Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart

Kory J Lavine, Slava Epelman, Keita Uchida, Kassandra J Weber, Colin G Nichols, Joel D Schilling, David M Ornitz, Gwendalyn J Randolph, Douglas L Mann, Kory J Lavine, Slava Epelman, Keita Uchida, Kassandra J Weber, Colin G Nichols, Joel D Schilling, David M Ornitz, Gwendalyn J Randolph, Douglas L Mann

Abstract

The mechanistic basis for why inflammation is simultaneously both deleterious and essential for tissue repair is not fully understood. Recently, a new paradigm has emerged: Organs are replete with resident macrophages of embryonic origin distinct from monocyte-derived macrophages. This added complexity raises the question of whether distinct immune cells drive inflammatory and reparative activities after injury. Previous work has demonstrated that the neonatal heart has a remarkable capacity for tissue repair compared with the adult heart, offering an ideal context to examine these concepts. We hypothesized that unrecognized differences in macrophage composition is a key determinant of cardiac tissue repair. Using a genetic model of cardiomyocyte ablation, we demonstrated that neonatal mice expand a population of embryonic-derived resident cardiac macrophages, which generate minimal inflammation and promote cardiac recovery through cardiomyocyte proliferation and angiogenesis. During homeostasis, the adult heart contains embryonic-derived macrophages with similar properties. However, after injury, these cells were replaced by monocyte-derived macrophages that are proinflammatory and lacked reparative activities. Inhibition of monocyte recruitment to the adult heart preserved embryonic-derived macrophage subsets, reduced inflammation, and enhanced tissue repair. These findings indicate that embryonic-derived macrophages are key mediators of cardiac recovery and suggest that therapeutics targeting distinct macrophage lineages may serve as novel treatments for heart failure.

