Role of innate and adaptive immune mechanisms in cardiac injury and repair.

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Role of innate and adaptive immune mechanisms in cardiac injury and repair Slava Epelman1, Peter P. Liu2 and Douglas L. Mann3

Abstract | Despite the advances that have been made in developing new therapeutics, cardiovascular disease remains the leading cause of worldwide mortality. Therefore, understanding the mechanisms underlying cardiovascular tissue injury and repair is of prime importance. Following cardiac tissue injury, the immune system has an important and complex role in driving both the acute inflammatory response and the regenerative response. This Review summarizes the role of the immune system in cardiovascular disease — focusing on the idea that the immune system evolved to promote tissue homeostasis following injury and/or infection, and that the inherent cost of this evolutionary development is unwanted inflammatory damage.

Toronto Medical Discovery Tower, 101 College Street, TMDT 3903 Toronto, Ontario, M5G 1L7, Canada. 2 University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa, Ontario, K1Y 4W7, Canada. 3 Center for Cardiovascular Research, Division of Cardiology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA. Correspondence to S.E. e-mail: slava.epelman@ uhnresearch.ca doi:10.1038/nri3800 1

The cardiovascular system evolved ~600 million years ago as a means to transport nutrients and cells within multicellular organisms1. Primitive organisms such as Drosophila possess a single chamber that functions as both a pumping tube and a simple vascular system2. More complex organisms have compartmentalized cardio vascular systems, comprising venous and arterial vascular systems that are connected to a multi-chamber muscular myocardium that continually receives and ejects blood components. Despite the more complex nature of the mammalian cardiovascular system, its primary functions remain the same, and its importance to health and dis ease is highlighted by the fact that cardiovascular disease is the leading cause of death worldwide, with an increasing burden over the past decade3. Therefore, understanding both how cardiac tissue is injured and how cardiac tissue regenerates is of prime importance to global health. The immune system evolved to enable host defence against invading pathogens but also to promote tissue growth and repair during development or following sterile tissue injury, such as that which can occur within the myocardium (BOX 1). In this Review, we detail the roles of individual immune cell subsets and immune signalling pathways in both sterile and infectious cardiac injury. We also use examples from the cardiac system to suggest the idea that the immune system evolved to promote tissue homeostasis, but that this beneficial activity comes at a cost of increased ‘bystander damage’ when the immune system overreacts to internal injury.

Immune anatomy of the resting heart As in most tissues, the primary immune cells that reside in the heart are macrophages, which are typically observed near endothelial cells or within the interstitial space, while very few, if any, monocytes are found within cardiac tissue4–7 (FIG. 1). Sparse populations of dendritic cells (DCs) have been found within cardiac tissue, some of which are localized in cardiac valves, where they presumably sample antigens5,8. Mast cells are found in resting cardiac tissue, and are thought to be important early triggers of immune responses9. A small number of B cells and regulatory T cell subsets are also present in cardiac tissue in resting conditions, whereas neutrophils are typically not found within non-inflamed cardiac tis sue5,10,11. Whether from sterile or infectious triggers, cardiac tissue injury initiates a dynamic cellular cascade that initially activates resident immune cells, and over time this evolves in a coordinated manner that leads to the recruitment of diverse leukocyte populations into inflamed tissue. Mechanisms of cardiac injury The myocardium can be injured by various pathophysio logical processes, which can be grouped broadly into ischaemic and non-ischaemic aetiologies. In terms of global disease burden, ischaemic injury is the primary pathophysiological mechanism of injury 3,12. Occlusion of a coronary vessel after acute plaque rupture can lead to two potential outcomes: a completed myocardial

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REVIEWS Box 1 | The immune system during tissue growth and regeneration A careful examination of examples from the natural world reveals temporal and phylogenetic characteristics that predict the ability of tissues to regenerate in diverse organisms. More primitive organisms such as invertebrates, reptiles and amphibians have a striking regenerative potential when compared with mammals. For example, both the zebrafish and newt heart can fully re‑grow after experiencing substantial injury, and the salamander can fully re‑grow its limbs after amputation — functions that are not possessed by adult mammals141–143. Very young mammals also retain this regenerative capacity. The neonatal heart can fully regenerate after apical resection of the left ventricle, myocardial infarction or necrotic injury, but this capacity is lost after the first weeks of life83–85. One important similarity between more primitive organisms and very young mammals is a more limited (primitive) immune system144. The macrophage is a specialized mononuclear phagocyte that resides in all tissues from the earliest stages of development57,145. Loss of macrophages leads to abnormalities in growth of complex vascular and neuronal networks, increased mortality and stunted growth146–152. Beyond supporting growth, macrophages also have an important and more generalized role in the clearance of senescent cells during embryonic development87,88. Importantly, non-selective depletion of all macrophages impairs the ability of primitive organisms and young mammals to regenerate, highlighting the crucial role these cells have in tissue growth and repair83–85. Although macrophages possess important regenerative functions, they can also mediate pathology. Excessive expansion of macrophage populations during ischaemic injury impairs tissue healing, indicating that certain macrophage subsets can interfere with the regenerative process78. Understanding when macrophages do or do not promote tissue repair is a crucial first step if we are to understand why the adult human heart has only a limited regenerative capacity.

infarction occurs if blood flow is not restored, result ing in permanent anoxic and low-nutrient tissue injury. If blood flow is re-established to ischaemic (yet viable) tissue, inflammatory ‘reperfusion injury’ can also occur (discussed below). Non-ischaemic cardiomyopathy is a composite diagnosis that includes myocarditis that occurs after viral or bacterial infections or toxin admin istration. In addition, there are cardiomyopathies that develop secondary to chronic hypertension. All these forms of injury are influenced by genetic predisposition, which can itself lead to early onset cardiac dysfunction13. Whether through acute ischaemic injury or through the gradual impairment of cardiac function following a variety of clinical pathologies, irreversible heart failure often develops. As we are coming to understand, the immune system can contribute both during the initial insult and during the chronic phase of cardiac injury — and despite the investment of substantial research into understanding the contribution of immune cells to cardiac injury and repair, much remains unknown.

