Absence of resident cardiac macrophages in Csf1rΔFIRE mice.

(A) Flow cytometry analysis of 3 month-old RankCreRosa26eYFP mice, showing single/CD45+/lin-(CD11c, Ter119, Tcrß, Nk1.1)/CD11b+ cardiac cells, eYFP expression in macrophages (CD64+/F4/80+) and percentage of eYFP+ blood monocytes, microglia and cardiac macrophages (n=3-5 each from an independent experiment). (B) Flow cytometry analysis of 3 month-old Flt3CreRosa26eYFP mice, showing macrophage expression of eYFP and percentage of eYFP+ blood monocytes, microglia and cardiac macrophages (n=4 each from an independent experiment). (C) Representative flow cytometry analysis of cardiac macrophages in control and ΔFIRE mice (D) Quantification of myeloid cells by flow cytometry (CD45+/lin-/CD11b+), macrophages (CD45+/lin-/CD11b+/CD64+/F4/80+), neutrophils (CD45+/lin-/CD11b+/CD64-/F4/80-/Ly6g+) and Ly6chi monocytes (CD45+/lin-/CD11b+/CD64-/F4/80-/Ly6g-/Ly6chi) (n=6 for control and n=5 for ΔFIRE mice, each single experiments). (E) Representative flow cytometry analysis of cardiac macrophages and their expression of eYFP in Csf1rΔFIRE/+RankCreRosa26eYFP and Csf1rΔFIRE/ΔFIRERankCreRosa26eYFP. (F) Representative immunohistological images showing macrophages (CD68+ cells in white and Hoechst in blue) in control and ΔFIRE hearts in 3 month-old mice at baseline conditions (scale bars represent 500µm). (F) Quantification of macrophages by histology ((n=3 for control and ΔFIRE mice). Either Fisher’s LSD test or unpaired t-test were performed and mean ± SD is shown.

Changes in the cardiac immune phenotype in Csf1rΔFIRE mice in baseline conditions.

(A) Experimental setup to analyze cardiac immune cells using scRNA-seq of sorted CD45+/live cells. (B) UMAPs of control and ΔFIRE in baseline conditions (n=3 for control and ΔFIRE) (C) Absolute difference (percentage points) in cluster abundance between control and ΔFIRE. (D) Contribution of EMP-derived (eYfp expressing) macrophages to the different macrophage clusters analysed by scRNA-seq analysis of immune cells harvested from a RankCreRosa26eYFP mouse. (E) Phagocytosis score projected on a UMAP displaying control and ΔFIRE immune cell subsets. Violin and box plots show the computed phagocytosis score combined in all macrophage clusters (n1/n2 represents number of cells from control/ ΔFIRE mice).

Adverse cardiac remodeling in Csf1rΔFIRE mice after I/R injury

(A) Schematic of the sequential analysis of cardiac function, dimensions and viability using positron-emission tomography 6 and 30 days after I/R injury in control and ΔFIRE with (B and C) showing the intraindividual changes in each parameter from d6 to d30 and (D-G) the individual timepoints on d6 and d30 (d6: n=11 for control and ΔFIRE, d30: n=10 for control and n=11 for ΔFIRE). (B and D) left ventricular ejection fraction (LVEF), (C and E) percentage of the viability defect, (F) stroke volume and (F) left-ventricular end-diastolic volume. (H) Representative immunohistological images showing the fibrotic area (WGA+ area) in hearts from control and ΔFIRE mice 30 days after I/R injury. Right panel shows the percentage of fibrotic area in the respective groups (n= 6 for each group). Student’s t-test was performed and mean ± SD is shown.

Recruitment of BM-derived macrophages into infarct zone of Csf1rΔFIRE mice.

