Fig. 3. Induction of DDIT4 by IL-10 inhibits mTOR signaling and maintains mitochondrial fitness.
BMDMs of the indicated strains were stimulated without (control) or with LPS in the presence or absence of IL-10, rapamycin, or antibody against IL-10Rα (anti-IL-10R) for 24 hours or the indicated times. (A and B) Comparison of mTORC1 activation in WT and Il10−/− BMDMs (A) or Stat3flox/flox (Stat3WT) and Stat3flox/flox LysM-Cre (Stat3M–KO) BMDMs (B) was analyzed by Western blotting. (C) Mitochondrial membrane potential (Δψm) was analyzed in Il10−/− BMDMs labeled with MitoTracker Green and MitoTracker Red. (D) Real-time changes in the OCR of Il10−/− BMDMs after treatment with oligomycin, FCCP, and rotenone were assessed by Seahorse assay. MRC, maximal respiratory capacity (double-headed arrow), is shown for rapamycintreated Il10−/− BMDMs at 12 hours. (E) Basal respiration and maximal respiratory piratory capacity of Il10−/− BMDMs, measured by real-time changes in OCR. (F) Heat map showing RNA-seq data, log2 (fold change) of a selected subset of genes encoding negative regulators of mTORC1 activation in Il10−/− BMDMs. (G) Induction of Ddit4 mRNA expression by IL-10 in Il10−/− or Stat3M–KO BMDMs was analyzed by quantitative PCR. Data are expressed as fold change. (H) Induction of DDIT4 protein expression by IL-10 in Il10−/− BMDMs. (I) Comparison of mTORC1 activation in WT and Ddit4−/− BMDMs. (J) ECAR and OCR in WT and Ddit4−/− BMDMs. (K and L) Δψm and mitochondrial ROS were analyzed in WT and Ddit4−/− BMDMs labeled with MitoTracker Green and MitoTracker Red (K) or with MitoSOX (L). All values are means ± SD of at least three independent experiments. Student’s t test (unpaired); **P < 0.05, **P < 0.01. *** P < 0.001.