Macrophages in cardiac homeostasis, injury responses and progenitor cell mobilisation

Stem Cell Research (2014) 13, 705–714

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REVIEW

Macrophages in cardiac homeostasis, injury responses and progenitor cell mobilisation Alexander R. Pinto a,⁎, James W. Godwin a , Nadia A. Rosenthal a,b a b

Australian Regenerative Medicine Institute/EMBL Australia, Monash University, Clayton, VIC 3800, Australia National Heart and Lung Institute, Imperial College London, W12 0NN, UK

Received 3 June 2014; accepted 26 June 2014 Available online 9 July 2014

Abstract Macrophages are an immune cell type found in every organ of the body. Classically, macrophages are recognised as housekeeping cells involved in the detection of foreign antigens and danger signatures, and the clearance of tissue debris. However, macrophages are increasingly recognised as a highly versatile cell type with a diverse range of functions that are important for tissue homeostasis and injury responses. Recent research findings suggest that macrophages contribute to tissue regeneration and may play a role in the activation and mobilisation of stem cells. This review describes recent advances in our understanding of the role played by macrophages in cardiac tissue maintenance and repair following injury. We examine the involvement of exogenous and resident tissue macrophages in cardiac inflammatory responses and their potential activity in regulating cardiac regeneration. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cardiac tissue macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Embryonic and adult origins of cardiac tissue macrophages. . . . . . . . . . . . . . . . . . . . . . . . . . . . Macrophage participation in cardiac homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Macrophages and regulation of epicardial progenitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Macrophage roles in cardiac injury responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Macrophage-mediated stem cell regulation — lessons from interactions between macrophages and non-cardiac progenitors Challenges and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

705 706 706 707 708 709 710 712 712 712

Introduction ⁎ Corresponding author. Fax: + 61 3 9902 9729. E-mail address: [email protected] (A.R. Pinto).

The term phagocytosis was first proposed in 1883 by Elie Metchikoff to describe the process whereby cells clear the

http://dx.doi.org/10.1016/j.scr.2014.06.004 1873-5061/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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body of disease-causing microbes (Nobelprize.org.). In his seminal observations, Metchikoff witnessed amoebic cells from translucent starfish larvae surround particles from a tangerine tree (Nobelprize.org.; Karnovsky, 1981; Tauber, 2003). Since these early observations, the macrophage – meaning big eater in Greek – has emerged as the principal phagocytic cell in mammals and other organisms, and is critical for the clearance of foreign antigens and cellular debris. Recent work, however, has extended our understanding of the role of macrophages and demonstrated the versatility of this unique cell type, which performs important functions in tissue homeostasis and injury resolution, especially in the heart. Through their interactions with other cardiac cell types, macrophages may also play an important role in the regeneration of the damaged myocardium. While macrophage ontogeny and actions are highly heterogeneous (Mabbott et al., 2010), all macrophages participate in the clearance of tissue debris and the detection of pathogens and tissue damage. Three key characteristics enable macrophages to perform these tissue housekeeping functions. First, they undertake extensive macropinocytosis to sample the local microenvironment for molecular signatures of tissue disturbance (Lim & Gleeson, 2011). Second, they present an array of pattern recognition receptors (PRRs) that recognise foreign- and cell damage-associated particles, including CD14 (the LPS receptor), a range of mannose receptors and Toll-like receptors (TLRs). This enables detection and processing of antigens, and also presentation of antigens to T-lymphocytes via MHC-II molecules, to activate adaptive immune responses. Third, macrophages secrete factors that further facilitate their role as tissue housekeepers and orchestrators of repair.

Cardiac tissue macrophages Cardiac tissue macrophages (cTMs), which are found in abundance in the murine heart (Fig. 1), display typical macrophage characteristics, undertaking extensive macropinocytosis and phagocytosis to sample the local microenvironment and express a wide array of PRRs such as CD14, CD64 and Lpar6 (Pinto et al., 2012) that enable rapid responses to a range of damage-associated molecular patterns. However, cTMs exhibit a distinct phenotype, closely resembling alternatively-activated M2 macrophages that are observed at late phases of tissue inflammation, secreting tissue salutary and immunomodulatory factors (Pinto et al., 2012). Moreover, cTMs

GFP Mrc1 Figure 1 Cardiac tissue macrophages (cTMs) in the uninjured heart. 40 μm maximum intensity projection of cTMs labelled with GFP (green) or Mrc1 (red) from an adult Cx3cr1GFP/+ mouse heart. External margin of the heart (epicardium) is visible (top right).

