Tissue-Resident Macrophage Ontogeny and Homeostasis

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Review Tissue-Resident Macrophage Ontogeny and Homeostasis Florent Ginhoux1,2,* and Martin Guilliams3,4,* Immunology Network (SIgN), A*STAR, 8A Biomedical Grove, Immunos Building, Level 3, Singapore 138648, Singapore Institute of Immunology, Shanghai JiaoTong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China 3Unit of Immunoregulation and Mucosal Immunology, VIB Inflammation Research Center, Ghent 9052, Belgium 4Department of Biomedical Molecular Biology, Ghent University, Ghent 9000, Belgium *Correspondence: [email protected] (F.G.), [email protected] (M.G.) http://dx.doi.org/10.1016/j.immuni.2016.02.024 1Singapore 2Shanghai

Defining the origins and developmental pathways of tissue-resident macrophages should help refine our understanding of the role of these cells in various disease settings and enable the design of novel macrophage-targeted therapies. In recent years the long-held belief that macrophage populations in the adult are continuously replenished by monocytes from the bone marrow (BM) has been overturned with the advent of new techniques to dissect cellular ontogeny. The new paradigm suggests that several tissue-resident macrophage populations are seeded during waves of embryonic hematopoiesis and self-maintain independently of BM contribution during adulthood. However, the exact nature of the embryonic progenitors that give rise to adult tissue-resident macrophages is still debated, and the mechanisms enabling macrophage population maintenance in the adult are undefined. Here, we review the emergence of these concepts and discuss current controversies and future directions in macrophage biology. Introduction Macrophages play a central role in both tissue homeostasis and inflammation, completing essential tissue-specific functions as well as protecting the organism from infection. However, they also contribute to the pathophysiology of multiple diseases including cancer and various inflammatory disorders. Understanding the origins, the developmental pathways, and the homeostatic processes that regulate tissue-resident macrophages is fundamental to enable the design of future intervention strategies to modulate macrophage functions at specific sites. For decades, it was believed that tissue-resident macrophages are continuously repopulated by blood-circulating monocytes, which arose from progenitors in the adult bone marrow (BM). This cellular hierarchy was a foundational concept in the definition of the ‘‘mononuclear phagocyte system’’ (MPS) by Van Furth and colleagues in the 1970s that grouped together promonocytes and their precursors in the BM, monocytes in the peripheral blood, and macrophages in the tissues (van Furth et al., 1972; Yona and Gordon, 2015). The most immature cell type included in the MPS at this time, the BM promonocyte, was thought to give rise to blood-circulating monocytes that in turn were constantly recruited to tissues where they differentiated into tissue-resident macrophages. In recent years, our knowledge of the ontogeny of the cell types within the MPS has changed dramatically. Several studies have now revealed that the homeostatic contribution of circulating monocytes to macrophage populations seems to be restricted to a few specific tissues including the gut, the dermis, and the heart with a turnover rate unique to each tissue in the steady state. Instead, many tissue-resident macrophage populations arise from embryonic precursors that take residence in these tissues prior to birth and maintain themselves locally throughout adulthood, independent of a major contribution from BM-derived precursors. Here, we review

the emergence of these new concepts and discuss remaining outstanding questions as well as further directions. Early Concepts of MPS Ontogeny Describing the MPS (van Furth et al., 1972), Van Furth and colleagues underlined that ‘‘although the promonocyte is the earliest identifiable cell in this system, there must be a more immature precursor cell feeding into the pool of promonocytes’’ (as reported in van Furth and Cohn [1968] and van Furth and Diesselhoff-Den Dulk [1970]). Three decades later, a clonogenic progenitor that gives rise to monocytes, macrophages, and dendritic cells (DCs) was identified in the BM (Fogg et al., 2006). In this study, adoptively transferred macrophage DC precursors (MDPs) in recipient mice exposed to irradiation injuries gave rise to monocytes, DCs, and several tissue-resident macrophage subsets (including those of spleen and lung). These results provide an important early illustration of the ontogenic relationships between monocytes, DCs, and macrophages, emphasizing monocyte potential to reconstitute tissue macrophage compartments under certain conditions. Since then, much research effort has been dedicated to deciphering the developmental lineages of monocytes, DCs, and macrophages leading to the identification of the CDP (common DC precursor), a DC-restricted precursor in the BM (Naik et al., 2007; Onai et al., 2007); the pre-DC, which is a CDP-derived circulating DC precursor that seeds the tissues to give rise to DCs (Liu et al., 2009; Ginhoux et al., 2009); and more recently the cMOP (common monocyte progenitor), a monocyte-restricted BM precursor (Hettinger et al., 2013). Both CDPs and cMOPs derive from MDPs and can be distinguished from MDPs by relatively lower c-Kit expression in the case of CDPs (Naik et al., 2007; Onai et al., 2007) and by the loss of CD135 expression in the case of cMOPs (Hettinger et al., 2013). cMOPs give rise to both monocyte subsets, the Ly6Chi and Ly6Clo populations, whose Immunity 44, March 15, 2016 ª2016 Elsevier Inc. 439