Innate sensing of cardiac injury Mammalian hearts use both innate and adaptive immunity to respond to tissue injury resulting from pathogens or environmental injury (for example, ischaemia or haemodynamic overloading). Resident cardiac immune cells are triggered by the detection of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) by a fixed number of germline encoded pattern recogni tion receptors (PRRs). Classic examples of pathogenassociated molecular patterns include the lipopolysac charides (LPSs) of Gram-negative microorganisms, the teichoic acids of Gram-positive microorganisms,

the zymosans of yeast, the glycolipids of mycobacterium, or the double-stranded RNAs of viruses (FIG. 2). More recently it has become clear that cardiac PRRs also rec ognize the molecular patterns of endogenous material released by dying or injured myocardial cells. Cells that die by accidental necrosis, regulated necrosis (that is, necroptosis) and/or secondary apoptosis release their cytosolic contents into the extracellular space, thereby initiating a brisk inflammatory response through the engagement of an ensemble of extracellular or intra cellular PRRs14. The time course of the inflamma tory response that ensues following tissue injury is remarkably consistent, irrespective of the specific cause of the injury, and it is associated with the rapid influx of neutrophils, and subsequently monocytes, into the area of tissue injury. This inflammatory response has been referred to as ‘sterile inflammation’, insofar as the inflam mation following tissue injury occurs in the absence of a known pathogenic infection15,16. Many PRRs encountering PAMPs and DAMPs trig ger signalling cascades that activate nuclear factor‑κB (NF‑κB), activator protein 1 (AP-1), and interferon regulatory factor (IRF) transcription factors, that in turn regulate target genes that encode pro-inflammatory cytokines and interferons (IFNs) in the heart 17. Another subset of PRRs in the heart trigger a distinct proinflammatory mechanism that requires assembly of cytosolic protein complexes called inflammasomes18. Canonical inflammasomes convert pro-caspase 1 into the catalytically active caspase 1 protease that is respon sible the production of interleukin‑1β (IL‑1β) and IL‑18 — cytokines that are sufficient to trigger inflammatory responses in the heart 18. PRRs can be subdivided into two major classes based on their subcellular localization. Toll-like recep tors (TLRs) and C‑type lectin receptors are found on plasma membranes or endosomes, where they can detect the presence of PAMPs or DAMPs. A second class of PRRs resides in intracellular compartments, and includes retinoic-acid inducible gene I (RIG-I)‑like receptors (RLRs), NOD-like receptors (NLRs) and absent-in‑melanoma 2 (AIM2) receptors19,20. Little is known of how TLR expression is regulated in the heart. However, TLR4 seems to be upregulated in the failing human heart and on circulating monocytes at the time of myocardial infarction21–23. In animal studies, loss of TLR4 in haematopoietic cells is protective in the setting of sepsis-induced cardiac dysfunction and the loss of TLR2 in haematopoietic cells is protective during ischaemic injury 24–26. The loss of TLR4 is also protective following ischaemic injury, although it is not known whether this is attributable to the absence of TLR4 activity in haematopoietic or other cell types25–28. During haemodynamic stress, mitochondria are typi cally damaged; if the degradation of mitochondrial DNA is inhibited in this setting, a TLR9‑dependent inflammation-induced cardiomyopathy develops29. NLRs function as cytosolic sensors of intracellular DAMPs and PAMPs. In humans, the NLR family is composed of 22 intracellular PRRs that share a central NACHT domain and a carboxy-terminal leucine-rich