DAPI PH3 GFP Figure 2 Local proliferation of cTMs. 3D perspective view of GFP+ cTM from an adult Cx3cr1GFP/+ mouse heart. Nuclear staining of mitosis marker phospho-histone H3 (PH3) is shown (red).

produce factors such as C1q and galectins that aid phagocytosis of cellular debris and simultaneously dampen inflammation. Therefore, a presumed function of cTMs is the inhibition of aberrant inflammatory reactions that may lead to cell death, and promotion of cell survival.

Embryonic and adult origins of cardiac tissue macrophages A recent revelation in the biology of tissue macrophages has been the discovery of their remarkable proliferative capacity (Sieweke & Allen, 2013). Using a range of approaches including novel transgenic mouse lines that enable inducible macrophage-specific lineage tracing, parabiosis and bone marrow transplant experiments, independent laboratories have demonstrated that macrophages in a range of tissues turn-over at steady state, and are populated by local proliferation (Hashimoto et al., 2013; Yona et al., 2013). In the heart, we and others have recently shown that cTMs are also proliferative (Fig. 2), dividing throughout life (Epelman et al., 2014; Heidt et al., 2014; Pinto et al., 2014). Macrophages that initially colonise the heart have heterogeneous ontogenic origins. Embryonic cTM subsets arrive within the myocardium early in development from yolk sac origins at embryonic day 7.5 (Epelman et al., 2014). Later macrophages are colonised by haematopoietic cells originating from the foetal liver and then bone marrow (Epelman et al., 2014). Once within the myocardium, a major mechanism of cTM population expansion and maintenance is by local proliferation (Epelman et al., 2014; Heidt et al., 2014; Pinto et al., 2014) with monocyte-derived cells contributing minimally towards the cTMs pool at steady-state (Epelman et al., 2014; Heidt et al., 2014). Intriguingly, not all cTM subsets maintain the same proliferative activity. Cx3cr1-expressing cells comprise the greatest proportion of proliferating cTMs (Pinto et al., 2014), corroborating other studies that show that not all cTM subsets have the same proliferative activity (Epelman et al., 2014). Currently, the basis for higher proliferative potential of different cTM subsets remains unknown. Stimulation by IL-4 signalling pathways may underlie the variable induction of cTM proliferation: while IL-4 has minimal effect on macrophage proliferation in vitro, even antagonising proliferation,

Macrophages in cardiac homeostasis, injury responses and progenitor cell mobilisation IL-4 stimulates macrophage proliferation in vivo (Jenkins et al., 2013; Jenkins et al., 2011). Further studies are needed to determine whether requisite components of IL-4 signalling, or other macrophage proliferation-inducing factors, are altered in various cTM subsets.

Macrophage participation in cardiac homeostasis While functional data examining the role of cTMs in adult cardiac homeostasis are limited, gene expression profiling experiments reveal a potentially important role of cTMs for many biological processes in the uninjured heart independent of tissue debris clearance (Pinto et al., 2012; Pinto et al., 2014). These include angiogenesis, fibrosis and the maintenance of immune quiescence. Supporting capillary homeostasis, cTMs express a number of pro-angiogenic factors such as Rentla/Fizz1 and IGF-1 (Pinto et al., 2012; Teng et al., 2003; Grant et al., 1993a; Nakao-Hayashi et al., 1992). However, unlike exogenous M2-macrophages, cTMs do not express VEGF and may promote angiogenesis by non-canonical VEGF-independent pathways (Nahrendorf et al., 2007; Rymo et al., 2011; Nucera et al., 2011). Moreover, cTMs may promote angiogenesis by direct interaction with the endothelium. In other tissues, neuropilin 1 (Nrp1) macrophages

A. Autocrine regulation C1q, LXA 4 , RvE1, RvD1, PD1, LGALS1

were demonstrated to physically interact with endothelial tip cells to bridge and mediate capillary anastomosis (Fantin et al., 2010). Macrophages are critical for both angiogenic growth (Sunderkotter et al., 1994) and inhibition to prevent vascular overgrowth by inducing endothelial cell death via Wntsignalling pathways (Stefater et al., 2011; Rao et al., 2007). In the heart, cTMs are in intimate contact with the dense cardiac vascular bed and express Nrp1 (Pinto et al., 2012). Nevertheless, whether cTMs or exogenous macrophages are required for the development of the dense capillary bed in the developing or injured heart remains to be determined. cTMs also contribute to cardiac fibrosis, a key hallmark of cardiac senescence, which accumulates with age (Pinto et al., 2014; Biernacka & Frangogiannis, 2011; Eghbali et al., 1989). cTMs secrete a number of pro-fibrotic proteins such as Retnla, IGF-1 and CCL24 and lipid mediators (such as LTC4) (Pinto et al., 2014) that may induce fibroblast proliferation and collagen deposition by both TGFβ1-dependent and independent pathways. In addition, cTM expression of these genes undergoes an age-dependent increase, particularly at the epicardium, suggesting that cTM-mediated ECM remodelling may alter cardiac ageing (Pinto et al., 2014). However, unlike M2-macrophages that also exhibit a fibrogenic phenotype (Murray & Wynn, 2011), cTMs do not directly produce arginase 1 (Arg1), which is required for the production of polyamine

