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Ontogeny of Tissue-Resident Macrophages

Ontogeny of Tissue-Resident Macrophages Review published: 22 September 2015 doi: 10.3389/fimmu.2015.00486 Ontogeny of tissue-resident macrophages Guillaume Hoeffel* and Florent Ginhoux* Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore The origin of tissue-resident macrophages, crucial for homeostasis and immunity, has remained controversial until recently. Originally described as part of the mononuclear phagocyte system, macrophages were long thought to derive solely from adult blood circulating monocytes. However, accumulating evidence now shows that certain mac- rophage populations are in fact independent from monocyte and even from adult bone marrow hematopoiesis. These tissue-resident macrophages derive from sequential seeding of tissues by two precursors during embryonic development. Primitive macro- Edited by: phages generated in the yolk sac (YS) from early erythro-myeloid progenitors (EMPs), Peter M. Van Endert, Université Paris Descartes, France independently of the transcription factor c-Myb and bypassing monocytic intermediates, Reviewed by: first give rise to microglia. Later, fetal monocytes, generated from c-Myb EMPs that Meredith O’Keeffe, initially seed the fetal liver (FL), then give rise to the majority of other adult macrophages. Burnet Institute for Medical Research, Australia Thus, hematopoietic stem cell-independent embryonic precursors transiently present in Jean M. Davoust, the YS and the FL give rise to long-lasting self-renewing macrophage populations. Institut National de la Santé et la Recherche Médicale, France Keywords: macrophages, monocytes, fetal liver, yolk sac, C-Myb, erythro-myeloid progenitors, hematopoiesis, hematopoietic stem cells *Correspondence: Guillaume Hoeffel and Florent Ginhoux, Singapore Immunology Network introduction (SIgN), Agency for Science, Technology and Research (A*STAR), Ilya (Elie) Metchnikoff first described the mechanism of phagocytosis and the cells responsible for 8A Biomedical Grove, IMMUNOS this process over a century ago. These professional phagocytic cells were named “macrophages” Building #3-4, Biopolis, (from the Greek derivation macro = large and phage = devouring, “large devouring cells”). These 138648 Singapore were separate from “microphages,” which included polymorphonuclear phagocytes (1). Determining guillaumehoeffel1@gmail.com; florent_ginhoux@immunol. the role of macrophages in pathogenic infections was one of the fundamental observations leading to a-star.edu.sg the concept of cellular immunity (2). Through this seminal work, Metchnikoff anticipated the central role of macrophages in tissue inflammation and homeostasis. We recommend an elegant historical Specialty section: review for more details about Metchnikoff ’s work by Yona and Gordon in this issue (3). This article was submitted to Antigen Since then, the definition of the phagocyte system has been continuously refined, and our under - Presenting Cell Biology, a section of standing of the wide-ranging functions of macrophages has been substantially expanded. It is now the journal Frontiers in Immunology clear that, in addition to their classical function in the activation and resolution of tissue inflamma - Received: 22 July 2015 tion, macrophages also play roles in tissue-specific functions, tissue remodeling during angiogenesis Accepted: 07 September 2015 and organogenesis, and wound healing, to name a few (4). Macrophages are exquisitely adapted to Published: 22 September 2015 their local environment, acquiring organ-specific functionalities during developmental stages and Citation: the steady state (4). Macrophages are able to support multiple tissue functions, integrating cues from Hoeffel G and Ginhoux F (2015) both the outside environment and their microenvironment to act as rheostatic cells of tissue function. Ontogeny of tissue-resident u Th s, tissue-resident macrophages represent an attractive target for modern medicine to treat a wide macrophages. spectrum of diseases in which they have been implicated, including atherosclerosis, autoimmune Front. Immunol. 6:486. doi: 10.3389/fimmu.2015.00486 diseases, neurodegenerative and metabolic disorders, and tumor growth (5–8). Understanding the Frontiers in Immunology | www.frontiersin.org 1 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny origin and developmental pathways of macrophages will help to rely on hematopoietic stem cell (HSC)-derived BM progeni- design novel intervention strategies targeting these cells in tissue- tors, certain macrophage populations possess the unique ability specific sites. to self-renew locally independently of circulating precursors A number of observations now indicate that certain (32–36). Initial studies describing the presence of macrophages macrophage populations derive from embryonic precursors in embryonic tissues suggested that tissue macrophages derived sequentially seeding tissues during development (9–13). Two from embryonic progenitors. In rodents, macrophage-like cells macrophage progenitors, yolk sac (YS) macrophages and fetal first described in the brain rudiment and in the developing skin monocytes, have been described in the embryo, but their exact (37, 38) were named “fetal macrophages” and found to exhibit nature and origin were not fully understood until recently (14, a high capacity for proliferation (39). These observations sug- 15). Here, we discuss recent developments in our understanding gested that adult macrophages derive from fetal macrophages of the origin of adult tissue-resident macrophages, exploring the established during early development. However, whether these sequence of progenitors generated during embryonic and adult fetal macrophages were maintained until adulthood or were hematopoiesis. We focus on the relative contributions of YS replaced postnatally was not addressed until recently. In addition, macrophages and fetal or adult monocytes, including a discus- the exact nature and the origin of fetal macrophage progenitors sion of our own recent data exploring the heterogeneity of fetal remained unclear. monocyte developmental pathways. e mbryonic Hematopoiesis e arly Concepts Mammalian embryos produce several transient waves of hemat- Macrophages form part of the mononuclear phagocyte system opoietic cells before the establishment of HSCs in the BM during late gestation (40, 41). The multiple embryonic waves are differ - (MPS), which also includes circulating monocytes and dendritic cells (16). Until recently, our vision of macrophage origin and entially regulated in time and space and exhibit distinct lineage potentials. Importantly, they contribute to hematopoietic popula- homeostasis was largely based on seminal studies that used in vivo radioisotope labeling and radiation chimera experiments. tions that persist until adulthood. These waves include primitive hematopoiesis in the YS, and definitive hematopoiesis, which es Th e studies led to the early dogma that resident macrophages comprises a transient definitive stage, generating multi-lineage were constantly replenished from circulating bone marrow (BM)- derived monocytes as a continuum of differentiation (17–19). erythro-myeloid progenitors (EMPs) and lympho-myeloid pro- genitors (LMPs), and a definitive stage characterized by the In agreement with that concept, studying the ontogeny of the MPS revealed that monocytes and macrophages derived from production of HSCs in the aorta-gonad-mesonephros (AGM). es Th e transient progenitors establish themselves transiently in macrophage and dendritic cell progenitors (MDPs) present in the BM, which are phenotypically defined as lineage-c-kit CX the fetal liver (FL) during the mid to late stages of hematopoiesis. + + + 3CR1 Flt3 CD115 (20). MDPs further differentiate through a e s Th equential waves of hematopoiesis can overlap in time and newly described common monocyte precursor (cMoP), pheno- space (Figure 1) and remain difficult to separate clearly, even with − + + − + typically defined as lineage c-kit CX3CR1 Flt3 CD115 (21), the most recent fate-mapping tools available. that gives rise to the two main subsets of circulating monocytes distinguished by the expression of Ly6C (22). Primitive Hematopoiesis Specific tissue macrophages, such as dermal, gut, and heart In mice, the first hematopoietic progenitors appear in the macrophages, seemed to follow the model of Van Furth, that extra-embryonic YS blood islands at around embryonic age 7.25 macrophages are derived from monocytes (23–25). However, this (E7.25), where primitive