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Cardiac macrophage biology in the steady-state heart, the aging heart, and following myocardial infarction

  • Yonggang Ma
    Affiliations
    Mississippi Center for Heart Research, Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Miss
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  • Alan J. Mouton
    Affiliations
    Mississippi Center for Heart Research, Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Miss
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  • Merry L. Lindsey
    Correspondence
    Reprint requests: Merry L. Lindsey, Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 North State St., Jackson, MS 39216-4505
    Affiliations
    Mississippi Center for Heart Research, Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Miss

    Research Service, G.V. (Sonny) Montgomery Veterans Affairs Medical Center, Jackson, Miss
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Open AccessPublished:October 13, 2017DOI:https://doi.org/10.1016/j.trsl.2017.10.001
      Macrophages play critical roles in homeostatic maintenance of the myocardium under normal conditions and in tissue repair after injury. In the steady-state heart, resident cardiac macrophages remove senescent and dying cells and facilitate electrical conduction. In the aging heart, the shift in macrophage phenotype to a proinflammatory subtype leads to inflammaging. Following myocardial infarction (MI), macrophages recruited to the infarct produce both proinflammatory and anti-inflammatory mediators (cytokines, chemokines, matrix metalloproteinases, and growth factors), phagocytize dead cells, and promote angiogenesis and scar formation. These diverse properties are attributed to distinct macrophage subtypes and polarization status. Infarct macrophages exhibit a proinflammatory M1 phenotype early and become polarized toward an anti-inflammatory M2 phenotype later post-MI. Although this classification system is oversimplified and needs to be refined to accommodate the multiple different macrophage subtypes that have been recently identified, general concepts on macrophage roles are independent of subtype classification. This review summarizes current knowledge about cardiac macrophage origins, roles, and phenotypes in the steady state, with aging, and after MI, as well as highlights outstanding areas of investigation.

      Abbreviations:

      AV (atrioventricular), CCL (chemokine C-C motif ligand), CCR (chemokine C-C motif receptor), CXCL (chemokine C-X-C motif ligand), CX3CR (chemokine C-X3-C motif receptor), ECM (extracellular matrix), GM-CSF (granulocyte macrophage colony-stimulating factor), HSC (hematopoietic stem cell), IFN (interferon), IL (interleukin), LPS (lipopolysaccharide), MERTK (myeloid epithelial reproductive tyrosine kinase), MHCII (major histocompatibility complex class II), MI (myocardial infarction), MMPs (matrix metalloproteinases), TGF-β1 (transforming growth factor-β1), TNF (tumor necrosis factor)

      Introduction

      Macrophages were first identified by Ilya Ilyich Mechnikov in 1882 and belong to the vertebrate first-line defense system against infection and injury.
      • Gordon S.
      The macrophage: past, present and future.
      With the advent of genetic fate mapping and tracing techniques (cell reporter lines, parabiosis, bone marrow transplant, and intravital microscopy), our understanding of macrophage physiology has been revolutionized over the past decade. We now know that macrophages reside in all organs in the steady state.
      • Epelman S.
      • Lavine K.J.
      • Randolph G.J.
      Origin and functions of tissue macrophages.
      Tissue resident macrophages persist from embryogenesis into adulthood and minimally rely on monocyte infiltration for renewal, with the exception of skin and gut macrophages that depend on monocyte entry to maintain numbers.
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      • Guilliams M.
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      Origins and functional specialization of macrophages and of conventional and monocyte-derived dendritic cells in mouse skin.
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      • Varol C.
      • Farache J.
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      Ly6C hi monocytes in the inflamed colon give rise to proinflammatory effector cells and migratory antigen-presenting cells.
      In response to infection or injury, circulating monocytes are mobilized to inflamed tissue and differentiate into macrophages, which constitute the majority of the macrophage population during the acute inflammatory phase.
      • Swirski F.K.
      • Robbins C.S.
      • Nahrendorf M.
      Development and function of arterial and cardiac macrophages.
      In the steady state, tissue resident macrophages exert homeostatic functions, including defending against infection and removing senescent or damaged cells. Moreover, macrophages exhibit distinct organ and tissue-specific physiological functions. For instance, skin macrophages participate in regulating salt-dependent extracellular volume and blood pressure homeostasis.
      • Machnik A.
      • Neuhofer W.
      • Jantsch J.
      • et al.
      Macrophages regulate salt-dependent volume and blood pressure by a vascular endothelial growth factor-C-dependent buffering mechanism.
      In adipose tissue, macrophages generate catecholamines to sustain adaptive thermogenesis and promote insulin resistance by nuclear receptor co-repressor–dependent mechanisms.
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      • Qiu Y.
      • Cui X.
      • et al.
      Alternatively activated macrophages produce catecholamines to sustain adaptive thermogenesis.
      • Li P.
      • Spann N.J.
      • Kaikkonen M.U.
      • et al.
      NCoR repression of LXRs restricts macrophage biosynthesis of insulin-sensitizing omega 3 fatty acids.
      Peritoneal macrophages orchestrate migration of immunoglobulin A-producing B cells to the intestine, where they play a key role in the early response to pathogens.
      • Okabe Y.
      • Medzhitov R.
      Tissue-specific signals control reversible program of localization and functional polarization of macrophages.
      Macrophages are also involved in erythrocyte removal and iron recycling in the liver, synaptic pruning and normal brain development, and hematopoietic control in the bone marrow and spleen.
      • Theurl I.
      • Hilgendorf I.
      • Nairz M.
      • et al.
      On-demand erythrocyte disposal and iron recycling requires transient macrophages in the liver.
      • Paolicelli R.C.
      • Bolasco G.
      • Pagani F.
      • et al.
      Synaptic pruning by microglia is necessary for normal brain development.
      • Dutta P.
      • Hoyer F.F.
      • Grigoryeva L.S.
      • et al.
      Macrophages retain hematopoietic stem cells in the spleen via VCAM-1.
      In addition to maintaining equilibrium, the macrophage plays an indispensable role in response to injury, including myocardial infarction (MI) both in the presence and absence of reperfusion. The importance of macrophages during post-MI remodeling has been highlighted by studies in which depletion of macrophages by clodronate liposomes compromises cardiac repair in mouse MI models.
      • Frantz S.
      • Hofmann U.
      • Fraccarollo D.
      • et al.
      Monocytes/macrophages prevent healing defects and left ventricular thrombus formation after myocardial infarction.
      • Ben-Mordechai T.
      • Holbova R.
      • Landa-Rouben N.
      • et al.
      Macrophage subpopulations are essential for infarct repair with and without stem cell therapy.
      Following MI, macrophages can secrete proinflammatory, anti-inflammatory, proangiogenic, or proreparative factors; can phagocytize dying cells; and can directly interact with other cell types to orchestrate the repair response.
      • Nahrendorf M.
      • Swirski F.K.
      Monocyte and macrophage heterogeneity in the heart.
      • Swirski F.K.
      • Nahrendorf M.
      Macrophage-stem cell crosstalk after myocardial infarction.
      The diverse functions of macrophages are partially attributed to their different phenotypes and polarization status. Macrophage polarization is a process by which macrophages exhibit vastly different gene expression profiles and functions in response to extremes in environmental signals. Post-MI macrophages show a proinflammatory M1 phenotype early and an anti-inflammatory M2 phenotype later, with these phenotypes playing distinct and even opposite roles.
      • Gombozhapova A.
      • Rogovskaya Y.
      • Shurupov V.
      • et al.
      Macrophage activation and polarization in post-infarction cardiac remodeling.
      • ter Horst E.N.
      • Hakimzadeh N.
      • van der Laan A.M.
      • Krijnen P.A.
      • Niessen H.W.
      • Piek J.J.
      Modulators of macrophage polarization Influence healing of the infarcted myocardium.
      In this review, we will discuss current literature regarding cardiac macrophage origins, roles, and phenotypes in the steady state, the aging heart, and post-MI, as well as emphasize outstanding areas of investigation to complete our understanding of macrophage polarization in the heart.

