Fig 2: Polarization of macrophages (Mf). Mouse immortalized bone marrow-derived Mf were treated with IFN-?/LPS or IL-4 for 18 h. (a) Representative flow cytometry images of the polarized subsets gated for the surface expression of CD11b and F4/80 (Mf markers, a) and then screened for increase in the expression of CD64 (b) and TNF-a (d) as the markers of classical/proinflammatory Mf and for CD206 (c) and arginase I (e) as the markers of alternative/anti-inflammatory Mf are shown. (b–e) Bar graphs show the median fluorescence intensity for the expression of CD64 (b), CD206 (c), TNF-a (d), and arginase I (e) in control (untreated) and IFN-?/LPS- or IL-4-polarized Mf after 18 h of incubation (n = 4-6 biological replicates per group per observation). (f) Cell supernatants were used to measure the release of nitric oxide by the Griess assay (n = 6 biological replicates per group, triplicate observations per sample). Data in bar graphs are presented as mean value ± SD. Data were analyzed by using InStat version 3 (GraphPad, La Jolla, CA) and analyzed by the Student t test (for comparison of 2 groups) or one-way analysis of variance (ANOVA) with Tukey's post hoc test (for comparison of multiple groups). The significance was accepted at p value = 0.05 (nontreated vs. treated).

Fig 3: Distinct Gene Expression Profiles in Ly6Chi and Ly6Clo MDM Subsets Differentiated In vitro in the Presence of Untreated or IL-17A-Treated Cardiac Fibroblasts(A) PCA analysis of microarray experiments. The principal components and their fraction of overall variability of the data (%) are shown on the x, y, and z axes.(B) Supervised hierarchical clustering highlighting differential gene expression profiles among three groups of in vitro MDMs, using a threshold of 2-fold change and p value < 0.05. Sample number scheme is identical to the legend in all heatmaps below.(C–F) Heatmaps showing relative fold changes in (C) genes associated with cytokines and growth factors, (D) NF?B pathway and antigen presentation, (E) chemokines and immune modulating activities, and (F) collagen production and matrix remodeling.(G) Gating of MHCII+CD64+ macrophages derived from monocyte-fibroblast co-culture. Frequencies of MHCII+ subset out of total in vitro co-culture MDMs were assessed by flow cytometry.(H) Frequencies of MHCII+ out of F4/80hiCD64+ macrophages were assessed by flow cytometry of the hearts of day 28 EAM mice.(B–F) All genes displayed on the heatmaps have one-way ANOVA p value < 0.05 among groups compared. Data are representative of three independent experiments with technical triplicates. (G) n = 3. (H) n = 8 – 9. (G) Groups were compared using one-way ANOVA followed by Dunnett test. **p < 0.01. (H) Groups were compared using Student’s t test. **p < 0.01. All data were presented as mean ± SD.See also Figure S6.

Fig 4: Infiltrating Monocytes Contribute to Three Subsets of Macrophages during Myocarditis(A) Schematics of parabiosis mice.(B) Representative images of H&E-stained heart sections of the median CD45.1 (day 21 EAM) and CD45.2 (non-EAM) mice parabionts. Scale bars: 100 µm.(C) Comparison of total grafted CD11b+Ly6G-myeloid cell counts between parabionts.(D) Comparison of total cardiac macrophage counts between parabionts.(E) Flow cytometry plots showing (top left) percentage of CD45.2+CD11b+Ly6G-Lineage (CD3e, B220, NKp46, CD90.2, and Ter119)- grafted cells infiltrating the CD45.1 EAM hearts; (top middle) percentage of grafted cells in the hearts that differentiated into macrophages or remained as monocytes; (top right) percentage of grafted MDM subsets; (bottom left) percentage of CD45.1+CD11b+Ly6G-Lineage grafted cells infiltrating the CD45.2 non-EAM hearts; (bottom middle) percentage of grafted cells in the hearts that differentiated into macrophages or remained as monocytes; and (bottom right) percentage of grafted MDM subsets.(F) Percentages of grafted MDMs out of total number of grafted CD11b+Ly6G-Lineage- myeloid cells.(G) Percentages of grafted Ly6Chi and Ly6Clo cells out of total number of grafted F4/80-CD64+ monocytes.(H) Comparison of grafted MDM subsets defined by CCR2 and MHCII expressions between parabionts. Data are representative of two independent experiments with biological triplicates. n = 3.(C, D, F, G) Groups were compared using Student’s t test. *p < 0.05. All data were presented as mean ± SD.See also Figure S1.

Fig 5: Loss of ZEB2 Affects Mac Phenotype and/or Number across Tissues(A) Expression of CD64, F4/80 and CD11b by live CD45+Ly6G-CD64+Ly6C-MHCII- SMs in Fcgr1-crexZeb2fl/fl and Zeb2fl/fl mice. SMs as a percentage of total live CD45+ cells, absolute number and percentage of CD11b+ and CD11b- SMs in Fcgr1-crexZeb2fl/fl or Zeb2fl/fl mice.(B) Expression of CD64, F4/80, CD11c, and CD11b by live CD45int microglia in Fcgr1-crexZeb2fl/fl and Zeb2fl/fl mice. Microglia as a percentage of total live CD45+ cells, absolute number and percentage of CD11c+ and CD11c- microglia in Fcgr1-crexZeb2fl/fl or Zeb2fl/fl mice.(C) Expression of Ly6C and MHCII (monocyte waterfall) by live CD45+CD11b+Ly6G-SiglecF- non cDCs in Fcgr1-crexZeb2fl/fl and Zeb2fl/fl mice. Percentage of live CD45+ and absolute number of Ly6C+MHCII-, Ly6C+MHCII+, and Ly6C- Macs in Fcgr1-crexZeb2fl/fl or Zeb2fl/fl mice. Data are pooled from two experiments with n = 8–11 per group. *p < 0.05, **p < 0.01, ***p < 0.001 Student’s t test. See also Figure S5.