hematopoiesis is initiated, producing mainly nucleated erythrocytes. This observation linked the model did not fit in all cases and evidence also emerged indicating that macrophages were long-lived cells, able to self-renew locally. myeloid progenitors observed in the YS at E7 with the emergence of YS macrophages aer E9.0 [ ft Figure 2; Ref. (42–45)]. Primitive Hashimoto was the first to speculate that Langerhans cells (LCs) represented a self-perpetuating “intraepithelial phagocytic sys- hematopoiesis was also shown to produce megakaryocyte pro- tem” (26). Performing a human skin transplantation assay onto genitors (46). The denomination “primitive” was given to reflect nude mice, Krueger et  al. described the remarkable longevity the production of embryonic erythroblasts, like those observed in of LCs, which were able to persist in the grafts for more than lower species such as fish, amphibians, and birds, and remaining 2  months (27). Their ability to self-renew through proliferation nucleated throughout their life span (47–49). This denomination was later described using DNA densitometry (28). Similar con- was extended to macrophages in the YS due to their concomitant clusions were drawn soon aer r ft egarding alveolar macrophages development prior to FL hematopoiesis. Interestingly, no clear (29). The dominant concept of “the monocytic origin” of tissue evidence of monocytic intermediates was reported at this stage, macrophages was also challenged through experiments in ani- although the seminal study of Cline and Moore did mention the mals with prolonged monocytopenia following strontium-89 existence of local intermediate cells between progenitors and monocyte depletion, in which liver Kuper ce ff lls were shown to functional macrophages (43). Studies by Naito and Takahashi maintain cell numbers by increasing local proliferation (30, 31). et  al. clarified the emergence of primitive macrophages in the More recently, the use of long-term parabiotic mice and sub- YS blood islands in the mouse and rat, observing an absence sequent fate-mapping models have challenged the MPS paradigm of endogenous peroxidase activity as a surrogate marker for an and revealed that, unlike all other hematopoietic cells, which absence of monocytic intermediates, such as those found in the Frontiers in Immunology | www.frontiersin.org 2 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny FiGURe 1 | Fetal hematopoiesis. Primitive, transient definitive, and definitive waves of fetal hematopoiesis sequentially generate progenitors able to seed the fetal liver. Primitive hematopoiesis starts at E7.0 in the blood islands of the extra-embryonic yolk sac (YS) and generates erythro-myeloid progenitors (EMPs). Early EMPs initially express CD41 and later, CSF-1R, a signature of myeloid/macrophage commitment. Concomitant to the establishment of the blood circulation at E8.5, the YS hemogenic endothelium (HE) generates late EMPs expressing C-Myb. At approximately E9.0, the intra-embryonic mesoderm generates additional HE and emerging progenitors with lymphoid potentials (LMPs) without long-term reconstitution (LTR) capacity. These C-Myb EMPs and LMPs constitute the so-called transient definitive wave. Finally, hematopoietic stem cells (HSCs) with LTR activity emerge from the main HE situated in the aorta-gonad-mesonephros (AGM) regions and in the placenta. BM (50–52), suggesting a unique developmental pathway for YS potential progenitors from E8.25 in the YS (45). Palis et al. first macrophages (53, 54). observed the emergence of definitive progenitors for mast cells and a bipotential granulocyte/macrophage progenitor. These Transient Definitive Hematopoiesis progenitors then migrated to the FL through the bloodstream The quest to elucidate the origins of embryonic HSCs led to after E8.5, once circulation was established ( 44, 56). From this the discovery of earlier lineage-restricted HSC-independent pattern of development, the authors concluded that definitive progenitors seeding the FL at E10.5. These progenitors arise hematopoietic progenitors arise in the YS, migrate through the concurrently with the transition of primitive to definitive bloodstream, and seed the FL to rapidly initiate the first phase erythropoiesis and were thus considered to form a transient of intra-embryonic hematopoiesis. Similarly, primitive and stage of definitive hematopoiesis (45, 47, 55). Transient defini- definitive erythropoiesis, associated with myelopoiesis, was also tive hematopoiesis consists of progenitors sequentially acquir- shown to emerge prior to HSC in the zebrafish embryo ( 57). ing myeloid, then lymphoid potential, without exhibiting the Bertrand et  al. showed that definitive hematopoiesis initiates long-term reconstitution potential of HSCs. Seminal work from in the posterior blood island with only transient proliferative Palis and colleagues on embryonic erythropoiesis in the YS potential. Because these HSC-independent definitive progeni - described the parallel emergence of multiple myeloid lineage tors were observed to produce definitive erythroid and myeloid Frontiers in Immunology | www.frontiersin.org 3 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny FiGURe 2 | Primitive hematopoiesis and yolk sac macrophage ontogeny. Early EMPs emerge in the YS around E7.5 before establishment of the blood circulation. They express CD41 and CSF-1R and are independent of the transcription factor C-Myb. Upon establishment of the blood circulation around E8.5, EMPs differentiate into primitive macrophages as well as primitive erythrocytes and granulocytes. Primitive macrophages seed all fetal tissues, in particular the head where they will give rise to future brain microglia that are able to continuously self-renew throughout adulthood. EMPs seeding the fetal liver briefly expand to generate a local macrophage population, likely important for sustaining enucleation of primitive erythrocytes passing through the sinusoid prior to the establishment of definitive hematopoiesis and the generation of fetal monocyte-derived macrophages in the fetal liver. cell types, but not to colonize the zebrafish thymus (implying FL with T cell, B cell, and macrophage potential (63), although that they are devoid of lymphoid potential), this population was the precise origin of these progenitors was not addressed. At the termed EMPs (57). Interestingly, EMPs can also emerge from same time, the team of Jacobssen identified Flt3 lympho-myeloid the hemogenic endothelium (HE) located in the placenta and progenitors (LMPs) devoid of erythrocyte and megakaryocyte umbilical cord (58) and colonize the FL from E9.5 (55) to par- capacity (64). Later, cells with myelo-erythroid and lymphoid ticipate in definitive hematopoiesis. Further studies advanced lineage potential, such as B-1 cells present in the adult spleen, the field significantly by identifying CD41 as an early marker were associated with E9.5 YS progenitors expressing AA4.1 and (pre-CD45) for defining hematopoietic progenitors, including CD19 (65). A year later, the same team also identified T cell EMPs, emerging from the YS (59, 60). Altogether, these impor- potential within the E9.5 YS progenitors (66). Using a Rag-1-Cre tant studies provided phenotypic and functional analyses of the fate-mapping model, Boiers et  al. confirmed that these LMPs first hematopoietic progenitors and demonstrated that defini- emerged at approximately E9.5 in the YS, seeding the FL by E11.5 tive hematopoiesis proceeds through two distinct waves during to give rise to T and B cells, as well as granulocytes and monocytes embryonic development (Figure 3). in the E14.5 FL, prior to HSCs (67). Finally, lympho-myeloid In parallel, several groups have also identified other multipo - progenitors isolated from the dorsal aorta at E9.0 were shown tential progenitors with lymphoid- or myelo-lymphoid-restricted to acquire long-term reconstitution capacity aer a f ft ew days of potential in the YS and the developing para-aortic splanchno- in  vitro culture with stromal cells, and were called immature pleura (P-Sp) prior to HSCs (61, 62). Lacaud et al. also described HSCs (68). Without preculture, these multipotential progenitors + −/− AA4.1 (CD93) multipotential progenitors present in the E14.5 can only engraft natural killer (NK)-deficient Rag2 γc mice. Frontiers in Immunology | www.frontiersin.org 4 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny FiGURe 3 | Fetal monopoiesis and macrophage ontogeny. At E8.5 as the blood circulation is established, the YS HE, possibly in conjunction with other hemogenic sites, generates EMPs