      Monocyte/Macrophage Markers

      Monocytes and macrophages have been assessed by multiple approaches using a variety of markers to label cells and cell subtypes. Table I provides a comprehensive list of monocyte and macrophage markers that have been used. Ly6C/Gr-1 is expressed in rodents, but not in humans, whereas all other markers in Table I are expressed in both rodents and humans. Single-marker labeling is commonly used in experiments with immunohistochemistry, immunoblotting, or immunofluorescence approaches. One underappreciated concept is the fact that the marker used to identify cell type by itself has biological functions. For instance, the most commonly used macrophage marker F4/80 has proinflammatory properties and can induce antigen-specific regulatory T cells (Tregs).
      • Lin H.H.
      • Faunce D.E.
      • Stacey M.
      • et al.
      The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance.
      Distinct gating strategies using flow cytometry can delineate monocyte and macrophage origin and subset types based on marker expression patterns. Table II summarizes current gating strategies to discriminate distinct blood and cardiac monocyte and macrophage phenotypes under steady state and after injury. In addition, the Macrophage Community Website (www.macrophages.com)
      • Robert C.
      • Lu X.
      • Law A.
      • Freeman T.C.
      • Hume D.A.
      Macrophages.com: an on-line community resource for innate immunity research.
      and the Immunological Genome Project (www.immgen.org) provide excellent macrophage database resources.
      Table IA list of known monocyte and macrophage markers
      MarkerLocationExpressed byCell physiological functionsReferences
      CCR2/CD192Cell surfaceMonocytes, macrophagesMediates Ly6Chigh monocyte recruitment and migration
      • Epelman S.
      • Lavine K.J.
      • Beaudin A.E.
      • et al.
      Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.
      • Tsou C.L.
      • Peters W.
      • Si Y.
      • et al.
      Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites.
      CD11 b/ITGAMCell surfaceMonocytes, macrophages, neutrophils, NK cellsCouples with CD18 to form integrin αMβ2 (also named Mac1 or complement receptor 3) to initiate immune responses
      • Todd 3rd, R.F.
      The continuing saga of complement receptor type 3 (CR3).
      • Zhou H.
      • Liao J.
      • Aloor J.
      • et al.
      CD11b/CD18 (Mac-1) is a novel surface receptor for extracellular double-stranded RNA to mediate cellular inflammatory responses.
      CD14Cell surfaceHuman monocytesMediates toll-like receptor 4 activation and production of IFN-β
      • Ma C.Y.
      • Chang W.E.
      • Shi G.Y.
      • et al.
      Recombinant thrombomodulin inhibits lipopolysaccharide-induced inflammatory response by blocking the functions of CD14.
      • Guillou C.
      • Freret M.
      • Fondard E.
      • et al.
      Soluble alpha-enolase activates monocytes by CD14-dependent TLR4 signalling pathway and exhibits a dual function.
      CD16/FCGR3Cell surfaceHuman monocytesBinds to the Fc portion of IgG antibodies, antigen presentation, anti-inflammatory cytokine production
      • Guilliams M.
      • Bruhns P.
      • Saeys Y.
      • Hammad H.
      • Lambrecht B.N.
      The function of Fcgamma receptors in dendritic cells and macrophages.
      CD64/FcγR1Cell surfaceMonocytes, macrophagesAntibody-dependent phagocytosis, recognizes the Fc region of IgG
      • Epelman S.
      • Lavine K.J.
      • Beaudin A.E.
      • et al.
      Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.
      • De Calisto J.
      • Villablanca E.J.
      • Mora J.R.
      FcgammaRI (CD64): an identity card for intestinal macrophages.
      • Mittal R.
      • Sukumaran S.K.
      • Selvaraj S.K.
      • et al.
      Fcgamma receptor I alpha chain (CD64) expression in macrophages is critical for the onset of meningitis by Escherichia coli K1.
      CD68/macrosialinEndosomal/lysosomal compartment, cell surfaceMonocytes, macrophagesAntigen processing and presentation, binds to oxidized low-density lipoprotein
      • Chistiakov D.A.
      • Killingsworth M.C.
      • Myasoedova V.A.
      • Orekhov A.N.
      • Bobryshev Y.V.
      CD68/macrosialin: not just a histochemical marker.
      CD163Cell surface, secreted (soluble)Macrophages, neutrophilsHemoglobin/haptoglobin scavenger receptor, anti-inflammatory
      • Etzerodt A.
      • Moestrup S.K.
      CD163 and inflammation: biological, diagnostic, and therapeutic aspects.
      • Rafatian N.
      • Westcott K.V.
      • White R.A.
      • Leenen F.H.
      Cardiac macrophages and apoptosis after myocardial infarction: effects of central MR blockade.
      CX3CR1Cell surfaceMonocytes, macrophagesMediates Ly6Clow monocyte recruitment, inhibits proliferation of local macrophages
      • Molawi K.
      • Wolf Y.
      • Kandalla P.K.
      • et al.
      Progressive replacement of embryo-derived cardiac macrophages with age.
      • Engel D.R.
      • Krause T.A.
      • Snelgrove S.L.
      • et al.
      CX3CR1 reduces kidney fibrosis by inhibiting local proliferation of profibrotic macrophages.
      F4/80/EMR1Cell surfaceMacrophagesPromotes proinflammatory factor production, induces antigen-specific efferent Treg cells
      • Lin H.H.
      • Faunce D.E.
      • Stacey M.
      • et al.
      The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance.
      Galectin 3/Mac2Cell surface, secretedMacrophagesInduces monocyte-macrophage differentiation, interferes with dendritic cell fate decision, regulates T cell apoptosis, inhibits B-lymphocyte differentiation into plasma cells
      • de Oliveira F.L.
      • Gatto M.
      • Bassi N.
      • et al.
      Galectin-3 in autoimmunity and autoimmune diseases.
      Ly6C/Gr-1
      Ly6C/Gr-1 is expressed in rodents, but not in humans, whereas all other markers in Table I are expressed in both rodents and humans.
      Cell surfaceMonocytesA specific marker for proinflammatory monocytes
      • Epelman S.
      • Lavine K.J.
      • Beaudin A.E.
      • et al.
      Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.
      Mac3Cell surfaceMacrophagesA glycoprotein
      • Ma Y.
      • Halade G.V.
      • Zhang J.
      • et al.
      Matrix metalloproteinase-28 deletion exacerbates cardiac dysfunction and rupture after myocardial infarction in mice by inhibiting M2 macrophage activation.
      • Ho M.K.
      • Springer T.A.
      Tissue distribution, structural characterization, and biosynthesis of Mac-3, a macrophage surface glycoprotein exhibiting molecular weight heterogeneity.
      MERTKCell surfaceMacrophages, phagocytesMediates phagocytosis, increases migration
      • Epelman S.
      • Lavine K.J.
      • Beaudin A.E.
      • et al.
      Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.
      • Tang Y.
      • Wu S.
      • Liu Q.
      • et al.
      Mertk deficiency affects macrophage directional migration via disruption of cytoskeletal organization.
      MHCIICell surfaceMacrophages, dendritic cells, B cellsMediates antigen presentation
      • Ting J.P.
      • Trowsdale J.
      Genetic control of MHC class II expression.
      * Ly6C/Gr-1 is expressed in rodents, but not in humans, whereas all other markers in Table I are expressed in both rodents and humans.
      Table IIGating strategies to label blood and cardiac monocytes and macrophages
      Gating strategyCells labeledSpeciesReferences
      Monocytes
      Ly6ChighCCR2highCX3CR1lowCD62 L+Classical monocytesMouse
      • Nahrendorf M.
      • Swirski F.K.
      Monocyte and macrophage heterogeneity in the heart.
      Ly6ClowCCR2lowCX3CR1highCD62LNonclassical monocytesMouse
      • Nahrendorf M.
      • Swirski F.K.
      Monocyte and macrophage heterogeneity in the heart.
      CCR2+Ly6ChighInflammatory blood monocytesMouse
      • Motomura Y.
      • Kanno S.
      • Asano K.
      • et al.
      Identification of pathogenic cardiac CD11c+ macrophages in Nod1-mediated acute Coronary Arteritis.
      CD14+CD16, CD14+CD16+Blood monocytesHuman
      • Ingersoll M.A.
      • Spanbroek R.
      • Lottaz C.
      • et al.
      Comparison of gene expression profiles between human and mouse monocyte subsets.
      B220F4/80+CD115+Ly6C,

      B220F4/80+CD115+Ly6C+
      Blood monocytesMouse
      • Ingersoll M.A.
      • Spanbroek R.
      • Lottaz C.
      • et al.
      Comparison of gene expression profiles between human and mouse monocyte subsets.
      MHCIIlowCCR2+Cardiac monocytesMouse
      • Lavine K.J.
      • Epelman S.
      • Uchida K.
      • et al.
      Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart.
      CD11 b+F4/80Ly6GLy6Chigh,

      CD11 b+F4/80Ly6GLy6Clow
      MonocytesMouse
      • Zlatanova I.
      • Pinto C.
      • Silvestre J.S.
      Immune modulation of cardiac repair and regeneration: the Art of Mending broken hearts.
      LineageCD11 b+F4/80lowLy6C+Cardiac monocytesMouse
      • Heidt T.
      • Courties G.
      • Dutta P.
      • et al.
      Differential contribution of monocytes to heart macrophages in steady-state and after myocardial infarction.
      CD11 b+CD11CMHCIICD68Ly6Clow,

      CD11 b+CD11CMHCIICD68Ly6Chigh
      Blood and cardiac monocytesMouse
      • Hilgendorf I.
      • Gerhardt L.M.
      • Tan T.C.
      • et al.
      Ly-6Chigh monocytes depend on Nr4a1 to balance both inflammatory and reparative phases in the infarcted myocardium.
      Macrophages
      CD45+CD11 b+F4/80+CD206M1 macrophagesMouse
      • Yan X.
      • Anzai A.
      • Katsumata Y.
      • et al.
      Temporal dynamics of cardiac immune cell accumulation following acute myocardial infarction.
      CD45+CD11 b+F4/80+CD206+M2 macrophagesMouse
      • Yan X.
      • Anzai A.
      • Katsumata Y.
      • et al.
      Temporal dynamics of cardiac immune cell accumulation following acute myocardial infarction.
      CD45+CD11 b+F4/80+Ly6ClowResident cardiac macrophagesMouse
      • Heidt T.
      • Courties G.
      • Dutta P.
      • et al.
      Differential contribution of monocytes to heart macrophages in steady-state and after myocardial infarction.
      CD11 b+F4/80+CD206+Alternatively activated macrophagesMouse
      • Shiraishi M.
      • Shintani Y.
      • Shintani Y.
      • et al.
      Alternatively activated macrophages determine repair of the infarcted adult murine heart.
      CD11 b+F4/80+CD64+Ly6C+MHCII+/−M1 like macrophagesMouse
      • Zlatanova I.
      • Pinto C.
      • Silvestre J.S.
      Immune modulation of cardiac repair and regeneration: the Art of Mending broken hearts.
      CD11 b+F4/80+CD64+Ly6CMHCII+/−M2 like macrophagesMouse
      • Zlatanova I.
      • Pinto C.
      • Silvestre J.S.
      Immune modulation of cardiac repair and regeneration: the Art of Mending broken hearts.
      F4/80+CD86+M1 macrophagesMouse
      • Ben-Mordechai T.
      • Holbova R.
      • Landa-Rouben N.
      • et al.
      Macrophage subpopulations are essential for infarct repair with and without stem cell therapy.
      F4/80+CD206+M2 macrophagesMouse
      • Ben-Mordechai T.
      • Holbova R.
      • Landa-Rouben N.
      • et al.
      Macrophage subpopulations are essential for infarct repair with and without stem cell therapy.
      • Ma Y.
      • Chiao Y.A.
      • Clark R.
      • et al.
      Deriving a cardiac ageing signature to reveal MMP-9-dependent inflammatory signalling in senescence.
      F4/80+CD206M1 macrophagesMouse
      • Ma Y.
      • Chiao Y.A.
      • Clark R.
      • et al.
      Deriving a cardiac ageing signature to reveal MMP-9-dependent inflammatory signalling in senescence.
      CD45+CD68+Cardiac, blood, and spleen macrophagesRat
      • de Couto G.
      • Liu W.
      • Tseliou E.
      • et al.
      Macrophages mediate cardioprotective cellular postconditioning in acute myocardial infarction.
      CD11 b+F4/80+CD68+Ly6Clow, CD11 b+F4/80+CD68+Ly6ChighMonocyte-derived cardiac macrophagesMouse
      • Hilgendorf I.
      • Gerhardt L.M.
      • Tan T.C.
      • et al.
      Ly-6Chigh monocytes depend on Nr4a1 to balance both inflammatory and reparative phases in the infarcted myocardium.
      CD14+CD64+MERTK+F4/80+CX3CR1+MHCII, CD14+CD64+MERTK+F4/80+CX3CR1+MHCII+, CD14+CD64+MERTK+F4/80+CX3CR1MHCII, CD14+CD64+MERTK+F4/80+CX3CR1MHCII+Resident cardiac macrophagesMouse
      • Molawi K.
      • Wolf Y.
      • Kandalla P.K.
      • et al.
      Progressive replacement of embryo-derived cardiac macrophages with age.
      F4/80+CD11 b+Ly6Clow, F4/80+CD11 b+Ly6Cmedium, F4/80+CD11 b+Ly6ClhighAlternatively activated macrophagesMouse
      • Falkenham A.
      • de Antueno R.
      • Rosin N.
      • et al.
      Nonclassical resident macrophages are important determinants in the development of myocardial fibrosis.
      CD45+CD11 b+F4/80+Ly6CMHCIIhigh, CD45+CD11 b+F4/80+Ly6CMHCIIlow, CD45+ CD11 b+F4/80+Ly6C+MERTK+CD206+, CD45+ CD11 b+F4/80+Ly6C+MERTKCD206Cardiac resident macrophagesMouse
      • Epelman S.
      • Lavine K.J.
      • Beaudin A.E.
      • et al.
      Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.
      CD45+F4/80+MHC-IIlowCCR2, CD45+F4/80+MHC-IIhighCCR2Cardiac resident macrophagesMouse
      • Lavine K.J.
      • Epelman S.
      • Uchida K.
      • et al.
      Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart.
      CD45+F4/80+MHCIIhighCCR2+Monocyte-derived cardiac macrophagesMouse
      • Lavine K.J.
      • Epelman S.
      • Uchida K.
      • et al.
      Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart.
      CD14+CD16+CD163+CD204+CD206+CD209Anti-inflammatory M2c macrophagesHuman
      • Zizzo G.
      • Hilliard B.A.
      • Monestier M.
      • Cohen P.L.
      Efficient clearance of early apoptotic cells by human macrophages requires M2c polarization and MerTK induction.

