IL-3 attracts monocytes by inducing cardiac macrophage chemokine production. (A) Flow cytometric analysis for IL-3 receptor α subunit (IL-3Rα: CD123)–positive cells in the inflamed heart on day 21. Representative flow dot plots for CD123 as well as the β subunit of the receptor (CD131) are shown. (B) Comparison of Ccl2, Ccl7, and Ccl12 gene expression in different leukocyte subsets sorted from WT myocarditis hearts on day 21. Values were normalized to that of Ly-6C^high monocytes (n = 4 per each population of two independent experiments). (C) Depiction of a lentiviral vector containing the sgRNA targeting IL-3Rα from a U6 promoter (U6) and Cas9 from a short EF1a promoter (EFS) with eGFP from a picornavirus-derived 2A autocleavage site (P2A). (D) Schematic diagram for experimental design. Lentiviral particles were transfected into BM lineage-negative (Lin⁻) cells, which were subsequently transferred into lethally irradiated WT mice, and myocarditis was induced 6 wk later. Il3rα^+/+ (GFP⁻) and Il3rα^−/− (GFP⁺) cardiac macrophages were sorted at the peak of inflammation. (E) Representative flow dot plots to identify GFP⁺ cells in blood after gating on Ly-6C^high monocytes. (F) Phosphorylation of STAT5 after IL-3 stimulation, demonstrating lack of IL-3 receptor signaling in blood GFP⁺ cells. (G) Representative flow dot plots of cardiac macrophages in the inflamed heart to identify GFP⁺ cells. (H) Gene expression of Ccl2, Ccl7, and Ccl12 in sorted IL-3Rα^+/+ (GFP^–) and IL-3Rα^−/− (GFP⁺) cardiac macrophages. (I) mRNA levels of indicated chemokines in the heart tissue of WT and Il3^−/− mice before and 21 d after the first immunization (n = 4–9 per group of two independent experiments). *, P < 0.05; **, P < 0.01. For statistical analysis, a two-tailed Mann-Whitney U test or unpaired t test was applied to compare two groups. Results are shown as mean ± SEM.