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Review relationship is still controversial (recently reviewed in Ginhoux and Jung, 2014). The identification of these dedicated precursors provide a basis for understanding the MPS on a cellular and molecular level, though the methods originally employed for the identification of most hematopoietic progenitors, mainly relying on minimal patterns of surface marker expression, have since been shown to have significant limitations. The notion that MDPs are the common restricted progenitor of macrophages, monocytes, and DCs has been challenged by a study that shows MDPs retain potential to develop into other hematopoietic lineages and fails to find evidence for a discrete macrophage-DC-restricted precursor as a major intermediate on the pathway to tissue-resident DC populations (Sathe et al., 2014). Single-cell RNA-seq-based unbiased approaches (Jaitin et al., 2014; Schlitzer et al., 2015; Paul et al., 2015) are now expanding our understanding of the MPS, revealing additional populations contained within previously defined populations that were not detectable by surface marker-based approaches alone. Such unsupervised approaches should help foster our ontogenic studies of the MPS. A foundational paradigm of the ontogeny of the MPS, as stated in the 1972 bulletin, was that ‘‘Monocytes in the circulation constitute a mobile pool of relatively immature cells on their way from the place of origin to the tissues’’ (van Furth et al., 1972). This notion has been supported by in vitro studies showing the differentiation of either BM cells or monocytes into macrophages, in vivo adoptive transfer experiments where labeled monocytes differentiate into tissue-resident macrophages, and the observation that blood leukocytes recruited into the inflamed peritoneum differentiated into macrophages. Although these data do suggest that circulating blood monocytes can differentiate into macrophages, the studies themselves are limited by the techniques available at the time and failed to address the extent to which blood monocytes do differentiate into macrophages. Thus the contribution of monocytes to adult macrophage homeostasis in the steady state remained unknown. In the early 2000s, studies started to suggest that the prevailing monocytes-to-macrophages dogma did not apply to all adult macrophage populations. Langerhans cells (the epidermal macrophage population) were shown to resist high doses of irradiation and to repopulate from the host after congenic BM transplantation, whereas monocytes were all of donor origin (Merad et al., 2002). Similar results have been also observed for microglia (Ajami et al., 2007; Ginhoux et al., 2010), suggesting that both these populations maintain themselves independently of the contribution of BM-derived circulating precursors, even after exposure to lethal doses of irradiation. Additional evidence for the BM or monocyte independence of adult macrophage populations come from experiments in parabiotic mice that share the same blood circulation leading to substantial mixing of circulating precursors. These studies reveal that, although monocytes were mixed in these animals, Langerhans cells (Merad et al., 2002), microglia (Ajami et al., 2007; Ginhoux et al., 2010), and alveolar macrophages (Hashimoto et al., 2013; Guilliams et al., 2013; Jakubzick et al., 2013) do not mix, whereas macrophage populations in the gut, dermis, and heart show some degree of mixing, suggesting some contribution from BM- or monocyte-derived cells (Tamoutounour et al., 440 Immunity 44, March 15, 2016 ª2016 Elsevier Inc.

2013; Epelman et al., 2014; Molawi et al., 2014, Bain et al., 2014). Work by Yona et al. (2013) using conditional CX3CR1 promoter-driven Cre recombinase expression to fate-map the murine monocyte and macrophage compartment confirmed that major tissue-resident macrophage populations, including microglia but also liver Kupffer cells, lung alveolar macrophages, and splenic macrophages, are established prior to birth and subsequently maintain themselves independently of replenishment by blood monocytes during adulthood. Altogether, these models show that monocytes differentially contribute to the maintenance of tissue-resident macrophages, with most macrophage populations being maintained almost entirely locally throughout the life of the animal. Distinct Waves of Embryonic Hematopoiesis The realization that tissue macrophages are maintained independently of adult BM progenitors together with the emergence of fate mapping technologies have led investigators to revisit the contribution of embryonic precursors to the adult macrophage pool. Here, we review the emergence of this conceptual breakthrough and discuss some of the outstanding questions on the precise identity of these embryonic macrophage progenitors. Several studies have reported that macrophages arise in embryos before the generation of the first hematopoietic stem cells (HSCs). In rodents, macrophages are present in the brain rudiment (for review, see Ginhoux et al., 2013) and developing skin (for review, see Ginhoux and Merad, 2010) as early as embryonic day (E)10.5. In the rat, the developing brain is the first organ to be colonized by highly proliferative fetal macrophages (Sorokin et al., 1992; Mizoguchi et al., 1992). Although the existence of fetal macrophage-like cells had been undisputed, they were not thought to contribute to the maintenance of the adult macrophage pool, although the potential contribution of fetal macrophages to adult brain macrophages was clearly suggested in several studies (Alliot et al., 1991, 1999). Despite the fact that the tools required to trace the progeny of embryonic precursors were critically lacking at the time, these early studies established that fetal macrophages develop before the development of HSCs and therefore are essential in the concept that macrophages might represent a lineage independent of HSCs. Defining the exact ontogeny of fetal macrophages is still extremely challenging because mammalian embryonic hematopoiesis is complex, occurring in successive waves that arise from both extra- and intra-embryonic sites, leading to the sequential acquisition of erythroid, myeloid, and lymphoid lineage potentials (Figure 1; for review, see Hoeffel and Ginhoux, 2015; Orkin and Zon, 2008). The first wave is termed primitive hematopoiesis and develops from the posterior plate mesoderm in the blood islands of the extra-embryonic yolk sac (YS) around E7.0, giving rise to primitive erythroblasts, megakaryocytes, and macrophages (Palis et al., 1999; Tober et al., 2007). A second wave of hematopoietic progenitors, called erythro-myeloid precursors (EMPs), arise from the YS hemogenic endothelium between E8.0 and E8.5. EMPs exhibit erythroid and broad myeloid, but not lymphoid, potential (for review, see Frame et al., 2013). Because this wave of EMPs is unable to persist upon transplant in immune-compromised animals, it has been called the ‘‘transient definitive’’ wave.

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Review Figure 1. Embryonic Hematopoiesis Three main successive waves of hematopoiesis occur during development. The first arises directly from the posterior plate mesoderm in the blood islands of the extra-embryonic yolk sac (YS) at E7.0, giving rise to progenitors as early as E7.25, which produces primitive erythroblasts and megakaryocytes and is termed primitive hematopoiesis. Progenitors giving rise to macrophages are poorly characterized. The second wave arises from the hemogenic endothelium formed between E8.0 and E8.25 in the YS and gives rise to the second wave of hematopoietic progenitors called eythro-myeloid precursors (EMPs) and is termed the transient definitive wave. Once the blood circulation is established, from E8.5 onward, EMPs migrate into the fetal liver (FL), where they expand and differentiate into multiple lineages including monocytes. Recently, using fate-mapping models, two waves of EMPs were shown to emerge in the YS. A first wave of ‘‘early’’ EMPs at E7.5 express the CSF-1R but not the transcription factor c-Myb and could represent primitive progenitors. A second wave of ‘‘late’’ EMPs emerges at E8.25, which express the transcription factor c-Myb and either give rise to YS macrophages locally or migrate to the FL through the blood circulation at E9.5 (Hoeffel et al., 2015) and could represent transient definitive progenitors. Lastly, almost concomitant with the emergence of the late EMPs at E8.5, the third wave arises in the embryo proper from hemogenic endothelium. It starts with the generation of immature HSCs in the para-aortic splanchnopleura (P-Sp) region and proceeds to give rise to fetal HSCs at E10.5 in the aorta, gonads, and mesonephros (AGM) regions (which themselves arise from the P-Sp). These precursors then colonize the FL where they establish definitive hematopoiesis and will also seed the fetal BM that will eventually lead to the generation of adult HSCs in the BM.