Keywords: cardiac repair; inflammation; macrophages.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Age-dependent susceptibility to DT-induced cardiac injury. (A) Schematic of the DT cardiomyocyte injury model (Left). β-Galactosidase staining of E12.5 (Middle) and adult (Right) Rosa26R-LacZMlc2v-cre hearts. (B) Kaplan-Meier graphs of P1, P7, P14, and adult animals subjected to DT-induced cardiac injury. Black, control; blue, one DT injection; red, three DT injections. (C) TUNEL staining of P1, P7, P14, and adult animals after three DT injections. (D) Quantification of the percentage TUNEL-positive area in injured P1, P7, P14, and adult animals. (E) Troponin I serum levels immediately after three DT injections. *P < 0.05 compared with control.
Fig. 2.
Fig. 2.
Distinct macrophage and monocyte subsets populate the neonatal and adult heart. (A) Quantification of F4/80+ cells in the injured neonatal and adult heart. (B) FACS analysis of monocyte and macrophage populations in the injured neonatal and adult heart. (C) MertK and CD64 staining demonstrating that MHC-IIlowCCR2−, MHC-IIhighCCR2−, and MHC-IIhighCCR2+ subsets are macrophages, whereas the MHC-IIlowCCR2+ subset are monocytes. Flt3-Cre lineage tracing of neonatal and adult macrophage populations. Gray, isotype control; blue, antibody or Rosa26td. (D) CD68 immunostaining of Flt3-Cre Rosa26td positive and negative macrophages and monocytes. 40× objective. (E) CD68 immunostaining of CSF1R-MerCre Rosa26td macrophages in the neonatal heart (Left). 40× objective. FACS analysis demonstrating that the vast majority of MHC-IIlowCCR2− macrophages in the neonatal heart are embryonic-derived (Right).
Fig. 3.
Fig. 3.
Distinct inflammatory responses after neonatal and adult cardiac injury. (A) Quantitative RT-PCR assays of proinflammatory chemokine and cytokines in the injured neonatal and adult heart. (B) Proinflammatory chemokine and cytokine expression in sorted neonatal CCR2− and adult CCR2+ macrophages subsets isolated from injured hearts. (C) MCP-1 (white), IL1β (white), and CD68 (red) immunostaining in the injured neonatal adult heart. (D) MCP-1 (white), IL6 (white), MHC-II+ (red), and CCR2+ (green) immunostaining in injured adult hearts. (E and F) CellRox (red) and DAPI (blue) staining, indicating increased oxidative stress in the injured adult heart. (G) Cyba1 mRNA expression after neonatal adult cardiac injury. (H) Ly6G immunostaining for neutrophils in the injured neonatal and adult heart. (I) Neonatal CCR2− macrophages produce less TNFα and IL1β in response to LPS compared with adult CCR2+ macrophages. (J and K) Matrigel endothelial tube assay showing that only conditioned media obtained from neonatal CCR2− macrophages possess proangiogenic activity. (L) BrdU ELISA demonstrating that only neonatal CCR2− macrophage conditioned media stimulated neonatal rat cardiomyocyte proliferation. (M) platelet endothelial cell adhesion molecule-1 immunostaining of neonatal and adult hearts after injury. (C–E) 40× objective. (H and M) 20× objective. *P < 0.05 compared with control; **P < 0.05 compared with all other groups.
Fig. 4.
Fig. 4.
The neonatal heart functionally recovers after DT-induced cardiac injury. (A) Echocardiographic analysis of neonatal (three DT injections) and adult (one DT injection) hearts immediately after and 6 wk after injury. (B) H&E staining of surviving control and Rosa26-DTRmlc2v-cre hearts 6 wk after DT-induced cardiac injury. (C) Picrosirius red staining (fibrosis) 6 wk after neonatal and adult cardiac injury. (D) Wheat germ agglutinin (WGA) staining showing a cardiomyocyte cross-sectional area after cardiac injury. (E and F) Quantification of Picrosirius red staining (E) and cardiomyocyte cross-sectional area (F). (G) Anp and Bnp mRNA expression 6 wk after cardiac injury. (H and I) Longitudinal area of cardiomyocytes isolated from control and surviving Rosa26-DTRmlc2v-cre mice 6 wk after neonatal cardiac injury. (J) Cardiomyocyte cell shortening and contraction velocity at baseline and after isoproterenol treatment. (B and C) 20× objective. (D) 40× objective. *P < 0.05 compared with control; **P < 0.05 compared with all other groups; #P < 0.05 compared with cardiomyocytes not treated with isoproterenol.
Fig. 5.
Fig. 5.
Macrophages are essential for cardiac recovery after neonatal cardiac injury. (A) Flow cytometry revealing that liposomal clodronate specifically inhibits the accumulation of MHC-IIlowCCR2− macrophages after neonatal cardiac injury. (B) BrdU (white) immunostaining showing that macrophages are essential for cardiomyocyte (green) proliferation after neonatal cardiac injury. (C) Perfused lectin staining revealing that macrophages mediate coronary angiogenesis after neonatal cardiac injury. (D) Kaplan-Meier plot demonstrating that macrophage depletion results in increased mortality after neonatal cardiac injury. (E and F) Low-magnification images of H&E stained sections (E) and Anp mRNA expression (F) from hearts of adult mice that underwent neonatal cardiac injury and macrophage depletion. (G and H) Increased cardiomyocyte size (WGA; G) and interstitial fibrosis (Picrosirius red; H) in adult mice that underwent macrophage depletion and neonatal cardiac injury. (B, C, G) 40× objective. (H) 20× objective. *P < 0.05 compared with control; **P < 0.05 compared with all other groups.
Fig. 6.
Fig. 6.
Inhibition of monocyte recruitment preserves embryonic-derived macrophage subsets and improves adult cardiac repair (A and B). Conditioned media obtained from adult MHC-IIlowCCR2− and MHC-IIhighCCR2− macrophages stimulates endothelial tube formation (A) and neonatal rat cardiomyocyte proliferation (B). (C) Quantification of CD68+ monocyte/macrophages after cardiac injury and CCR2 inhibition. (D) Flow cytometry revealing that CCR2 inhibition blocked CCR2+ monocyte and macrophage recruitment to the injured heart and preserved CCR2− resident macrophage subsets. (E and F) Immunostaining (E) and quantitative RT-PCR (F) assays measuring cardiac expression of IL1β, IL6, MCP-1, and TNFα after cardiac injury and CCR2 inhibition. (G and H) Ly6G immunostaining (G) and quantitative RT-PCR (H) showing that CCR2 inhibition reduced neutrophil influx, Cxcl1, and Cyba1 mRNA expression after cardiac injury. (I) Perfused lectin staining showing that CCR2 inhibition preserves coronary microvascular integrity after cardiac injury. *P < 0.05 compared with control; **P < 0.05 compared with all other groups.