Cardiac tissue macrophage

B. Inhibition of leukocyte infiltration C1q, RvE1, GDF15 LGALS1

707

D. Paracrine cTM regulation IL -27, IL-10

C. Paracrine regulation C1q, LXA 4 , LGALS1

Cardiac cells E. Indirect inhibition of leukocyte infiltration IL -27, IL-10

Infiltrating leukocytes

Figure 3 Indirect and direct mechanisms of cardiac immune quiescence mediated by cTMs. Different mechanisms of immunesuppression are indicated in orange boxes. (A) Autocrine regulation of cTM phenotype. cTM secreted factors regulate macrophage responses to phagocytosis to mediate non-phlogistic phagocytosis (that does not trigger inflammation), production of pro-inflammatory mediators and polarisation of cTMs to M2-macrophage-like phenotype. (B) cTMs directly inhibit leukocyte influx to the myocardium from vasculature. This is achieved by targeting chemotaxis-associated proteins, and inhibition of leukocyte activation and differentiation. (C) cTM-expressed factors modulate local cells by paracrine mechanisms. This includes production of macrophage trophic factors (D) and other anti-inflammatory elements that inhibit leukocyte infiltration (E). Examples of molecules involved in the different mechanisms are shown in grey boxes. These include, complement component 1q (C1q), resolvin E1 (RvE1), resolvin E2 (RvE2), protectin D1 (PD1), lipoxin A4 (LXA4) and galectin 1 (LGALS1), growth and differentiation factor 15 (GDF15), interleukin-27 (IL-27) and interleukin-10 (IL-10).

708 precursors for collagen deposition (Odegaard & Chawla, 2011), or the potent fibrotic factor TGFβ1 (Wynn & Barron, 2010). One of the most important activities in which cTMs are likely to participate is immune quiescence (Fig. 3). cTMs produce a wide array of immune-dampening molecules, including secreted proteins complement component 1q (C1q), growth and differentiation factor 15 (GDF15), galectin-1 (LGALS1) and lipid mediators dependent on 5-lipoxygenase (ALOX5) enzyme expression such as lipoxin A4 (LXA4), resolvin E1 and D1 (RvE1 and RvD1, respectively) and protectin D1 (PD1) (Pinto et al., 2012; Pinto et al., 2014). A principle mechanism whereby cTM-secreted factors contribute to immune quiescence is by facilitating nonphlogistic phagocytosis (phagocytosis that does not signal inflammation) of cellular debris and dead cells that may cause inflammation by the release of intracellular contents. C1q mediates this process by directly binding apoptotic debris and antibody-bound targets for phagocytosis by macrophages (Ezekowitz et al., 1984) and inhibiting macrophage production of inflammatory cytokines interleukin-1α, -6 (IL-1α and IL-6, respectively) and tumour necrosis factor α (TNFα) (Fraser et al., 2010). In contrast, LXA4 and RvE1 and PD1 promote non-phlogistic phagocytosis by directly stimulating macrophages to undertake phagocytosis, and simultaneously inhibiting the production of inflammatory cytokines interleukin-6, -8 (IL-6 and IL-8, respectively), monocyte chemoattractant protein-1 (MCP-1) or interferon-γ (IFN-γ) (Schwab et al., 2007; Godson et al., 2000). Dampening of inflammatory cytokines is further consolidated by cTM production of LGALS1, which also directly inhibits macrophage production of inflammatory cytokines TNFα, IL-1β, IL-12, and IFNγ and reduces acute inflammatory responses (Santucci et al., 2003). LGALS1 also promotes apoptosis of potentially self-reactive T cells during development and activated T cells in the periphery (Santucci et al., 2003; Perillo et al., 1995; Rabinovich et al., 1999), minimising the potential of tissue destructive immune responses. Indeed, mice deficient in LGALS1 have increased leukocyte infiltration following myocardial injury with impaired cardiac function and postinfarction ventricular remodelling (Seropian et al., 2013). To further counter inflammation, cTM-secreted factors also induce macrophages and other cells to generate anti-inflammatory factors. For example, C1q, LXA4 and LGALS1 induce production of anti-inflammatory cytokines such as interleukin-27 and -10 (IL-27 and IL-10, respectively) (Schwab et al., 2007; Son et al., 2012; Benoit et al., 2012), which have potent paracrine activities to further dampen inflammation. Moreover, RvD1 induces macrophage M2 polarisation (Titos et al., 2011). Another immunomodulatory process potentially modulated by cTMs is inhibition of leukocyte infiltration. RvE1, LGALS1 and GDF15 inhibit leukocyte cell activation and infiltration, by multiple mechanisms including targeting chemotactic proteins such as leukocyte integrin and selectins (Ariel et al., 2005; Kempf et al., 2011; Cooper et al., 2008; Dona et al., 2008). Depletion of these factors results in increased leukocyte tissue infiltration and inflammation following inflammatory stimulus (Seropian et al., 2013; Kempf et al., 2011). Moreover, C1q treatment of cultured human monocytes limits monocyte differentiation and activation (Son et al., 2012). In summary, the suite of factors secreted by cTMs are