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REVIEWS Immune cell activation in cardiac injury Acute ischaemic injury is the best characterized model of cardiac injury and repair. Following injury, necrotic Cardiomyocyte cell death leads to the activation of tissue-resident immune and non-immune cells. These cells produce pro-inflammatory cytokines and chemokines that are Macrophage responsible for recruiting inflammatory leukocytes from the blood into the area of tissue injury 35. The initial Blood DC vessel inflammatory phase is followed by a proliferative phase that is characterized by the expansion of neutrophil Endothelial TReg cell and macrophage populations that are responsible for cell Mast cell removing dead cells and matrix debris, as well as releas ing cytokines and growth factors that lead to the forma Neutrophil tion of a highly vascularized granulation tissue, which is B cell composed of connective tissue and new blood vessels. The final maturation phase is characterized by fibroblast activation and endothelial cell proliferation, culminating in reparative myocardial fibrosis and angiogenesis36. During the very early stages of ischaemic injury, mast cells and soluble complement proteins become impor tant initiators of inflammation — a process that is ampli Figure 1 | Immune cells in the resting heart. The majority of immune cells in the resting fied following coronary reperfusion. Immediately after heart are macrophages, which are found primarily surrounding endothelial but are Nature Reviewscells | Immunology blood flow is restored, resident cardiac mast cells release also seen in the interstitium amongst cardiomyocytes. Mast cells, dendritic cells (DCs), pre-formed pro-inflammatory mediators (for example, B cells and regulatory T (TReg) cells are found sparsely in cardiac tissue, while neutrophils tumour necrosis factor (TNF), histamine and mast cell and monocytes are not observed within myocardial tissue, but can be observed as contaminants found in the vasculature during steady state5. proteases) that initiate an amplification loop involving adjacent cells, such as endothelial cells, resident macro phages and, subsequently, infiltrating neutrophils9. 30 repeat region . Analysis of human tissue has revealed that Timely restoration of blood flow to viable tissue is cru NOD1, NOD2 and the NLR family members NOD-, LRR- cial to prevent cardiomyocyte death; however, reperfu and pyrin domain-containing 2 (NLRP2) and NLRP3 sion comes at the cost of exposing complement proteins are expressed in the heart. Both NOD1 and NLRP3 have to injured endothelial cells and myocardium (see REF. 37 been shown to activate canonical inflammasomes in for a review). Activated complement proteins trigger the heart, and these molecules have an important role further mast cell degranulation, release of histamine in the adverse cardiac remodelling events that follow and vasogenic oedema37. Cleaved complement proteins ischaemia–reperfusion injury and myocardial infarction; such as C5a both attract neutrophils and induce their however, the cell types involved are not known18,31. transendothelial migration into the injured tissue via the The RLR family is composed of RIG‑I, melanoma CD11b–CD18 complex 38 (FIG. 3a). If reperfusion is not differentiation-associated gene 5 (MDA5) and LGP2. established, cardiomyocyte cell death ensues through RLRs are localized in the cytoplasm and recognize the a variety of pathways that lead to additional DAMP genomic RNA of double-stranded RNA (dsRNA) viruses release. The exact role of resident mast cells is in part and the dsRNA that is generated as the replication inferred, as models of mast cell deficiency generally use intermediate of single-stranded RNA (ssRNA) viruses. Kit−/− animals, which have other immune-cell deficits39–41. MDA5 is the best characterized receptor in this family, However, mast cells, the complement cascade, oxidative and the loss of MDA5 expression in cardiomyocytes stress and pro-inflammatory cytokine and chemokine leads to uncontrolled viral replication and rapid death in production immediately after injury initiate a complex mice infected with encephalomyocarditis virus (EMCV), interplay between innate and adaptive immune cells. whereas over-expression of MDA5 in the heart protects against lethal myocarditis32,33. A final set of PRRs that Neutrophil recruitment during cardiac injury are expressed in human and mouse heart tissue are the Following either cardiac ischaemic injury or pres C‑type lectin receptors, which are calcium-dependent sure overload, neutrophils are the first innate immune carbohydrate-binding receptors34. However, very little is cells recruited to the myocardium in large numbers42. known about their roles in cardiac tissue injury. Patients deficient in neutrophils or neutrophil function One important issue that has been only partially suffer from devastating disseminated bacterial infec addressed is the delineation of cell-type-specific roles tions, indicating a clear requirement for this cell type to Granulation tissue for PRRs. Beyond the few examples given here, it is not prevent expansion of otherwise harmless pathogens43. This is tissue that arises after cardiac tissue injury, clear what differential roles various PRRs have within However, their role in the response to cardiac injury is when the replacement of individual immune and non-immune cell subsets almost entirely pathological and, as such, neutrophils cardiomyocytes with collagen during the process of cardiac tissue injury and repair, are the best example of a cell type that promotes overall and extracellular matrix occurs which represents an important avenue for further longevity, but in the setting of sterile injury, they have no in order to maintain the integrity of the myocardial wall. investigations. known protective role. Monocyte



REVIEWS Heart

Cell necrosis

DAMPs • dsRNA • CpG • Uric acid crystal • HMGB1 • ATP • HSP • IL-1α

Receptors • TLR3 • TLR9 • NLRP3 •TLR2–TLR4–RAGE • P2Y11 and P2X7 • TLR4 • IL-1R

ECM degradation

DAMPs • Hyaluronan fragment • Heparan sulphate

Receptors • CD44–TLR4 • TLR4

Thrombus

Ischaemic area

Immune cell recruitment and activation

Figure 2 | Cardiac injury and sensing damaged tissue. The figure shows a coronary artery occlusion (black) that leads to ischaemic tissue injuryNature (grey zone). From within the Reviews | Immunology ischaemic area, cell necrosis, extracellular matrix (ECM) degradation and recruitment of immune cells all lead to the production of specific damage-associated molecular patterns (DAMPs), which are recognized by pattern recognition receptors. This leads to the generation of inflammatory responses to internal injury signals. CpG, CpG dinucleotides; dsRNA, double-stranded RNA; HMGB1, high-mobility group box 1; HSP, heat shock protein; IL, interleukin; IL-1R, IL-1 receptor; NLRP3, NOD-, LRRand pyrin domain-containing 3; P2Y, P2Y purinoceptor; P2X, P2X purinoceptor; RAGE, receptor for advanced glycation end-products; TLR, Toll-like receptor.

Neutrophil recruitment is mediated in two phases (FIG. 3a); the first phase is peripheral activation prior to infiltration. Mitochondria in all cell types, including cardiomyocytes, contain formylated peptides and mito chondrial DNA, both of which are structurally similar to bacterial components. In an analogous system (skel etal muscle necrosis), these mitochondrial DAMPs are released. Formylated peptides and mitochondrial DNA are sensed by formyl peptide receptor 1 (FPR1) and TLR9, respectively, and the triggering of these receptors promotes neutrophil activation and their recruitment to inflamed tissues29. It is important to confirm whether similar mechanisms are involved in neutrophil activation following cardiomyocyte necrosis. The ability to sense mitochondrial motifs is not surprising as mitochondria are endosymbionts and related to microorganisms in many ways. However, the exact role of mitochondrial motifs in cardiac injury remains to be defined. The second phase of neutrophil activation depends on cardiac endothelial cells. Pro-inflammatory mediators, such as TNF, IL‑1β and histamine, activate endothelium and induce the upregulation of adhesion molecules that enable neutrophil transmigration between and through endothelial cells to reach the site of tissue injury 9,44. Detailed imaging studies in other organ systems have aided our understanding of how neutrophils enter damaged tissue. For example, following necrotic tissue injury in the liver, neutrophils adhere to more remote

sites, where viable tissue is present. Initially, migration is dependent on chemokines, and subsequently neutrophils follow necrotic signals, such as liberated intracellular ATP (which is a DAMP), in order to precisely home to the site of tissue injury 45. The reason neutrophils (and perhaps other recruited immune cells) take this more convoluted path is that necrotic tissue is usually non- (or under) perfused, and transendothelial migration at the site of injury is not possible. Unfortunately, such detailed imaging of migrating neutrophils has yet to be done in the injured beating heart, but techniques to achieve this are being developed46. Such studies will be highly inform ative for understanding the spatial dynamics of resident macrophages, recruited neutrophils and monocytes during the progression of ischaemic cardiac injury. An important early mediator of tissue injury seems to be IL‑6, which is produced in an autocrine fashion by cardiomyocytes and recruited myeloid cells (both neutro phils and macrophages)47,48. IL‑6 upregulates intracellular adhesion molecule 1 (ICAM1) expression on cardio myocytes, inducing neutrophil binding and stimulating cytotoxic activity 49–51. The role of neutrophils following cardiac injury may be more pronounced when major areas of the myocardium are at risk of cell death (but have not yet died), such as following ischaemia–reperfusion injury. Neutrophils produce numerous proteases that contribute to injury, and blockade of neutrophil recruit ment seems to be most effective during more limited episodes of ischaemia, rather than during longer episodes in which cardiomyocyte death is largely secondary to anoxia and nutrient deprivation52–54.

