Figure 7.

mTOR is a key mediator of TLR/MyD88-induced macrophage metabolic and functional reprogramming. (A) Western blotting of phosphorylated S6 kinase (p-S6) and total S6K (S6) on protein lysates from BMDMs treated for 24h with the TLR agonists (1 µg/mL for MPLA and CpG, 10 µg/mL for Poly I:C) compared to unstimulated negative controls (left). Blots are representative of three repeated experiments which were quantified by densitometry analysis (right). (B-E) BMDMs were treated with 100 nM Rapamycin (R) or vehicle (V) 1h prior to the addition of TLR agonists which remained in culture until washed off 24h later and cells were rested for 3 days prior to assays. (B) Glycolysis stress test was performed using Seahorse Xfe96 which measured extracellular acidification rate (ECAR) for vehicle-treated (left) and rapamycin-treated (middle) BMDMs, and peak glycolysis was analyzed (right). (C) Mitochondrial stress test was performed under the same experimental parameters as glycolysis stress test, and oxygen consumption rate (OCR) is presented for vehicle-treated (left), rapamycin-treated (middle), and peak respiration was measured (right). (D) Mitochondrial content was assessed using MitoTracker Green staining. (E) Phagocytic capacity was indicated by fluorescence emitted upon phagocytosis of pHrodo-tagged S. aureus bioparticles and analyzed at peak activity. Data are shown as mean ± SEM, n = 2-3/group *p < 0.05, **p < 0.01, ***p < 0.001 by ANOVA followed by Dunnett’s post-hoc multiple comparison test.