Once the blood circulation is established, from E8.5 onward, EMPs migrate into the fetal liver (FL) (Palis and Yoder, 2001), where they expand and differentiate into cells of multiple lineages, including monocytes. In line with these studies, Bertrand et al. (2005) have shown via in vitro cloning assays that a first wave of monopotent progenitors in the YS gives rise to macrophages and a second wave is responsible for generating granulocytes, monocytes, and macrophages. Accordingly, we have identified during the transient definitive stage (Hoeffel et al., 2015) a first wave of ‘‘early’’ EMP-like cells at E7.5 that mainly give rise to macrophages and might represent primitive progenitors and a second wave of ‘‘late’’ EMPs at E8.25, which gives rise to YS macrophages locally and migrates to the FL at E9.5 to generate progenitors with broader myeloid cell potential, including FL monocytes (Figure 1). Almost concomitant with the emergence of the late EMPs at E8.5, a new wave of hematopoietic progenitors arises from the intraembryonic hemogenic endothelium, which begins with the generation of immature HSCs in the para-aortic splanchnopleura (P-Sp) region and proceeds to give rise to fetal HSCs in the aorta, gonads, and mesonephros (AGM) regions at E10.5 (which themselves arise from the P-Sp) (Figure 1; for review, see Cumano and Godin, 2007; Godin and Cumano, 2002). These precursors then colonize the FL where they establish definitive hematopoiesis, but will also seed the fetal BM where these cells will eventually lead to the generation of adult BM HSCs. From E12.5, the FL becomes the major hematopoietic organ within the embryo and contains progenitors of different origins and varied potentials, which together will give rise to the emergent immune system.

The presence of these different waves of embryonic precursors has fueled many hypotheses in the field. Although the embryonic ontogeny of macrophages is now accepted, the precise identity of the progenitors that give rise to fetal macrophages, the pathway of differentiation from these progenitors to mature cells, and the transcription factor requirements of the process remain under debate or unknown. Are tissue-resident macrophages mainly derived from EMPs, and if so are they early EMPs or late EMPs? And how much do fetal HSCs or adult HSCs contribute to tissue-resident macrophage populations? As a result, there are now three different models of macrophage embryonic ontogeny (Figure 2). The established facts from which these models have been proposed and the persisting open areas of research are discussed below. Contribution of YS Macrophages to Tissue-Resident Macrophage Populations Only recently have advances in fate-mapping models allowed researchers to trace the contribution of embryonic progenitors to adult tissue-resident macrophage populations. The first fatemapping model used to probe the contribution of embryonic precursors to adult tissue-resident macrophages have traced the progeny of embryonic Runt-related transcription factor 1 (RUNX1)+ hematopoietic cells (Ginhoux et al., 2010) using the tamoxifen-inducible Runx1-Mer-cre-Mer fate mapping model (Samokhvalov et al., 2007). RUNX1 expression is necessary for the sequential emergence of EMPs and HSCs from the hemogenic endothelium during embryogenesis (Tober et al., 2013). RUNX1+ hematopoietic precursors are restricted to YS-derived Immunity 44, March 15, 2016 ª2016 Elsevier Inc. 441

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Review Figure 2. Three Different Models of Fetal Macrophage Ontogeny The first model corresponds to the work of Gomez Perdiguero et al. (2015), the second to the work of Hoeffel et al. (2015), and the last to Sheng et al. (2015). Red arrow indicates the proposed major path of ontogeny and differentiation in each model. Cell colors are matched to their proposed origins. For example, whereas model 1 considers the contribution of FL monocytes unlikely, models 2 and 3 propose that these cells represent the main precursor of fetal macrophage populations, with the exception of microglia, which arise predominantly from c-Myb-independent YS macrophages.

cells between E7.0 and E8.0 whereas RUNX1 starts to be expressed by definitive hematopoietic precursors around E8.5 (Samokhvalov et al., 2007). Thus, YS progenitors and their progeny are labeled when tamoxifen is administered at E7.0–E7.5, whereas administration after E8.5 labels definitive hematopoietic precursors and their progeny. We have found that upon early E7.0 tamoxifen administration, labeled macrophages infiltrate the whole embryo, contributing to macrophages in all tissues between E10.0 and E13.0 (Ginhoux et al., 2010). Of note, labeled microglia persist in adult brains, whereas most resident macrophage populations are unlabeled in adult tissues, suggesting that they have been replaced by non-labeled precursors. In contrast, when RUNX1+ cells are tagged after E8.0, the relative number of tagged microglia decrease dramatically, whereas 442 Immunity 44, March 15, 2016 ª2016 Elsevier Inc.

the relative number of tagged leukocytes (including monocytes) and other tissueresident macrophages increase progressively. We therefore propose that microglia is the only adult macrophage population that is derived from E7.25 RUNX1+ YS-derived hematopoietic progenitors, with little contribution from hematopoietic progenitors arising later in embryonic development (Ginhoux et al., 2010). However, it remains possible that a minor population of adult microglia, perhaps in a defined region of the brain, might derive from non-YS progenitors recruited later during development. Geissmann and colleagues have confirmed these findings, but proposed that these YS-derived macrophages represent the main precursors for most tissue-resident macrophages (Schulz et al., 2012; Gomez Perdiguero et al., 2015; Perdiguero and Geissmann, 2015). Indeed, Schulz et al. (2012) have observed that CD11bhi myeloid cells are absent in tissues of Myb–/– embryos whereas F4/80hi macrophages are still present in early embryos, and concluded that fetal macrophages arise from a c-Myb-independent lineage in the YS, whereas most hematopoietic cells arise from c-Myb+ progenitors. A subsequent study has suggested that EMPs that arise in the YS are the main precursors of adult macrophages including microglia, Langerhans cells, and Kupffer cells (Gomez Perdiguero et al., 2015). In tamoxifen-inducible Tek-Mer-cre-Mer mice (also named Tie2-cre), in which tamoxifen (40 OHT) administration labels TIE2+ hemogenic endothelia and their progeny, including EMPs and fetal HSCs, the authors have shown that tamoxifen injection at E7.5 labels a higher proportion of adult tissue-resident macrophages than leukocytes; injection at E8.5 labeled comparable proportions of both cell types; and from E9.5 leukocyte populations are relatively more frequently labeled than tissue-resident macrophages. Altogether these findings indicate that fetal and adult macrophages originate predominantly from a progenitor cell type that expresses Tie2 at E7.5 but not after