Cardiac macrophages The dominant view for the past half century has been that bone marrow-derived haematopoietic stem cells (HSCs) give rise to circulating blood monocytes, which enter into tissues and become tissue macrophages55. However, in the past few years a series of more definitive publications have drastically revised our understand ing of monocyte and macrophage origin by showing that many tissue-resident macrophage populations are established during embryonic development, and they are subsequently maintained by self-renewal, rather than through blood monocyte input 5,56–61. Given the impor tance of monocytes and macrophages to cardiovascular disease, we review recent insights into the origin of functions of these subsets and how they contribute to cardiac tissue injury and repair. There are two main subsets of circulating mono cytes in mice, LY6Chi monocytes and LY6Clow monocytes. LY6C+ monocyte progenitors give rise to LY6Chi mono cytes and, through a nuclear receptor subfamily 4 group A member 1 (NR4A1)-dependent transcrip tional programme, LY6C hi monocytes differentiate into LY6Clow monocytes58,62,63. Global transcriptional profiling has shown these monocyte subsets are con served in humans64 and they have very different roles in vivo. LY6Clow monocytes adhere to and move along the endothelium, both clearing damaged cells and triggering inflammatory responses without entering tissue65,66. During cardiac stress, LY6Chi monocytes are



REVIEWS a 24 hours post ischaemic injury

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Endothelial cell activation Histamine, TNF and IL-1β

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IL-17A promotes neutrophil production in bone marrow

IL-17A induces apoptosis Apoptotic cardiomyocyte Apoptotic neutrophil

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Monocytes ingest apoptotic cells and release anti-inflammatory cytokines

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CCR2-dependent monocyte recruitment

Innate B cell Innate B cells activated in response to DAMPs

Fibroblast TGFβ induces fibrosis Nature Reviews | Immunology

Figure 3 | Immune response to ischaemic injury in adult animals. a | Early after ischaemic injury (within 24 hours), endogenous danger-associated molecular patterns (DAMPs) are released from necrotic cardiomyocytes and activate resident mast cells, causing mast cell degranulation and release of preformed pro-inflammatory cytokines and vasogenic compounds (such as histamine, tumour necrosis factor (TNF) and interleukin‑1β (IL‑1β)), which activate endothelial cells. Necrotic cardiomyocytes also release mitochondrial DAMPs (such as formylated peptides and mitochondrial DNA) into the circulation, which causes systemic neutrophil activation. Activated neutrophils adhere to activated endothelium and transmigrate into the cardiac tissue following a chemokine gradient. Neutrophils secrete proteases that digest tissue (and also activate chemoattractants, such as complement component C5a), which further potentiates leukocyte recruitment. Neutrophils are directed to ischaemic areas by following gradients of DAMPs (such as ATP). Neutrophils may then phagocytose dying cells, but they can also induce apoptosis in healthy cardiomyocytes themselves through the release of reactive oxygen species. b | In the later phases of ischaemic injury (3–4 days), there is recruitment of LY6Chi monocytes from the blood into ischaemic cardiac tissue. Some of these monocytes originate from ‘reservoirs’ in the spleen. Innate B cells that express immunoglobulins IgM and IgD are also recruited into the myocardium and produce CC‑chemokine ligand 7 (CCL7), which promotes further monocyte recruitment10. Innate B cell activation is MYD88 (myeloid differentiation primary response protein 88) dependent (suggesting Toll-like receptor and DAMP involvement). Recruited monocytes secrete pro-inflammatory cytokines and chemokines, and they drive inflammatory processes. A proportion of recruited monocytes ingest apoptotic cells, including neutrophils, which increases the secretion of anti-inflammatory cytokines, such as transforming growth factor-β (TGFβ) and IL‑10, and thereby decrease leukocyte recruitment. Monocytes produce IL‑23, which drives the production of IL‑17A by γδ T cells. IL‑17A has two main roles in perpetuating the inflammatory response: it drives neutrophil production in the bone marrow and causes cardiomyocyte death. As inflammatory responses diminish, less IL‑23 is produced in the tissue. CCR2, CC-chemokine receptor 2; TReg, regulatory T cell. NATURE REVIEWS | IMMUNOLOGY


REVIEWS the primary subset recruited to the heart, either follow ing ischaemic injury or hypertensive stress; whereas LY6Clow monocytes do not seem to be directly recruited into the myocardium5,59,61,67,68. Monocytes recruited into ischaemic myocardium are found in the blood, but the spleen is also a monocyte reservoir that can be used when blood and bone marrow stores are insufficient69,70. Splenic monocytes also seem to have a protective role, as splenectomy leads to impaired infarct healing 71. Monocyte recruitment is in part dependent on innate B cells, which are also recruited to the ischaemic myocardium, and these B cells drive monocyte population expansion through a CC‑chemokine ligand 7 (CCL7)-dependent process10 (FIG. 3b). Once monocytes enter the tissue they begin to differentiate into macrophages. For many years, it has been very challenging to separate recruited mono cytes from resident macrophages and, as a result, they have been analysed as a single population. However, as we describe below, these populations have different ontological lineages, and this has important functional implications.

Innate B cells These cells are thought to become rapidly activated in the absence of classical T cell-dependent antigen presentation mechanisms. They are mobilized by other cell types and/or inflammatory triggers.

