Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia

Tobias Weinberger; Denise Messerer; Markus Joppich; Max Fischer; Clarisabel Garcia; Konda Kumaraswami; Vanessa Wimmler; Sonja Ablinger; Saskia Räuber; Jiahui Fang; Lulu Liu; Wing Han Liu; Julia Winterhalter; Johannes Lichti; Lukas Tomas; Dena Esfandyari; Guelce Percin; Sandra Martin Salamanca; Andres Hidalgo; Claudia Waskow; Stefan Engelhardt; Andrei Todica; Ralf Zimmer; Clare Pridans; Elisa Gomez-Perdiguero; Christian Schulz

doi:10.7554/eLife.89377.1

Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia

Medical Clinic I., Department of Cardiology, University Hospital, Ludwig Maximilian University, 81377 Munich, Germany
Institute of Surgical Research at the Walter-Brendel-Centre of Experimental Medicine, University Hospital, Ludwig Maximilian University, 81377 Munich, Germany
DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, 80336 Munich, Germany
Institut Pasteur, Unité Macrophages et Développement de l’Immunité, Département de Biologie du Développement et Cellules Souches, Paris, France
LFE Bioinformatik, Department of Informatics, Ludwig Maximilian University, 80333 Munich, Germany
Department of Neurology, Medical Faculty, Heinrich Heine University of Düsseldorf, 40225 Düsseldorf, Germany
Institute of Pharmacology and Toxicology, Technical University Munich, 80802 Munich, Germany
Immunology of Aging, Leibniz-Institute on Aging - Fritz-Lipmann-Institute (FLI), 07745 Jena, Germany
Area of Cell & Developmental Biology, Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain
Vascular Biology and Therapeutics Program and Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA
Faculty of Biological Sciences, Friedrich-Schiller-University, 07737 Jena, Germany
Department of Nuclear Medicine, Ludwig Maximilian University, 81377 Munich, Germany
University of Edinburgh Centre for Inflammation Research, The Queen’s Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom
Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom

https://doi.org/10.7554/eLife.89377.1

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Figures and data

Absence of resident cardiac macrophages in *Csf1r^ΔFIRE* mice.
**(A)** Flow cytometry analysis of 3 month-old *Rank^CreRosa26^eYFP* 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 *Flt3^CreRosa26^eYFP* mice, showing macrophage expression of eYFP and percentage of eYFP⁺ blood monocytes, microglia and cardiac macrophages (n=4 each from an independent experiment). **(C)** Number of myeloid cells (CD45⁺/lin^-/CD11b⁺), macrophages (CD45⁺/lin^-/CD11b⁺/CD64⁺/F4/80⁺), neutrophils (CD45⁺/lin^-/CD11b⁺/CD64^-/F4/80^-/Ly6g⁺) and Ly6c^hi monocytes (CD45⁺/lin^-/CD11b⁺/CD64^-/F4/80^-/Ly6g-/Ly6c^hi) (*n=6 for control* and n=5 for *ΔFIRE* mice, each single experiments). **(D)** 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). **(E)** Representative flow cytometry analysis of cardiac macrophages and **(F)** their expression of eYFP in *Csf1r^ΔFIRE/+Rank^CreRosa26^eYFP and Csf1r^{ΔFIRE/ΔFIRE}Rank^CreRosa26^eYFP*. Either Fisher’s LSD test or unpaired t-test were performed and mean ± SD is shown.

Changes in the cardiac immune phenotype in *Csf1r^ΔFIRE* 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 *Rank^CreRosa26^eYFP* 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 *Csf1r^ΔFIRE* 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 *Csf1r^ΔFIRE* 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 *Flt3^CreRosa26^mT/mG*mice 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 *Rank^CreRosa26^RFP* 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 *Flt3^CreRosa26^mT/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 Mx1^CreMyb^flox/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. (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 *Csf1r^ΔFIRE* 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, Ccr2^lo ly6c^lo 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 Ccr2^lo 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.

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Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia

Figures and data

Absence of resident cardiac macrophages in Csf1r^ΔFIRE mice.

Changes in the cardiac immune phenotype in Csf1r^ΔFIRE mice in baseline conditions.

Adverse cardiac remodeling in Csf1r^ΔFIRE mice after I/R injury

Recruitment of BM-derived macrophages into infarct zone of Csf1r^ΔFIRE mice.

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

Altered inflammatory patterns and immune cell communication in Csf1r^ΔFIRE mice.

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

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Figures and data

Absence of resident cardiac macrophages in Csf1rΔFIRE mice.

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

Adverse cardiac remodeling in Csf1rΔFIRE mice after I/R injury

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

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

Altered inflammatory patterns and immune cell communication in Csf1rΔFIRE mice.

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

Absence of resident cardiac macrophages in Csf1r^ΔFIRE mice.

Changes in the cardiac immune phenotype in Csf1r^ΔFIRE mice in baseline conditions.

Adverse cardiac remodeling in Csf1r^ΔFIRE mice after I/R injury

Recruitment of BM-derived macrophages into infarct zone of Csf1r^ΔFIRE mice.

Altered inflammatory patterns and immune cell communication in Csf1r^ΔFIRE mice.