Abstract

Macrophages promote both injury and repair after myocardial infarction, but discriminating functions within mixed populations remains challenging. Here we used fate mapping, parabiosis and single-cell transcriptomics to demonstrate that at steady state, TIMD4+LYVE1+MHC-IIloCCR2 resident cardiac macrophages self-renew with negligible blood monocyte input. Monocytes partially replaced resident TIMD4LYVE1MHC-IIhiCCR2 macrophages and fully replaced TIMD4LYVE1MHC-IIhiCCR2+ macrophages, revealing a hierarchy of monocyte contribution to functionally distinct macrophage subsets. Ischemic injury reduced TIMD4+ and TIMD4 resident macrophage abundance, whereas CCR2+ monocyte-derived macrophages adopted multiple cell fates within infarcted tissue, including those nearly indistinguishable from resident macrophages. Recruited macrophages did not express TIMD4, highlighting the ability of TIMD4 to track a subset of resident macrophages in the absence of fate mapping. Despite this similarity, inducible depletion of resident macrophages using a Cx3cr1-based system led to impaired cardiac function and promoted adverse remodeling primarily within the peri-infarct zone, revealing a nonredundant, cardioprotective role of resident cardiac macrophages.

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Data availability

Single-cell sequence data that support the findings of this study are available through the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) (GSE119355). The Agilent gene array data that support the findings of this study are available through the NCBI GEO (GSE119515).

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Acknowledgements

This work was supported by the Canadian Institutes of Health Research (S.E., PJT364831; J.A.M. and S.A.D.), Heart and Stroke Foundation (S.E.), March of Dimes (S.E.), Ted Rogers Centre for Heart Research (S.E., S.A.D. and J.A.M.), the Peter Munk Cardiac Centre (S.E.) and the National Institutes of Health (S.E. K08HL112826). We thank D. Mann for his insight and advice, and S. Wilson for editorial assistance. Thanks to N. Winegarden and N. Khuu for help with processing single-cell RNA sequencing samples.

Author information

Author notes

  1. These authors contributed equally: Sarah A. Dick, Jillian A. Macklin

Affiliations

  1. Toronto General Hospital Research Institute, University Health Network (UHN), Toronto, Canada

    • Sarah A. Dick
    • , Jillian A. Macklin
    • , Sara Nejat
    • , Abdul Momen
    • , Xavier Clemente-Casares
    • , Marwan G. Althagafi
    • , Crystal Kantores
    • , Siyavash Hosseinzadeh
    • , Laura Aronoff
    • , Anthony Wong
    • , Rysa Zaman
    • , Iulia Barbu
    • , Rickvinder Besla
    • , Mansoor Husain
    • , Myron I. Cybulsky
    • , Clinton S. Robbins
    •  & Slava Epelman
  2. Ted Rogers Centre for Heart Research, Toronto, Canada

    • Sarah A. Dick
    • , Jillian A. Macklin
    • , Mansoor Husain
    •  & Slava Epelman
  3. Department of Medicine, University of Toronto , Toronto, Canada

    • Jillian A. Macklin
    • , Mansoor Husain
    •  & Slava Epelman
  4. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

    • Jillian A. Macklin
    • , Marwan G. Althagafi
    • , Siyavash Hosseinzadeh
    • , Laura Aronoff
    • , Rickvinder Besla
    • , Mansoor Husain
    • , Myron I. Cybulsky
    • , Clinton S. Robbins
    •  & Slava Epelman
  5. Singapore Immunology Network(SIgN), Agency for Science Technology and Research (A*STAR), Singapore, Singapore

    • Jinmiao Chen
    •  & Florent Ginhoux
  6. Shanghai Institute of Immunology, Shanghai JiaoTong University School of Medicine , Shanghai, China

    • Jinmiao Chen
    •  & Florent Ginhoux
  7. Department of Immunology, University of Toronto, Toronto, Canada

    • Anthony Wong
    • , Rysa Zaman
    • , Iulia Barbu
    • , Clinton S. Robbins
    •  & Slava Epelman
  8. Division of Cardiology, Washington University School of Medicine, St Louis, MO, USA

    • Kory J. Lavine
    •  & Babak Razani
  9. John Cochran VA Medical Center, St Louis, MO, USA

