Differential contribution of monocytes to heart macrophages in steady-state and after myocardial infarction

Abstract

Rationale: Macrophages populate the steady-state myocardium. Previously, all macrophages were thought to arise from monocytes; however, it emerged that, in several organs, tissue-resident macrophages may self-maintain through local proliferation.

Objective: Our aim was to study the contribution of monocytes to cardiac-resident macrophages in steady state, after macrophage depletion in CD11b(DTR/+) mice and in myocardial infarction.

Methods and results: Using in vivo fate mapping and flow cytometry, we estimated that during steady state the heart macrophage population turns over in ≈1 month. To explore the source of cardiac-resident macrophages, we joined the circulation of mice using parabiosis. After 6 weeks, we observed blood monocyte chimerism of 35.3±3.4%, whereas heart macrophages showed a much lower chimerism of 2.7±0.5% (P<0.01). Macrophages self-renewed locally through proliferation: 2.1±0.3% incorporated bromodeoxyuridine 2 hours after a single injection, and 13.7±1.4% heart macrophages stained positive for the cell cycle marker Ki-67. The cells likely participate in defense against infection, because we found them to ingest fluorescently labeled bacteria. In ischemic myocardium, we observed that tissue-resident macrophages died locally, whereas some also migrated to hematopoietic organs. If the steady state was perturbed by coronary ligation or diphtheria toxin-induced macrophage depletion in CD11b(DTR/+) mice, blood monocytes replenished heart macrophages. However, in the chronic phase after myocardial infarction, macrophages residing in the infarct were again independent from the blood monocyte pool, returning to the steady-state situation.

Conclusions: In this study, we show differential contribution of monocytes to heart macrophages during steady state, after macrophage depletion or in the acute and chronic phase after myocardial infarction. We found that macrophages participate in the immunosurveillance of myocardial tissue. These data correspond with previous studies on tissue-resident macrophages and raise important questions on the fate and function of macrophages during the development of heart failure.

Keywords: heart; macrophages; monocytes; myocardial infarction.

Figure 1. Resident cardiac macrophages in the healthy heart

A, Gating strategy for identification of CD45⁻ non-leukocyte cells, lymphocytes (identified as CD45⁺, CD11b⁻ and SSC^low) and heart macrophages (identified as CD45^high F4/80^high Ly6C^low) by flow cytometry. Frequencies within the entire heart are provided as mean±SEM. B, GFP expression in Cx3cr1^GFP/+ mice in different cell types, compared to C57BL/6 mice. C, Immunofluorescence microscopy of healthy heart tissue showing co-staining of MAC3 or F4/80 (D) with nuclear staining (DAPI) and the Cx3cr1^GFP/+ reporter (right). E, Immunofluorescence microscopy of healthy myocardium in a Cx3cr1^GFP/+ αSMA^RFP/+ dual reporter mouse. F, Immunofluorescence microscopy of healthy myocardium in a Cx3cr1^GFP/+ reporter mouse, stained for the fibroblast reporter DDR2. F, Immunohistochemical staining for CD68 in human myocardium. Scale bars indicates 5μm.

Figure 2. Resident cardiac macrophages turn over slowly in the steady-state

Mice were given daily BrdU injections for 4 weeks. BrdU incorporation in tissue resident macrophages (heart, lung and spleen) was measured immediately and 21 days after the last BrdU injection (n=6 per group, mean±SEM, *p<0.05 versus day 1, **p<0.01). One control mouse per cohort was not injected with BrdU to serve as a staining control (no BrdU).

Figure 3. Resident cardiac macrophages self-maintain through local proliferation

A, C57BL/6 CD45.2⁺ and CD45.1 mice were put in parabiosis for 6 weeks. Plots show representative data for chimerism of parabiont-derived cells from blood monocytes and heart macrophages. Upper dot plots are gated on CD11b⁺ CD115⁺ cells. B, Bar graph depicts relation of donor-derived chimerism (n=8, mean±SEM). C, Recipient Ubc^GFP+ mice were lethally irradiated and transplanted with 100 Lin^neg cKit⁺ Sca-1⁺ CD48⁻ CD150⁺ hematopoietic stem cells obtained from CD45.2⁺ mice. Bar graph displays donor-derived CD45.2⁺ cells among blood monocytes and heart macrophages 18 weeks later (n=8, mean±SEM). D, Dot plots show BrdU⁺ incorporation in heart macrophages 2 hours after one dose of BrdU (upper panel) compared to the blood monocytes (middle panel) and bone marrow monocytes (lower panel, n=4 each, mean±SEM). A mouse without BrdU injection served as staining control (left panels). E, Cell cycle analysis with Ki67/DAPI staining on heart macrophages (left: isotype control, right: Ki67/DAPI, n=5, mean±SEM).

