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.
Highlights
The mechanistic target of rapamycin is an integrative serine/threonine kinase in the PI3K family
Glycolytic capacity was increased significantly in non-stimulated target of rapamycin complex 2 (TORC2)−/− dendritic cells (DC) compared to Ctrl DC, and in LPS-stimulated TORC2−/− DC compared to Ctrl DC
While there was no significant difference in adenosine triphosphate (ATP) production between Ctrl and TORC2−/− DC following inhibition of OXPHOS with oligomycin, Ctrl and TORC2−/− DC displayed significantly decreased ATP production when glycolysis was inhibited with 2-DG
Summary
The mechanistic target of rapamycin (mTOR) is an integrative serine/threonine kinase in the PI3K family. In response to environmental cues, mTOR regulates cell growth/proliferation, and metabolism [1, 2], and immune cell function [3, 4]. Assembled mTORC1 phosphorylates and activates the translational proteins ribosomal S6 kinase β-1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), and regulates cellular processes in a nutrient-dependent fashion [7]. The function of mTORC1 in dendritic cells (DC) has been studied extensively using the immunosuppressive prodrug rapamycin (RAPA) [8,9,10]. While little had been known previously about the function of RAPA-insensitive mTORC2 (referred to subsequently as TORC2) in DC, we have shown recently that functional TORC2 deletion in these antigen-presenting cells (APC) leads to both an enhanced proinflammatory DC phenotype and Th1/Th17 allogeneic T cell polarization and proliferation [13]. The mechanisms underlying these enhanced DC functions remain undefined
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