    • Babak Razani
  10. Peter Munk Cardiac Centre, Toronto, Canada

    • Mansoor Husain
    • , Myron I. Cybulsky
    • , Clinton S. Robbins
    •  & Slava Epelman

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Contributions

S.A.D. and J.A.M. designed and performed experiments with the help of X.C-C., S.H., C.K., M.G.A., A.W., L.A., R.Z., R.B. and I.B. A.M. performed all surgeries. M.H., K.J.L., B.R., F.G., M.I.C. and C.S.R. provided expertise and feedback. S.N. performed the bioinformatics analyses. J.C. performed the Mpath analysis. S.E. conceived the study, obtained funding and wrote the manuscript with S.A.D., S.N. and J.A.M.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Slava Epelman.

Integrated supplementary information

  1. Supplementary Figure 1 Gene-expression analysis of cardiac macrophage clusters defined by single-cell RNA sequencing reveals five distinct populations.

    a) Cardiac mononuclear phagocytes were sorted from adult c57bl/6j mice (~20 weeks old). Total macrophages (MFs) (R2; CD45+CD64lo–hiCD11b+) and total dendritic cells (DCs) (R3; CD45+CD64CD11c+MHC-IIint–hi). The two sorted populations were pooled in a 1:1 ratio before processing for scRNA-seq on the 10x Genomics Chromium platform. N=8 (4 male; 4 female); one experiment. b) The top ~65 differentially expressed genes in each cluster were input into the ImmGEN online portal for "My Geneset" http://rstats.immgen.org/MyGeneSet_New/index.html to generate a similarity score. We included all MF, monocyte and dendritic cell populations in the V1 array database for the assessment to generate a means-normalized expression scatter plot. Colored boxes highlight similarity in dendritic cell (light green), macrophage (dark green) and monocyte (red) populations to the sorted populations contributed to ImmGen. c) Violin plots of cluster defining genes in the 11 clusters defined in Fig. 1c. *p<0.01 (adjusted P value). Statistical analysis of scRNA-seq is defined in the material and methods, and was performed using the MAST method.

  2. Supplementary Figure 2 Cx3cr1CreER:R26Td mice retain the ability to distinguish resident macrophages from recruited macrophages following irradiation and adoptive transplant.

    a) Cx3cr1CreER–YFP:R26Td mice were used to determine Td reporter expression in cardiac macrophages (CD45+CD64+CD11b+), brain microglia (CD45lowCD64+CD11b+), liver Kupffer cells (CD45+CD64+CD11b+) and blood monocytes (CD115+CD11b+) in the absence of tamoxifen administration (No TAM) at the indicated times (from E17.5 to 77 days post-natal). Recombination in tissue macrophages without TAM was compared to pregnant mice that were given a TAM pulse at E18.5 in utero (via i.p. injection of 2mg TAM). N=3; center value, mean; error bars, SEM; one representative experiment, repeated twice. b) A second fate-mapping system was also used (Cx3cr1CreERT2)30 and bred to the same reporter R26Td line to assess for Td expression in cardiac macrophages (heart) and microglia (brain) at the indicated times either without TAM (No TAM) or following TAM administration at E18.5 (+TAM). N=3; center value, mean; one experiment. c) Bone marrow from adult Cx3cr1CreER–YFP:R26Td mice (Donor BM, CD45.2), not previously pulsed with tamoxifen, was isolated and the %Td+ macrophages was determined in blood CD115+CD11b+ monocytes and total CD45+ bone marrow. Donor Bone marrow was transplanted into non-lethal irradiated CD45.1 mice (Recipients). After 6 weeks, chimerism rates in the recipient were ~95%. Blood and cardiac tissue was isolated from the CD45.1 recipient. A subset of recipient mice was given a pulse of tamoxifen (TAM) (i.p. 2mg) 2 days prior to isolation to confirm the responsiveness of chimeric CD45.2 cells in the recipient compared to without TAM administration (No TAM). The %Td+ cells was determined in total CD45.2+ blood monocytes (CD115+CD11b+) and total CD45.2+ cardiac macrophages (MFs) (CD64+CD11b+). N=3,2; center value, mean; error bars, SEM; one experiment.