A.R. Pinto et al. likely to contribute substantially to immune homeostasis in the heart.

Macrophages and regulation of epicardial progenitors The mammalian epicardial niche hosts multi-potent progenitor cells with the capacity to form endothelial cells, fibroblasts and smooth muscle cells (Smart et al., 2011; Chong et al., 2011). Although the relationship between macrophages and epicardial progenitors has not been characterised, the epicardium is in close contact with cTMs that are likely to modulate the activity of epicardial progenitors in the un-injured and injured heart (Pinto et al., 2014). cTMs constitutively express a number of genes involved in epicardial development and stem cell homeostasis including thymosin β4 and CEBP/β, suggesting that cTMs may play a role in epicardial development from an early stage and following injury (Pinto et al., 2014; Huang et al., 2012; Smart et al., 2007). Following cardiac injury, both resident cTMs and exogenous macrophages may be important for epicardial responses through paracrine mechanisms. Supporting this hypothesis, studies have indicated that regulation of the post-injury epicardium inflammatory milieu may be beneficial for cardiac repair and function. Indeed, injection of supernatants from ex vivo cultured epicardial progenitors improve cardiac repair after injury (Zhou et al., 2011). Moreover, adenoviral targeting of CEBP/β, a transcription factor expressed in macrophages, at the epicardium also improves cardiac repair (Huang et al., 2012). Both reports propose that modulation of epicardial paracrine environment is potentially key for the observed enhanced myocardial repair (Huang et al., 2012; Zhou et al., 2011), underscoring the potentially important role of cTMs and exogenous macrophages in regulating the post-MI inflammatory milieu and tissue repair. The finding that macrophage signalling, particularly from M2-like macrophages, enhances MSC survival, engraftment and potentially also differentiation (Ben-Mordechai et al., 2013; Freytes et al., 2013), suggests that the role of macrophage signalling may be also important for the regulation of epicardial progenitor cells to repair the damaged myocardium. However, cTM contribution to inflammatory responses at the epicardium is likely to change with age. We recently showed that cTM phenotype changes as a function of age within the epicardium resulting in a reduction of expression in chemokine/injury-sensing receptors including Cx3cr1, Lpar6, CD9, Cxcr4, Itga6 and Tgfβr1 in addition to downstream signal transduction elements (Pinto et al., 2014). Indeed, cTMs at the epicardium from aged mice (N 30 weeks old) are almost completely lacking Cx3cr1, which regulates macrophage responses to tissue injury (Lee et al., 2010). Therefore age-dependent loss of Cx3cr1 and other cTM injury sensing receptors may alter epicardial stem cell mobilisation and cardiac tissue repair. Moreover, with age, cTMs increase expression of pro-fibrotic elements that may alter the epicardial extracellular matrix (ECM). These include leukotriene c4 synthase, and Mmp9 which are associated with the promotion of fibrosis, collagen deposition and fibroblast proliferation (Pinto et al., 2014). ECM modification is important for stem cell homeostasis and function. ECM stiffness can regulate stem cell homing and