ApoE-deficient mice These mice are used to model atherosclerosis. They have increased total plasma cholesterol levels and increased bone marrow production of monocytes and heightened inflammatory responses.

Cardiac macrophage subsets. Recently, studies using genetic fate mapping, parabiosis and adoptive transfer techniques have defined resident cardiac macrophage populations in much more detail. Rather than being a homogenous population, resident cardiac macrophages comprise three discrete subsets that have different ori gins and functions5 (FIG. 4a). These three macrophage subsets are defined by their differential expression of MHC class II and CC chemokine receptor 2 (CCR2). MHC class IIhi and MHC class IIlow cardiac macrophages are both CCR2− and are, numerically, the dominant subsets in the heart 5. These macrophage subsets are primarily derived from embryonic progenitors, with a substantial number arising from embryonic yolk sac precursors, and they renew through in situ proliferation, rather than through monocyte input. After birth, there is some dilution of embryonically derived macrophages by recruited monocyte-derived macrophages in the heart, but in adult mice the majority of resident cardiac macrophages remain of embryonic origin5,72. The third cardiac macrophage subset is made up of CCR2+ macrophages, which are derived from, and slowly replenished by, circulating blood monocytes. Detailed studies during hypertensive stress suggest that embryonically derived macrophage populations expand solely through in situ proliferation, whereas monocytederived macrophages require monocyte input prior to proliferative expansion in the tissue5. CCR2+ macro phages express high levels of pro-inflammatory genes, including those associated with the NLPR3 inflamma some, which is required to process and deliver IL‑1β to the heart during cardiac stress5. Inflammasome acti vation promotes adverse cardiac remodelling follow ing ischaemic injury, genetic cardiac hypertrophy and hypertensive cardiac disease18,73,74; it is likely that CCR2+ cardiac macrophages are involved in inflammasome acti vation in each of these settings. The robust pro-inflam matory gene signature of CCR2+ cardiac macrophages suggests that they are important for immune protection

against invading pathogens, but their inappropriate activation in the setting of sterile inflammation may lead to unwanted pathology 75,76. Prior studies have used dichotomous expression of two classical myeloid cell markers (F4/80 and CD11b) in order discern embry onic versus adult monocyte origin57. However, differ ent cardiac macrophage lineages (embryonic and adult) cannot be distinguished by these cell surface markers alone and, as such, genetic fate mapping remains the most accurate method for lineage discrimination of cardiac macrophages in adult mice5,72. Functions of cardiac macrophages. One of the interest ing challenges in the setting of cardiac tissue injury is to define the roles of recruited monocytes and monocytederived macrophages. Non-selective depletion strategies have shown that in the absence of both monocytes and macrophages, scar formation is impaired after cardiac ischaemic injury, with decreased collagen production, decreased angiogenesis and increased mortality due to myocardial rupture6,77. However, experiments using ApoE‑deficient mice (apolipoprotein-E-deficient mice) sug gest that excessive expansion of macrophage populations can have a detrimental effect on infarct healing, leading to excessive inflammation and impaired cardiac function78. Furthermore, studies of cardiac injury in Ccr2−/− mice, which lack circulating monocytes, indicate that monocyte recruitment and the associated inflammatory response leads to increased cardiac pathology 79–81. The loss of LY6Chi monocytes also prevents hypertension-induced cardiac fibrosis and improves left ventricle function after myocardial infarction. Taken together, these data suggest that recruited monocytes have a pathological role in the setting of sterile cardiac injury, but that resident embry onically derived macrophages are likely to have important roles in the tissue-repair response6,67,82. Interestingly, as already mentioned, the neonatal heart has a remarkable capacity to regenerate in response to multiple forms of tissue injury, but the regenerative pro cess is lost if resident embryonically derived macrophages are eliminated83–85. In fact, the neonatal heart supports the expansion of tissue-resident embryonically derived macrophage populations following injury, as opposed to the recruitment of monocytes, and this seems to be a fundamental difference between the neonatal and adult heart. Neonatal cardiac macrophages promote endothelial cell activation and cardiomyocyte growth, and they gener ate minimal inflammation after stimulation through TLR and inflammasome pathways85 (FIG. 4b). These data suggest that that the neonatal heart avoids excessive inflamma tory responses, which may be a crucial factor that aids the regenerative process, although this benefit may come at the cost of being more susceptible to infections86. Macrophage subsets that are primarily derived from embryonic precursors are more efficient at internaliz ing debris and engulfing apoptotic cardiomyocytes, sug gesting they have important homeostatic roles. Indeed, these functions are reminiscent of embryonic macrophage functions during development and could suggest that some of these functions are ‘hard-wired’ into the adult macrophages5,87,88. The uptake of dead or dying cells is



REVIEWS a

Early in embryonic development

MHC class IIlow yolk sacCardiomyocyte derived macrophage

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Adult heart MHC class IIhi macrophage MHC class IIlow macrophage

IIlow fetal

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Monocyte

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Developing heart seeded by yolk sac-derived macrophages

Embryonically derived macrophages proliferate in situ MHC class IIhi macrophages develop from MHC class IIlow populations

Heart seeded by macrophages derived from fetal monocytes

Adult monocytes give rise to MHC class IIhi and MHC class IIlow macrophages and short-lived CCR2+ macrophages

b

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Adult heart Receptive neonatal cardiomyocyte Quiescent adult cardiomyocyte

Tissue injury

↑ ↑ ↓

Expansion of resident macrophage populations

Angiogenesis Cardiomyocyte proliferation Inflammation

Tissue injury

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Regeneration of cardiac tissue

Angiogenesis Cardiomyocyte proliferation Inflammation • Impeded regeneration of cardiac tissue • Scar formation