expressing CD41 and c-Myb. These late EMPs seed the fetal liver around E9.5 where they expand rapidly to give rise to CSF-1R myeloid progenitors that are able to generate fetal monocytes through cMoP intermediates at E12.5. Fetal monocytes then spread via the blood circulation to all tissues, with the exception of the brain, which is isolated by the establishment of the blood–brain barrier at approximately E13.5. In the tissues, fetal monocytes begin to differentiate into macrophages, progressively outnumbering the previously established primitive macrophages. These fetal monocyte-derived macrophages maintain the capacity to self-renew throughout adulthood in certain tissues, such as the liver or the lung, where they will not be replaced by adult BM-derived monocytes. Whether these immature HSCs arise from LMPs or represent To conclude, commitment to hematopoietic fates begins dur- a distinct wave of progenitors remains to be clarified. However, ing gastrulation in the YS, which represents the only site of primi- these seminal studies provided strong evidence that lymphoid tive erythropoiesis and also serves as the first source of transient potential can emerge from the YS, prior to HSC-budding from definitive hematopoietic progenitors. HE develops from the YS the AGM (69). to various intra-embryonic sites, and acquires myeloid and then Because the emergence of EMPs and LMPs overlaps in time lymphoid lineage potentials in overlapping waves, highlighting and space, they could not be distinguished clearly until recently. the complexity of the hematopoietic output. Whether some of Previous reports had suggested that lymphoid potential was these progenitors arise from independent sources or represent restricted to the CD41-negative cell fraction (59, 65). However, different maturation stages of a shared hematopoietic wave, cul- CD41 is also expressed in a sub-fraction of FL HSCs, and so this minating with the generation of HSCs, needs to be further clari- phenotypical distinction spread some confusion (47). Finally, fied. However, it is tempting to speculate that the clear contrasts a recent report from the group of Palis clarified this point by in differentiation/lineage potential do not reside in their intrinsic showing that co-expression of c-kit, CD41, and CD16/32 defines potential, but rather in the extrinsic signals provided by the local EMPs and allows their separation from other progenitors with environment. lymphoid potential, such as those giving rise to the B-1 cell (70). McGrath et  al. extended the notion of EMPs by showing their Definitive Hematopoiesis potential to generate neutrophils, megakaryocytes, macrophages, e co Th mplex hierarchy of stem and progenitor cells in the BM and erythrocytes. Finally, transplantation of EMPs in immune- is first established during embryonic development starting compromised adult mice can also provide transient adult red with the emergence of small numbers of HSCs from the AGM blood cell reconstitution (70). at E10.5 in murine embryos or at 5  weeks in human embryos Frontiers in Immunology | www.frontiersin.org 5 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny (71, 72). Aer E9.5 in t ft he mouse, with the determination of the e co Th ntribution of HSCs to FL hematopoiesis is complex intra-embryonic mesoderm toward a hematopoietic lineage, new to evaluate, partly because of the lack of specific fate-mapping waves of hematopoietic progenitors emerge within the HE of the models, and also the relatively limited knowledge regarding embryo proper (Figure 4), first in the P-Sp region and the umbili - embryonic HSC maintenance and homeostasis in this environ- cal and vitelline arterial regions of the embryo, then in the AGM ment. The capacity for long-term reconstitution, which defines region and the placenta (55, 73, 74). e h Th ematopoietic activities functional HSCs, is present in the AGM by E10.5 (76). However, of the P-Sp and AGM first generate immature HSCs and then lineage-specific commitment may not occur in  vivo imme- mature HSCs, which are defined by their capacity to reconstitute diately aer r ft eaching the FL environment. A number of other adult conventional mice (long-term reconstitution; LTR). Both progenitors generated during transient definitive hematopoiesis, immature and mature HSCs seed the FL at approximately E10.5 as discussed above, are already present and able to give rise to (68, 71, 75, 76) to establish definitive hematopoiesis ( 40, 77, 78). almost all cell lineages, which could prevent HSC consumption A maturation step seems necessary for immature HSCs to express and differentiation ( Figure  5). Evaluation of HSC contribution their LTR activity in full, which is then maintained until adult- has long been based on the assumption that all hematopoietic hood (68). However, further investigations using a fate-mapping cells in the FL were derived from HSCs as is the case in the BM system would be necessary to confirm this model. (81). Many multipotential progenitors share the same phenotype e FL b Th ecomes the major hematopoietic organ aer E11.5, ft with pre-HSC and HSCs, such as the expression of CD41 and generating all hematopoietic lineages. Importantly, the FL AA4.1 (60), adding to this confusion. e Th combination of the itself does not produce progenitors de novo, but rather recruits marker Sca-1 and new markers such as those from the SLAM progenitors derived from the YS and other hemogenic sites, to family (82) have greatly helped to clarify the characterization of − + + + − − ckit Sca-1 CD150 CD48 CD244 . initiate definitive hematopoiesis (79) in parallel with the expan- HSCs, defined now as Lin sion of the definitive HSC population before their migration to However, no specific fate-mapping model exists to characterize embryonic HSC progeny with the exception of the Flt3-Cre the spleen and BM (80). FiGURe 4 | Transition between fetal and adult hematopoiesis. Hemogenic endothelial cells from extra and intra-embryonic hematopoietic tissues generate C-Myb-dependent multipotential progenitors, such as LMPs and pre-HSCs, between E9.0 and E10.5, culminating with the emergence of mature HSCs with long-term reconstitution-bearing potential. CD93 (AA4.1) expression is associated with the emergence of lymphoid potential, whereas Sca-1 is the hallmark of HSCs. These progenitors seed the fetal liver around E10/E11, expanding and giving rise to the various lineages of the hematopoietic system, including fetal monocytes. These late fetal monocytes continue to participate in the tissue-resident macrophage network until hematopoiesis switches completely from the fetal liver to the bone marrow. Once adult hematopoiesis begins to take place in the bone marrow generating monocytes, certain tissues, such as the dermis, heart peritoneum, and the gut, continue to recruit adult monocytes to generate resident macrophages and replace with time the embryonic-derived macrophages. Frontiers in Immunology | www.frontiersin.org 6 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny FiGURe 5 | Timeline of fetal and adult hematopoiesis. The primitive hematopoiesis is initiated in the yolk sac independently of C-Myb activity, and generates early CSF-1R EMPs that give rise to YS macrophages without monocytic intermediates during a short time window and will establish the brain microglia. The transient definitive hematopoiesis and then the definitive hematopoiesis are both dependent on C-Myb activity and generate progenitors that differentiate in the fetal liver. The transient definitive wave, which include EMPs and then LMPs, give rise in particular to fetal monocytes that seed the tissues prior to birth to establish the self-renewing tissue-resident macrophage network. Although only HSCs, which result from the definitive hematopoiesis, seem to be maintained in the bone marrow in adults, the relative contribution of the transient definitive wave to the adult immune system remains unclear. model (83), which was used until now with the assumption that colonize various tissues (52, 93–95). Primitive macrophages may embryonic and adult HSCs follow similar differentiation path - contribute to many fundamental processes during mid and late ways. Our recent report suggests that the Flt3-Cre model can embryogenesis, such as clearance of dead cells or tissue matura- also be used to follow the progeny of LMPs (15). Furthermore, tion. In this regard, the developmental process of interdigital in the nascent BM, the long-term repopulation (LTR) capacity cell death removal during the mouse footplate remodeling that that characterizes functional HSCs is only observed