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Review E9.5, and the authors propose this progenitor to be YS EMPs (Figure 2, model 1). Contribution of Late EMP-Derived FL Monocytes to Tissue-Resident Macrophage Populations The decoupling of macrophage and leukocyte labeling seen in the Tie2-based fate mapping study has been also observed in the Runx1-Mer-cre-Mer model (Ginhoux et al., 2010; Hoeffel et al., 2012, 2015). We have also concluded that fetal macrophages arise mostly from EMPs generated in the YS in an HSC-independent fashion but have since exploited the exceptional resolution of the Runx1-Mer-cre-Mer model to uncover a further level of complexity: we now know that two waves of temporally separated and functionally distinct EMPs emerge in the YS between E7.5 and E8.5 (Figure 2, model 2). The first wave emerges from E7.5 and these ‘‘early’’ CSF-1Rhi-c-Myb EMPs give rise mostly to local YS macrophages (without monocytic intermediates), and by consequence to microglia. In agreement with data of Kierdorf et al. (2013) that defined early E8 primitive uncommitted F4/80c-Kit+ EMPs as microglia progenitors, this early wave might consist of primitive progenitors. Whether these early EMPs are more related to primitive YS progenitors than to EMPs according to the traditional definition remains to be formally established. The recently proposed EMP phenotype approach (McGrath et al., 2015) might be able to shed some light on this situation. The second wave of ‘‘late’’ CSF-1Rlo-c-Myb+ EMPs emerges in the YS from E8.5. These cells can also give rise locally to YS macrophages but most of these cells migrate into the FL through the blood circulation at E9.5. Once seeded in the FL, these cells generate cells of multiple lineages, including FL monocytes. These late EMPs could represent the real transient definitive progenitors and we propose that these late EMPs represent the main precursors for most tissue-resident macrophages through a monocytic intermediate (Figure 2, model 2; Hoeffel et al., 2015). The notion of EMP heterogeneity is supported by the differential labeling at closely spaced time points of tamoxifen administration in both Runx1 and Csf1r fate mapping models (Hoeffel et al., 2015). In the Runx1-Mer-cre-Mer embryos, induction of labeling at E7.5 tags E10.5 YS EMPs but not intra-embryonic EMPs, whereas induction at E8.5 tags both YS EMPs and intra-embryonic EMPs with the same efficiency. Similarly, in Csf1r-Mer-cre-Mer mice, induction at E8.5 efficiently tags E10.5 EMPs and YS macrophages. However, increased decoupling between the tagging frequencies of EMPs and macrophages occurs with time, suggesting that early EMPs are not maintained but are rapidly replaced by late EMPs. These observations indicate that early EMPs differentiate locally, predominantly generating YS macrophages before the onset of blood circulation, and late EMPs can spread through the blood circulation, as reported (Palis and Yoder, 2001). In addition to the temporal separation between early and late EMPs, it seems that both cells are functionally distinct. This is supported by their differential expression of CSF-1R and c-Myb. Whereas early EMPs express CSF-1R but little c-Myb, late EMPs do not express CSF-1R but exhibit higher c-Myb expression (Hoeffel et al., 2015; Kierdorf et al., 2013). As a consequence, injection of 40 OHT at E8.5 or E9.5 in the Csf1r-Mer-cre-Mer model does not efficiently tag late EMPs or FL monocytes, but does effi-

ciently label YS macrophages that derive from early EMPs (Hoeffel et al., 2015). It remains unclear whether early EMPs and late EMPs are distinct progenitor cell types arising from independent sources, or rather a single population that exists along a continuum of maturation stages. If the latter is true, it is tempting to speculate that the differences in differentiation potential between the early and late EMP waves are not cell intrinsic but rather arise from the fact that late EMPs might receive different extrinsic signals when they seed the FL and if such is the case, it will be important to identify the FL factors that activate the multi-potency in late EMPs. Once seeded in the FL, late EMPs generate cells of multiple lineages, including FL monocytes. The late EMP-derived generation of FL monocytes and the extent to which FL monocytes contribute to adult macrophage populations remains debated. Fetal monocytes were first described in 1990 (Naito et al., 1990). These cells emerge in the FL around E12.5, are released into the blood from E13.5, and colonize all tissues except the brain around E14.5 (Naito et al., 1996; Hoeffel et al., 2012, 2015). Although fetal monocytes share a similar phenotype to their adult counterparts, they do not require CSF-1R expression for their differentiation (Ginhoux et al., 2010; Hoeffel et al., 2012), possess high proliferative potential in tissues, and express few genes related to pathogen recognition and immune activation (Hoeffel et al., 2015; van de Laar et al., 2016). We propose that late EMP-derived FL monocytes constitute the main precursor of adult macrophage populations. Our hypothesis has its roots in observations made in Runx1-Mer-cre-Mer and Csf1r-Mercre-Mer mice, where pulse labeling at E7.5 or E8.5, respectively, leads initially to comparable labeling of both tissue macrophages and microglia by E10.5, but from E13.5 onward the extent of tissue macrophage labeling gradually decreases whereas microglia remain abundantly labeled (Hoeffel et al., 2012, 2015), suggesting dilution of the initial YS macrophage wave by a second wave of unlabeled progenitors, shown later to be fetal monocytes (Figure 2, model 2). Of note, a similar observation has been made in a recent study from Gomez Perdiguero et al. (2015) in Csf1r-Mer-cre-Mer mice. This is also supported by FL monocyte fate-mapping based on their specific expression of S100A4 (S100 calcium-binding protein A4), which is not shared by either YS macrophages or differentiated fetal macrophages (Hoeffel et al., 2015). In this constitutive S100a4-cre model, all tissue macrophage populations (with the exception of microglia) are labeled from E14.5 onward, at a ratio that plateaus from E17.5 and is maintained into adulthood. Importantly, the ratio of S100A4 tagging is lower than that of circulating monocytes (excluding BM monocytes as main precursor) and higher than YS macrophages (excluding YS macrophages as main precursor for tissue-resident macrophages, except microglia that, like YS macrophages, exhibit a low baseline ratio of labeling). Accordingly, several studies have found that FL monocytes contribute to many adult macrophage populations: in utero adoptive transfer of FL monocytes demonstrated in situ differentiation of fetal monocytes into adult Langerhans cells (Hoeffel et al., 2012); fetal monocytes injected intranasally have been revealed as the precursor of adult macrophages in lung alveoli (Guilliams et al., 2013; Schneider et al., 2014); and fetal monocytes were further shown to be involved in the generation of adult macrophages in the heart (Epelman et al., 2014). Immunity 44, March 15, 2016 ª2016 Elsevier Inc. 443

