Figure 9.

Activation of TLR/MyD88 signaling induces training in human monocyte-derived macrophages. (A) PBMCs were enriched from human buffy coat samples and monocytes were isolated using immunomagnetic CD14+ positive selection. Monocytes were differentiated for 7d with recombinant human M-CSF (rhM-CSF) resulting in human monocyte-derived macrophages (hMDMs). hMDMs were treated for 24h with MPLA (10 µg/mL), CpG (10 µg/mL), or Poly I:C (100 µg/mL) or left unstimulated (US) as negative controls. Agonist-treated hMDMs were washed, media was replaced, and cells were rested for 3 days prior to assays (i.e. 3 days post-treatment; 3dp). (B) Extracellular acidification rate (ECAR) was measured as an indicator of glycolytic capacity and (C) Peak glycolysis was analyzed. (D) Oxygen Consumption Rate (OCR) was measured and (E) peak mitochondrial respiration were analyzed. (F) Mitochondrial content was assessed by MitoTracker staining. (G) Phagocytic capacity was measured by fluorescence of pHrodo-labeled S. aureus. (H) ROS production was determined by relative dihydrorhodamine 123 (DHR 123) fluorescence. N=6-7/group, *p < 0.05 and **p < 0.01 by ANOVA followed by Dunnett’s post-hoc multiple comparison test. .