at around occurs between E12.5 and E14.5 is of interest as the interdigit E17.5 (84). Considering the time required to initiate full HSC regions become heavily populated by macrophages and most of differentiation, these data suggest that proper adult HSC-derived the dead cells were shown to be rapidly engulfed by macrophages hematopoiesis does not take place in the BM until a few days (96). However, mouse models devoid of primitive macrophages aer b ft irth. Characterization of the functional specificities and such as the colony-stimulating factor 1 receptor (CSF-1R) KO regulatory pathways of HE that give rise to HSCs versus those (Florent Ginhoux, unpublished data,) and PU.1 KO (97) appears that generate EMPs and other multipotential progenitors could to exhibit a normal interdigit web tissue. Wood et  al. observed aid the development of new fate-mapping models and improve that interdigit web tissue in PU.1 KO was only slightly retarded, our understanding of this process (85). Use of other fate-mapping suggesting that other cell type such as neighboring mesenchymal models such as the Runx1-Mer-Cre-Mer (Runx1-iCre) (86), cells were compensating (97). In addition, we recently showed Tie2-Mer-Cre-Mer mice (14), and the c-kit-Mer-Cre-Mer mice that depletion of primitive macrophages and hence of embryonic (87) provided complementary results, although a careful analysis microglia, ae ff cted the progression of dopaminergic axons in the of the targeted cells in time and space is not yet fully available forebrain and the laminar positioning of subsets of neocortical for the last two models. We present here our best interpretation interneurons, likely through phagocytic mechanisms (98). of the data provided in these two recent studies that have used Schulz et al. highlighted further differences between primitive these models in light of the literature and our own results and and definitive hematopoiesis, showing that the latter relies on experience using the Runx1-iCre model (Figure 6). the transcription factor Myb, while YS-derived macrophages are Myb-independent, and are instead dependent on PU.1 (12). This e mbryonic and Adult Precursors of Adult again reinforces the view that YS-derived macrophages constitute an independent lineage, distinct from the progeny of definitive Tissue-Resident Macrophages HSCs. Schulz et  al. exploited the differential dependence of Yolk Sac Macrophages primitive versus definitive hematopoiesis on the transcription Yolk sac macrophages first appear in the YS blood islands at E9 factor c-Myb and reported that E16.5 tissue macrophage popula- (albeit in small numbers) with a unique pattern of differentiation tions were not ae ff cted by the loss of c-Myb. Using a CSF-1R-iCre that bypasses the monocytic intermediate stage seen in adult mac- fate-mapping model of YS macrophages, they also reported the rophages (50, 52). YS-derived primitive macrophages spread into persistence of YS macrophages progeny in adult tissue-resident the embryo proper through the blood as soon as the circulatory macrophage populations (lung, liver, and pancreas, as well as in system is fully established (from E8.5 to E10) (56), and migrate the brain and skin), although the level of labeling was minimal to various tissues, including the brain. Importantly, this occurs (below 3–5%) and decreased with time. The authors concluded before the onset of fetal monocyte production by the FL, which that tissue-resident macrophages were therefore derived from starts around E11.5/E12.5 (92). These primitive macrophages a c-Myb-independent lineage via YS macrophages (12), data retain the high proliferative potential observed in the YS as they supporting the initial report showing that microglia arise from Frontiers in Immunology | www.frontiersin.org 7 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny FiGURe 6 | Fate-mapping systems. (A) The Runx-iCre fate-mapping model (86) used in our study targets the hemogenic transition (88), hence, labeling specifically pr ogenitors in the process of budding out from the hemogenic endothelium. The Runx1 expression decreases in progenitors once they start to express Vav thus reducing the chances of tagging released progenitors from precedent waves (88). As a consequence, Runx-iCre tagging is restricted to a short time window in the lifespan of a given progenitor and allows a sharp definition of each hematopoietic wave. However, this model also restricts the tagging to only a small fraction of the targeted progenitor wave. (B) Tie2 is expressed in all endothelial cells that constitute the hemogenic endothelia even before the hemogenic transition (89). Thus, all endothelial cells and their progeny (non-hematopoietic and hematopoietic cells) will be labeled after tamoxifen injection using the Tie2-iCre model. As a consequence, an early tamoxifen injection (such as at E7.5) will result in the tagging of all hematopoietic cells emerging before the time of analysis. This will include progenitors from the primitive, the transient definitive, and the definitive waves if, for example, the analysis is done at E11.5. A late injection (such as at E10.5) will restrict the tagging to only the latest hematopoietic stem cells wave as they are just budding from HEs (90). Thus, this model might not be suitable to clearly separate the primitive from the transient definitive waves of hematopoiesis. However, this model could be important to study late HSC progeny as no other progenitors than HSCs emerge from HE after E10.5 (91). (C) C-kit is expressed by all hematopoietic progenitors and does not label endothelial cells that constitute the HEs (89). An early tamoxifen injection (such as at E7.5) will restrict the labeling to early progenitors making suitable the c-kit-iCre model to study the primitive hematopoiesis. However, the FL recruits progenitors of each hematopoietic wave from E8.5 until E11 (79). These progenitors still express c-kit and coexist after seeding the FL during the time necessary for their differentiation (47, 55). A later tamoxifen injection (such as at E9.5) might thus result in the cumulative labeling of undifferentiated primitive and definitive progenitors, including the transient wave of EMPs and LMPs. Thus, such model may not be suitable to resolve the complexity of the different embryonic hematopoietic waves characterized by short time windows of emergence and strong overlapping tendencies. Primitive hematopoietic progenitors are rapidly consumed and the engagement of EMPs and LMPs in FL hematopoiesis reduces the expression of c-kit on their surface. Thus, later tamoxifen injection (such as at E11.5) could restrict the labeling to newly derived HSCs expressing high level of c-kit without labeling precedent progenitor waves (87). Such model might be interesting to study the progeny of late HSCs although the risk of tagging the progeny of EMPs and LMPs or later committed progenitors derived from HSCs remains high and difficult to exclude. Further analysis would be necessary to clarify the potential of such model. YS macrophages (9). Embryonic origin of macrophages was although the exact nature of this precursor was not elucidated. In further supported by the work of Yona et al. and Hashimoto et al. fact, both YS macrophages and fetal monocytes express CX3CR1 showing that adult monocytes do not substantially contribute (9, 11, 15) and could therefore correspond to the unidentified to tissue macrophages under steady-state conditions (13, 33). precursors suggested by Yona et  al. However, using a CSF-1R- Furthermore, Yona et al. suggested the existence of a CX3CR1 iCre fate-mapping model, also used by Schulz et al. (12), another precursor for some of the monocyte-independent macrophages, study noted that the YS macrophage contribution in the brain, Frontiers in Immunology | www.frontiersin.org 8 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny the adult liver, and the heart was maintained although at a mini- stage, direct differentiation of fetal monocytes into macrophages mal level that decreased with time (99). Interestingly, the level of in vivo had not been demonstrated (100). labeling was always higher in microglia than that in the liver or To investigate the developmental event leading to the emer- the heart, suggesting that the level of YS macrophage contribu- gence of tissue-specific macrophages, we initially focused on tion may differ between tissues and that YS macrophages may the LC, the specialized myeloid population of the epidermis. be differently replaced over time by later waves of progenitors, While YS macrophages seed the embryonic skin before