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Review Altogether, we thus propose that most tissue-resident macrophages arise from HSC-independent EMPs along two distinct developmental programs. (1) Through early EMPs that give rise to YS macrophages (without monocytic intermediate). These cells that could be related to primitive precursors represent the main precursor for microglia. (2) Through late EMPs that give rise, among others lineages, to FL monocytes, which subsequently colonize all fetal tissues (except the brain) and differentiate into tissue-resident macrophages that outcompete early EMP-derived YS macrophages (Figure 2, model 2). Contribution of Fetal HSCs to Tissue-Resident Macrophage Populations The contribution of EMPs (whether early or late) to adult tissue macrophages remains debated. Sheng et al. (2015) have recently suggested a major contribution from fetal HSCs to the macrophage pool in various tissues. The authors have developed a fate-mapping strain in which tamoxifen administration induces irreversible tagging of cells expressing the stem-cellfactor receptor c-Kit (CD117) and their progeny. In this Kit-Mer-cre-Mer model, 40 OHT injection at E7.5 leads to robust labeling of adult microglia and, to a lesser extent, epidermal Langerhans cells, but fails to label peripheral hematopoietic cells. In contrast, injection of 40 OHT at E8.5 labels all adult peripheral hematopoietic cells including adult tissue-resident macrophages. These results led to the hypothesis that adult macrophages, with the exception of microglia and, partially, epidermal Langerhans cells, arise from definitive fetal HSCs (Figure 2, model 3). These fetal HSCs, rather than late EMPs, would represent the main source of FL monocytes in this model. However, the same observations would be made if tissue-resident macrophages and leukocytes arose from distinct c-Kit+ progenitors that were both independently labeled to the same extent by 40 OHT administration at E8.5. Indeed, the data of Sheng et al. (2015) could equally support the existence of the two waves of EMPs: because E7.5 labeling in the Kit-Mer-creMer mouse tags microglia and some Langerhans cells, but not adult tissue-resident macrophages, this might represent tagging of the early EMPs; and the E8.5 labeling in the Kit-Mer-cre-Mer model that tags adult tissue-resident macrophages and HSCderived leukocytes in the FL, but not microglia, might represent the labeling of both late EMPs and fetal HSCs. The equal labeling of E10.5 EMPs and AGM multipotent progenitors (MPPs) after E8.5 40 OHT administration that has been also reported (Sheng et al., 2015) further supports such an interpretation and also suggests that the Kit-Mer-cre-Mer model is not adapted to distinguish between late EMPs and fetal HSCs given their temporal promiscuity. A major challenge for the field is now to begin to reconcile the data generated in different fate-mapping models. Toward this goal, analysis of the observations made in Tek-Mer-cre-Mer and Kit-Mer-cre-Mer mice reveals possible further support for the existence of the two waves of EMPs we have discovered via the Runx1-Mer-cre-Mer model. In all three models, activation at E7.5 results in abundant tagging of adult microglia, which according to our data arise from early EMPs and not of other tissue-resident macrophage populations, consistent with the idea of distinct origins. Conversely, in all mice, activation at E9.5 induces no or low labeling of microglia. Taken together, the 444 Immunity 44, March 15, 2016 ª2016 Elsevier Inc.

abundant labeling of microglia after E7.5 activation in these mice suggests that they arise from a population of early c-Kit+ Tie2+ progenitors and that a distinct population of late c-Kit+ Tie2+ progenitors also exists that gives rise to other fetal macrophages. In summary, murine fate-mapping models have an incredible capacity to advance our knowledge of cellular ontogeny, but their intrinsic limitations need to be taken into account during experimental design and data interpretation. One of the important limitations of fate-mapping studies is that labeling of multiple downstream populations tells us only that both of their progenitors express the inducible transgene at the time of activation of recombination, and not necessarily that their progenitor is shared. Nonetheless, although the findings generated in different models cannot be compared quantitatively, qualitative comparisons can be useful and allow broad and robust conclusions. Among the different models, we consider that the Runx1 model has key advantages for studying murine embryogenesis. Runx1 tagging helps distinguish between late EMPs and HSCs, because tagging at E8.5 predominantly labels late EMPs and few HSCs, whereas at E9.5 the majority of labeled cells are HSCs in the FL (Hoeffel et al., 2015). Moreover, because Runx1 expression is necessary for progenitor cells to emerge from the hemogenic endothelium, but decreases rapidly after their emergence (Chen et al., 2009), activation of labeling occurs only during a short time window, reducing the risk of tagging preceding waves and allowing a sharper definition of each hematopoietic wave. Further experiments will be required to integrate the findings of the various fate-mapping models to establish a robust and consistent model of immune cell ontogeny from the early embryonic stage right through to the steady-state adult. c-Myb Dependence c-Myb is a transcription factor required for the expansion and differentiation of all hematopoietic cell lineages (Ramsay and Gonda, 2008). Although primitive hematopoiesis can occur independently of c-Myb (Clarke et al., 2000), definitive hematopoiesis depends upon its expression in EMPs (Mucenski et al., 1991; Palis et al., 1999; Sumner et al., 2000; Yoder and Hiatt, 1997). Taking advantage of the differential c-Myb dependence of primitive and definitive hematopoiesis, Schulz et al. (2012) have shown that fetal F480hiCD11b+ cells are still detected in the lung, pancreatic, spleen, and skin rudiment of E16.5 c-Myb-deficient animals, whereas CD45+c-Kit+ cells (including EMPs) and F480loCD11b+ cells are absent. Based on these results, the authors have hypothesized that most fetal macrophages and adult macrophages arise from c-Myb-deficient precursors. Alternatively, the macrophages present in c-Myb-deficient mice might mainly derive from an early c-Myb-independent EMP wave that persists as a compensatory mechanism only because the macrophages cannot be replaced by macrophages arising from a later wave of c-Myb-dependent EMPs. Recent findings from a converse approach support the latter hypothesis. Injection of a blocking anti-CSF-1R antibody into E6.5 embryos depletes all YS macrophages by transiently inhibiting the CSF-1R signaling pathway (Squarzoni et al., 2014). CSF-1R blockade depleted all macrophages between E10.5 and E14.5. Nonetheless, the macrophage pool is restored around E17.5