Figure 4 | Role of embryonically derived macrophages during tissue injury and repair. a | During embryonic development, extra-embryonic yolk-sac derived macrophages (shown in blue) initially seed the developing heart. The cells express low levels of MHC class II molecules. Later during development, fetal monocyte-derived macrophages (shown in orange) also infiltrate the heart, and both of these embryonic macrophage populations expand further during development, and after birth, by in situ proliferation. MHC class IIhi macrophages develop from these populations of MHC class IIlow macrophages after weaning5,72. During this time, haematopoietic stem cell-derived monocytes infiltrate the heart and also differentiate into MHC class IIhi macrophages (dark green) and MHC class IIlow macrophages (light green). MHC class IIhi and MHC class IIlow macrophages persist in the tissue as an ontologically mixed population, primarily composed of embryonically derived macrophages5. Monocytes that infiltrate the heart can also become short-lived CC-chemokine receptor 2 (CCR2)+ macrophages, which are entirely derived from blood monocytes5. b | The figure shows a comparison of the macrophage response to tissue injury in neonatal animals (which regenerate cardiac tissue)

and adult animals (where there is minimal tissue regeneration). In the neonate, Nature Reviews | Immunology resident embryonically derived macrophage populations expand without notable monocyte input. Embryonically derived macrophages promote angiogenesis, cardiomyocyte proliferation and drive minimal inflammation when stimulated by damage-associated molecular patterns (DAMPs). These properties of neonatal macrophage populations facilitate cardiac regeneration85. Following cardiac injury in the adult, there is marked expansion of CCR2 + monocyte and macrophage populations. Notably, these macrophages possess a limited ability to promote angiogenesis and cardiomyocyte population expansion, but have notable capacity to drive inflammatory responses, which impedes tissue regeneration5,85. Importantly, neonatal cardiomyocytes are primed to divide and therefore are receptive to regenerative signals84,153,154. Adult cardiomyocytes are much less receptive to regenerative signals, and growth signals from embryonically derived cardiac macrophages are one of many important components of cardiac regeneration promotion in adult tissues. A key therapeutic goal would be to recapture the neonatal response to injury in the injured adult heart.



REVIEWS an important function in the setting of ischaemic injury. Expression of the MER tyrosine kinase (MERTK) — a phagocytic receptor that is a highly specific marker of tissue macrophages — is upregulated on cardiac macrophages after myocardial infarction, and loss of MERTK expression leads to the accumulation of apop totic cardiomyocytes, increased neutrophil persistence and decreased levels of the anti-inflammatory cytokine IL‑10 in the myocardium89,90. This ongoing inflamma tion ultimately results in decreased cardiac function89,90. Phagocytosis of apoptotic cells (efferocytosis) in skeletal muscle results in the transition of macrophages to a more anti-inflammatory state through the upregulation of the intracellular signalling kinase AMPK, which is associated with downregulation of pro-inflammatory genes (such as Tnf) and the upregulation of anti-inflammatory genes, such as Il10 (REFS 91,92). Together, these data suggest that strategies that target newly recruited monocytes while sparing resident macrophages may yield therapeutic benefit during cardiac injury.

Adaptive immune cells and heart injury Lymphocytes have been reported to have diverse roles during cardiac tissue injury and repair. CD4‑deficient mice, MHC class II‑deficient mice and mice that express a single transgenic T cell receptor show impaired wound healing and increased monocyte population expansion after ischaemic injury. This suggests that CD4+ T cells have protective functions during cardiac injury, and that auto-antigens may be presented to CD4+ T cells by MHC class II‑expressing cells (such as macrophages and DCs), which then drives immunosuppressive responses in the myocardium93. Interestingly, it may be that regu latory T cells are the key CD4+ T cell subset involved here, and regulatory T cells have been shown to promote myocardial recovery through an IL‑10‑dependent path way 94,95. Another study found that recombination acti vating gene (RAG)-deficient mice (which lack T cells and B cells) have decreased infarct sizes after ischaemic injury, and that the transfer of CD4+ T cells into the RAGdeficient mice had a negative effect on infarct healing. Therefore, in certain situations CD4+ T cells may have pathogenic roles following ischaemic injury96. In addi tion, RAG-deficient mice have decreased levels of circu lating IgG antibodies, and these antibodies are known to have immunosuppressive effects (as reviewed in REF. 97). CD8+ T cells have also been shown to have regula tory roles following ischaemic injury. CD8+ T cells express type 2 angiotensin II receptor (AGTR2), which is known to drive anti-inflammatory responses98,99. Angiotensin II is part of the renin–angiotensin system, which has long been known to be a crucial pathological pathway in heart disease100,101. Angiotensin II is thought to primarily induce cardiomyocyte hypertrophy and increase vascular tone by signalling through AGTR1, in a process that is amplified by blockade of AGTR2 (as reviewed in REF. 102). After ischaemia–reperfusion injury AGTR2‑expressing CD8+ T cells, inhibit inflam mation and decrease infarct size by producing IL‑10 in an angiotensin II‑dependent fashion, and this subsequently dampens the immune response98.

In addition to these roles of T cells, other lympho cytes have been shown to shape inflammatory responses in cardiac tissue. Natural killer (NK) cells were reported to infiltrate the myocardium and have protective effects following ischaemic injury 103. The adoptive transfer of activated NK cells into mice was shown to protect against left ventricle dysfunction following ischaemia by pre venting fibrosis and enhancing angiogenesis through a contact-dependent interaction with endothelial cells.