      Macrophages in the Steady State Heart

       Macrophage origins

      The earlier dogma that macrophages are exclusively derived from circulating monocytes generated by the bone marrow and spleen has been challenged.
      • van Furth R.
      • Cohn Z.A.
      The origin and kinetics of mononuclear phagocytes.
      In the past decade, a growing body of literature demonstrates that tissue resident macrophages, in the brain, spleen, liver, lung, bone marrow, kidney, pancreas, peritoneum, and heart are established prenatally, persist throughout the life span, and self-renew locally.
      • Nahrendorf M.
      • Swirski F.K.
      Innate immune cells in ischaemic heart disease: does myocardial infarction beget myocardial infarction?.
      • Ginhoux F.
      • Jung S.
      Monocytes and macrophages: developmental pathways and tissue homeostasis.
      In the steady state, resident cardiac macrophages in mice are reported to account for approximately 5%–10% of nonmyocytes in the heart.
      • Heidt T.
      • Courties G.
      • Dutta P.
      • et al.
      Differential contribution of monocytes to heart macrophages in steady-state and after myocardial infarction.
      • Pinto A.R.
      • Ilinykh A.
      • Ivey M.J.
      • et al.
      Revisiting cardiac cellular composition.
      Resident macrophages adopt a spindle-like shape and intermingle closely with myocytes, endothelial cells, and fibroblasts.
      • Heidt T.
      • Courties G.
      • Dutta P.
      • et al.
      Differential contribution of monocytes to heart macrophages in steady-state and after myocardial infarction.
      Genetic fate mapping and lineage-tracing studies reveal that the vast majority of resident cardiac macrophages originate from embryonic yolk sac and fetal liver progenitors (Fig 1).
      • Epelman S.
      • Lavine K.J.
      • Beaudin A.E.
      • et al.
      Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.
      Replenishment occurs at the rate of about once per month via proliferation.
      • Epelman S.
      • Lavine K.J.
      • Beaudin A.E.
      • et al.
      Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.
      • Heidt T.
      • Courties G.
      • Dutta P.
      • et al.
      Differential contribution of monocytes to heart macrophages in steady-state and after myocardial infarction.
      In terms of subpopulations, CCR2+ macrophages are replenished by blood monocyte recruitment and local proliferation, whereas CCR2 macrophages are repopulated largely by local proliferation (Fig 1).
      • Epelman S.
      • Lavine K.J.
      • Beaudin A.E.
      • et al.
      Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.
      Fig 1
      Fig 1Macrophage origins in the steady-state heart and the aging heart. In the steady state, the vast majority of resident cardiac macrophages originate from the yolk sac and fetal liver progenitors, with minimal dependence on blood monocytes as a source. In terms of subpopulations, CCR2+ macrophages are replenished by blood monocyte recruitment and local proliferation, whereas CCR2 macrophages are repopulated largely by local proliferation. With age, self-renewal of resident cardiac macrophage declines, and blood monocytes increasingly contribute to the cardiac macrophage population.

       Macrophage roles

      Cardiac macrophages in the healthy state closely resemble alternatively activated anti-inflammatory M2 macrophages, expressing a plethora of M2-designated markers.
      • Swirski F.K.
      • Robbins C.S.
      • Nahrendorf M.
      Development and function of arterial and cardiac macrophages.
      • Pinto A.R.
      • Paolicelli R.
      • Salimova E.
      • et al.
      An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile.
      This is logical, as M2 macrophages promote tissue rebuilding after injury and thus help to re-establish homeostasis. In terms of cell physiology, resident macrophages can engulf fluorescently labeled bacteria, indicating the capacity to phagocytose dying cells.
      • Heidt T.
      • Courties G.
      • Dutta P.
      • et al.
      Differential contribution of monocytes to heart macrophages in steady-state and after myocardial infarction.
      The Nahrendorf et al. recently revealed a novel function for macrophages in the healthy mouse heart. Using specific macrophage reporter lines in combination with optical clearing techniques and confocal microscopy, they for the first time demonstrated that macrophages are abundant in the atrioventricular (AV) node, and these AV nodal macrophages intervene with cardiomyocytes through connexin-43-containing gap junctions to accelerate myocyte repolarization and electrical conduction.
      • Hulsmans M.
      • Clauss S.
      • Xiao L.
      • et al.
      Macrophages facilitate electrical conduction in the heart.
      This is supported by the observation that deleting connexin-43 in macrophages delays AV conduction, and macrophage deletion induces AV block.
      • Hulsmans M.
      • Clauss S.
      • Xiao L.
      • et al.
      Macrophages facilitate electrical conduction in the heart.
      Therefore, the macrophage is involved in myocardial conduction, representing a novel target to treat cardiac arrhythmias. Although this study only evaluated the steady state, it raises interesting questions as to whether macrophages are involved in arrhythmia generation after injury and whether macrophages help to regulate myocyte contraction under normal conditions.

       Macrophage phenotypes

      In the steady state, resident cardiac macrophages are heterogeneous in origin. Different laboratories have divided macrophages into subpopulations using different markers. Four populations expressing varying levels of Ly6C and major histocompatibility complex class II (MHCII) have been identified in the mouse heart.
      • Ben-Mordechai T.
      • Palevski D.
      • Glucksam-Galnoy Y.
      • Elron-Gross I.
      • Margalit R.
      • Leor J.
      Targeting macrophage subsets for infarct repair.
      Of these populations, the Ly6CCCR2 population comprises the vast majority, which originates from the yolk sac and contains MHCIIhigh and MHCIIlow subsets. The third (Ly6C+CCR2) and the fourth (Ly6C+CCR2+) subsets are both derived from hematopoiesis.
      • Epelman S.
      • Lavine K.J.
      • Beaudin A.E.
      • et al.
      Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.
      • Ben-Mordechai T.
      • Palevski D.
      • Glucksam-Galnoy Y.
      • Elron-Gross I.
      • Margalit R.
      • Leor J.
      Targeting macrophage subsets for infarct repair.
      • Cohen H.B.
      • Mosser D.M.
      Cardiac macrophages: how to mend a broken heart.
      The exact roles for these macrophage subsets are incompletely understood. MHCIIhigh cardiac macrophages more efficiently present antigen to T lymphocytes, whereas MHCIIlow cells have higher phagocytic capability. CCR2+ macrophages express high levels of NLRP3-inflammasome associated genes, implying a proinflammatory role for this subtype.
      • Epelman S.
      • Lavine K.J.
      • Beaudin A.E.
      • et al.
      Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.
      Cardiac macrophages in mice can also be divided into 4 populations based on CX3CR1 and MHCII expression: CX3CR1MHCII, CX3CR1MHCII+, CX3CR1+MHCII, and CX3CR1+MHCII+.
      • Molawi K.
      • Wolf Y.
      • Kandalla P.K.
      • et al.
      Progressive replacement of embryo-derived cardiac macrophages with age.
      Molawi et al. demonstrated that almost all macrophages at birth were CX3CR1+MHCII, and with age, there was a progressive increase in MHCII+ cells and a decrease of CX3CR1+ population, leading to a more even distribution of these cell populations by adulthood.
      • Molawi K.
      • Wolf Y.
      • Kandalla P.K.
      • et al.
      Progressive replacement of embryo-derived cardiac macrophages with age.
      Combined, the above studies showed the existence of MHCII and MHCII+ macrophages. However, the question about the relationship between CCR2 and CX3CR1 lineages remains to be addressed.