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Review by CSF1R-independent monocytes in these mice. These results suggest that niche access and competition are the key factors driving the contribution of the different embryonic waves to each tissue in these models. Though the completion of definitive hematopoiesis depends upon c-Myb expression in EMPs (Palis et al., 1999; Sumner et al., 2000; Yoder and Hiatt, 1997), recent data indicate that expression of this transcription factor undergoes important temporal variations within EMP populations. Kierdorf et al. (2013) showed that the early YS EMPs barely express c-Myb; other reports found that c-Myb is expressed in the YS only from E8.25 (Palis et al., 1999) and that its expression in EMPs increases with time (Palis et al., 1999; Sumner et al., 2000; Yoder and Hiatt, 1997). These observations are consistent with the notion that the early EMPs (which emerge from the YS and give rise to all macrophages in early embryos, including microglia) do not express nor depend upon c-Myb (Clarke et al., 2000), which explains why early EMP-derived fetal macrophages persist in c-Myb-deficient embryos. In contrast, late EMPs display higher c-Myb expression and give rise to fetal monocytes in a c-Myb-dependent manner. Although the c-Myb dependency of late EMPs remains to be formally proven, the fact that far fewer c-Kit+ cells (which include EMPs) (Schulz et al., 2012) and fewer monocytes (Mucenski et al., 1991; Schulz et al., 2012) were present in the FL of Myb/ embryos compared to wild-type suggests that c-Myb might help control late EMP persistence, expansion, and differentiation. Contribution of Circulating Monocytes to Macrophage Homeostasis in Adult Tissues Although all tissues at birth are populated with fetal macrophages, gradual replacement of these cells to a greater or lesser extent by HSC-derived progenitors can occur with time at specific sites. The contribution of HSCs to adult resident macrophages has been revealed using fate mapping model of HSCs or their immediate progeny as discussed below. These studies have revealed that the contribution of adult HSCs to tissue-resident macrophages differs among organs and increases with age. Further evidence for the contribution of adult precursors to some tissue-resident macrophages and continuous turnover in adulthood come from parabiosis experiments that reveal the recruitment of circulating Ly6Chi monocytes and their differentiation into tissue macrophages (for review, see Scott et al., 2014). Tissue macrophages that are maintained by adult circulating precursors include the intestine (Bain et al., 2014), dermis (Tamoutounour et al., 2013), heart (Epelman et al., 2014; Molawi et al., 2014), and pancreas macrophages (Calderon et al., 2015). In the case of the intestine and dermis, recruitment of Ly6Chi monocytes is CCR2 dependent, as indicated by the fact that macrophage populations in these sites are significantly reduced in mice lacking this chemokine receptor (Bain et al., 2014; Platt et al., 2010; Zigmond et al., 2012) and originate almost exclusively from wild-type BM-derived cells in WT:Ccr2/ competitive chimeras (Tamoutounour et al., 2013). Of note, differentiation to macrophages or DCs is not an obligatory route for monocytes entering tissues, because monocytes have been shown to retain their undifferentiated phenotype in the lung (Jakubzick et al., 2013). Other tissue-resident macrophage populations such as microglia, epidermal Langerhans cells, liver

Kupffer cells, and alveolar macrophages exhibit negligible need for replacement in adulthood. However, the origin of macrophages remains puzzling in some tissues such as the peritoneum or the fat. Using the Cx3cr1-fate mapping system, it has been proposed that there is only a minimal contribution of circulating monocytes to the peritoneal macrophage pool (Yona et al., 2013). This has been confirmed by parabiosis data (Hashimoto et al., 2013). However, via a Kit-based fate-mapping model, it has recently been proposed that adult HSCs contribute substantially to the pool of peritoneal macrophages (Sheng et al., 2015). This is supported by their relatively high tagging in the Flt3-crebased fate-mapping model (Epelman et al., 2014). Note that peritoneal macrophages consist of F4/80lo and F4/80hi macrophages (Okabe and Medzhitov, 2014), which might explain some of these discrepancies. The cellular origin of adipose tissue macrophages also remains unclear. Although the massive expansion of adipose tissue macrophages in obese mice (Weisberg et al., 2003) has been attributed both to in situ proliferation of resident macrophages (Amano et al., 2014; Haase et al., 2014) and to CCR2-mediated recruitment of monocytes and differentiation into macrophages (Kanda et al., 2006; Weisberg et al., 2006), little is known about the ontogeny of these cells in healthy animals. The molecular mechanisms that promote local macrophage turnover in some tissues and their repopulation by circulating precursors in others are still very much unknown and are particularly exciting avenues of research. It is also worth noting that among tissues where monocytes differentiate into macrophages in the steady state, there is a marked difference in kinetic of replacement of these cells. Intestinal macrophages have an estimated half-life of 4–6 weeks and are not capable of selfmaintenance, relying on a constant replenishment by BM-derived monocytes (Bain et al., 2014). Similarly, dermal MHCIIhi macrophage populations contain almost no embryonic macrophages (Epelman et al., 2014; McGovern et al., 2014) and also have an estimated half-life of 4–6 weeks (Scott et al., 2014; Tamoutounour et al., 2013). In the heart, cardiac macrophages derived from monocytes seem to have a longer half-life as compared to dermis and intestinal macrophages and based on the various fate-mapping tools utilized, it is likely to be about 8–12 weeks (Epelman et al., 2014; Molawi et al., 2014). The existence of tissues with such a slow macrophage turnover highlights the need to analyze the contribution of the BM over prolonged periods of time. A period that would also deserve more attention is the neonatal period. BM-derived monocytes were recently shown to differentiate into arterial macrophages immediately after birth and to self-renew locally from this point (Ensan et al., 2016). We also have found that monocytes can differentiate into Kupffer cells in this period (Scott et al., 2016). Given that, in adulthood, monocytes do not contribute to Kupffer cell (Gomez Perdiguero et al., 2015; Hoeffel et al., 2015; Schulz et al., 2012; Yona et al., 2013) or arterial macrophage (Ensan et al., 2016) populations, it appears that these tissues are somehow only temporarily ‘‘open’’ at birth but remain ‘‘closed’’ during adulthood. Adult tissues can thus be classified into those that are closed, with no steady-state monocyte recruitment (brain, epidermis, lung, and liver); open tissues with fast steady-state recruitment (gut and dermis); and open tissues with slow steady-state recruitment (heart and pancreas) (Figure 3). This Immunity 44, March 15, 2016 ª2016 Elsevier Inc. 445

