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 4

NOTCH regulates mtDNA transcription.

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NOTCH regulates mtDNA transcription. (A) Immunoblot of NICD1 of mitochon...
(A) Immunoblot of NICD1 of mitochondrial proteins from control or OF+Alc HMacs and Raw 264.7 cells treated with or without LPS or IFN-γ for 24 hours. VDAC1 served as a loading control, and the absence of β-tubulin or histone H3 validates the purity of mitochondrial proteins. (B) Fluorescent confocal microscopy shows the colocalization of NICD1 (green) and MitoTracker (red) in Raw 264.7 cells treated with LPS plus LI for 24 hours. Results are representative of 5 different experiments. Scale bars: 5 μm. (C) Electron microscopy image (original magnification, ×150,000) of immunogold-NICD1 in mitochondria (MT) of Raw 264.7 cells stimulated with LPS for 24 hours, as indicated by arrows. Results are representative of 3 different experiments. (D) ChIP-seq analysis on mtDNA from Raw 264.7 cells stimulated with LPS for 24 hours. Integrative Genomics Viewer genome browser tracks show the levels of NOTCH1 ChIP sample (blue) over input (red). Different genomic coordinates and genome window size for ChrM (chr17:23,695,786–23,710,730; 15 kb) are shown along with mm9 RefSeq data. (E) Mitochondrial ChIP-qPCR with primers specific for the D-loop region (15,752–15,903 bp) of the mitochondrial genome in Raw 264.7 cells stimulated with LPS for 24 hours. Values are fold enrichments relative to control or LPS IgG and represent 1 pooled mtDNA sample from 10 × 10 cm plates of control cells and 2 pooled mtDNA samples from 10 × 10 cm plates per each LPS-treated cell.