References

    1. Glaros T, Larsen M, Li L. Macrophages and fibroblasts during inflammation, tissue damage and organ injury. Front Biosci. 2009;14:3988–3993.
    1. Frantz S, Nahrendorf M. Cardiac macrophages and their role in ischaemic heart disease. Cardiovasc Res. 2014 doi: 10.1093/cvr/cvu025.
    1. Nahrendorf M, et al. The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions. J Exp Med. 2007;204:3037–3047.
    1. Schulz C, et al. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science. 2012;336:86–90.
    1. Hashimoto D, et al. Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes. Immunity. 2013;38:792–804.
    1. Yona S, et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity. 2013;38:79–91.
    1. Ginhoux F, et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 2010;330:841–845.
    1. Hoeffel G, et al. Adult Langerhans cells derive predominantly from embryonic fetal liver monocytes with a minor contribution of yolk sac-derived macrophages. J Exp Med. 2012;209:1167–1181.
    1. Epelman S, et al. Embryonic and Adult-Derived Resident Cardiac Macrophages Are Maintained through Distinct Mechanisms at Steady State and during Inflammation. Immunity. 2014;40:91–104.
    1. Porrello ER, et al. Transient regenerative potential of the neonatal mouse heart. Science. 2011;331:1078–1080.
    1. Haubner BJ, et al. Complete cardiac regeneration in a mouse model of myocardial infarction. Aging. 2012;4:966–977.
    1. Mahmoud AI, Porrello ER, Kimura W, Olson EN, Sadek HA. Surgical models for cardiac regeneration in neonatal mice. Nat Protoc. 2014;9:305–311.
    1. Porrello ER, et al. Regulation of neonatal and adult mammalian heart regeneration by the miR-15 family. Proc Natl Acad Sci USA. 2013;110:187–192.
    1. Aurora AB, et al. Macrophages are required for neonatal heart regeneration. J Clin Invest. 2014;124:1382–1392.
    1. Dewald O, et al. Development of murine ischemic cardiomyopathy is associated with a transient inflammatory reaction and depends on reactive oxygen species. Proc Natl Acad Sci USA. 2003;100:2700–2705.
    1. Chen J, et al. Selective requirement of myosin light chain 2v in embryonic heart function. J Biol Chem. 1998;273:1252–1256.
    1. Buch T, et al. A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration. Nat Methods. 2005;2:419–426.
    1. Gautier EL, et al. Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nat Immunol. 2012;13:1118–1128.
    1. Boyer SW, Schroeder AV, Smith-Berdan S, Forsberg EC. All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells. Cell Stem Cell. 2011;9:64–73.
    1. Pinto AR, et al. An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile. PLoS ONE. 2012;7:e36814.
    1. Tauber AI. Metchnikoff and the phagocytosis theory. Nat Rev Mol Cell Biol. 2003;4:897–901.
    1. Kaikita K, et al. Targeted deletion of CC chemokine receptor 2 attenuates left ventricular remodeling after experimental myocardial infarction. Am J Pathol. 2004;165:439–447.
    1. Madisen L, et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci. 2010;13:133–140.
    1. Qian BZ, et al. CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis. Nature. 2011;475:222–225.
    1. Seiler P, et al. Crucial role of marginal zone macrophages and marginal zone metallophils in the clearance of lymphocytic choriomeningitis virus infection. Eur J Immunol. 1997;27:2626–2633.

Source: PubMed

赞助者和合作者

医疗条件

药物干预

3
订阅