      Macrophages in the Aging Heart

       Macrophage origins

      Aging is a major risk factor for cardiac morbidity and mortality. Cardiac aging is characterized by myocardial sarcopenia, hypertrophy, vascular hyperpermeability, inflammation, fibrosis, and mild cardiac physiology impairment.
      • Lin J.
      • Lopez E.F.
      • Jin Y.
      • et al.
      Age-related cardiac muscle sarcopenia: combining experimental and mathematical modeling to identify mechanisms.
      • Yabluchanskiy A.
      • Ma Y.
      • Chiao Y.A.
      • et al.
      Cardiac aging is initiated by matrix metalloproteinase-9-mediated endothelial dysfunction.
      • Lindsey M.L.
      • Goshorn D.K.
      • Squires C.E.
      • et al.
      Age-dependent changes in myocardial matrix metalloproteinase/tissue inhibitor of metalloproteinase profiles and fibroblast function.
      In mice, blood pressure does not increase with age, and thus changes in the heart due to age can be attributed to direct changes on the myocardium rather than alterations in ventricular pre-load or after-load.
      • Chiao Y.A.
      • Ramirez T.A.
      • Zamilpa R.
      • et al.
      Matrix metalloproteinase-9 deletion attenuates myocardial fibrosis and diastolic dysfunction in ageing mice.
      In mouse studies, young (<9 months), middle-aged (12–15 months), old (18–24 months), and senescent (>26 months) mice are commonly used to define different age groups.
      • Ma Y.
      • Chiao Y.A.
      • Clark R.
      • et al.
      Deriving a cardiac ageing signature to reveal MMP-9-dependent inflammatory signalling in senescence.
      • Chiao Y.A.
      • Ramirez T.A.
      • Zamilpa R.
      • et al.
      Matrix metalloproteinase-9 deletion attenuates myocardial fibrosis and diastolic dysfunction in ageing mice.
      We have previously reported that the number of cardiac macrophages in mice increases beginning at about 18 months, and numbers positively correlate with age.
      • Ma Y.
      • Chiao Y.A.
      • Clark R.
      • et al.
      Deriving a cardiac ageing signature to reveal MMP-9-dependent inflammatory signalling in senescence.
      • Chiao Y.A.
      • Ramirez T.A.
      • Zamilpa R.
      • et al.
      Matrix metalloproteinase-9 deletion attenuates myocardial fibrosis and diastolic dysfunction in ageing mice.
      • Ma Y.
      • Chiao Y.A.
      • Zhang J.
      • Manicone A.M.
      • Jin Y.F.
      • Lindsey M.L.
      Matrix metalloproteinase-28 deletion amplifies inflammatory and extracellular matrix responses to cardiac aging.
      By genetic fate mapping and parabiotic approaches, Molawi et al. have reported that with age, self-renewal of resident cardiac macrophages in mice declines, and blood monocytes increasingly contribute to the cardiac macrophage population.
      • Molawi K.
      • Wolf Y.
      • Kandalla P.K.
      • et al.
      Progressive replacement of embryo-derived cardiac macrophages with age.
      Although this study used young mice (2- to 9-month old),
      • Molawi K.
      • Wolf Y.
      • Kandalla P.K.
      • et al.
      Progressive replacement of embryo-derived cardiac macrophages with age.
      it is likely that in the aging heart, macrophages derive from both mechanisms (self-renewal and blood monocyte differentiation) (Fig 1).

       Macrophage roles

      Aging involves an upregulation in the basal inflammatory response, a process termed inflammaging.
      • Baylis D.
      • Bartlett D.B.
      • Patel H.P.
      • Roberts H.C.
      Understanding how we age: insights into inflammaging.
      • Wang M.
      • Shah A.M.
      Age-associated pro-inflammatory remodeling and functional phenotype in the heart and large arteries.
      The macrophage is a key contributor, evidenced by increased numbers of cardiac macrophages and enhanced levels of proinflammatory molecules such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, matrix metalloproteinases (MMPs), and chemokine C-C motif ligand-2 (CCL2)/monocyte chemoattractant protein-1 during cardiac aging.
      • Chiao Y.A.
      • Dai Q.
      • Zhang J.
      • et al.
      Multi-analyte profiling reveals matrix metalloproteinase-9 and monocyte chemotactic protein-1 as plasma biomarkers of cardiac aging.
      Macrophages produce MMP-9 and CCL2, both of which positively correlate with the increase in LV dimensions, indicating a role for macrophages in cardiac aging.
      • Chiao Y.A.
      • Dai Q.
      • Zhang J.
      • et al.
      Multi-analyte profiling reveals matrix metalloproteinase-9 and monocyte chemotactic protein-1 as plasma biomarkers of cardiac aging.
      Immunosenescence refers to the gradual deterioration of the immune system with age, with concomitant higher incidences of infection, neoplasia, autoimmune, and cardiovascular diseases, as well as a worse prognosis after infection or injury.
      • Linton P.J.
      • Thoman M.L.
      Immunosenescence in monocytes, macrophages, and dendritic cells: lessons learned from the lung and heart.
      • Aw D.
      • Silva A.B.
      • Palmer D.B.
      Immunosenescence: emerging challenges for an ageing population.
      • Pawelec G.
      Immunosenescence: impact in the young as well as the old?.
      Compared to young controls (10–12 week old), splenic macrophages from 18-20 month old mice exhibit reduced responses to a variety of proinflammatory or anti-inflammatory stimuli, indicative of age-induced desensitization.
      • Mahbub S.
      • Deburghgraeve C.R.
      • Kovacs E.J.
      Advanced age impairs macrophage polarization.
      Old macrophages also display impaired phagocytic capacity and reduced production of nitric oxide and hydrogen peroxide.
      • Khare V.
      • Sodhi A.
      • Singh S.M.
      Effect of aging on the tumoricidal functions of murine peritoneal macrophages.
      • Ding A.
      • Hwang S.
      • Schwab R.
      Effect of aging on murine macrophages. Diminished response to IFN-gamma for enhanced oxidative metabolism.
      Macrophages from old mice produce higher levels of immunosuppressive prostaglandin E2, which contributes to dysregulated immune function.
      • Beharka A.A.
      • Wu D.
      • Han S.N.
      • Meydani S.N.
      Macrophage prostaglandin production contributes to the age-associated decrease in T cell function which is reversed by the dietary antioxidant vitamin E.
      Taken together, aging induces immune senescence to increase the susceptibility to and poor prognosis after cardiovascular disease.
      • Dace D.S.
      • Apte R.S.
      Effect of senescence on macrophage polarization and angiogenesis.

       Macrophage phenotypes

      How the different subpopulations of resident cardiac macrophages in the steady state change with age in mice has been evaluated. Using flow cytometry, our laboratory has shown that there is a linear increase in cardiac proinflammatory M1 (F4/80+CD206) macrophages and a decrease in anti-inflammatory M2 macrophages (F4/80+CD206+) with age.
      • Ma Y.
      • Chiao Y.A.
      • Clark R.
      • et al.
      Deriving a cardiac ageing signature to reveal MMP-9-dependent inflammatory signalling in senescence.
      This is prevented by MMP-9 deletion, indicating that MMP-9 modifies aging related macrophage polarization.
      • Ma Y.
      • Chiao Y.A.
      • Clark R.
      • et al.
      Deriving a cardiac ageing signature to reveal MMP-9-dependent inflammatory signalling in senescence.
      The fact that in vitro MMP-9 alone activates young macrophages to an M1/M2 mid-transition phase implies that other unknown factors contribute to in vivo age-induced macrophage M1 polarization.
      • Ma Y.
      • Chiao Y.A.
      • Clark R.
      • et al.
      Deriving a cardiac ageing signature to reveal MMP-9-dependent inflammatory signalling in senescence.
      Increased inflammatory macrophages during cardiac aging may be a result of exaggerated monocyte recruitment, alterations in monocyte fate specification, or changes in resident macrophage behavior. The precise functions of aging associated M1 and M2 macrophages and how many additional macrophage phenotypes there are need to be determined.

      Macrophages in the MI heart

       Macrophage origins

      Following MI, abundant blood monocytes infiltrate the ischemic and border regions and differentiate into macrophages, which is the major source of infarct macrophages during the first 7 days post-MI (Fig 2).
      • Heidt T.
      • Courties G.
      • Dutta P.
      • et al.
      Differential contribution of monocytes to heart macrophages in steady-state and after myocardial infarction.
      Immediately on exposure to ischemia, resident cardiac macrophages begin to die and are almost completely removed by 24 h post-MI in mice; and their numbers begin to recover by day 4 post-MI. After the acute phase of monocyte recruitment in the first 2 weeks post-MI, resident macrophages in the infarct regain independence from blood monocytes and can proliferate locally.
      • Heidt T.
      • Courties G.
      • Dutta P.
      • et al.
      Differential contribution of monocytes to heart macrophages in steady-state and after myocardial infarction.
      This is perhaps caused by the differentiation of local progenitor or stem cells into tissue resident macrophages.
      Fig 2
      Fig 2Macrophage origins in the postmyocardial infarction (MI) heart. Following MI, bone marrow hematopoiesis and extramedullary hematopoiesis by the spleen produce abundant numbers of monocytes, which translocate to the circulation and are recruited to the ischemic heart. In the infarct area, the vast majority of macrophages in the first 3 days are derived from recruitment of blood monocytes, and the renewal of resident macrophages is trivial; in contrast, in the nonischemic remote myocardium, macrophages arise from both local renewal of resident macrophages and recruitment of blood monocytes.
      The images of cells and organs were obtained from Servier Medical Art (www.servier.com).
      Remote nonischemic myocardium also exhibits alterations in inflammation and macrophage numbers after MI, albeit with lower and delayed changes compared to the infarct area. We have previously shown that the remote region has more inflammation than the infarct region at day 28 post-MI, indicating a secondary inflammatory response that occurs at a late time point and distant location.
      • Ramirez T.A.
      • Iyer R.P.
      • Ghasemi O.
      • et al.
      Aliskiren and valsartan mediate left ventricular remodeling post-myocardial infarction in mice through MMP-9 effects.
      Sager et al. recently reported that macrophage numbers in the mouse remote myocardium increased 2.9-fold at 8 weeks after MI, which resulted from both local macrophage renewal and blood monocyte recruitment (Fig 2).
      • Sager H.B.
      • Hulsmans M.
      • Lavine K.J.
      • et al.
      Proliferation and recruitment contribute to myocardial macrophage expansion in chronic heart failure.
      More importantly, inhibition of monocyte extravasation into the cardiac tissue by silencing 5 endothelial cell adhesion molecules (Icam1, Icam2, Vcam1, E-selectin, and P-selectin) decreased macrophage numbers and improved cardiac physiology, suggesting that macrophages contribute to adverse remodeling of the remote myocardium.
      • Sager H.B.
      • Hulsmans M.
      • Lavine K.J.
      • et al.
      Proliferation and recruitment contribute to myocardial macrophage expansion in chronic heart failure.
      In this study, the authors also revealed that mechanical strain, the deformation of the heart, could elicit macrophage proliferation.
      • Sager H.B.
      • Hulsmans M.
      • Lavine K.J.
      • et al.
      Proliferation and recruitment contribute to myocardial macrophage expansion in chronic heart failure.
      During the acute inflammatory phase, the majority of macrophages recruited to the ischemic area derive from the differentiation of peripheral blood monocytes, which stem from 2 sources: bone marrow and spleen. The following paragraphs provide detailed information on these sources.