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Review Figure 3. Heterogeneity of Tissue-Resident Macrophage Ontogeny in Adult Tissues in the Steady State

A Brain

B Epidermis

C Lung

D

Tissue-resident macrophages in closed tissues might arise only from YS macrophages (A, microglia), from both YS macrophages and fetal liver (FL) monocytes (B, Langerhans cells that have been shown to have a mixed contribution with around 20% of YS macrophages and 80% FL monocytes), or mostly from FL monocytes (C, alveolar macrophages and D, Kupffer cells). Note that for Kupffer cells, a minor contribution of neonatal monocytes and YS macrophages was suggested. For open tissues, bone marrowderived monocytes are recruited and differentiate into macrophages with a kinetic specific to each tissue, with slow (heart and pancreas) and fast (gut and dermis) kinetics of replacement evidenced.

Liver

suggests a major role of the tissue itself in controlling the persistence of macrophages and the recruitment and differentiation of monocytes, but very little is known about the precise mechanisms governing these processes. Moreover, how these mechanisms evolve and change from fetal development into the neonatal period and through the phases of adulthood remains unknown. Nature versus Nurture: Does Ontogeny Matter? The existence of multiple macrophage progenitors raises the question of whether ontogeny of macrophages dictates their functional properties. Several studies have shown that macrophage populations exhibit distinct transcriptional signatures (Gautier et al., 2012; Lavin et al., 2014) and epigenetic marks (Gosselin et al., 2014; Lavin et al., 2014) that are specific to their tissue of residence. The molecular mechanisms underlying tissue imprinting of macrophage function are now starting to be unraveled (for reviews, see Amit et al., 2015; Lavin et al., 2015; Okabe and Medzhitov, 2015). Given that macrophages can derive from embryonic progenitors or from BM monocytes and that they exhibit immense heterogeneity both in vitro (Xue et al., 2014) and in vivo (Gautier et al., 2012; Lavin et al., 2014), understanding how influences from their origin and environment are integrated to define gene expression profiles and functional capacities remains unanswered. Using a model of genotoxic irradiation to induce the depletion of embryonic macrophages and their subsequent repopulation by BM monocyte-derived macrophages, Lavin et al. (2014) have compared the gene expression profile of the repopulating macrophage population in different tissues. BM-derived lung and peritoneal macrophages acquire 90% of the transcriptional profile of their embryonic macrophage counterparts, but 446 Immunity 44, March 15, 2016 ª2016 Elsevier Inc.

BM-derived Kupffer cells acquire less than 50% of the tissue-specific enhancers of embryonic Kupffer cells. Similarly, by using a model of microglial depletion coupled with genotoxic irradiation, monocyte-derived microglia that colonize the irradiated brain possess more than 2,000 genes that are differentially expressed compared with embryonic microglia (Bruttger et al., 2015). These results might suggest that either a substantial component of the Kupffer cell- and microglia-specific gene signature is determined by their embryonic origin, or alternatively that irradiation can disrupt liver and brain homeostasis and thereby prevent normal imprinting of colonizing macrophages. Indeed, when lungs are protected from irradiation injuries in BM chimeric animals, Gibbings et al. (2015) has reported only a handful of genes whose expression differed in embryonic alveolar macrophages compared to BM-derived alveolar macrophages. In addition, upon conditional deletion of embryonic Kupffer cells, we found that monocytes differentiating into Kupffer cells acquire an almost identical gene expression profile to embryonic Kupffer cells (Scott et al., 2016). These findings highlight the key role of tissue factors in the imprinting of the macrophage transcriptional program. One aspect of macrophage biology that appears to be tightly linked to ontogeny is self maintenance. A recent study has revealed that embryonic-derived cells might have a unique ability to resist genotoxic stress, providing a survival advantage compared to adult-derived macrophages (Price et al., 2015). FL monocytes and YS macrophages seem to be imprinted with self-maintenance capacity even before reaching their organ of residence, because these cells display higher expression of proliferation genes as compared to BM monocytes (Hoeffel et al., 2015; van de Laar et al., 2016). Moreover, in vitro GM-CSF-cultured FL monocytes, but not BM-derived monocytes, give rise to macrophages that can proliferate for weeks in culture, despite the fact that both cell types exhibit comparable GM-CSF-receptor responsiveness (van de Laar et al., 2016). In addition, monocytes recruited to inflamed tissues often fail to stably engraft once inflammation resolves (Ajami et al., 2007, 2011; Hashimoto et al., 2013; Zigmond et al., 2014). Together