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Figure 4 Diverse role of macrophages in injury resolution. (A) Macrophage mediated removal of cellular and tissue debris by phagocytosis. (B) Angiogenesis mediated by macrophage paracrine factors and direct interaction of macrophage with endothelial tip cells. (C) Induction of tissue fibrosis by various paracrine factors. Macrophage-derived factors can induce fibroblast proliferation and myofibroblast differentiation, and can directly and indirectly contribute to extracellular matrix (ECM) deposition. (D) Macrophage subtypes (‘M1’ and ‘M2’) can promote and suppress inflammation, respectively by enhancing or inhibiting leukocyte infiltration and secretion of inflammatory mediators by local cells. (E) Various macrophage-derived factors can promote cell survival. Abbreviations: insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF2), tumour necrosis factor α (TNFα), interleukin 1β (IL-1β), nitric oxide (NO), matrix metallopeptidases, resistin-like molecule alpha (Retnla), transforming growth factor β1 (TGFβ1), fibronectin 1 (FN1), tissue inhibitor of matrix metalloproteinases (TIMPs), interleukin-10 (IL-10), interleukin-1 receptor antagonist (IL-1ra), complement component 1q (C1q), C–C motif ligand 2 (CCL2) and interleukin-6 (IL-6).

differentiation (reviewed Discher et al., 2009) and composition and bioavailability of growth factors and cytokines (Schultz & Wysocki, 2009). Thus, cTM-mediated ECM remodelling of the epicardium is another potential mechanism whereby cTMs may regulate epicardial progenitors.

Macrophage roles in cardiac injury responses While cTMs may play an important role in cardiac homeostasis, they are likely to be amongst the first cell types to react to cardiac tissue damage. Following injury, a robust inflammatory response involves secretion of various cytokines and infiltration and mobilisation of multiple cell types. Inflammation recruits exogenous monocytes/macrophages that have diverse functions (Fig. 4), distinct developmental origins and bear a different phenotype to cTMs, and the ways in which cTMs and invading monocytes/macrophages interact to progress the wound healing response and influence each other's phenotype are yet to be identified. A major factor initiating the injury response cascade is the necrosis-dependent release of intracellular contents and

cellular debris, which is recognised by various cells presenting PRRs, including cTMs (Pinto et al., 2012). These factors are likely to override any intrinsic immune-dampening elements and signal entry of neutrophils, the first exogenous myeloid cells that peak in number within 24 h after injury. Neutrophils phagocytose tissue debris and degranulate to release inflammatory mediators before submitting to phagocytosis by macrophages. Monocytes next enter the lesion by homing to soluble chemotactic cues such as MCP-1 and Cx3cl1, and differentiate to macrophages (Nahrendorf et al., 2007; Nahrendorf & Swirski, 2013). MCP-1 is a key monocyte/ macrophage chemoattractant and is produced by a range of cardiac cells, including endothelial cells, macrophages and fibroblasts, within hours following cardiac injury (Dewald et al., 2005). Ablation of the Mcp-1 receptor Ccr2 results in the severe impairment of the monocyte influx into damaged tissue (Majmudar et al., 2013; Kaikita et al., 2004). After cardiac injury, two classes of macrophage are sequentially predominant within damaged myocardium: the classically activated ‘M1’, and alternatively activated ‘M2’ macrophages. The M1 and M2 paradigm, while an

