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 3

NOTCH reprograms M1 Macs to glucose mitochondrial oxidation through upregulation of PDP1.

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NOTCH reprograms M1 Macs to glucose mitochondrial oxidation through upre...
(A) Increased glucose uptake and lactate production by M1 Hmacs is attenuated with DAPT (n = 6). *P < 0.001, #P < 0.05, 1-way ANOVA. (B) Primary HMacs were cultured with 99.9% [U-13C6]-glucose (1 g/l), with or without DAPT. Percentage glucose flux to the TCA cycle was determined by mass spectrometry (n = 6). *P < 0.001, #P < 0.05, 1-way ANOVA. (C) Seahorse analysis of OCR in HMacs. ATP synthase inhibitor oligomycin (Oligo), mitochondrial uncoupling agent FCCP, and ETC inhibitors antimycin and rotenone (AR) were given at indicated times (n = 5). *P < 0.05 vs. control or OF+Alc+DAPT, t test. (D) Immunoblot of PDP1, PDK, and total and phospho–PDH-E1α (pSer293) in HMacs isolated from WT and Notch1 KO mice with OF+Alc feeding. ImageJ quantification of the pPDH-E1α/PDH-E1α ratio is shown (n = 3). *P < 0.05, t test. (E) PDH activity in Raw 264.7 cells infected with lentiviral scrambled or Notch1 shRNA, treated with or without LPS. Values are relative activity to the control (n = 6). *P < 0.05 vs. sh-Scr, #P < 0.05 vs. sh-Scr + LPS, 2-way ANOVA. (F) ChIP-qPCR for NICD1 (N1) at the CSL site of Pdp1 promoter in Raw 264.7 cells stimulated with or without LPS for 4 hours. Values are fold enrichments relative to IgG (n = 3). *P < 0.05 vs. control NICD1, t test. (G) Expression of Pdp1 in Raw 264.7 cells infected with scrambled or Notch1 shRNA, stimulated with or without LPS for 24 hours (n = 3). *P < 0.05 vs. scrambled control, #P < 0.05 vs. scrambled LPS, 2-way ANOVA.