Figure 4. Macrophages phagocytose Staphylococcus aureus

Mice were injected with fluorescent staphylococcus aureus bioparticles into the left ventricular myocardium. Two hours later, fluorescence within cardiac macrophages was analyzed by flow cytometry (right panel, n=5, mean±SEM). One mouse which was not injected served as staining control (left panel). Gated on CD45⁺ Lineage⁻ CD11b⁺ cardiac cells.

Figure 5. Monocytes contribute to macrophage recovery following induction of macrophage apoptosis

CD11b^DTR/⁺ mice were injected with a single dose of diphtheria toxin (DT, n=3 per time point). FACS plots show depletion and recovery in blood (A) and the heart (B). C, Percentages of F4/80^low Ly6C^high monocytes and F4/80^high Ly6C^low macrophages in the heart after one DT injection, gated on CD45⁺ Lin⁻ CD11b⁺ cells. D, Macrophages were depleted in CD11b^DTR/⁺ mice 2 weeks after establishing parabiosis with Ubc^GFP+ mice. Percentages of donor GFP⁺ cells in peripheral blood monocytes and cardiac macrophages were assessed in parabionts after 6 days (n=4).

Figure 6. Monocytes repopulate macrophages in ischemic tissue

A, Dot plots show kinetics of F4/80^low Ly6C^high monocytes and F4/80^high Ly6C^low macrophages in the heart after MI. The graph indicates percentages of Lin⁻ CD11b⁺ F4/80^low Ly6C^high monocytes and F4/80^high Ly6C^low macrophages in the MI (n=3–4 per time point). B, MI was induced in C57BL/6 CD45.2⁺ mice after parabiosis with Ubc^GFP+ mice. Percentages of donor GFP⁺ cells in peripheral blood monocyte subsets and heart macrophages were assessed in parabionts 4 days (n=6 pairs) and 1 month after MI (n=5 pairs). C, C57BL/6 CD45.2⁺ and Ubc^GFP+ mice were put in parabiosis 2 weeks after MI. Percentages of GFP⁺ cells among peripheral blood monocytes and heart macrophages were assessed in parabionts after 4 weeks (n=8) and 16 weeks (n=4 mean±SEM, **p<0.01 versus blood Ly6C^high monocytes).

Figure 7. Macrophage death

A, TUNEL⁺ MAC3⁺ macrophages (arrow) were observed in ischemic myocardium 12 hours after MI but not in the remote zone (n=3 mice, 10 fields of view per mouse, mean±SEM, ***p<0.001). Arrow head: TUNEL⁺ MAC3⁻ cell. Scale bar indicates 5μm. B, FACS analysis of myocardium 2 hours after MI, compared to non-MI controls. Representative histograms show the increase in propidium iodide (PI) positive, i.e. dead macrophages. Prior gates include CD45⁺ leukocytes and F4/80⁺ macrophages (n=2 controls, 4 mice with MI, mean±SEM, *p<0.05).

Figure 8. Macrophage exit

DiO lipophilic membrane dye was injected into the myocardium of C57BL/6 mice prior to coronary ligation. Mice injected with the dye but without MI or mice injected with PBS were used as controls. A, Twenty four hours later, reduced frequencies of cardiac macrophages are observed in mice with MI. The procedure resulted in DiO⁺ macrophages (B) which also were reduced in frequency after MI (C). MI did not change the frequency of DiO⁺ macrophages in mediastinal lymph nodes (D) but in spleen (E) and in the bone marrow (F). (G) The dye was not found in bone marrow Ly6C^high monocytes, cells that served as controls as they are not observed in the steady state myocardium (n=4 mean±SEM, *p<0.05 and **p<0.01).