E13.5, we which follow tissue-distinct kinetics (discussed below). Our discovered that the major fraction of adult LCs is in fact derived own report using the Runx1-iCre fate-mapping model (86) from fetal monocytes that are generated in the FL from E12.5 and indicated that only microglia, specialized macrophages of the are then recruited into fetal skin at E14.5 (11). These cells share central nervous system, were derived solely from primitive a similar phenotype to their adult counterparts; however, they macrophages while all other tissue macrophages derived from are generated independently of CSF-1R expression (9, 11). They definitive hematopoiesis (9 ). possess high proliferative potential, and, in contrast to their adult To understand whether YS macrophages might be the sole counterparts, express few genes related to pathogen recognition progenitors of every other adult macrophages, we asked what and immune activation (15). Further studies should clarify impact their in  utero depletion would have on the subsequent whether such differences reflect monocyte immaturity imposed generation of fetal tissue macrophages. CSF-1R is expressed on by a sterile fetal environment, or rather dedicated functional YS macrophages and fetal monocytes, but only the development specializations that have yet to be unraveled. In  utero adoptive of the former is actively dependent on CSF-1R (9, 11). Thus, we transfers combined with fate-mapping studies unequivocally attempted to deplete YS macrophages by transiently inhibiting confirmed in situ differentiation of fetal monocytes into adult LCs the CSF-1R signaling pathway using a blocking anti-CSF-1R (11). Fetal monocytes were then demonstrated to be the precur antibody, as recently described (98). Importantly, aer ft complete sor of adult macrophages in lung alveoli by intranasal injection (10, 101). Fetal monocytes were also shown to be involved in depletion of primitive YS macrophages in E10.5 embryos and thus of most macrophages in treated embryos at E14.5, tissue mac- the generation of adult macrophages of the heart (99). In fact, fetal monocytes become the major leukocyte within the blood rophages (including microglia) were able to repopulate to normal levels before birth. These data suggest that YS macrophages are circulation aer E13.5, s ft preading to all tissues. This occurred independently of the CCL2/CCR2 axis (15), suggesting an dispensable for the generation of tissue-resident macrophages in the embryo, and that another CSF-1R-independent embryonic alternative mechanism of exit from the FL and/or recruitment precursor can functionally replace YS macrophages during by fetal tissues. Moreover, we were able to fate-map, from before development (15, 98). Using a combination of both the CSF-1R- birth to adulthood, the local differentiation of fetal monocytes iCre and the Runx1-iCre fate-mapping models, we noted that into resident macrophages, by taking advantage of the specific although YS macrophages infiltrate all tissues (including lung, expression of S100a4 in fetal monocytes compared to YS mac- liver, kidney, skin, gut, heart, pancreas, and stomach) until E13.5, rophages (15). Only the brain remained free from fetal monocyte a second wave of precursors, with a monocytic morphology and infiltration, possibly resulting from the isolation of the brain by phenotype, supersedes them aer E14.5 w ft ith the exception of the nascent blood–brain barrier as early as E13.5 (15, 102). Thus, the brain where YS macrophages are maintained until adulthood these data now reveal that fetal monocytes are the major circulat- (15). A fuller understanding of this process may help to resolve ing embryonic precursor for all macrophages, with the exception some of the earlier discrepancies regarding the contribution of of the brain. The absence of monocyte precursor contribution to YS macrophages. the microglial pool could result from a lack of intrinsic potential or a lack of access to the developing brain due to the nascent Fetal Monocytes blood–brain barrier. Interestingly, we observed a major influx of Fetal monocytes were described by Naito et  al. (92). Focusing monocytes in the brain at E14.5 in our YS macrophage depletion their study on liver Kuper ce ff lls (the resident macrophages of the model, and preliminary data using our fetal monocyte S100a4- liver) during embryonic development, they exploited the endog- Cre/WT fate-mapping model combined with in  utero depletion enous peroxidase activity of monocytes and pro-monocytes of YS macrophages suggest that fetal monocytes are capable of granules described earlier by van Furth et  al. (18, 92). Naito giving rise to microglia under certain conditions (Hoeffel & et al. observed the transient appearance of peroxidase activity, a Ginhoux, personal communication). Whether this atypical fetal signature for monocyte and pro-monocyte granule activity, dur- monocyte infiltration reflects a compensatory mechanism to ing the in  vitro generation of macrophages from a preparation fulfill an empty niche in the brain or results from a disruption of of FL-dissociated cells (92). In the YS and at early stages of FL the blood–brain barrier remains to be investigated. development, no peroxidase activity was observed, suggesting Adult Monocytes that primitive macrophages first seed the FL. At a later stage, the peroxidase activity increased, suggesting the presence of BM-derived circulating monocytes were considered the only precursors for all tissue-resident macrophages since the seminal monocytic intermediates. In  vitro clonal expansion assays con- firmed the existence of two types of colonies, those containing work of van Furth et  al. (17–18). Although this dogma was entirely revisited recently with the emergence of sophisticated fetal monocytes and those devoid of them. This provided early evidence for the existence of two distinct developmental path- fate-mapping tools as well as parabiotic models, the physi- ological contribution of circulating adult monocytes to the adult ways leading to the generation of Kuper ce ff lls, although at this Frontiers in Immunology | www.frontiersin.org 9 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny macrophage network remains valid at least in certain tissues. (108). The high level of foreign antigens passing through the LN e co Th ntinuous recruitment of circulating monocytes to the during the lifespan, support the model of a constant replenish- dermis has been shown to shape the adult dermal macrophage ment of the local macrophage pool by circulating adult mono- network (25). Although this study did not employ fate-mapping cytes. However, the work of Jakubzick et  al. suggests otherwise techniques, Tamoutounour’s data suggest the existence in the as tissue-patrolling monocytes at steady state seem to enter the dermis of both a prenatal pool of macrophages and a second LN without any sign of local differentiation to macrophages or pool derived from adult blood monocytes. The authors argue dendritic cells (34). Further studies using fate-mapping systems that the dermis, in contrast to the epidermis, continues to recruit should be addressed to clarify this point. Spleen macrophages are circulating monocytes in adulthood, most likely facilitated by generated prenatally (13, 33). However, red pulp macrophages its high level of vascularization. The macrophage network in the and marginal zone macrophages seem highly dependent, respec- intestine follows a similar model. Data from Bain et  al. suggest tively, on the transcription factor SPI-C (109) and on the nuclear that embryonic macrophages do not persist in adulthood in the receptor LXR (110), also expressed by circulating monocytes and gut, and are replaced constantly by circulating adult monocytes suggest again that embryonic-derived macrophages are replaced (23), convincingly showing that adult monocytes are the source over time by adult monocytes-derived macrophages. e Th use of of intestine-resident macrophages. e Th role of commensal the S100a4-Cre fate-mapping model in our hands supports these microbiota in this process is supported by the observation that observations and similar conclusions were obtained for BM and the use of germ-free animals or treatment with broad-spectrum peritoneal macrophages (15). Although tissue microenviron- antibiotics results in a significant reduction in the recruitment ment shapes certain macrophage functional specificities ( 111), of Ly6C monocytes to the colon (23). e Th macrophage network through an ontogenic point of view, the composition of each of the heart has also been shown to contain a