Fig. 2. IL-10 prevents accumulation of dysfunctional mitochondria and production of mitochondrial ROS via the induction of mitophagy.

(A to E) WT or Il10^−/− BMDMs stimulated without (control) or with LPS in the presence or absence of IL-10 for 24 hours. Total mitochondrial mass was analyzed by flow cytometry in cells labeled with MitoTracker Green (A). Mitochondrial membrane potential (Δψ_m) and ROS were analyzed in cells labeled with MitoTracker Green and MitoTracker Red (B), or with MitoSOX (C) and (D), respectively. Representative microscopic images show mitochondrial ROS production in Il10^−/− BMDMs labeled with MitoTracker Green and MitoSOX (E). Scale bars, 10 μm. (F and G) Quantification of LC3-GFP punctate formation in IL-10–sufficient (Il10^+/−) or –deficient (Il10^−/−) LC3-GFP tg BMDMs stimulated as in (A) or with rapamycin for 6 hours. Representative microscopic images show LC3-GFP punctate in higher magnification of the indicated area (box) (F). Quantification based on counting LC3-GFP punctate per cell in the field of view (G). (H) OCR in BMDMs generated from Atg5^flox/flox (Atg5^WT) or Atg5^flox/flox LysM-Cre (Atg5^M–KO) mice and stimulated as in (A) for the indicated times. All values are means ± SD of at least three independent experiments. Student’s t test (unpaired); *P < 0.05, **P < 0.01.