  3. Supplementary Figure 3 Tracking resident cardiac macrophages post myocardial infarction.

    a-f) 3-week-old Cx3cr1CreER–YFP:R26Td mice were fed tamoxifen (TAM)- containing chow for 10 days, and then mice were either left as non-infarcted controls for an additional 6 weeks before a myocardial infarction was performed (MI). a) Ischemic tissue was isolated at Day 2 and Day 28 post-MI and total resident cardiac macrophages (CD45+CD64+CD11b+Td+) were analyzed as TIMD4+ and TIMD4 subsets per mg/tissue compared to non-infarcted controls (No MI). N=3; repeated independently 2 times with similar results. b) Total CD45+CD64+CD11b+ cardiac macrophages (MFs) were partitioned into TIMD4+, CCR2+ and TIMD4CCR2 MF subsets. LYVE1 was detected by flow cytometry. N=3; repeated independently 2 times with similar results. c) Representative images of hearts isolated, fixed and sectioned longitudinally for immunofluorescence from Cx3cr1CreER–YFP:R26Td mice post-MI. Td expression was induced as in panel (a) above. Cardiac macrophages (CD68+, white) were counted based on their coexpression for the residency marker Td (red) and LYVE1 (green) in the infarct and peri-infarct regions, defined in Fig. 4a, from 3 individual images/region/mouse. N=3; one experiment. d) Hearts were isolated from the ischemic zone of Cx3cr1CreER–YFP:R26Td/DTR mice day 4 post-MI after a 2 hr-BrdU pulse. Total Td+ cardiac macrophages were subdivided into TIMD4+ and TIMD4 MF populations and %BrdU+ macrophages was quantified. N=3,3,4,4; one experiment. e) Heat map of the 20 most differentially expressed genes from clusters from Fig. 5b. Overlapping clusters (#1 to #6a), represent macrophages from control and post-MI samples that clustered together. For the overlapping clusters both the control and MI cells from each cluster are aligned next to each other in the heatmap. f) Means-normalized expression scatter plot produced by comparison of the differentially expressed genes from each unique cluster (7, 8, 9, 10 and 11) in Fig. 5b to the ImmGen data set. For all: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; two-tailed Student’s T test; center value, mean; error bars, SEM.

  4. Supplementary Figure 4 Chimeric Cx3cr1CreER–YFP:R26Td monocytes retain the ability to distinguish resident macrophages from recruited macrophages post myocardial infarction in the setting of parabiosis and shielded irradiation.

    a) Cx3cr1CreER–YFP:R26Td mice (Donor CD45.2) were fed tamoxifen (TAM)-containing chow at 3 weeks of age for 1 week, TAM was then discontinued for additional 10 weeks. At this time, Cx3cr1CreER–YFP:R26Td mice were paired with recipient CD45.1 mice. Two weeks after pairing, recipient CD45.1 mice were infarcted by surgical ligation of the left anterior descending artery (MI). At day 5 post-MI, blood and cardiac tissue (ischemic zone) was quantified for %Td+ in chimeric CD45.2 macrophages (MFs) (CD45+CD64+CD11b+) and blood monocytes (CD115+CD11b+) in the CD45.1 recipient mouse. N=3. One experiment. b) Bone marrow was isolated from adult Cx3cr1CreER–YFP:R26Td mice (Donor BM, CD45.2) treated as in panel (a). The bone marrow cells were transplanted into non-lethally irradiated CD45.1 mice (Recipient) that had a lead shield covering the heart and thoracic cavity to minimize loss of resident cardiac macrophages. Following 6 weeks of reconstitution, surgical MI was performed on the recipient CD45.1 mouse and it recovered for an additional 4 weeks. Blood and cardiac tissue (separated into ischemic and remote zone, see Fig. 4a) was taken for flow cytometric analysis. A subset of mice was given a pulse of TAM (i.p.) 2 days prior to isolation to confirm the responsiveness of the CD45.2 cells to activate the TAM-inducible Td reporter compared to No TAM controls. Gated on total CD45.2+ blood monocytes (CD115+CD11b+) and total CD45.2+ cardiac macrophages (CD64+CD11b+) in the CD45.1 recipient and %Td+ was quantified. N=3; one experiment. For all: center value, mean; error bars, SEM.