710 oversimplification, describes two broad and heterogeneous macrophage classes at the extremes of a continuum of maturation states (Mosser & Edwards, 2008). Extensive research has established this model in wound healing and inflammation in many tissues (Jenkins et al., 2011; Nahrendorf et al., 2007; Arnold et al., 2007). Instrumental for the study of M1 and M2 macrophages, particularly in the mouse, has been the identification of cell surface markers that have enabled their discrimination, such as Ly6C (where M1 and M2 are Ly6Chigh and Ly6Clow, respectively), Mrc1 (also known as CD206; where M1 and M2 macrophages are Mrc1− and Mrc1+, respectively) and Cx3cr1 (where M1 and M2 macrophages are Cx3cr1low and Cx3cr1high, respectively) (Nahrendorf et al., 2007; Arnold et al., 2007). Within the first 5 days (the wound healing phase) and peaking at approximately 3 days after cardiac injury, the M1 macrophage subtype predominates, characterised by their fibrolytic, phagocytic and inflammatory properties, releasing inflammatory mediators such as TNFα, IL-6, IL-1β, Ccl2, Ccl5 and nitric oxide (NO) (Nahrendorf et al., 2007; Murray & Wynn, 2011). M1 macrophages undertake extensive phagocytic activity to clear necrotic and apoptotic debris including remnant neutrophils, and release fibrolytic proteases such a MMP-1, -2, -7, -9 and -12 which facilitates cells to penetrate towards the injury lesion, paving the way for tissue granulation. Following the infiltration of M1 macrophages, approximately 7 days post-MI the injury-resolution phase begins with M2 macrophages becoming predominant (Nahrendorf et al., 2007). M2 macrophages are characterised as anti-inflammatory, salutary, and fibrogenic (Murray & Wynn, 2011). Anti-inflammatory factors including IL-10, IGF-1 and lipid mediators such as lipoxins, resolvins and protectins (discussed above) drive resolution of the acute inflammatory response. M2 macrophage-derived factors, such as FGF2, IGF-1, PDGF, TGFβ1 and VEGF are angiogenic, limiting cell death due to lack of blood supply (Nakao-Hayashi et al., 1992; Nahrendorf et al., 2007; Grant et al., 1993b; Roberts et al., 1986). Angiogenesis is also supported by M2 macrophage production of ECM modulating factors such as matrix metallopeptidases (MMPs) (Zijlstra et al., 2004) and serine proteases (u-PA, t-PA) that liberate ECM-bound growth factors and regulate both angiogenesis and mobilisation of other cell types (Eming & Hubbell, 2011). While M2 macrophages may be the principal drivers of angiogenesis associated with injury resolution, M1 macrophages are highly fibrolytic, which may facilitate angiogenesis, and secrete pro-angiogenic factors such as TNFα and IL-1β, and nitric oxide (Sunderkotter et al., 1994; Cooke & Losordo, 2002). Therefore, both injury-associated macrophage subsets may contribute to blood vessel growth, which is critical for the minimization of cell death due to hypoxia. The fibrogenic phenotype of M2 macrophages is a critical element of cardiac healing by conferring cardiac tensile strength and prevention of fatal cardiac rupture (Nahrendorf & Swirski, 2013; Frangogiannis, 2012). M2 macrophages secrete a number of pro-fibrotic factors, such as IGF-1, fibronectin 1 (FN1), Arg1, Retnla, TGFβ1, FGF2 and others that promote fibrosis (Wynn & Barron, 2010; Bitterman et al., 1983; Goldstein et al., 1989). While macrophage-derived FN1, collagen and other molecules may directly contribute to ECM deposition, a major

A.R. Pinto et al. mechanism of macrophage-stimulated fibrosis is the activation of cardiac fibroblasts. M2 macrophage derived factors such as IGF-1, Retnla, TGFβ1 and FGF2 can stimulate fibroblast proliferation and also myofibroblast differentiation (Bitterman et al., 1983; Goldstein et al., 1989). While both phases of macrophage infiltration are critical for resolving myocardial injury, it is increasingly becoming evident that shifting the balance of macrophages to an M2-like phenotype is salutary (Frangogiannis, 2012). For example, inhibition of Ccr2-dependent entry of M1 macrophage forming monocytes after MI, by genetic ablation of Ccr2 or RNAi, improves cardiac healing (Majmudar et al., 2013; Kaikita et al., 2004). Indeed, cardiac-specific genetic overexpression or direct delivery of M2-associated growth factors such as IGF-1 or IL-10 is cardioprotective (Burchfield et al., 2008; Santini et al., 2007).

Macrophage-mediated stem cell regulation — lessons from interactions between macrophages and non-cardiac progenitors In recent years, increased attention focused on macrophages for stem cell activation in regenerating and neoplastic tissues has underscored their role as guardians of the stem cell niche that may be modulated to induce tissue regeneration (Table 1). These studies offer valuable insights regarding the potential role of macrophages, particularly cTMs, in regulating the activities of intrinsic progenitor cells within the heart. Our rapidly growing understanding of macrophages in these contexts has relied on approaches that permit macrophage depletion within defined temporal windows (see Chow et al., 2011a) for comprehensive review). Clodronate-loaded liposomes (Clo-Lip) specifically target phagocytic cells for destruction (Van Rooijen & Sanders, 1994), whereas myeloid/ macrophage-specific promoters drive genetic cassettes that enable inducible cell death (for example diphtheria toxin receptor (DTR) or Fas-ligands). Using these approaches, macrophages have been identified as a critical component in tissue regeneration. For example, Clo-Lip treatment following hepatic injury prevents liver regeneration in mice (Boulter et al., 2012). Similarly, Clo-Lip depletion of macrophages following mouse skeletal muscle injury leads to derailment of the regenerative programme and tissue scarring (Arnold et al., 2007), comparable to the genetic blockade of M2 macrophage generation (Ruffell et al., 2009). In salamanders, which undergo efficient regeneration of whole body parts, transient depletion of macrophages using Clo-Lip within the first four days following limb amputation alters the cytokine profile and ECM composition preventing initiation of a successful regeneration programme (Godwin et al., 2013). Depletion of macrophages at later phases has no effect, suggesting the early requirement for specific macrophagederived regenerative cues for regeneration. Blockade of regeneration is not, however, permanent; reamputation of the limb stump at a more proximal position, after replenishment of macrophages, restores epimorphic regeneration of the limb (Godwin et al., 2013). These observations confirm the evolutionarily conserved role played by macrophages in the initiation of tissue regeneration.

