Increased erythropoiesis in LysM-Atg5^−/− mice. (A) Representative example of LysM-Atg5^−/− and WT mouse spleens and ratios (%) of spleen weight to body weight (bw). Data are presented as histograms of the mean and standard error values, with individual measurements (n = 14-15 in each group). (B) Macrophage localization and cell count. Immunofluorescence with a phycoerythrin-conjugated anti-F4/80 antibody for macrophage staining (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue) for nuclear staining; flow cytometric analysis results depicted as dot plot–type histograms (mean values and standard errors; n = 6) showing the F4/80⁺/CD11b⁻ macrophage and CD11b⁺/F4/80⁻ monocyte populations in the spleens of LysM-Atg5^−/− and WT mice (Leica fluorescence microcopy imaging; original magnification, 20×; scale bars, 20 μm; gating strategy shown in supplemental Figure 3A). (C) Spleen (i) and BM (ii) histology and blood cell analysis (iii) with flow cytometric plots showing erythroid lineage cells from LysM-Atg5^−/− and WT mice. (Ci) Hematoxylin and eosin (H&E)–stained sections of spleen tissue (NanoZoomer scan imaging; original magnification, 5×; scale bars, 200 μm; representative from n = 10 per group). The insets in the spleen panels show enlarged images of the boxed regions (original images, 40×; scale bars, 20 μm) showing brown hemosiderin deposits in WT mice and erythroblastic islands consisting of a central nurse macrophage surrounded by a ring of developing RBC precursors in LysM-Atg5^−/− mice. (Cii) H&E-stained bone sections (Leica microcopy imaging; original magnification, 20×; scale bars, 20 μm; representative from n = 3 per group). (Ciii) Peripheral blood smears stained with May-Grunwald-Giemsa stain; arrows indicate hematoxylin blue–stained reticulocytes (NanoZoomer scan imaging; original magnification, 20×; scale bars, 20 μm). Representative flow cytometric plots for spleen, BM, and blood showing erythroid precursors based on CD71 and Ter119 markers used to separate the erythroid progenitors (gating strategy is shown in supplemental Figure 3A). The relative percentages of cells in stages I to IV of erythropoiesis with stage I single-positive CD71⁺ Ter119⁻ early-stage proerythroblasts (region I), stage II double-positive CD71⁺ Ter119⁺ late-stage basophilic erythroblasts (region II), CD71^low Ter119⁺ polychromatic and orthochromatic erythroblasts (regions III and IV), and single-positive CD71⁻ Ter119⁺ CD11b⁺ (region IV) in LysM-Atg5^−/− and WT mice are indicated (representative histograms of flow cytometry from n = 8-10 per group are shown in supplemental Figure 3B). ****P < 10⁻⁴. RP, red pulp; WP, white pulp.