       Peripheral blood monocytes

      Mouse blood monocytes are heterogeneous and have 2 subsets based on Ly6C expression. Ly6Chi monocytes are inflammatory and express large amounts of chemokine C-X-C motif receptor (CCR) 2 and low chemokine C-X3-C motif receptor (CX3CR) 1 level; and Ly6Clow monocytes low CCR2 and high CX3CR1.
      • Gombozhapova A.
      • Rogovskaya Y.
      • Shurupov V.
      • et al.
      Macrophage activation and polarization in post-infarction cardiac remodeling.
      In the steady state, 50%–60% of circulating mouse monocytes are Ly6ChiCCR2highCX3CR1lowCD62 L+, and have a relatively short life span.
      • Nahrendorf M.
      • Swirski F.K.
      Monocyte and macrophage heterogeneity in the heart.
      Ly6Clow monocytes arise from Ly6Chi cell conversion, instead of from different progenitors.
      • Yona S.
      • Kim K.W.
      • Wolf Y.
      • et al.
      Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis.
      • Sunderkotter C.
      • Nikolic T.
      • Dillon M.J.
      • et al.
      Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.
      Likewise, the circulating monocyte pool in humans is heterogeneous and divided into 3 phenotypes based on CD14 and CD16 expression. CD14++CD16 monocytes, which resemble mouse Ly6Chi monocytes, account for 80%–90% of total circulating monocytes and are proinflammatory. CD14+CD16++ cells are anti-inflammatory and resemble mouse Ly-6Clow population in terms of function.
      • Ingersoll M.A.
      • Spanbroek R.
      • Lottaz C.
      • et al.
      Comparison of gene expression profiles between human and mouse monocyte subsets.
      • Cros J.
      • Cagnard N.
      • Woollard K.
      • et al.
      Human CD14dim monocytes patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors.
      The third population of CD14++CD16+ cells has proinflammatory features and can secrete proinflammatory TNF-α after exposure to lipopolysaccharide (LPS).
      • Dutta P.
      • Nahrendorf M.
      Monocytes in myocardial infarction.

       Bone marrow hematopoiesis

      Circulating monocytes derive from hematopoietic stem cells (HSCs) residing in the bone marrow. HSCs sequentially differentiate into common myeloid progenitors, granulocyte macrophage progenitors, macrophage monocyte progenitors, common monocyte progenitors, and ultimately monocytes in the bone marrow, a process termed hematopoiesis.
      • Geissmann F.
      • Manz M.G.
      • Jung S.
      • Sieweke M.H.
      • Merad M.
      • Ley K.
      Development of monocytes, macrophages, and dendritic cells.
      • Frantz S.
      • Nahrendorf M.
      Cardiac macrophages and their role in ischaemic heart disease.
      Numerous housekeeping cells, including mesenchymal stem cells, endothelial cells, CD169+ macrophages, nerve cells, and osteoblasts reside in the hematopoietic niche and regulate blood cell production. They produce growth factors and fate-regulating signals such as granulocyte colony-stimulating factor, angiopoietin-1, chemokine C-X-C motif ligand (CXCL) 12, and stem cell factor.
      • Frantz S.
      • Nahrendorf M.
      Cardiac macrophages and their role in ischaemic heart disease.
      • Ehninger A.
      • Trumpp A.
      The bone marrow stem cell niche grows up: mesenchymal stem cells and macrophages move in.
      • Scadden D.T.
      The stem-cell niche as an entity of action.
      CXCL12 facilitates quiescent HSC retention in the bone marrow.
      • Sugiyama T.
      • Kohara H.
      • Noda M.
      • Nagasawa T.
      Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches.
      Lower CXCL12 levels after MI lead to the liberation of HSCs from the bone marrow niche, which then migrate to the spleen and differentiate into monocytes that are subsequently recruited to the infarct.
      • Dutta P.
      • Courties G.
      • Wei Y.
      • et al.
      Myocardial infarction accelerates atherosclerosis.
      CCR2 and β-adrenergic receptor signaling cascade also mediate monocyte mobilization from the bone marrow to circulation (Fig 2).
      • Dutta P.
      • Courties G.
      • Wei Y.
      • et al.
      Myocardial infarction accelerates atherosclerosis.
      • Serbina N.V.
      • Pamer E.G.
      Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2.

       Spleen extramedullary hematopoiesis

      The spleen is an additional reservoir for monocytes that actually outnumbers their equivalents in circulation and contributes to the blood monocyte pool. The spleen can generate new monocytes by a process called extramedullary hematopoiesis.
      • Robbins C.S.
      • Chudnovskiy A.
      • Rauch P.J.
      • et al.
      Extramedullary hematopoiesis generates Ly-6C(high) monocytes that infiltrate atherosclerotic lesions.
      • Leuschner F.
      • Rauch P.J.
      • Ueno T.
      • et al.
      Rapid monocyte kinetics in acute myocardial infarction are sustained by extramedullary monocytopoiesis.
      Splenic monocytes are located in the subcapsular red pulp of the spleen and resemble circulating counterparts.
      • Nahrendorf M.
      • Swirski F.K.
      Monocyte and macrophage heterogeneity in the heart.
      Splenic hematopoiesis occurs during embryogenesis and in disease settings but not in the steady state after birth. Post-MI, the spleen produces sufficient monocytes to enter the blood and infiltrate the ischemic myocardium (Fig 2). This process is at least partially angiotensin II-dependent and can be attenuated by angiotensin converting enzyme inhibitors.
      • Leuschner F.
      • Panizzi P.
      • Chico-Calero I.
      • et al.
      Angiotensin-converting enzyme inhibition prevents the release of monocytes from their splenic reservoir in mice with myocardial infarction.
      • Swirski F.K.
      • Nahrendorf M.
      • Etzrodt M.
      • et al.
      Identification of splenic reservoir monocytes and their deployment to inflammatory sites.
      Extramedullary hematopoiesis shrinks the mouse spleen weight by 50% and depletes the number of splenic monocytes by 24 h post-MI.
      • Nahrendorf M.
      • Swirski F.K.
      Monocyte and macrophage heterogeneity in the heart.
      IL-1β, IL-3, and granulocyte macrophage colony-stimulating factor (GM-CSF) can modulate the production of splenic monocytes.
      • Leuschner F.
      • Rauch P.J.
      • Ueno T.
      • et al.
      Rapid monocyte kinetics in acute myocardial infarction are sustained by extramedullary monocytopoiesis.
      More importantly, abrogation of extramedullary monocytopoiesis in mice exacerbates adverse cardiac remodeling and heart failure progression, indicating that spleen-derived monocytes are essential for post-MI cardiac repair.
      • Leuschner F.
      • Rauch P.J.
      • Ueno T.
      • et al.
      Rapid monocyte kinetics in acute myocardial infarction are sustained by extramedullary monocytopoiesis.