(A) Representative immunohistology of hearts from ΔFIRE mice 30 days after I/R injury showing macrophages (CD68+ cells in red and Hoechst in blue) in the infarct, border and remote zone. Right panel shows number of cardiac macrophages in the respective area (n=5 control and n=3 for ΔFIRE). (B) Flow cytometry analysis of Flt3CreRosa26mT/mGmice 2 days after I/R injury, (left) representative flow cytometry showing expression of tomato and GFP in macrophages in the remote and ischemic myocardium and (right) number of tomato+ and GFP+ cardiac macrophages in the respective area (n=3, each individual experiments). (C) Flow cytometry analysis of RankCreRosa26RFP mice 2 days after I/R injury, (left) representative flow cytometry showing expression of RFP in macrophages in the remote and ischemic myocardium and (right) number of RFP- and RFP+ cardiac macrophages in the respective area (n=3, each individual experiments). (D) Histological analysis of Flt3CreRosa26mT/mG mice 30 days after I/R injury in the infarct, border and remote zone, (left) representative immunohistology of the infarct and border zone and (right) number of GFP- and GFP+ cardiac macrophages in the respective areas (n=4). Fishers LSD test was performed for all experiments and mean ± SD is shown.

Transcriptional landscape of resident versus recruited macrophages in I/R injury

(A) Experimental setup to generate non-irradiation BM chimera using CD45.2 Mx1CreMybflox/flox and transplantation of CD45.1 BM. I/R injury was induced 4 weeks after BM-transplantation and CD45.1+ and CD45.2+ macrophages were sorted from the remote and ischemic myocardium 2 days after I/R injury and RNA-sequencing was performed on bulk cells (n=3). (B and C) Volcano plot showing differential gene expression analysis results of recruited CD45.1 vs. resident CD45.2 macrophages in the (B) remote and (C) ischemic zone. (D) Gene ontology enrichment analysis showing specific biological processes enriched in CD45.1 and CD45.2 macrophages in the ischemic and remote zone.

Altered inflammatory patterns and immune cell communication in Csf1rΔFIRE mice.

(A) Experimental setup to analyze transcriptional changes in cardiac immune cells on a single cell level 2 days after I/R injury in ΔFIRE mice. (B) UMAPs of control and ΔFIRE 2 days after I/R injury (n=2 for control and ΔFIRE). (C) Absolute difference (percentage points) in cluster abundance between control and ΔFIRE. (D) Inflammasome score projected on a UMAP displaying control and ΔFIRE immune cell subsets after I/R injury. Violin and box plots show the computed inflammasome score in neutrophil clusters (n1/n2 represents number of cells from control/ ΔFIRE mice). (E) Ligand–receptor interactions of antigen-presenting, Ccr2lo ly6clo and homeostatic macrophages (highlighted) with other immune cell clusters. Shown are the aggregated communication scores (width of interactions) for all cell types. Only communication scores larger than 6 are considered. (F) Number of interactions (with communication score > 6) outgoing from homeostatic, antigen presenting and Ccr2lo macrophages to other immune cell clusters.

Ablation of resident and recruited macrophages severely impacts on cardiac healing after I/R injury

(A) Schematic of the analysis of cardiac function and infarct size in mice treated with PLX5622 7 days prior and 30 days after I/R injury. (B) Number of cardiac macrophages and neutrophils in the remote and ischemic myocardium 2 days after I/R injury in mice fed control chow (n=6) or PLX5622 (n=7). (C) Representative immunohistology of hearts 30 days after I/R injury showing macrophages (CD68+ cells in red and Hoechst in blue) in the infarct, border and remote zone and (D) number of cardiac macrophages in the respective area (n=4 for control chow and n=4 for PLX5622). (E) Left ventricular ejection fraction (LVEF), (F) viability deficit, (G) stroke volume and (H) end-diastolic volume measured using positron-emission tomography 6 and 30 days after I/R injury (n=6 for control chow, n=8 for PLX5622). (I) Representative immunohistological images showing the fibrotic area (WGA+ area) in hearts 30 days after I/R injury from mice fed control chow or PLX5622. Percentage of fibrotic area in the respective groups (n= 4 for each group). Student’s t-test or Fishers LSD test was performed and mean ± SD is shown.