Fate-mapping infiltrating monocytes following experimental myocardial infarction revealsdifferentiation trajectories in the infarcted heart.

Figures

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  Figure 1. Recruited monocyte-derived populations persist in the heart after injury. (A) Representative flow cytometry plots demonstrating that a single dose of tamoxifen administered to Ccr2CreERT2Rosa26tdTomato mice results in 80% labeling efficiency in monocytes in the blood, while resident cardiac macrophages are not labeled. (B) Schematic of experimental strategy for monocyte lineage tracing after MI. (C) Bar graphs demonstrating tdTomato labeling of blood monocytes (left) and cardiac monocyte and macrophages. Single inj., single dose labeling; cont, continuous dose labeling. Brown-Forsythe 1-way ANOVA.

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  Figure 2. Recruited monocyte-derived populations are concentrated within the infarcted area. (A) Quantification of tdTomato+ macrophages relative to total CD68-positive macrophages measured by IHC. (B) Representative IHC images. Magnification ×20. Each data point represents an individual animal. Brown-Forsythe 1-way ANOVA.

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  Figure 3. Recruited monocyte-derived populations are transcriptionally diverse. (A) Clustered UMAP projection of combined monocyte lineage tracing single-cell libraries from each time point. (B) Feature plots of calculated z-scores for each cluster. Blue box, list of genes in each z-score. (C) Dot plot of z-scores by cluster.

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  Figure 4. Monocyte-derived populations dynamically shift as a function of time after MI. (A) Gaussian kernel plots indicating cell density at each time point after MI (left) and proportion of cells in each cluster split by time point (right). (B) UMAP plot of Tomato+ cells. (C) Line graph of proportion of tdTomato+ cells in each cluster across time points.

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  Figure 5. Monocyte-derived populations exist independent of cardiac resident macrophages. (A) Schematic of experimental strategy for resident macrophage lineage tracing (left) and FACS gating strategy for constructing tdTomato+ (resident) and tdTomato– (recruited) libraries. (B) Combined UMAP plot of resident and recruited libraries. Red indicates cells derived from cardiac resident macrophages. (C) Heatmap of genes differentially expressed in cells derived from either cardiac resident macrophages or recruited monocytes. (D) Feature plot of tdTomato expression split by library. (E) Violin plot of tdTomato expression split by library.

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  Figure 6. Monocyte-derived populations are conserved between mice and humans. (A) UMAP plot of monocytes, macrophages, and dendritic cells from human donor (n = 2) and heart failure samples (n = 5). (B)Schematic of analysis strategy (left) and dot plot of denoting the expression of genes enriched in each mouse monocyte, macrophage, and dendritic cell population (displayed as a z-score) in corresponding human monocyte, macrophage, and dendritic cell populations. Genes with mouse and human homologues are included in this analysis. (C) UMAP feature plots of each mouse z-score signature in the human object.

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  Figure 7. Monocyte-derived populations are temporally distinct. (A and B) Palantir tSNE projection of combined time points labeled by cluster with superimposed RNA velocity information (A) and split by time point (B). (C) Palantir tSNE projection labeled by pseudotime and differentiation potential. (D) Feature plots of monocyte, macrophage, and dependent cell populations z-scores overlayed on the tSNE projection.

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  Figure 8. Monocyte-derived populations are spatially distinct. Spatial transcriptomic expression plots of monocyte, macrophage and dependent cell populations, and cardiomyocyte z-scores at day 7 after MI. Images were acquired with a 10X objective.

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  Figure 9. Monocyte fate specifications is initiated prior to myocardial infiltration. (A) Experimental strategy for single-cell RNA-seq of intravascular (anti-CD45 BV421+) and extravascular (anti-CD45 BV421–) compartments. Anti-CD45 BV421 was administered via retro-orbital injection 5 minutes prior to collection. (B) Representative FACS plots and gating strategy demonstrating anti-CD45 BV421 antibody staining in cardiac monocytes and macrophages. (C) Clustered UMAP plots of intravascular (BV421+) and extravascular (BV421–) libraries and stacked bar chart showing distribution of monocyte, macrophage, and dendritic cell populations in each compartment. (D) Violin plot of monocyte signature gene expression (expressed as a z-score) in monocytes and macrophages located within the intravascular and extravascular compartments. 2 tailed t test with Welch’s correction.

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  Figure 10. IFN is activated in recruited monocytes during extravasation. (A and B) Representative FACS plots and quantification of GFP+ monocytes and macrophages within the heart and blood of Mx1GFP mice after MI. Intravascular and extravascular cardiac monocytes and macrophages expressing GFP is shown. Two tailed t-test with Welch’s correction. (C and D) Experimental strategy for heterotopic heart transplantation and representative images demonstrating Mx1GFP-positive recruited macrophages (arrowheads) in surface vessels of the WT donor heart. *P < 0.05, **P < 0.01. Scale bars 40 μM (D, left); 10 μm (D, right).

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  Figure 11. Type 1 IFN signaling in monocytes attenuate post-MI remodeling. (A) Experimental strategy for deletion of Ifnar in recruited monocytes and their progeny. (B) Representative long-axis echocardiographic images at end diastole showing enhanced LV remodeling in Ccr2ERT2CreIfnarFl mice compared to controls. (C) Quantification of echocardiography measurements of LV end systolic volume, ejection fraction, and akinetic area 4 weeks after MI. Two tailed t-test with Welch’s correction. (D) Measurement of cardiomyocyte size by WGA staining 4 weeks after MI. Two tailed t-test with Welch’s correction. (E) Measurement of infarct size assessed by fibrosis as a percentage of LV diameter 4 weeks after MI. Two tailed t-test with Welch’s correction. (F) Representative images of WGA staining at the border zone and trichrome staining in control and Ccr2ERT2CreIfnarFl hearts. Scale bar: 100 μM. (G) Spatial transcriptomic expression plots of Irf7 and Irf3.

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  Figure 12. Type 1 IFN–activated macrophages give rise to MHCIIhi macrophages. (A) Experimental strategy for deletion of Ifnar in recruited monocytes and their progeny. (B) UMAP projection and stacked bar chart comparing distribution of cells in control and Ccr2ERT2CreIfnarFl hearts. (C) Representative images and bar chart quantifying MHCII+ macrophages per 20 × field in the infarct of control and Ccr2ERT2CreIfnarFl hearts. 2 tailed t test with Welch’s correction. Scale bar: 100 μM. (D and E) FACS plots of elicited peritoneal macrophages isolated from Mx1GFP mice stimulated with either PBS or IFN-β showing GFP and MHC-II expression (D). Quantification of the percent GFP+ and MHCII+ macrophages (E). 2 tailed t test with Welch’s correction. (F and G) Quantification and representative FACs plots demonstrating Mx1ERT2CreRosa26LSL–tdTomato labeling in circulating monocytes, macrophages in the heart, and percent of tdTomato+ macrophages which were MHCIIhi.