       Macrophage roles

      As early as 30 min following MI, blood monocytes infiltrate the infarct, initially outnumbering neutrophils.
      • Jung K.
      • Kim P.
      • Leuschner F.
      • et al.
      Endoscopic time-lapse imaging of immune cells in infarcted mouse hearts.
      • Ma Y.
      • Yabluchanskiy A.
      • Iyer R.P.
      • et al.
      Temporal neutrophil polarization following myocardial infarction.
      Recruitment of monocytes is dependent on activation of the CCL2/CCR2 signaling pathway.
      • Dewald O.
      • Zymek P.
      • Winkelmann K.
      • et al.
      CCL2/Monocyte Chemoattractant Protein-1 regulates inflammatory responses critical to healing myocardial infarcts.
      • Kaikita K.
      • Hayasaki T.
      • Okuma T.
      • Kuziel W.A.
      • Ogawa H.
      • Takeya M.
      Targeted deletion of CC chemokine receptor 2 attenuates left ventricular remodeling after experimental myocardial infarction.
      On arrival at the tissue, these monocytes begin to differentiate into macrophages. Some monocytes, however, do not undergo differentiation; these monocytes serve similar tissue roles as their macrophage counterparts.
      • Nahrendorf M.
      • Swirski F.K.
      • Aikawa E.
      • et al.
      The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions.
      Delineation between monocytes and macrophages in mice has been shown based on the expression of F4/80/I-Ab/CD11c. Monocytes are (F4/80/I-Ab/CD11c)low, whereas macrophages are (F4/80/I-Ab/CD11c)high.
      • Nahrendorf M.
      • Swirski F.K.
      • Aikawa E.
      • et al.
      The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions.
      CD64 and myeloid epithelial reproductive tyrosine kinase (MERTK) can also distinguish cardiac monocytes from cardiac macrophages, with macrophages expressing both CD64 and MERTK and monocytes expressing CD64 and not MERTK.
      • Epelman S.
      • Lavine K.J.
      • Beaudin A.E.
      • et al.
      Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.
      • Gautier E.L.
      • Shay T.
      • Miller J.
      • et al.
      Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages.
      In the mouse MI model, macrophages are in the infarct peak at days 5–7 after MI.
      • Ma Y.
      • Halade G.V.
      • Zhang J.
      • et al.
      Matrix metalloproteinase-28 deletion exacerbates cardiac dysfunction and rupture after myocardial infarction in mice by inhibiting M2 macrophage activation.
      • Yan X.
      • Anzai A.
      • Katsumata Y.
      • et al.
      Temporal dynamics of cardiac immune cell accumulation following acute myocardial infarction.
      • Troidl C.
      • Mollmann H.
      • Nef H.
      • et al.
      Classically and alternatively activated macrophages contribute to tissue remodelling after myocardial infarction.
      MI patients show similar but delayed kinetics of macrophage infiltration compared to rodents. Timely reperfusion reduces leukocyte numbers accumulated in the infarct, shifts the peak of the innate immune response earlier, and blunts the adaptive immune response.
      • Yan X.
      • Anzai A.
      • Katsumata Y.
      • et al.
      Temporal dynamics of cardiac immune cell accumulation following acute myocardial infarction.
      Although inflammation is essential for orchestrating post-MI cardiac repair, timely resolution is necessary for favorable cardiac repair. Following MI, there is a burst of acute inflammation over the first 5 days. After this period, inflammation gradually wanes. Targeting monocyte recruitment to attenuate inflammation is protective by enhancing myocardial repair.
      • Sager H.B.
      • Hulsmans M.
      • Lavine K.J.
      • et al.
      Proliferation and recruitment contribute to myocardial macrophage expansion in chronic heart failure.
      • Leuschner F.
      • Dutta P.
      • Gorbatov R.
      • et al.
      Therapeutic siRNA silencing in inflammatory monocytes in mice.
      Phagocytosis of apoptotic myocytes and neutrophils by macrophages is a prerequisite for the resolution of inflammation. Impaired macrophage phagocytic capacity prolongs inflammation and impedes post-MI cardiac repair.
      • Wan E.
      • Yeap X.Y.
      • Dehn S.
      • et al.
      Enhanced efferocytosis of apoptotic cardiomyocytes through myeloid-epithelial-reproductive tyrosine kinase links acute inflammation resolution to cardiac repair after infarction.
      Macrophages play pivotal roles in the post-MI wound healing response. Macrophage depletion compromises wound healing and accelerates adverse remodeling, and adoptive transfer of activated macrophages improves cardiac repair.
      • Frantz S.
      • Hofmann U.
      • Fraccarollo D.
      • et al.
      Monocytes/macrophages prevent healing defects and left ventricular thrombus formation after myocardial infarction.
      • Ben-Mordechai T.
      • Holbova R.
      • Landa-Rouben N.
      • et al.
      Macrophage subpopulations are essential for infarct repair with and without stem cell therapy.
      • van Amerongen M.J.
      • Harmsen M.C.
      • van Rooijen N.
      • Petersen A.H.
      • van Luyn M.J.
      Macrophage depletion impairs wound healing and increases left ventricular remodeling after myocardial injury in mice.
      • Leor J.
      • Rozen L.
      • Zuloff-Shani A.
      • et al.
      Ex vivo activated human macrophages improve healing, remodeling, and function of the infarcted heart.
      Similarly, clinical findings demonstrate that patients with high inflammatory CD14+CD16 blood monocyte counts at the onset of MI have larger cardiac dilation at follow-up, and the peak levels of CD14+CD16 monocytes negatively correlate with the extent of myocardial salvage.
      • Tsujioka H.
      • Imanishi T.
      • Ikejima H.
      • et al.
      Impact of heterogeneity of human peripheral blood monocyte subsets on myocardial salvage in patients with primary acute myocardial infarction.
      • Maekawa Y.
      • Anzai T.
      • Yoshikawa T.
      • et al.
      Prognostic significance of peripheral monocytosis after reperfused acute myocardial infarction:a possible role for left ventricular remodeling.
      The macrophage coordinates each phase of the remodeling process, including the acute inflammatory, reparative, and maturation phases. Macrophage roles include: (1) secreting an extensive array of inflammatory cytokines, chemokines, growth factors, and MMPs to regulate inflammation and degrade the extracellular matrix; (2) phagocytizing dead cell and tissue debris to clean up the wound; (3) producing proangiogenic and proreparative factors (eg, vascular endothelial growth factor and transforming growth factor [TGF]-β1) to facilitate neoangiogenesis and scar building; and (4) presenting antigen to lymphocytes to induce an adaptive immune response.
      • Ben-Mordechai T.
      • Palevski D.
      • Glucksam-Galnoy Y.
      • Elron-Gross I.
      • Margalit R.
      • Leor J.
      Targeting macrophage subsets for infarct repair.
      Neonate mice depleted of macrophages lose their myocardial regenerative capacity,
      • Aurora A.B.
      • Porrello E.R.
      • Tan W.
      • et al.
      Macrophages are required for neonatal heart regeneration.
      and these reparative macrophages are embryonic and not monocyte-derived.
      • Lavine K.J.
      • Epelman S.
      • Uchida K.
      • et al.
      Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart.
      These findings indicate that macrophages may mediate myocardial regeneration in the neonatal heart. This field is controversial, and the results need to be further validated. The multi-functional capacity of macrophages is, at least partially, attributed to different cell polarization phenotypes.

       Macrophage phenotypes

      Macrophages demonstrate high plasticity and adaptability, both in vitro and in vivo. They can adopt differential phenotypes in response to varying stimuli or when residing in varying environments. Macrophages have been classified into classically activated (M1) and alternatively activated (M2) subsets.
      • Sica A.
      • Mantovani A.
      Macrophage plasticity and polarization: in vivo veritas.
      Macrophage subsets are further divided based on the in vitro stimuli to which they are exposed. For instance, M1 macrophages can be divided into M1a if stimulated with toll-like receptors or M1b subsets if stimulated with high-mobility group protein B1.
      • Ben-Mordechai T.
      • Palevski D.
      • Glucksam-Galnoy Y.
      • Elron-Gross I.
      • Margalit R.
      • Leor J.
      Targeting macrophage subsets for infarct repair.
      Subsets also have distinct cell physiology; for example, M1b is less phagocytic than M1a.
      M2 macrophages are further subdivided into M2a if stimulated with IL-4 or IL-13 and M2b if stimulated with immune complexes in combination with IL-1β and M2c if stimulated with IL-10, TGF-β, or glucocorticoids.
      • Gombozhapova A.
      • Rogovskaya Y.
      • Shurupov V.
      • et al.
      Macrophage activation and polarization in post-infarction cardiac remodeling.
      • Martinez F.O.
      • Sica A.
      • Mantovani A.
      • Locati M.
      Macrophage activation and polarization.
      M2a and M2c macrophages are primarily responsible for coordinating adaptive immune response, whereas M2b macrophages suppress inflammation.
      • Ben-Mordechai T.
      • Palevski D.
      • Glucksam-Galnoy Y.
      • Elron-Gross I.
      • Margalit R.
      • Leor J.
      Targeting macrophage subsets for infarct repair.
      • Lindsey M.L.
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      Knowledge gaps to understanding cardiac macrophage polarization following myocardial infarction.
      More recently, an M4 phenotype has been proposed to describe monocytes exposed to CXCL4.
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      CXC chemokine ligand 4 induces a unique transcriptome in monocyte-derived macrophages.
      Moreover, different phenotypes can mutually convert under in vitro conditions. For example, M1 macrophages could switch to the M2 phenotype after stimulation with pro-M2 factors, and vice versa.
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      Dynamics of macrophage polarization reveal new mechanism to inhibit IL-1beta release through pyrophosphates.
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      Macrophages in the infarcted heart are heterogeneous. M1 macrophages dominate at days 1–3 post-MI, whereas M2 macrophages are the major cell at days 5–7 post-MI in the mouse heart.
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      • Katsumata Y.
      • et al.
      Temporal dynamics of cardiac immune cell accumulation following acute myocardial infarction.
      Proinflammatory M1 macrophages secrete cytokines, chemokines, growth factors, and MMPs to help clear the cell debris and degrade extracellular matrix (Table III).
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      Modulators of macrophage polarization Influence healing of the infarcted myocardium.
      However, the prolonged presence of M1 macrophages can lead to expansion of infarct size and impede the resolution of inflammation and scar formation.
      • Leuschner F.
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      • et al.
      Therapeutic siRNA silencing in inflammatory monocytes in mice.
      In contrast, anti-inflammatory M2 macrophages are proreparative.
      • ter Horst E.N.
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      • Krijnen P.A.
      • Niessen H.W.
      • Piek J.J.
      Modulators of macrophage polarization Influence healing of the infarcted myocardium.
      M2 macrophages can produce anti-inflammatory, proangiogenic, and proreparative factors (eg, IL-10, vascular endothelial growth factor, and TGF-β1) and engulf apoptotic cells to facilitate neoangiogenesis and scar repair (Table III). Shifting the balance from M1 to M2 macrophages improves myocardial repair and function post-MI.
      • Harel-Adar T.
      • Ben Mordechai T.
      • Amsalem Y.
      • Feinberg M.S.
      • Leor J.
      • Cohen S.
      Modulation of cardiac macrophages by phosphatidylserine-presenting liposomes improves infarct repair.
      • Courties G.
      • Heidt T.
      • Sebas M.
      • et al.
      In vivo silencing of the transcription factor IRF5 reprograms the macrophage phenotype and improves infarct healing.
      • Zhou L.S.
      • Zhao G.L.
      • Liu Q.
      • Jiang S.C.
      • Wang Y.
      • Zhang D.M.
      Silencing collapsin response mediator protein-2 reprograms macrophage phenotype and improves infarct healing in experimental myocardial infarction model.
      • Weirather J.
      • Hofmann U.D.
      • Beyersdorf N.
      • et al.
      Foxp3+ CD4+ T cells improve healing after myocardial infarction by modulating monocyte/macrophage differentiation.
      Likewise, our laboratory has shown that MMP-9, MMP-28, and IL-10 regulate post-MI cardiac remodeling by affecting the M1/M2 balance.
      • Ma Y.
      • Halade G.V.
      • Zhang J.
      • et al.
      Matrix metalloproteinase-28 deletion exacerbates cardiac dysfunction and rupture after myocardial infarction in mice by inhibiting M2 macrophage activation.
      • Jung M.
      • Ma Y.
      • Iyer R.P.
      • et al.
      IL-10 improves cardiac remodeling after myocardial infarction by stimulating M2 macrophage polarization and fibroblast activation.
      • Yabluchanskiy A.
      • Ma Y.
      • DeLeon-Pennell K.Y.
      • et al.
      Myocardial infarction Superimposed on aging: MMP-9 deletion promotes M2 macrophage polarization.
      • DeLeon-Pennell K.Y.
      • de Castro Bras L.E.
      • Iyer R.P.
      • et al.
      P. gingivalis lipopolysaccharide intensifies inflammation post-myocardial infarction through matrix metalloproteinase-9.
      • Zamilpa R.
      • Ibarra J.
      • de Castro Bras L.E.
      • et al.
      Transgenic overexpression of matrix metalloproteinase-9 in macrophages attenuates the inflammatory response and improves left ventricular function post-myocardial infarction.
      • Zamilpa R.
      • Kanakia R.
      • Cigarroa 4th, J.
      • et al.
      CC chemokine receptor 5 deletion impairs macrophage activation and induces adverse remodeling following myocardial infarction.
      Table IIICharacteristics of MI-associated proinflammatory and anti-inflammatory macrophages
      • Gombozhapova A.
      • Rogovskaya Y.
      • Shurupov V.
      • et al.
      Macrophage activation and polarization in post-infarction cardiac remodeling.
      • Yan X.
      • Anzai A.
      • Katsumata Y.
      • et al.
      Temporal dynamics of cardiac immune cell accumulation following acute myocardial infarction.
      • Ben-Mordechai T.
      • Palevski D.
      • Glucksam-Galnoy Y.
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      • Margalit R.
      • Leor J.
      Targeting macrophage subsets for infarct repair.
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      • Mantovani A.
      Macrophage plasticity and polarization: in vivo veritas.
      • Jung M.
      • Ma Y.
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      • et al.
      IL-10 improves cardiac remodeling after myocardial infarction by stimulating M2 macrophage polarization and fibroblast activation.
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      • Yoshikawa T.
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      Effect of granulocyte-macrophage colony-stimulating factor inducer on left ventricular remodeling after acute myocardial infarction.
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      Hydrogen Sulfide Mitigates myocardial infarction via promotion of Mitochondrial Biogenesis-dependent M2 polarization of macrophages.
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      • Li P.
      • Hu F.
      • Wang Y.
      • Chai X.
      • Zhang Y.
      IL-33 attenuates cardiac remodeling following myocardial infarction via inhibition of the p38 MAPK and NF-kappaB pathways.
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      • et al.
      Systemic and cardiac depletion of M2 macrophage through CSF-1R signaling inhibition Alters cardiac function post myocardial infarction.
      • Tugal D.
      • Liao X.
      • Jain M.K.
      Transcriptional control of macrophage polarization.
      ProinflammatoryAnti-inflammatory
      StimuliGM-CSF, IFN-γ, TNF-α, IL-1βHydrogen sulfide, IL-4, IL-10, IL-13, IL-33, TGF-β1, M-CSF
      Transcription factorsAP-1, HIF-1α, IRF3, IRF5, NF-κB, STAT1c-Maf, c-Myc, IRF4, JMJD3, KLF4, PPAR-γ, STAT3, STAT6
      MarkersCCL2 (MCP1), CCL3 (MIP1a), CCL4 (MIP1b), CCL5 (RANTES), CCL7, CCL8, CCR2, CD80, CD86, CXCL1, CXCL2, CXCL6, CXCL8 (IL-8), CXCL9, CXCL10, CXCL11, CXCL16, IL-1β, IL-6, IL-12, IL-23, iNOS, MHCII, RNS, ROS, S100a8, S100a9, TNF-αArg1, CCL1, CCL16, CCL17, CCL18, CCL22, CCL24, CXCL13, CXCL17, CXCL22, CXCL24, CXCR1, CXCR2, CD163, CD206 (MRC1), CD280 (MRC2), Cd301a (Clec10a, Mgl1), Cd301 b (Mgl2), Dectin-1, Fizz1 (Retnla, Relmα), IL-10, PGE2, Spp1 (osteopontin), Stabilin1, TGF-β1, VEGF, Ym1 (Chi3l3)
      Cell physiologyProinflammation; proteolysis; phagocytosis of debris; antigen presentation to lymphocytesAnti-inflammation and resolution of inflammation; phagocytosis of apoptotic cells; pro-angiogenesis; ECM production and scar formation
      Abbreviations: AP-1, activator protein 1; HIF-1α, hypoxia-inducible factor-1α; IRF, interferon-regulatory factor; NF-κB, nuclear factor-κB; STAT, signal transducer and activator of transcription; KLF4, Kruppel-like factor 4; PPAR-γ, peroxisome proliferator-activated receptor-γ; iNOS, inducible nitric oxide synthase; RNS, reactive nitrogen species; ROS, reactive oxygen species; M-CSF, macrophage colony-stimulating factor; Arg1, arginase 1; CXCR, C-X-C chemokine receptor; Fizz1, found in inflammatory zone1; VEGF, vascular endothelial growth factor.

