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MTORC2 Deficiency Alters the Metabolic Profile of Conventional Dendritic Cells.

ABSTRACT: In myeloid dendritic cells (DC), deletion of the mechanistic target of rapamycin complex 2 (TORC2) results in an augmented pro-inflammatory phenotype and T cell stimulatory activity; however, the underlying mechanism has not been resolved. Here, we demonstrate that mouse bone marrow-derived TORC2-deficient myeloid DC (TORC2^-/- DC) utilize an altered metabolic program, characterized by enhanced baseline glycolytic function compared to wild-type WT control (Ctrl) DC, increased dependence on glycolytic ATP production, elevated lipid content and higher viability following stimulation with LPS. In addition, TORC2^-/- DC display an increased spare respiratory capacity (SRC) compared to WT Ctrl DC; this metabolic phenotype corresponds with increased mitochondrial mass and mean mitochondrial DNA copy number, and failure of TORC2^-/- DC mitochondria to depolarize following LPS stimulation. Our data suggest that the enhanced metabolic activity of TORC2^-/- DC may be due to compensatory TORC1 pathway activity, namely increased expression of multiple genes upstream of Akt/TORC1 activity, including the integrin alpha IIb, protein tyrosine kinase 2/focal adhesion kinase, IL-7R and Janus kinase 1(JAK1), and the activation of downstream targets of TORC1, including p70S6K, eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) and CD36 (fatty acid translocase). These enhanced TORC1 pathway activities may culminate in increased expression of the nuclear receptor peroxisome proliferator-activated receptor γ (Pparγ) that regulates fatty acid storage, and the transcription factor sterol regulatory element-binding transcription factor 1 (Srebf1). Taken together, our data suggest that TORC2 may function to restrain TORC1-driven metabolic activity and mitochondrial regulation in myeloid DC.

SUBMITTER: Watson AR

PROVIDER: S-EPMC6626913 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Recent advances reveal that metabolic reprogramming is required for adequate antiviral responses of dendritic cells (DCs) that possess the capacity to initiate innate and adaptive immune responses. Several reports indicate that Toll-like receptor (TLR) stimulation of DCs is accompanied by a rapid induction of glycolysis; however, the metabolic requirements of retinoic-acid inducible gene I (RIG-I)-like receptor (RLR) activation have not defined either in conventional DCs (cDCs) or in plasmacytoid DCs (pDCs) that are the major producers of type I interferons (IFN) upon viral infections. To sense viruses and trigger an early type I IFN response, pDCs rely on endosomal TLRs, whereas cDCs employ cytosolic RIG-I, which is constitutively present in their cytoplasm. We previously found that RIG-I is upregulated in pDCs upon endosomal TLR activation and contributes to the late phase of type I IFN responses. Here we report that TLR9-driven activation of human pDCs leads to a metabolic transition to glycolysis supporting the production of type I IFNs, whereas RIG-I-mediated antiviral responses of pDCs do not require glycolysis and rather rely on oxidative phosphorylation (OXPHOS) activity. In particular, TLR9-activated pDCs show increased extracellular acidification rate (ECAR), lactate production, and upregulation of key glycolytic genes indicating an elevation in glycolytic flux. Furthermore, administration of 2-deoxy-D-glucose (2-DG), an inhibitor of glycolysis, significantly impairs the TLR9-induced secretion of type I IFNs by human pDCs. In contrast, RIG-I stimulation of pDCs does not result in any alterations of ECAR, and type I IFN production is not inhibited but rather promoted by 2-DG treatment. Moreover, pDCs activated via TLR9 but not RIG-I in the presence of 2-DG are impaired in their capacity to prime allogeneic naïve CD8+ T cell proliferation. Interestingly, human monocyte-derived DCs (moDC) triggered via RIG-I show a commitment to glycolysis to promote type I IFN production and T cell priming in contrast to pDCs. Our findings reveal for the first time, that pDCs display a unique metabolic profile; TLR9-driven but not RIG-I-mediated activation of pDCs requires glycolytic reprogramming. Nevertheless, the metabolic signature of RIG-I-stimulated moDCs is characterized by glycolysis suggesting that RIG-I-induced metabolic alterations are rather cell type-specific and not receptor-specific.

Project description:Adhesion G Protein-Coupled Receptor L4 (ADGRL4/ELTD1) is an endothelial cell adhesion G protein-coupled receptor (aGPCR) which regulates physiological and tumour angiogenesis, providing an attractive target for anti-cancer therapeutics. To date, ADGRL4/ELTD1's full role and mechanism of function within endothelial biology remains unknown, as do its ligand(s). In this study, ADGRL4/ELTD1 silencing, using two independent small interfering RNAs (siRNAs), was performed in human umbilical vein endothelial cells (HUVECS) followed by transcriptional profiling, target gene validation, and metabolomics using liquid chromatography-mass spectrometry in order to better characterise ADGRL4/ELTD1's role in endothelial cell biology. We show that ADGRL4/ELTD1 silencing induced expression of the cytoplasmic metabolic regulator ATP Citrate Lyase (ACLY) and the mitochondria-to-cytoplasm citrate transporter Solute Carrier Family 25 Member 1 (SLC25A1) but had no apparent effect on pathways downstream of ACLY (fatty acid and cholesterol synthesis or acetylation). Silencing induced KIT expression and affected the Notch signalling pathway, upregulating Delta Like Canonical Notch Ligand 4 (DLL4) and suppressing Jagged Canonical Notch Ligand 1 (JAG1) and Hes Family BHLH Transcription Factor 2 (HES2). The effect of ADGRL4/ELTD1 silencing on the cellular metabolic profile was modest but several metabolites were significantly affected. Cis-aconitic acid, uridine diphosphate (UDP)-glucoronate, fructose 2,6-diphosphate, uridine 5-diphosphate, and aspartic acid were all elevated as a result of silencing and phosphocreatine, N-acetylglutamic acid, taurine, deoxyadenosine triphosphate, and cytidine monophosphate were depleted. Metabolic pathway analysis implicated ADGRL4/ELTD1 in pyrimidine, amino acid, and sugar metabolism. In summary, this study shows that ADGRL4/ELTD1 impacts core components of endothelial metabolism and regulates genes involved in endothelial differentiation/homeostasis and Notch signalling.

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MTORC2 Deficiency Alters the Metabolic Profile of Conventional Dendritic Cells.

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