component of YS tissue-resident macrophage pool evolves throughout life and the macrophages and fetal monocyte-derived macrophages, both of respective origins of each macrophage population may account which are maintained in adulthood (99). However, similar to the for some of their key functions and cellular behaviors in a given dermis and the gut, adult monocytes seem to replace embryonic tissue. Hence, a new challenge is to understand if an embryonic macrophages progressively over time (24). The decreasing capac - or adult origin matters for the function and the activation states ity for self-renewal of embryonic macrophages with age observed of tissue-resident macrophages. by Molawi et  al. may explain the requirement for continuous Origin and Development of YS recruitment of monocyte-derived macrophages to the heart in the absence of inflammation. It remains to be clarified whether Macrophages and Fetal Monocytes this phenomenon occurs in other tissues as a result of aging. In Origin of YS Macrophages agreement, proliferation of YS macrophages and fetal monocytes is very high during development (20–40% before E14.5) but Bertrand et  al., in line with the seminal work of Palis (45), described two sequential myeloid waves within the early YS decreases progressively to 10% few days aer b ft irth in most tis - sues and decreases to almost undetectable levels in adults (15). (42). Using an in  vitro culture reporter system, Bertrand et  al. observed a first wave of monopotent progenitors that gave rise Interestingly, macrophage turnover seems different from one tissue to another. Following BrdU incorporation at steady state, only to macrophages, followed by a second wave that gave rise to a mix of granulocytes, monocytes, and macrophages. More almost no proliferation was observed in adult gut macrophages (23), while 2–5% was measured in adult heart macrophages recently, Kierdorf et  al. revisited the work of Bertrand et  al. (24). Macrophage proliferation activity can also be mobilized exploiting organotypic embryonic brain slices to demonstrate upon inflammation. For example, peritoneal macrophages can that microglial cells derived from YS EMPs (112). Kierdorf et al. increase their proliferation rate from 1 to 9% in response to also showed that these EMPs did not express the transcription parasite infection or in response to IL-4 stimulation (35), while factor c-Myb, associating them with the progenitors reported enhanced local proliferation of macrophages in atherosclerotic by Schulz et  al. (12), although a direct link with the generation lesions sustain disease progression (103). The characterization of of microglia in  vivo in adulthood was not conclusively demon- local signals regulating macrophage proliferation as well as the strated. More recently, Perdiguero et  al. used the CSF-1R-iCre presence of specialized tissue niches that sustain macrophage fate-mapping model to show that YS macrophages are derived EMPs (14). Hence, these two studies suggest survival, proliferation, or even “stemness” will be fundamental to from CSF-1R better understand their tissue homeostasis. that YS macrophages, and thus microglia, would originate from c-Myb-independent CSF-1R EMPs. Furthermore, Perdiguero e m Th acrophage network of the lymphoid system seems to follow a similar pattern than in the gut and dermis. Although et al. demonstrated that CSF-1R EMPs were able to seed the FL by E10.5, suggesting that these progenitors could later populate the lymph nodes (LN) start to develop very early in the embryo (104), they become functionally active only within the first week other tissue niches and produce YS-like macrophage later during development in others tissues. Nevertheless, these data do not aer b ft irth recruiting and organizing B and T cell areas when fol - licles start to shape with connections to aer ff ent lymphatics via explain the low percentage of labeled adult macrophages observed by Schulz et al. using the CSF-1R-iCre fate-mapping model (12). the subcapsular sinus (105). Although macrophages are known to participate in lymphangiogenesis during development, notably Later observations by Epelman et al. (99), and more recently by by the production of VEGF (106, 107), the precise origin of the our group using the same fate-mapping model (15), indicated different LN macrophage populations remain poorly understood that the ability of CSF-1R EMP to reach the FL could explain Frontiers in Immunology | www.frontiersin.org 10 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny the surprising maintenance of primitive macrophages until E16.5 derivation from the recently described YS-derived LMPs (67). in c-Myb null embryos, where primitive macrophages generated u Th s, LMPs may be important for the generation of a small but in the YS as well as in the FL would be able to fulfill the empty significant proportion of fetal monocytes prior to the expansion niche left by the absence of c-Myb-dependent myeloid cells, that of mature HSCs (Figure  4). Further investigations using more include fetal monocytes. However, this may not reflect the physi - specific fate-mapping models will be necessary to elucidate the ological situation and may instead result from a compensatory exact contribution of LMPs as well as the hematopoietic transi- mechanism to ensure the presence of macrophages in all tissues tion between the FL and the BM. in the absence of c-Myb activity and fetal monocytes. Using the Importantly, we observed that fetal monocytes were not same CSF-1R-iCre fate-mapping model (15), we were able to tagged with the CSF-1R-Cre model that label early CSF-1R EMPs, suggesting that fetal monopoiesis is not dependent on follow the maintenance of microglia in the brain by self-renewal + + from E10.5 until adulthood, linking them with CSF-1R EMPs CSF-1R EMPs, consistent with our previous data (9, 11) and and confirming the previous observations of Perdiguero et  al. with our YS macrophage depletion results (15, 98). Furthermore, (14). However, for all other macrophage populations, the reduc- the Runx1-iCre fate-mapping model allowed us to identify two tion of fate-mapping reporter labeling aer E13.5 co ft nfirmed the waves of EMPs that arise sequentially before LMPs in the YS. progressive replacement of YS macrophages by another unlabeled es Th e included an early wave, arising at E7.5 that differentiates precursor arising from a different hematopoietic wave. locally into YS macrophages; and a later wave tagged at E8.5, that We previously showed that Runx1 YS progenitors that migrates and seeds the FL following the establishment of the blood emerged at E7.5 give rise to YS macrophages and microglia (9, 11). circulation before E9.0. Early EMPs tagged at E7.5 were therefore Using both the Runx1-iCre and the CSF-1R-iCre fate-mapping related to those described previously by Kierdorf and Perdiguero models, we showed that these E7.5 Runx1 YS progenitors were (14, 112). The late EMPs tagged at E8.5, however, expressed in fact the same CSF-1R EMPs described by Perdiguero et  al. c-Myb, expanded more efficiently in the FL, and differentiated and Kierdorf et  al., which contributed to the generation of YS in vivo into fetal cMoPs, constituting the major component of the macrophages and, to a lesser extent, those seeding the FL (14, 15, fetal monocyte population as well as the fetal monocyte-derived 112). However, we also observed their disappearance from the FL macrophage population (Figure  3), which was able to maintain aer E11.5 in ft dicative of a rapid local consumption/differentiation itself in all tissues tested (15). rather than long-term maintenance. Our results also suggest that e exi Th stence of two distinct EMP waves is in agreement these early CSF-1R EMPs are able to contribute to a short-term with Bertrand et al. who reported an early wave of macrophage maintenance of macrophages in the FL (Figure  2), but do not progenitors restricted to the YS, and a second wave that was able contribute to other tissue macrophages as evidenced by their rapid to reach the FL to participate in definitive hematopoiesis ( 42). disappearance from the blood circulation aer E14.5 ( ft 15). This e diff Th erential expression of c-Myb between early and late EMPs transient population in the FL may be due to a local immediate is in agreement with previous reports indicating that primitive requirement for macrophages, at least during the onset of FL hematopoiesis can occur in the absence of c-Myb, especially for hematopoiesis, to perform efficient enucleation of primitive eryth - the generation of monopotent macrophage progenitors (114), rocytes passing through the FL sinusoids (100, 113). Combining whereas EMPs from definitive hematopoiesis express and are historical evidences showing their direct lineage connection with dependent on c-Myb activity (45, 62, 115). the emergence of YS macrophages and recent findings showing Notably, a previous study showed that c-Myb ablation strongly their independence with c-Myb activity, we propose that CSF-1R compromises definitive hematopoiesis ( 116). Palis et al. observed EMPs should be designated as primitive EMPs. that c-Myb is expressed prior to and during the early develop- ment of definitive erythrocyte progenitors (45). u Th s, late EMPs Origin of Fetal Monocytes and LMPs, as well as HSCs, express c-Myb (15, 45, 61, 62), Because adult monocytes are derived from HSCs in the BM, it suggesting that the entire fetal monopoiesis machinery is reliant hi lo would be reasonable to assume that embryonic HSCs might also F480 on this transcription factor. In agreement, the CD11b give rise to fetal monocytes in the developing liver. In agreement population, which in our hands contains fetal monocytes, was with this hypothesis, we have identified a population in the FL completely absent in the c-Myb-deficient embryo ( 12, 116). As a similar to adult MDPs that have the potential to generate fetal consequence, the contribution of c-Myb-dependent progenitors cMoPs and monocytes following in vitro culture (15). Exploiting to tissue-resident macrophage populations could not be evalu- the Flt3-Cre tomato fate-mapping model (83), we then fol- ated in c-Myb-deficient embryos, where c-Myb-independent YS macrophages maintain themselves as a compensatory mecha- lowed the progeny of embryonic HSCs. However, the poor labeling observed between E14.5 and E17.5 in FL monocytes and nism due to the absence of c-Myb-dependent fetal monocytes macrophages contrasted with the strong labeling of FL MDPs, that normally outcompete them. Because c-Myb expression is suggesting that HSCs had limited involvement in the generation upregulated during the successive steps of fetal monopoiesis of fetal monocytes (15). Nonetheless, the limited but significant (15), the switch in EMP localization between the YS and the labeling in fetal monocytes and macrophages at birth suggested FL may indeed be orchestrated by c-Myb. As a consequence, an increasing derivation from fetal HSCs, assuming that fetal most tissue-resident macrophages derived from fetal monocytes HSCs follow a similar Flt3-dependent differentiation pathway as would therefore rely on c-Myb activity. Altogether we propose EMPs giving rise to the first circulating monocytes adult HSCs. In parallel, gene array analysis highlighted a strong that c-Myb lymphoid signature within fetal MDPs (15), indicative of their should be designated as definitive EMPs. Frontiers in Immunology | www.frontiersin.org 11 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny understanding of the mechanisms that control the tissue-specific Conclusion functions of macrophages in the steady state, and thus may Recent reports have drastically changed the view of the develop- uncover new therapeutic opportunities in diverse pathological ment of the MPS and shed light on the multiple layers that define settings such as metabolic diseases, fibrosis, and carcinogenesis. fetal hematopoiesis. It is now evident that fetal monocytes form the major precursors of most adult tissue-resident macrophages, Funding and further investigations are now necessary to clarify how they shape macrophage heterogeneity. Examining how tissues This work was supported by the Singapore Immunology Network imprint specific fates in these circulating precursors will aid our (SIgN) core grant. 19. Virolainen M. 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Ontogeny of Tissue-Resident Macrophages

Frontiers in Immunology , Volume 6 – Sep 22, 2015

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Abstract

Review published: 22 September 2015 doi: 10.3389/fimmu.2015.00486 Ontogeny of tissue-resident macrophages Guillaume Hoeffel* and Florent Ginhoux* Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore The origin of tissue-resident macrophages, crucial for homeostasis and immunity, has remained controversial until recently. Originally described as part of the mononuclear phagocyte system, macrophages were long thought to derive solely from adult blood circulating monocytes. However, accumulating evidence now shows that certain mac- rophage populations are in fact independent from monocyte and even from adult bone marrow hematopoiesis. These tissue-resident macrophages derive from sequential seeding of tissues by two precursors during embryonic development. Primitive macro- Edited by: phages generated in the yolk sac (YS) from early erythro-myeloid progenitors (EMPs), Peter M. Van Endert, Université Paris Descartes, France independently of the transcription factor c-Myb and bypassing monocytic intermediates, Reviewed by: first give rise to microglia. Later, fetal monocytes, generated from c-Myb EMPs that Meredith O’Keeffe, initially seed the fetal liver (FL), then give rise to the majority of other adult macrophages. Burnet Institute for Medical Research, Australia Thus, hematopoietic stem cell-independent embryonic precursors transiently present in Jean M. Davoust, the YS and the FL give rise to long-lasting self-renewing macrophage populations. Institut National de la Santé et la Recherche Médicale, France Keywords: macrophages, monocytes, fetal liver, yolk sac, C-Myb, erythro-myeloid progenitors, hematopoiesis, hematopoietic stem cells *Correspondence: Guillaume Hoeffel and Florent Ginhoux, Singapore Immunology Network introduction (SIgN), Agency for Science, Technology and Research (A*STAR), Ilya (Elie) Metchnikoff first described the mechanism of phagocytosis and the cells responsible for 8A Biomedical Grove, IMMUNOS this process over a century ago. These professional phagocytic cells were named “macrophages” Building #3-4, Biopolis, (from the Greek derivation macro = large and phage = devouring, “large devouring cells”). These 138648 Singapore were separate from “microphages,” which included polymorphonuclear phagocytes (1). Determining guillaumehoeffel1@gmail.com; florent_ginhoux@immunol. the role of macrophages in pathogenic infections was one of the fundamental observations leading to a-star.edu.sg the concept of cellular immunity (2). Through this seminal work, Metchnikoff anticipated the central role of macrophages in tissue inflammation and homeostasis. We recommend an elegant historical Specialty section: review for more details about Metchnikoff ’s work by Yona and Gordon in this issue (3). This article was submitted to Antigen Since then, the definition of the phagocyte system has been continuously refined, and our under - Presenting Cell Biology, a section of standing of the wide-ranging functions of macrophages has been substantially expanded. It is now the journal Frontiers in Immunology clear that, in addition to their classical function in the activation and resolution of tissue inflamma - Received: 22 July 2015 tion, macrophages also play roles in tissue-specific functions, tissue remodeling during angiogenesis Accepted: 07 September 2015 and organogenesis, and wound healing, to name a few (4). Macrophages are exquisitely adapted to Published: 22 September 2015 their local environment, acquiring organ-specific functionalities during developmental stages and Citation: the steady state (4). Macrophages are able to support multiple tissue functions, integrating cues from Hoeffel G and Ginhoux F (2015) both the outside environment and their microenvironment to act as rheostatic cells of tissue function. Ontogeny of tissue-resident u Th s, tissue-resident macrophages represent an attractive target for modern medicine to treat a wide macrophages. spectrum of diseases in which they have been implicated, including atherosclerosis, autoimmune Front. Immunol. 