Research Paper Volume 11, Issue 24 pp 11955—11974

Figure 3. Cardiac macrophages had different inflammatory phenotypes in older WT and Plt-β2M^-/- mice. (A) WT and Plt-β2M^-/- mice had equal numbers of cardiac macrophages, but old Plt-β2M^-/- mice had more monocyte derived macrophages. WT old mice had a trend towards increase in monocyte derived macrophages compared to young genotype control. Flow cytometry of single cell suspensions isolated from hearts at 4 and 14 mos old mice (mean ± SEM, **P<0.01, one-way ANOVA with Bonferroni correction). (B) Hearts from Plt-β2M^-/- mice had more M2-like macrophages. Flow cytometry of single cell suspensions isolated from hearts at 4 and 14 mos (mean ± SEM, *P<0.05, one-way ANOVA with Bonferroni correction). Representative gating strategy is shown. (C) Hearts from Plt-β2M^-/- mice had more M2-like macrophages. Immunohistochemistry was performed for Arginase-1. Positive staining was observed by brown staining and quantified as mean gray value using ImageJ. Images were collected at 10x from 7 mice of WT young, WT old and Plt-β2M^-/- young and 9 mice of Plt-β2M^-/- old. Representative images shown at 20x, scale bar 30 μm (mean ± SEM, *P<0.05, 1-way ANOVA with Bonferroni correction). (D) Plt-β2M^-/- mice had greater M2-like macrophage marker gene expression. qRT-PCR for Il10, Chil3, Nos2 were performed (N=3, mean ± SD, *P<0.05, **P<0.01, one-way ANOVA with Bonferroni correction). (E) Old Plt-β2M^-/- monocytes/macrophages in the heart, had increased pSMAD2/3 nuclear localization. ImageStream analysis was performed using single cell heart suspensions. Representative images of monocyte derived macrophages shown. Nuclear localization quantified for overlap of pSMAD2/3 with nuclear DAPI staining. Quantified images were pooled from 3 mice of young genotypes and 4 mice of old genotypes (mean ± SEM, **P<0.01, one-way ANOVA with Bonferroni correction).