Macrophages in cardiac homeostasis, injury responses and progenitor cell mobilisation

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Table 1 In vivo regulation of stem cells by macrophages. Summary of in vivo studies implicating direct or indirect regulation of stem cells by macrophages in tissue stress and homeostasis. Tissue

Stem cell

MU targeting approach

Effect of MU depletion

MU paracrine mediators

Reference

Liver

Hepatic progenitors

Clo-Lip

Wnt3a

Boulter et al. (2012)

Bone marrow

Haematopoietic

Cxcl12

Heart

Impaired therapeutic effect of MSCs

Unspecified

Chow et al. (2011b) and Winkler et al. (2010) Ben-Mordechai et al. (2013)

Tumour

Transplanted mesenchymal stem cells (MSCs) Tumour stem cell

Clo-Lip, CD169-DTR 1, MaFIA 2 Clo-Lip

Impairment of liver regeneration, re-specification of progenitor differentiation Stem cell mobilisation to blood

Unspecified

Yang et al. (2013)

Mammary gland

Mammary stem cells

Csf1op/op 3, Clo-Lip

Unspecified

Gyorki et al. (2009)

Gut

Colonic epithelial progenitors (ColEPs) Alveolar epithelial type II cells (AE2)

Csf1op/op

Induction of cancer stem cell phenotype Impaired mammary outgrowth of ectopic mammary glands Loss of ColEP proliferation after inflammatory stimulus Loss of AE2 cells; lung injury

Unspecified

Pull et al. (2005)

Unspecified

Miyake et al. (2007)

Lung

Clo-Lip

LysM-DTR 4

1

Transgenic mouse line with diphtheria toxin receptor (DTR) driven by the CD169 promoter (Chow et al., 2011b). Transgenic mouse line expressing Fas-based cell death gene driven by the c-fms promoter, enabling inducible macrophage depletion (Burnett et al., 2004). 3 Mutant mouse line with deficiency in macrophage colony stimulating factor (CSF1) and macrophages. 4 Transgenic mouse line expressing DTR driven by the LysM promoter (Miyake et al., 2007). 2

A key mechanism whereby macrophages may affect tissue repair and regeneration is by instructing the stem cell niche by paracrine mechanisms. In the mouse skeletal muscle injury model, macrophage-derived paracrine factors promote satellite cell survival, proliferation and differentiation in vitro and in vivo (Cantini et al., 1994; Chazaud et al., 2003). In the mouse hepatic injury model, phagocytic macrophages activate HPCs via Wnt3a signalling, to induce HSP proliferation and differentiation (Boulter et al., 2012). Depletion of macrophages sharing the HPC niche results in re-specification of HPCs to generate peri-portal biliary structures instead of hepatocytes (Boulter et al., 2012). Macrophages also occupy the haematopoietic stem cell (HSC) niche (Chow et al., 2011b; Ludin et al., 2012; Winkler et al., 2010). Macrophage depletion using either Clo-Lip, macrophage-specific genetic or antibody-mediated depletion strategies, results in egression of haematopoietic stem cells (HSCs). Here, also, paracrine signalling is important with Cxcl12 playing a prominent role (Chow et al., 2011b; Ludin et al., 2012; Winkler et al., 2010; Christopher et al., 2011). Depletion of bone marrow macrophages leads to a significant decrease in Cxcl12 production (Chow et al., 2011b; Ludin et al., 2012; Winkler et al., 2010; Christopher et al., 2011), a critical factor for HSC homeostasis (Sugiyama et al., 2006). Moreover, macrophage G-CSFR is important for regulation of Cxcl12 and HSC mobilisation, with macrophage-restricted G-CSFR expression sufficient for G-CSF mediated HSC egression (Christopher et al., 2011). These observations indicate that macrophages, in particular subepicardial

