Figure 1.

Activation of the autophagic flux in dendritic cells under atherogenic conditions. A, Representative flow chart showing expression of LC3 (microtubule-associated protein 1A/1B-light chain 3)-GFP (green fluorescent protein) in CD11c^high cells in the spleen of Ldlr^−/− (low-density lipoprotein receptor–deficient) mice transplanted with LC3-GFP bone marrow and put under high-fat diet (HFD) for 8 wk. The inset represents the same staining from a wild-type mouse (not expressing the LC3-GFP). B, Quantification of LC3-GFP mean fluorescence intensity (MFI) on CD11b⁺ dendritic cells (DCs) in the spleen of Ldlr^−/− mice transplanted with LC3-GFP bone marrow and left on chow diet (CD) or put under HFD for 8 wk. *P=0.0317 CD11b⁺ DCs chow diet vs CD11b⁺ DCs HFD, Mann-Whitney test. C, Representative histogram of flow cytometry showing the expression of LC3-GFP in CD8α⁺ and CD11b⁺ DC under CD or HFD. D, Flow charts depicting the gating strategy to analyze macrophages (CD45⁺CD11b⁺F4/80⁺) and DCs (CD45⁺F4/80⁻CD11c⁺MHC [major histocompatibility complex] II^high) subsets (CD11b⁺ DCs [CD11b⁺CD103⁻], double negative (DN) DCs [CD11b⁻CD103⁻], CD103⁺ DCs [CD11b⁻CD103⁺]) as well as their expression of LC3-GFP, from the aorta of Ldlr^−/− mice transplanted with LC3-GFP and kept under HFD for 8 wk. A–D, n=5 mice/group. E, Representative confocal images of DCs (CD11c⁺MHCII⁺, white arrows) and LC3 staining in atherosclerotic lesions from the aortic root of Ldlr^−/− mice kept under HFD for 8 wk. F, Representative flow histograms and quantification of the expression of LC3-GFP in splenic DC subsets (CD8α⁺ and CD11b⁺ DCs) of Ldlr^−/− mice transplanted with LC3-GFP bone marrow and put on HFD for 8 wk. Chloroquine (CQ) was injected intraperitoneally 48 and 24 h before the end of the experiment to prevent the degradation of GFP by lysosomes. *P=0.0379 CD11b⁺ DCs HFD vs CD11b⁺ DCs HFD+CQ, Mann-Whitney test. G, Representative flow histograms and quantification of the expression of LC3-GFP in aortic macrophages and DC subsets (CD11b⁺, DN and CD103⁺ DCs) of Ldlr^−/− mice transplanted with LC3-GFP bone marrow and put on HFD for 8 wk. CQ was administered as in F. F and G: n=7 mice/group, *P=0.0216 CD103⁺ DCs HFD vs CD103⁺ DCs HFD+CQ, Mann-Whitney test, data were obtained from 1 experiment. FMO indicates fluorescence minus one control.