Transition from acute inflammation to fibrosis One of the first important steps that limits tissue injury and promotes the transition to tissue healing is the resolution of neutrophil recruitment. Macrophages are believed to initiate the process through two key mechanisms that have been linked to their phagocytic capacity. First, it has been well described in vitro and in vivo that macrophages that have ingested apoptotic cells increase their production of anti-inflammatory and pro-fibrotic cytokines, such as IL‑10 and TGFβ, while they decrease production of pro-inflammatory cytokines, such as IL‑1β and TNF 104,105. Following ischaemic injury, if all cardiac macrophages are absent, or they if they lack the phagocytic receptor MERTK, there is neutrophil persistence within infarcted myo cardial tissue and ongoing inflammation. This suggests that the activation of cardiac macrophage phagocytic pathways limits inflammation, and that the loss of the phagocytic macrophages results in an accumulation of apoptotic material in the infarct zone5,6,89.The transi tion from inflammatory to pro-fibrotic macrophages has been classically thought to involve polarization changes (from an ‘M1’ to an ‘M2’ macrophage pheno type); however, it is becoming clear that macrophages are very heterogeneous and fit along a broad spectrum of possibilities, rather than into clearly defined subsets. Further work is required to define where individual cardiac macrophage subsets fit into the M1 and M2 framework106. The second mechanism involves a complex cytokine cascade that depends on γδ T cells and their production of IL‑23 and IL‑17A. The production of IL‑17A leads to neutrophil production and release from bone marrow stores107. Interestingly, IL‑17A is regulated by secretion of IL‑23 by macrophages, and the ingestion of apoptotic neutrophils decreases IL‑23 production, which leads to decreased IL‑17A levels and decreased neutrophil pro duction in the bone marrow 107–109. Following ischaemic cardiac injury, γδ T cells and neutrophils themselves seem to be important sources of IL‑17A production within the myocardium, and mice that lack IL‑23 have reduced IL‑17A levels in cardiac tissue, reduced neutrophil accu mulation, increased γδ T cell population expansion and improved infarct healing 110. Indeed, IL‑17A seems to also be directly toxic to cardiomyocytes, and neutralization of IL‑17A improves infarct healing 111 (FIG. 3b). Additional work has shown an important link between IL‑17A production and activation of fibrotic pathways within the heart that lead to collagen deposition, both fol lowing ischaemia and haemodynamic strain112,113. In this context, and in contrast to the work described above, the



REVIEWS Coxsackievirus B

TLR2 or TLR4 MYD88

CAR

DAF Cardiomyocyte

FYN or ABL

LCK Endosome

Viral entry

Viral nucleic acids MDA5

RIG-I

Activation of cytosolic PRRs IRAK4

NF-κB

TLR7

TLR3 TRIF

Activation of endosomal PRRs

IRF3

p50 p65 NF-κB activation

IRF activation

Type I IFN production

Activation of immunopathological response, tissue damage

Protective immune response

Figure 5 | Interaction of coxsackievirus B with the host innate and acquired immune system. Coxsackievirus B (CVB) engages the internalizingNature receptor coxsackievirus and Reviews | Immunology adenovirus receptor (CAR), and the receptor-associated tyrosine kinases FYN and ABL can facilitate viral remodelling of the cell cytoskeleton to promote viral cell entry. This process is further aided by the co‑receptor decay accelerating factor (DAF) and its associated tyrosine kinase LCK. Viral components can be detected by the intracellular pattern recognition receptors (PRRs) melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG‑I) leading to the activation of nuclear factor‑κB (NF‑κB)‑dependent inflammatory pathways. NF-κB‑dependent responses can also be activated following the engagement of CVB with cell surface-expressed Toll-like receptors (TLRs), which signal through the myeloid differentiation primary response protein 88 (MYD88) adaptor and downstream signal intermediates such as IL‑1-receptorassociated kinase 4 (IRAK4) and TNF receptor-associated factor 6 (TRAF6). These pathways facilitate viral proliferation and host immune-mediated tissue damage. By contrast, activation of interferon regulatory factor 3 (IRF3) following the triggering of the TIR-domain-containing adapter-inducing IFNβ (TRIF) pathway by CVB leads to the production of protective type I interferons (IFNs). There is mutual counter-regulation of the MYD88 –IRAK4 and TRIF–IRF3 pathways. The subsequent maturation of CD4+ and CD8+ T cell subsets is also detrimental for the host, whereas regulatory T cells are host-protective. Cross-talk between acquired and innate immune signalling pathways also modulates the host response to CVB infection.

loss of IL‑23 was demonstrated to impair infarct healing, which was mechanistically linked to a failure to activate fibroblasts, leading to impaired scar formation and myo cardial rupture113. These data are reminiscent of the rela tionship between monocyte influx and tissue healing after

myocardial ischaemic injury that we discussed earlier, in which either too few or too many infiltrating monocytes impaired infarct healing 6,78. These data indicate that there are yet-to-be determined, context-dependent factors that modulate whether a particular cell type or pathway mediates cardiac tissue repair or damage. The activation of cardiac fibroblasts and subse quent extracellular matrix deposition is an important component of cardiac ischaemic injury and repair. Interestingly, cardiac fibroblasts can also become acti vated in more subtle forms of cardiac stress that also lead to impaired cardiac function owing to increased myocardial stiffness (such as hypertension caused by infusion of angiotensin II). In this setting there is rapid recruitment of neutrophils and monocytes into the myocardium and also expansion of resident car diac macrophage populations5,114–118. Cardiac fibroblasts become activated, leading to interstitial cardiac fibrosis, a process dependent on input and possible differen tiation from monocytes117. The activation of cardiac fibroblasts is also amplified by CD4+ and CD8+ T cells through an IFNγ-dependent pathway 119,120 (reviewed in detail in REF. 121).

Myocarditis as a model of cardiac inflammation Myocarditis is an excellent model for dissecting the inflammatory processes that occur in the heart. It shows how the balance of innate and adaptive immune mechanisms that occur following tissue injury deter mine whether there is progression to heart failure or to repair of cardiac tissue. Myocarditis is most often induced by infection with viruses, although other infec tious pathogens can also be involved, such as bacteria or the protozoan parasite Trypanosoma cruzii, which is the cause of Chagas disease, the commonest cause of heart failure in South America122,123. Myocarditis can also result from non-infectious triggers of DAMP release, which activate the innate immune response leading to sterile inflammation, such as allergic reactions to drugs or chemicals124. Myocarditis accounts for about one in nine cases of heart failure and remains one of the most common reasons for heart transplantation worldwide, as spe cific treatments are lacking 125. The most common viral triggers of myocarditis include enteroviruses, such as coxsackievirus B3 (CVB3) and CVB4, and adenovi ruses. Virus-induced myocarditis shows a periodicity in its prevalence in human populations, which is pos sibly related to herd immunity. European studies also show a substantial contribution of parvovirus B19, which shows tropism for endothelial cells and the bone marrow 126. Coxsackieviruses and adenoviruses target host tissues, including the immune, cardiovascular and neurological systems, through their binding and inter nalization by coxsackievirus and adenovirus receptor (CAR) — which is an immune-regulated tight junc tion protein127,128. Internalization of the virus is assisted by co‑binding to decay accelerating factor (DAF; also known as CD55), a ubiquitously expressed host pro tein that inhibits complement activation129 (FIG. 5). Loss of CAR expression in cardiomyocytes prevent cardiac



