with nothing (WT) or scrambled (Scr) or Notch1 (N1) shRNA, with or without LPS for 4 hours (n = 5–8). *P < 0.05 vs. WT, #P < 0.05, 1-way ANOVA. (E) Nos2 promoter activity in Raw 264.7 cells overexpressing 3xflag-YFP or 3xflag-NICD1 (n = 5). *P < 0.05, t test. (F) Western blots of nuclear proteins NICD1, p65, and HIF-1α. (G and H) Effects of HRE, NF-κB, or CSL site mutation on Nos2 promoter activity under (G) normoxia and (H) hypoxia. Raw 264.7 cells transfected with WT or mutant Nos2 promoter luciferase reporters were treated with or without DAPT or LPS for 4 hours (n = 5–8). *P < 0.05 vs. untreated WT, #P < 0.05 between the treatments, t test.