       Macrophage polarization mechanisms

      The exact mechanisms regarding post-MI in vivo macrophage polarization remain poorly understood. Nahrendorf et al. previously identified sequential infiltration of 2 distinct monocyte subsets into the ischemic heart.
      • Nahrendorf M.
      • Swirski F.K.
      • Aikawa E.
      • et al.
      The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions.
      Ly6Chigh monocytes predominate at days 1–4 post-MI due to selective expansion, whereas cardiac Ly6Clow cells dominate from days 5 onward due to increased migration capacity. Using genetically modified mice, they also showed that early Ly6Chigh subset recruitment relies on CCR2, whereas later Ly6Clow accumulation depends on CX3CR1 (Fig 3). In addition, Ly6Chigh monocytes can differentiate into Ly6Clow monocytes during the reparative phase and proliferate locally.
      • Hilgendorf I.
      • Gerhardt L.M.
      • Tan T.C.
      • et al.
      Ly-6Chigh monocytes depend on Nr4a1 to balance both inflammatory and reparative phases in the infarcted myocardium.
      • Dutta P.
      • Nahrendorf M.
      Monocytes in myocardial infarction.
      Ly6Chigh monocytes are proinflammatory, phagocytic, and proteolytic; in contrast, Ly6Clow monocytes are anti-inflammatory, proangiogenic, and proreparative.
      • Nahrendorf M.
      • Swirski F.K.
      • Aikawa E.
      • et al.
      The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions.
      The monocyte time course reported by Nahrendorf et al. is completely consistent with the time course of macrophages reported by Yan et al.
      • Yan X.
      • Anzai A.
      • Katsumata Y.
      • et al.
      Temporal dynamics of cardiac immune cell accumulation following acute myocardial infarction.
      As both Ly6Chigh and Ly6Clow cells circulate in the blood, and recruitment is the major source of infarct macrophages, it is reasonable to conclude that infarct proinflammatory M1-like and anti-inflammatory M2-like macrophages are derived from blood Ly6Chigh and Ly6Clow monocytes, respectively. This hypothesis, however, ignores the impact of the local microenvironment on macrophage polarization. Inflammatory monocytes can switch their phenotype to an anti-inflammatory subset and further differentiate into M2-like macrophages in models of skeletal muscle injury and allergic skin.
      • Arnold L.
      • Henry A.
      • Poron F.
      • et al.
      Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis.
      • Egawa M.
      • Mukai K.
      • Yoshikawa S.
      • et al.
      Inflammatory monocytes recruited to allergic skin acquire an anti-inflammatory M2 phenotype via basophil-derived interleukin-4.
      Peritoneal macrophages acquire features of pulmonary macrophages after adoptive transfer to the lung.
      • Lavin Y.
      • Winter D.
      • Blecher-Gonen R.
      • et al.
      Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment.
      These findings highlight a deterministic role for the microenvironment in guiding polarization of monocytes and macrophages. The infarct microenvironment is filled with early pro-M1 mediators (eg, IFN-γ and GM-CSF) and later pro-M2 factors (eg, IL-10 and TGF-β1), which likely direct macrophage polarization.
      • Jung M.
      • Ma Y.
      • Iyer R.P.
      • et al.
      IL-10 improves cardiac remodeling after myocardial infarction by stimulating M2 macrophage polarization and fibroblast activation.
      • Christia P.
      • Bujak M.
      • Gonzalez-Quesada C.
      • et al.
      Systematic characterization of myocardial inflammation, repair, and remodeling in a mouse model of reperfused myocardial infarction.
      However, the short life span of post-MI monocytes (∼20 h) suggests that phenotypic conversion has to be very rapid if it occurs in the MI setting. Alternatively, conversion may account for a small percentage (∼20%) of the macrophage pool. Although it is likely that the 2 mechanisms combined determine macrophage polarization (Fig 3), these ideas need to be validated in future experiments. Table III lists known characteristics of MI-associated M1 and M2 macrophages. Additional studies are warranted to systematically identify pathway networks that coordinate post-MI macrophage polarization.
      Fig 3
      Fig 3Proposed post-MI macrophage polarization mechanisms. The integration of 2 mechanisms determines the polarization status of macrophages in the MI heart. (1) M1 and M2 macrophages originate from circulating Ly6Chigh and Ly6Clow monocytes, respectively; Recruitment of Ly6Chigh monocytes depends on CCR2 signaling, whereas recruitment of Ly6Clow monocytes is CX3CR1 dependent; and (2) the mix of pro-M1 and pro-M2 factors existing in the MI myocardium orchestrates macrophage polarization status. MI, myocardial infarction.
      The images of cells and organs were obtained from Servier Medical Art (www.servier.com).