REVIEWS dysfunction and inflammation following viral infection, suggesting that entry into cardiomyocytes is required for tissue damage130. However, although viral entry and subsequent viral proliferation trigger disease, they are not necessarily the primary determinants of disease out comes. Both the innate and adaptive immune responses have crucial roles in the progression of viral myocar ditis, as shown by the fact that genetic deletion of the TLR-signalling proteins myeloid differentiation pri mary response protein 88 (MYD88) or IL‑1R-associated kinase 4 (IRAK4), or deletion of the T cell receptorassociated tyrosine kinase LCK, ameliorates myocardial inflammation and improves survival in mice, despite increased levels of viral proliferation131–134. Following viral entry into the target cell, such as an immune cell or cardiomyocyte, the virus can engage intracellular NLRs, including RIG‑I and MDA5. The endosomal degradation of the virus can also lead to the activation of TLR3 and TLR732,133. The signalling cas cades induced by such PRR activation can have detri mental consequences for the host. As stated above, the MYD88–IRAK4 pathway, showed a very marked protec tive benefit in mice during CVB3 infection. At least four different mechanisms are thought to mediate protection in this setting. First, the loss of MYD88–IRAK4 signal ling decreases downstream activation of TNF receptorassociated factor 6 (TRAF6) and nuclear translocation of the NF‑κB complexes, resulting in reduced pro-inflam matory cytokine production and T cell activation135. Second, increased IRF3 or IRF5 homodimerization and phosphorylation of signal transducers and activators of transcription 1 (STAT1) and STAT5 occurs in the absence of MYD88 or IRAK4, leading to increased production of protective antiviral type I IFNs133–135. Third, IRAK4 dele tion facilitates the mobilization of protective CCR5+ monocytes from the bone marrow into the myocar dium133. Fourth, the absence of MYD88 or IRAK4 leads to downregulation of CAR expression and decreased viral proliferation135. However, the genetic deletion of IRF3, which is activated by the TIR-domain-containing adaptor-inducing IFNβ (TRIF)-dependent TLR path way, leads to much worse outcome during virus-induced myocarditis, with mice showing increased mortality and greater levels of viral proliferation136. Mechanistically, this is associated with decreased type I interferon production and increased activation and translocation of NF-κB136. These observations have several important implica tions. The first is that increased expression of viral entry receptors and enhanced viral proliferation are, paradoxi cally, facilitated by the host innate immune response — probably an evolutionarily selected advantage for the pathogenic virus at the expense of the host. The second is that intracellular immune signalling pathways seem to induce functional responses that regulate trafficking of inflammatory cells, such as CCR5+ monocyte-derived macrophages, from the bone marrow. Indeed this is also the case in sterile inflammation post myocardial infarction, in which IRAK4 signalling seems to regulate the traffick ing and maturation of dendritic cells into the myocardium, with subsequent orchestration of host inflammatory responses that dictate tissue remodelling and host survival.

The activation of innate immune signalling path ways also sets the stage for subsequent T cell maturation and participation in the host inflammatory response. Genetic deletion of LCK, which impairs T cell matura tion, results in almost complete protection of the host against CVB3 infection131. A similar outcome is observed following CD4+ or CD8+ T cell subset deletion, or fol lowing deletion of the leukocyte tyrosine phosphatase CD45, which is associated with upregulation of type I IFNs132,137. Adoptive transfer of regulatory T cells into CVB3‑infected hosts also had a marked protective effect, again associated with a decrease in viral prolif eration and upregulation of type I IFNs138. Surprisingly, there was a general downregulation of molecules asso ciated with TLR signalling pathways and even TLR4 itself. Moreover, beyond their role during infections, regulatory T cells also promote recovery through an IL‑10‑dependent pathway following ischaemic injury 95. These data suggest that there is close cross-talk between the regulatory T cell subsets and the innate immune sig nalling pathways that operate during both infectious and sterile injury. The above progress in understanding myocarditic processes has moved the field forward from the ear lier failed attempts to use immunosuppression to treat patients with myocarditis139. Subsequently, a Phase II trial has demonstrated that in patients with persistent viral proliferation, intravenous delivery of IFNβ clears the virus and improves symptoms of myocarditis140. Targeted immunosuppressive therapy may be indi cated for patients with persistent immune activation, in which the viral proliferation phase has already passed. Nevertheless, there are still large gaps in our knowledge, including why there are periodic enteroviral outbreaks in the population. Furthermore, why do some patients develop severe viral myocarditis that requires transplan tation, while others are able to rebalance the immune response and promptly recover from viral mycocarditis? Are there biomarkers to permit one to predict an indi vidual patient’s susceptibility and, for those at high risk, could vaccination be a viable option?

Future directions The ultimate goal in terms of understanding how the immune system directs inflammatory and reparative programmes following cardiac injury is the develop ment of therapeutic strategies that promote tissue regeneration. Evolutionary pressures drove both the development of primitive phagocytic cells that promote tissue growth and wound healing, but also drove the development of innate and adaptive immune mecha nisms that promote survival of the host in the face of infectious threats. Adaptations that both enhance pathogen clearance and promote tissue repair may result in a zero-sum game, whereby the very adapta tions that enhance host defence result in a loss of repar ative potential owing to excessive inflammation. The ability to selectively suppress excessive inflammation while preserving reparative functions is a novel thera peutic avenue that can enhance recovery in patients with a wide range of cardiovascular diseases.



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Competing interests statement

The authors declare no competing interests.


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