Mapping macrophage polarization over the myocardial infarction time continuum

Fig. 1. Distinct time-dependent gene expression profiles in post-MI macrophages. a Of 23,847 genes sequenced, 7152 did not meet quality control standards and were removed. Of the remaining 16,695 genes, 8109 were differentially expressed (DE) by one-way ANOVA (FDR adjusted p value < 0.05). b Macrophages from different post-MI days analyzed by principal component analysis. Day 1 macrophages were most distinct from the other times. Day 3 and 7 macrophages were distinct from day 0 and day 1 and showed overlap. c One-way ANOVA plot showing significant genes in red and d heat map of all differentially expressed genes. e Fold change analysis of differentially expressed genes at each day post-MI (fold change threshold of 2, FDR adjusted p value < 0.05) and Venn diagrams of upregulated and downregulated genes showing distinction and overlap in gene expression among the times

Fig. 2. Gene expression pattern clustering. a Markov clustering analysis generated three distinct major clusters representing different post-MI gene expression patterns. Each node represents a single gene, and genes within the same cluster (color) show similar gene expression patterns. Genes representing each cluster are highlighted in red. b Enrichment analysis and expression patterns for each cluster. Cluster 1 (yellow) contained 565 genes increased at day 1 and was enriched for pro-inflammatory processes. Cluster 2 genes (blue, 1965 genes) were increased at day 3 and 7 and were enriched for translation processes. Cluster 3 (green, 1222 genes) were increased at day 7 and enriched for ECM processes. *p < 0.05 versus day 0, #p < 0.05 versus day 1, $p < 0.05 versus day 3

Fig. 3. Day 1 post-MI macrophages showed a pro-inflammatory profile. a Volcano plot with all values normalized to day 0 no MI controls and representative genes highlighted (left). Enrichment analysis of upregulated and downregulated genes (right). b Flow cytometry analysis of macrophage cell surface markers. At day 1 post-MI, total myeloid cells, neutrophils, and monocytes significantly increased in the infarct region, while resident macrophages decreased. c Top five upregulated day 1 post-MI genes ranked by fold change (left) and p value (right). *p < 0.05 versus day 0

Fig. 4. Post-MI macrophage signaling profiles. a Principal component analysis of genes involved in ubiquitous signaling pathways indicates that day 1 macrophages display a unique signaling profile compared to days 0, 3, and 7. b Heat maps grouped by significance (left, non-significant genes (p > 0.05); right, significant genes (p < 0.05) by unpaired two-tailed t test)

Fig. 5. Day 3 post-MI macrophages showed a phagocytic, proliferative, and metabolic reprogramming profile. a Volcano plot with representative genes highlighted and enrichment analysis of upregulated and downregulated genes. All values are normalized to day 0 no MI controls and representative genes are highlighted. b Representative images of phagocytic (top) and proliferative (bottom) macrophages. Phagocytic capacity significantly increased at day 3 post-MI, whereas proliferation was similar to day 0. c In vivo turnover. At day 3 post-MI, ~ 5% of macrophages were remaining from day 1. d Top five upregulated day1 genes ranked by fold change (left) and p value (right). *p < 0.05 versus day 0

Fig. 6. Day 7 post-MI macrophages showed a pro-reparative profile. a Volcano plot with representative genes highlighted and enrichment analysis of upregulated and downregulated genes. All values are normalized to day 0 no MI controls and representative genes are highlighted. b ECM genes that clustered together. Fold change values for genes upregulated at day 7 compared to day 1 and 3 post-MI (top) or remained downregulated at day 7 (bottom). c Top five upregulated day 7 genes ranked by fold change (left) and p value (right). *p < 0.05 versus day 0, #p < 0.05 versus day 1, $p < 0.05 versus day 3

Fig. 7. Macrophage expression of Col1a1 and Postn in LV infarct region. Numbers of cells expressing Postn and Col1a1 mRNA in LV infarct tissue at day 7 post-MI were determined by in situ hybridization. Both Emr1 (a; red, Cy5, macrophages) and Acta2 (b; green, Cy5, fibroblasts) positive cells were assessed for expression of Col1a1 (yellow, Cy3) and Postn (magenta, FITC). Emr1 and Ccr2 positive cells (c; yellow, Cy3) were assessed for expression of Postn (magenta, FITC). d Pie charts displaying proportion of LV infarct Emr1 + (macrophages) and Acta2 + (fibroblasts) cells expressing Col1a1 and Postn. Nuclei are stained with DAPI (cyan)

Fig. 8. Map of macrophage polarization over the MI time continuum. Post-MI macrophage profiles in terms of inputs, signaling, outputs, and cell physiology over the three major post-MI remodeling phases (inflammation, proliferation, and scar maturation). DAMPs damage-associated molecular patterns, ECM extracellular matrix