      Future Directions in our Understanding of Post-MI Macrophage Polarization

      The M1 and M2 nomenclature has been helpful for appreciating the heterogeneity of macrophages. The M1/M2 paradigm, however, was originally based on the in vitro stimuli used, surface marker expression, and production of inflammatory associated factors. For instance, LPS + IFN-γ induce macrophage production of proinflammatory Ccl3, IL-1β, IL-6, and TNF-α; and thus, this macrophage is termed M1. IL-4 elicits macrophages to produce anti-inflammatory Cd206, Arg1, Fizz1, and Ym1, namely M2 macrophage markers.
      • Ma Y.
      • Halade G.V.
      • Zhang J.
      • et al.
      Matrix metalloproteinase-28 deletion exacerbates cardiac dysfunction and rupture after myocardial infarction in mice by inhibiting M2 macrophage activation.
      The main limitation with this nomenclature system is that the simple in vitro setting does not reflect the complex in vivo microenvironment. In the ischemic heart, there is a complex mixture of both pro-M1 and pro-M2 stimuli. Defining M1 or M2 phenotype based only on 1 marker (eg, CD206), or even a combination of several M1/M2 markers, does not reflect the in vivo situation. One simple example is that CD206 could not distinguish pre-MI resident vs post-MI M2 macrophages, as both express high levels of CD206.
      • Shiraishi M.
      • Shintani Y.
      • Shintani Y.
      • et al.
      Alternatively activated macrophages determine repair of the infarcted adult murine heart.
      Second, it is arbitrary to force in vivo data onto an in vitro M1/M2 spectrum.
      • Nahrendorf M.
      • Swirski F.K.
      Abandoning M1/M2 for a network model of macrophage function.
      Frequently, there is a mixture of M1 or M2 markers that may not follow the simplified in vitro pattern. Macrophages overall may have more total M1 markers, while displaying divergence in particular markers (eg, less TNF-α or IL-10). If only TNF-α and IL-10 are measured and are lower in the comparison group, one might conclude these cells were M2, which would not be accurate based on the other M1 markers. Third, one assumption this classification system makes is that all stimuli induce macrophages to the same phenotype. For example, although LPS + IFN-γ and GM-CSF both trigger an M1 phenotype, transcriptional profiles induced by these 2 stimuli vary, indicating that M1 does not equal M1.
      • Mantovani A.
      • Sica A.
      • Sozzani S.
      • Allavena P.
      • Vecchi A.
      • Locati M.
      The chemokine system in diverse forms of macrophage activation and polarization.
      • Lehtonen A.
      • Ahlfors H.
      • Veckman V.
      • Miettinen M.
      • Lahesmaa R.
      • Julkunen I.
      Gene expression profiling during differentiation of human monocytes to macrophages or dendritic cells.
      In view of the limitations stated previously about the current M1/M2 polarization paradigm, we discuss here 3 outstanding areas of investigation needed to better understand macrophage polarization in the post-MI LV.
      • Lindsey M.L.
      • Saucerman J.J.
      • DeLeon-Pennell K.Y.
      Knowledge gaps to understanding cardiac macrophage polarization following myocardial infarction.
      (1) The polarization phenotypes of cardiac macrophages at day 0 (before MI) and at varying time points post-MI (eg days 1, 3, and 7) need to be systematically mapped. We propose that there are likely differences in cell phenotypes along the post-MI continuum that span beyond the simple M1/M2 paradigm. As mentioned previously, although day 1 and day 3 macrophages have similar M1 phenotypes in terms of some markers, they are likely different in terms of transcriptional programs and cell physiology. In addition, individual cell phenotypes at a given time may be different. At day 3 post-MI, for example, M1 macrophages may also be heterogeneous, reflecting the exact cytokine and chemokine environments they are exposed to on entry into the infarct region, an environment that is in rapid flux over the first days post-MI. Therefore, we need to know the continuous phenotypes across the time course of MI and the variability across individual cell phenotypes at the same time. The first thing we need to know is what markers distinguish phenotypes. This could be addressed by globally examining transcriptional profiles of macrophages isolated from different time point post-MI using RNA sequencing. Flow cytometry could further distinguish individual cell phenotypes.
      The use of a novel nomenclature system on the basis of post-MI time when macrophages are activated may be a better way to define macrophage phenotypes. For instance, cM(MI-D1) could be used to denote cardiac macrophages at day 1 post-MI. This system could be used for in vitro macrophages stimulated by different factors as well. cM(IL-4) represents resident cardiac macrophages stimulated with IL-4. The advantage of this classification system is that we can more clearly distinguish what cell type is under examination.
      (2) Computational models mimicking post-MI macrophage polarization have not been established.
      • Lindsey M.L.
      • Saucerman J.J.
      • DeLeon-Pennell K.Y.
      Knowledge gaps to understanding cardiac macrophage polarization following myocardial infarction.
      Mathematical algorithms can provide a means to predict outcomes that integrate complex in vivo factors at molecular, cellular, organ, and systemic levels as well as reduce complexity.
      • Yang J.H.
      • Saucerman J.J.
      Computational models reduce complexity and accelerate insight into cardiac signaling networks.
      Algorithms for macrophage physiology have recently been established for some biological processes, such as the acute inflammatory response, chronic wound inflammation, cholesterol efflux, tumor, and iron release.
      • Vodovotz Y.
      • Clermont G.
      • Chow C.
      • An G.
      Mathematical models of the acute inflammatory response.
      • Day J.
      • Friedman A.
      • Schlesinger L.S.
      Modeling the immune rheostat of macrophages in the lung in response to infection.
      • Nagaraja S.
      • Wallqvist A.
      • Reifman J.
      • Mitrophanov A.Y.
      Computational approach to characterize causative factors and molecular indicators of chronic wound inflammation.
      • Gaus K.
      • Gooding J.J.
      • Dean R.T.
      • Kritharides L.
      • Jessup W.
      A kinetic model to evaluate cholesterol efflux from THP-1 macrophages to apolipoprotein A-1.
      • Owen M.R.
      • Sherratt J.A.
      Modelling the macrophage invasion of tumours: effects on growth and composition.
      • Potdar A.A.
      • Sarkar J.
      • Das N.K.
      • et al.
      Computational modeling and analysis of iron release from macrophages.
      These models do not incorporate macrophage polarization nor have models been developed to describe macrophages in the infarcted heart. Our team has previously developed cellular models of macrophage polarization and myocardial remodeling on a limited scale.
      • Jin Y.F.
      • Han H.C.
      • Berger J.
      • Dai Q.
      • Lindsey M.L.
      Combining experimental and mathematical modeling to reveal mechanisms of macrophage-dependent left ventricular remodeling.
      • Wang Y.
      • Yang T.
      • Ma Y.
      • et al.
      Mathematical modeling and stability analysis of macrophage activation in left ventricular remodeling post-myocardial infarction.
      A more complete computational map that includes macrophage activation factors, signaling network, and phenotypic information is warranted. Building these algorithms requires the building of an initial framework to ensure the establishment of optimum models.
      • Ma Y.
      • Iyer R.P.
      • Jung M.
      • Czubryt M.P.
      • Lindsey M.L.
      Cardiac fibroblast activation post-myocardial infarction: current knowledge gaps.
      Macrophage polarization has been defined for the most part by single-stimulus responses; we need to examine how macrophages respond to mixed stimuli. Initial models could focus on short-term in vitro treatment of macrophages with different factors known to regulate macrophage polarization and post-MI remodeling. The structure of the computational algorithm could be based on previously known pathways in conjunction with bridging these inputs to specific downstream genes, secreted proteins, and cell physiology outputs.
      • Lindsey M.L.
      • Saucerman J.J.
      • DeLeon-Pennell K.Y.
      Knowledge gaps to understanding cardiac macrophage polarization following myocardial infarction.
      • Ma Y.
      • Iyer R.P.
      • Jung M.
      • Czubryt M.P.
      • Lindsey M.L.
      Cardiac fibroblast activation post-myocardial infarction: current knowledge gaps.
      Conversion of these initial models into logic-based distinct equations will provide a window for simulations with other key players that may be identified to build on the existing framework.
      • Ma Y.
      • Iyer R.P.
      • Jung M.
      • Czubryt M.P.
      • Lindsey M.L.
      Cardiac fibroblast activation post-myocardial infarction: current knowledge gaps.
      The in silico integration of complex data sets can help define key trigger point responses, combined with bioinformatics analysis to provide a more comprehensive evaluation. Subsequent model iterations could incorporate comprehensive evaluations of transcriptome and secretome profiles, allowing inference of novel players in these processes. Computational models generated for post-MI macrophage polarization could be used in the future to understand cardiac remodeling patterns, which would allow predictions of new therapeutic interventions to be tested, validated, and refined.
      (3) There is a need to know how to modulate endogenous and exogenous targets to generate predictable macrophage polarization subsets.
      • Lindsey M.L.
      • Saucerman J.J.
      • DeLeon-Pennell K.Y.
      Knowledge gaps to understanding cardiac macrophage polarization following myocardial infarction.
      Interfering with endogenous signaling cascades will tell us whether the developed models have successfully defined the key drivers of macrophage polarization and accurately dissected their roles in cardiac remodeling. Similarly, modifying exogenous pathways will tell us whether modifying phenotypes could affect outcomes in a predictable manner. Imitating effects of individual components and combinations on macrophage polarization will provide a systematic picture of the in vivo complexity.

      Conclusions

      Our understanding of macrophage ontogeny, polarization, and cell physiology has greatly expanded over the past decade. Basic and pre-clinical studies have shown the promising potential of targeting macrophages to prevent adverse cardiac remodeling and physiological deterioration in the post-MI LV. With emerging knowledge of the beneficial and detrimental functions of macrophages, future studies can be aimed at targeting specific detrimental functions while preserving beneficial roles. Establishing the progression of post-MI macrophage polarization and signaling patterns will provide mechanistic insight into how macrophages coordinate cardiac repair and help us identify novel intervention targets. Developing predictable computational models that incorporate the macrophage phenotype continuum will help to achieve this goal.

      Acknowledgments

      Conflicts of Interest: All authors have read the journal authorship agreement and policy on disclosure of potential conflicts of interest and have nothing to disclose.
      The authors acknowledge funding from the American Heart Association under award number 15SDG22930009 , from the National Institutes of Health under award numbers GM104357 , GM114833 , and GM115428 , HL051971 , HL075360 , HL105324 , HL129823 , and from the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development under award number 5I01BX000505 .
      The content is solely the responsibility of the authors and does not necessarily represent the official views of the American Heart Association, the National Institutes of Health, or the Veterans Administration.
      All authors have reviewed and approved the article.

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