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Review with the fact that intestinal, dermal, and cardiac macrophages that derive from BM precursors fail to self maintain, it is possible that the capacity for self-maintenance is restricted to macrophages of embryonic origin. Accordingly, monocyte-derived cells have been proposed as ‘‘passenger myeloid cells,’’ as opposed to their ‘‘resident’’ counterparts of embryonic origin (Perdiguero and Geissmann, 2015). To directly assess the selfmaintenance capacity of macrophages derived from adult versus embryonic precursors, we transferred YS macrophages, FL monocytes, and BM monocytes into Csf2r/ animals, which cannot form alveolar macrophages and therefore have an empty alveolar macrophage niche. Of note, all progenitors differentiated into alveolar macrophages, but FL monocytes were more efficient than YS macrophages in this regard, in line with the FL monocyte origin of AM macrophages in steady-state mice. In addition, all macrophage progeny were able to self-renew locally, independently of their YS macrophage or FL or BM monocyte origin. Similarly, depletion of Kupffer cells induce the differentiation of BM monocytes into Kupffer cells that self maintained for months (Scott et al., 2016). Thus, BM origin per se does not preclude the development of self-maintaining tissueresident macrophages. Further research will thus be required to understand what mechanisms govern the fact that BM monocytes can acquire self maintenance in some tissues but not in others and how this is regulated during inflammation. Concluding Remarks Despite some remaining controversies, the embryonic origin of key tissue-resident macrophage populations is now fully recognized. This represents a paradigm shift in the field and also reveals an added level of complexity to the functional heterogeneity of immune cells in the tissues. We now know that multiple populations of macrophage-like cells co-exist: in the steady state, embryonic macrophages, newborn monocyte-derived macrophages, and adult monocyte-derived macrophages function alongside one another, and in inflamed tissues adult monocyte-derived cells with features of macrophages and dendritic cells (Guilliams et al., 2014) are added to the already-diverse population. Defining the functional consequences for tissue repair, homeostasis, and function when embryonic macrophages are replaced by adult BM-derived cells upon infection or inflammation is one of the key future questions that will have crucial implications for our understanding of inflammatory diseases and cancer. Whether adult-derived replacing macrophages developing during inflammation can recapitulate all functions and homeostatic features of pre-existing embryonic macrophages remains to be investigated, especially in response to chronic or repetitive challenges. In addition, studying how specific tissue macrophage identities are established and maintained will help to reveal to what extent immune cell identities are dictated by the environment, as opposed to by cell-intrinsic qualities of the precursors. To answer these questions we will require high-resolution markers of cellular origin and improved genetic tools to dissect the function of tissue-resident and inflammatory macrophages as discussed recently (Ginhoux et al., 2015). Modeling in vitro macrophage tissue specificity through the use of induced pluripotent stem cell (iPSC)-derived macrophage-like cells in co-culture with iPSC-derived tissue-specific cells might help in answering some of these questions. Finally,

translating these findings to human macrophage biology will be essential and will help with the design of future intervention strategies for inflammatory disease and cancer where macrophages play a central role in determining clinical outcome. ACKNOWLEDGMENTS This work was supported by core grants of the Singapore Immunology Network and the Shanghai Institute of Immunology to F.G. M.G. is supported by a Marie Curie Reintegration grant, an Odysseus grant, and FWO grants of the Flemish Government. We thank Lucy Robinson for editing the manuscript. We thank Alicia Wong for the design of the figures. REFERENCES Ajami, B., Bennett, J.L., Krieger, C., Tetzlaff, W., and Rossi, F.M. (2007). Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nat. Neurosci. 10, 1538–1543. Ajami, B., Bennett, J.L., Krieger, C., McNagny, K.M., and Rossi, F.M. (2011). Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool. Nat. Neurosci. 14, 1142–1149. Alliot, F., Lecain, E., Grima, B., and Pessac, B. (1991). Microglial progenitors with a high proliferative potential in the embryonic and adult mouse brain. Proc. Natl. Acad. Sci. USA 88, 1541–1545. Alliot, F., Godin, I., and Pessac, B. (1999). Microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain. Brain Res. Dev. Brain Res. 117, 145–152. Amano, S.U., Cohen, J.L., Vangala, P., Tencerova, M., Nicoloro, S.M., Yawe, J.C., Shen, Y., Czech, M.P., and Aouadi, M. (2014). Local proliferation of macrophages contributes to obesity-associated adipose tissue inflammation. Cell Metab. 19, 162–171. Amit, I., Winter, D.R., and Jung, S. (2015). The role of the local environment and epigenetics in shaping macrophage identity and their effect on tissue homeostasis. Nat. Immunol. 17, 18–25. Bain, C.C., Bravo-Blas, A., Scott, C.L., Gomez Perdiguero, E., Geissmann, F., Henri, S., Malissen, B., Osborne, L.C., Artis, D., and Mowat, A.M. (2014). Constant replenishment from circulating monocytes maintains the macrophage pool in the intestine of adult mice. Nat. Immunol. 15, 929–937. Bertrand, J.Y., Jalil, A., Klaine, M., Jung, S., Cumano, A., and Godin, I. (2005). Three pathways to mature macrophages in the early mouse yolk sac. Blood 106, 3004–3011. Bruttger, J., Karram, K., Wo¨rtge, S., Regen, T., Marini, F., Hoppmann, N., Klein, M., Blank, T., Yona, S., Wolf, Y., et al. (2015). Genetic cell ablation reveals clusters of local self-renewing microglia in the mammalian central nervous system. Immunity 43, 92–106. Calderon, B., Carrero, J.A., Ferris, S.T., Sojka, D.K., Moore, L., Epelman, S., Murphy, K.M., Yokoyama, W.M., Randolph, G.J., and Unanue, E.R. (2015). The pancreas anatomy conditions the origin and properties of resident macrophages. J. Exp. Med. 212, 1497–1512. Chen, M.J., Yokomizo, T., Zeigler, B.M., Dzierzak, E., and Speck, N.A. (2009). Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter. Nature 457, 887–891. Clarke, D., Vegiopoulos, A., Crawford, A., Mucenski, M., Bonifer, C., and Frampton, J. (2000). In vitro differentiation of c-myb(-/-) ES cells reveals that the colony forming capacity of unilineage macrophage precursors and myeloid progenitor commitment are c-Myb independent. Oncogene 19, 3343–3351. Cumano, A., and Godin, I. (2007). Ontogeny of the hematopoietic system. Annu. Rev. Immunol. 25, 745–785. Ensan, S., Li, A., Besla, R., Degousee, N., Cosme, J., Roufaiel, M., Shikatani, E.A., El-Maklizi, M., Williams, J.W., Robins, L., et al. (2016). Self-renewing resident arterial macrophages arise from embryonic CX3CR1(+) precursors and circulating monocytes immediately after birth. Nat. Immunol. 17, 159–168. Epelman, S., Lavine, K.J., Beaudin, A.E., Sojka, D.K., Carrero, J.A., Calderon, B., Brija, T., Gautier, E.L., Ivanov, S., Satpathy, A.T., et al. (2014). Embryonic and adult-derived resident cardiac macrophages are maintained through

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