cTMs, may act as gatekeepers regulating progenitor cell mobilisation. Macrophages likewise positively regulate mesenchymal stem cells. In vitro experiments on cultured human MSCs demonstrate that macrophage-derived growth factors enhance MSC growth, viability, motility and secretion of paracrine factors (Freytes et al., 2013; Anton et al., 2012). MSC mobilising factors include IL-8, Mcp-1 and CCL5, which are chemotactic for MSCs (Anton et al., 2012). However, macrophage-MSC interactions are not uni-directional. MSCs transplanted to the injured myocardium induce a shift in the balance of macrophages to an M2-like phenotype (Ben-Mordechai et al., 2013). Similarly, in a spinal cord injury model, MSC transplantation shifts macrophage phenotype to an M2-like phenotype, leading to improved functional recovery (Nakajima et al., 2012). These findings are consistent with in vitro evidence that supernatants from cultured human MSCs polarise human monocyte-derived macrophages towards M2-like cells (Kim & Hematti, 2009). Finally, macrophages play a significant role in promoting cancer stem cell proliferation and activation (De Palma & Lewis, 2013). Tumour associated macrophages (TAMs), closely resembling M2 macrophages (Pucci et al., 2009; Mantovani et al., 2002), release a number of paracrine factors that regulate cancer stem cells (CSCs) (De Palma & Lewis, 2013), including those that induce tumour cells to acquire cancer stem cell-like phenotypes (Yang et al., 2013; Jinushi et al., 2011). Depletion of macrophages by either using Clo-Lip or synthetic inhibitors of macrophage colony stimulating factor 1 receptor results in a reduction in the number and activity of CSCs within tumours

712 and increased sensitivity to chemotherapeutic agents (Yang et al., 2013; Mitchem et al., 2013). Taken together, these examples underscore the importance of macrophage-stem cell cross talk for stem cell homeostasis and mobilisation. Considering the close interaction of cTMs with the epicardium (Pinto et al., 2014), these observations indicate that cTMs may be important for epicardial progenitor cell homeostasis and potential maintenance of the progenitor cell phenotype.

Challenges and future perspectives While a significant body of work indicates that macrophages are critical for tissue homeostasis, repair and regeneration, much work is still required to delineate the precise contribution of macrophages in these processes particularly in the heart. Indeed, recent work has demonstrated that the regeneration of the mouse neonatal heart and adult axolotl heart is dependent on macrophages (Aurora et al., 2014; Godwin, Pinto, Rosenthal, unpublished data). However, the basic questions of whether macrophages are merely required for clearance of tissue debris, an essential pre-requisite process for tissue injury resolution, or whether they play a greater role in directing cell fate and organogenesis remain to be unequivocally demonstrated. Addressing these primary questions or more focussed questions such as macrophage-derived molecular mechanisms is hampered by current technical limitations. Utilisation of macrophage depletion and modulation strategies such as Clo-Lip or genes driven by myeloid-specific promoters, mainstays of macrophage biology, is not specific to injury lesions. Therefore, whether the effects of perturbation derive from local modulation of macrophages in the site of injury or secondary effects in remote tissues (such as the spleen, liver or bone marrow, for instance) is not clear. Deciphering the precise molecular pathways for the activation of progenitor cells in regenerative and non-regenerative tissues will therefore necessitate the generation of new genetic tools. In addition to identifying novel macrophage-mediated regenerative signalling pathways, increasing macrophage numbers in the injured heart may yield salutary results. As cTMs are anti-inflammatory and modulation of inflammation by MSCs or other mechanisms improve cardiac repair, cTM transplantation or expansion in situ may be therapeutically beneficial following injury. Moreover, strategies to reverse age-dependent changes in cTM gene expression (Pinto et al., 2014), particularly in the epicardium, may improve inflammatory signalling profiles in response to injury or stress. The last ten years has seen a dramatic expansion in our understanding of this most versatile cell type. However, while important work has been done regarding the role of exogenous macrophages in cardiac injury, our understanding of the role of cTMs for cardiac development and homeostasis is currently superficial and limited to interpretation of gene expression data. It is unknown what role cTMs may play in cardiac development at the structural and functional level. Moreover, the contribution of macrophages to cardiac inflammatory responses is unclear and the dynamic interplay between cTMs and cardiac progenitors is unresolved. New approaches are required to modulate macrophage numbers and gene expression in the heart with greater specificity. Nevertheless,

A.R. Pinto et al. evidence gathered to date suggests that the manipulation of macrophages in the heart is worthy of further investigation, and may be pivotal for reducing morbidity and mortality following cardiac injury.

Acknowledgments This work is supported by Stem Cells Australia, a grant (DP130100294) from the Australian Research Council and a Grant-in-Aid (G 12M 6627) from the National Heart Foundation of Australia. The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government. N.A.R. is an NH&MRC Australia Fellow.

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