  5. Supplementary Figure 5 Similar cardiac macrophage subsets exist in human cardiomyopathy.

    Left ventricular cardiac tissue was obtained from human patients with end-stage cardiomyopathy during implantation of left ventricular assist devices. a) Tissue was digested and analyzed by flow cytometry [gated similar to mouse (CD45+CD64+MerTK+CD14+)] or b) sorted for Hema-3 staining analysis and visualization. c) Cardiac macrophage (MF) populations were sorted from human tissue based on MHC-II and CCR2 expression. RNA was extracted, amplified, and gene expression was determined by gene array (Agilent). Genes that were most differentially expressed in the 5 Seurat MF clusters identified by scRNA-seq in the mouse (Fig. 1d) were compared to the gene expression across the three human MF subsets isolated to generate heatmaps. N=6 patient samples; one experiment.

  6. Supplementary Figure 6 Resident cardiac macrophages proliferate post myocardial infarction in the remote myocardium, with resident macrophage depletion leading to increased cardiomyocyte hypertrophy without cardiac fibrosis.

    3-week-old Cx3cr1CreER–YFP:R26Td mice were fed tamoxifen (TAM)- containing chow for 10 days, and then mice were either left as non-infarcted controls for an additional 6 weeks. a) Diphtheria toxin (DT) was then administered to Cx3cr1CreER–YFP:R26Td/DTR mice for 2 weeks at steady state. Blood monocytes (CD115+CD11b+Ly6C+), neutrophils (CD45+CD11b+CD64Ly6G+), lymphocytes (CD45+CD11b+CD64), TdCCR2+ cardiac macrophages, and Td+ cardiac macrophages were quantified by flow cytometry, normalized to DT-injected Cx3cr1+/+:R26Td/DTR control mice. N=5; repeated independently 3 times with similar results. b) Representative flow plots of resident macrophage depletion during steady state (Fig. 6a) from blood and cardiac tissue following a myocardial infarction demonstrating effective depletion of resident macrophages. c) Naive wild-type mice were given chronic PBS or 250ng DT injections (i.p.) for 35 days. Whole hearts were either cut longitudinally and sectioned to be stained with Fast Green for interstitial fibrosis, quantified as % Fibrosis using ImageJ software, or quantified by flow cytometry for the number of neutrophils or cardiac macrophages normalized per mg tissue. N=3, 7; one experiment. d) Hearts were isolated from adult TAM-fed Cx3cr1CreER–YFP:R26Td/DTR mice (as above) day 5 post-MI, comparing non-depleted or depleted mice (DT treated daily). Recruited macrophages (MF), monocytes, and neutrophils were quantified and normalized per mg tissue in the ischemic and remote cardiac zones. N=6,5,4; repeated independently 2 times with similar results. e-f) Hearts were isolated from adult Cx3cr1CreER–YFP:R26Td mice following TAM-induction (as above), and mice received BrdU injection 2 hours prior to harvest. Resident MFs (Td+), recruited MFs (Td) and monocytes were normalized per mg tissue in the remote zone at various time points (e), or %BrdU+ was determined (f), compared to an uninfarcted control (No MI). N(Td+)=13, 13, 6, 3, 10. N(Td, mono)= 12, 10, 7, 3, 6; Repeated 3 times for day 2, 2 times for day 4 and 28, 1 time for day 7, 6 times for control with similar results. g) Change in fractional shortening (contractile function) from day 7 to day 28 post-MI in the remote zone comparing mice with or without chronic resident macrophage depletion (Paired T-test) through the use of M-mode echocardiography. N=11; repeated 2 times independently with similar results. h-i) Longitudinally cut hearts from Cx3cr1CreER–YFP:R26Td/DTR mice (as above) isolated day 35 post-MI to compare post-MI control and depleted groups. Cardiomyocyte hypertrophy was detected using Wheat Germ Agglutinin staining in the remote zone. Hearts imaged at 20x magnification and cardiomyocytes measured in ImageJ, 8 images/zone/heart at 2 cutting levels. N=4, 6, 6; repeated once with similar results (h); Cardiac tissue fibrosis was detected using Fast Green staining. Percentage fibrotic area was quantified with ImageJ analysis, 4 images/zone/heart at 2 cutting levels. N = 9; repeated once with similar results. For all: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Two-tailed Student’s T test; center value, mean; error bars, SEM.

Supplementary information

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    Supplementary Figures 1-6 Supplementary Table 1

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  3. Supplementary Table 2

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https://doi.org/10.1038/s41590-018-0272-2