6:486. doi: 10.3389/fimmu.2015.00486 diseases, neurodegenerative and metabolic disorders, and tumor growth (5–8). Understanding the Frontiers in Immunology | www.frontiersin.org 1 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny origin and developmental pathways of macrophages will help to rely on hematopoietic stem cell (HSC)-derived BM progeni- design novel intervention strategies targeting these cells in tissue- tors, certain macrophage populations possess the unique ability specific sites. to self-renew locally independently of circulating precursors A number of observations now indicate that certain (32–36). Initial studies describing the presence of macrophages macrophage populations derive from embryonic precursors in embryonic tissues suggested that tissue macrophages derived sequentially seeding tissues during development (9–13). Two from embryonic progenitors. In rodents, macrophage-like cells macrophage progenitors, yolk sac (YS) macrophages and fetal first described in the brain rudiment and in the developing skin monocytes, have been described in the embryo, but their exact (37, 38) were named “fetal macrophages” and found to exhibit nature and origin were not fully understood until recently (14, a high capacity for proliferation (39). These observations sug- 15). Here, we discuss recent developments in our understanding gested that adult macrophages derive from fetal macrophages of the origin of adult tissue-resident macrophages, exploring the established during early development. However, whether these sequence of progenitors generated during embryonic and adult fetal macrophages were maintained until adulthood or were hematopoiesis. We focus on the relative contributions of YS replaced postnatally was not addressed until recently. In addition, macrophages and fetal or adult monocytes, including a discus- the exact nature and the origin of fetal macrophage progenitors sion of our own recent data exploring the heterogeneity of fetal remained unclear. monocyte developmental pathways. e mbryonic Hematopoiesis e arly Concepts Mammalian embryos produce several transient waves of hemat- Macrophages form part of the mononuclear phagocyte system opoietic cells before the establishment of HSCs in the BM during late gestation (40, 41). The multiple embryonic waves are differ - (MPS), which also includes circulating monocytes and dendritic cells (16). Until recently, our vision of macrophage origin and entially regulated in time and space and exhibit distinct lineage potentials. Importantly, they contribute to hematopoietic popula- homeostasis was largely based on seminal studies that used in vivo radioisotope labeling and radiation chimera experiments. tions that persist until adulthood. These waves include primitive hematopoiesis in the YS, and definitive hematopoiesis, which es Th e studies led to the early dogma that resident macrophages comprises a transient definitive stage, generating multi-lineage were constantly replenished from circulating bone marrow (BM)- derived monocytes as a continuum of differentiation (17–19). erythro-myeloid progenitors (EMPs) and lympho-myeloid pro- genitors (LMPs), and a definitive stage characterized by the In agreement with that concept, studying the ontogeny of the MPS revealed that monocytes and macrophages derived from production of HSCs in the aorta-gonad-mesonephros (AGM). es Th e transient progenitors establish themselves transiently in macrophage and dendritic cell progenitors (MDPs) present in the BM, which are phenotypically defined as lineage-c-kit CX the fetal liver (FL) during the mid to late stages of hematopoiesis. + + + 3CR1 Flt3 CD115 (20). MDPs further differentiate through a e s Th equential waves of hematopoiesis can overlap in time and newly described common monocyte precursor (cMoP), pheno- space (Figure 1) and remain difficult to separate clearly, even with − + + − + typically defined as lineage c-kit CX3CR1 Flt3 CD115 (21), the most recent fate-mapping tools available. that gives rise to the two main subsets of circulating monocytes distinguished by the expression of Ly6C (22). Primitive Hematopoiesis Specific tissue macrophages, such as dermal, gut, and heart In mice, the first hematopoietic progenitors appear in the macrophages, seemed to follow the model of Van Furth, that extra-embryonic YS blood islands at around embryonic age 7.25 macrophages are derived from monocytes (23–25). However, this (E7.25), where primitive hematopoiesis is initiated, producing mainly nucleated erythrocytes. This observation linked the model did not fit in all cases and evidence also emerged indicating that macrophages were long-lived cells, able to self-renew locally. myeloid progenitors observed in the YS at E7 with the emergence of YS macrophages aer E9.0 [ ft Figure 2; Ref. (42–45)]. Primitive Hashimoto was the first to speculate that Langerhans cells (LCs) represented a self-perpetuating “intraepithelial phagocytic sys- hematopoiesis was also shown to produce megakaryocyte pro- tem” (26). Performing a human skin transplantation assay onto genitors (46). The denomination “primitive” was given to reflect nude mice, Krueger et  al. described the remarkable longevity the production of embryonic erythroblasts, like those observed in of LCs, which were able to persist in the grafts for more than lower species such as fish, amphibians, and birds, and remaining 2  months (27). Their ability to self-renew through proliferation nucleated throughout their life span (47–49). This denomination was later described using DNA densitometry (28). Similar con- was extended to macrophages in the YS due to their concomitant clusions were drawn soon aer r ft egarding alveolar macrophages development prior to FL hematopoiesis. Interestingly, no clear (29). The dominant concept of “the monocytic origin” of tissue evidence of monocytic intermediates was reported at this stage, macrophages was also challenged through experiments in ani- although the seminal study of Cline and Moore did mention the mals with prolonged monocytopenia following strontium-89 existence of local intermediate cells between progenitors and monocyte depletion, in which liver Kuper ce ff lls were shown to functional macrophages (43). Studies by Naito and Takahashi maintain cell numbers by increasing local proliferation (30, 31). et  al. clarified the emergence of primitive macrophages in the More recently, the use of long-term parabiotic mice and sub- YS blood islands in the mouse and rat, observing an absence sequent fate-mapping models have challenged the MPS paradigm of endogenous peroxidase activity as a surrogate marker for an and revealed that, unlike all other hematopoietic cells, which absence of monocytic intermediates, such as those found in the Frontiers in Immunology | www.frontiersin.org 2 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny FiGURe 1 | Fetal hematopoiesis. Primitive, transient definitive, and definitive waves of fetal hematopoiesis sequentially generate progenitors able to seed the fetal liver. Primitive hematopoiesis starts at E7.0 in the blood islands of the extra-embryonic yolk sac (YS) and generates erythro-myeloid progenitors (EMPs). Early EMPs initially express CD41 and later, CSF-1R, a signature of myeloid/macrophage commitment. Concomitant to the establishment of the blood circulation at E8.5, the YS hemogenic endothelium (HE) generates late EMPs expressing C-Myb. At approximately E9.0, the intra-embryonic mesoderm generates additional HE and emerging progenitors with lymphoid potentials (LMPs) without long-term reconstitution (LTR) capacity. These C-Myb EMPs and LMPs constitute the so-called transient definitive wave. Finally, hematopoietic stem cells (HSCs) with LTR activity emerge from the main HE situated in the aorta-gonad-mesonephros (AGM) regions and in the placenta. BM (50–52), suggesting a unique developmental pathway for YS potential progenitors from E8.25 in the YS (45). Palis et al. first macrophages (53, 54). observed the emergence of definitive progenitors for mast cells and a bipotential granulocyte/macrophage progenitor. These Transient Definitive Hematopoiesis progenitors then migrated to the FL through the bloodstream The quest to elucidate the origins of embryonic HSCs led to after E8.5, once circulation was established ( 44, 56). From this the discovery of earlier lineage-restricted HSC-independent pattern of development, the authors concluded that definitive progenitors seeding the FL at E10.5. These progenitors arise hematopoietic progenitors arise in the YS, migrate through the concurrently with the transition of primitive to definitive bloodstream, and seed the FL to rapidly initiate the first phase erythropoiesis and were thus considered to form a transient of intra-embryonic hematopoiesis. Similarly, primitive and stage of definitive hematopoiesis (45, 47, 55). Transient defini- definitive erythropoiesis, associated with myelopoiesis, was also tive hematopoiesis consists of progenitors sequentially acquir- shown to emerge prior to HSC in the zebrafish embryo ( 57). ing myeloid, then lymphoid potential, without exhibiting the Bertrand et  al. showed that definitive hematopoiesis initiates long-term reconstitution potential of HSCs. Seminal work from in the posterior blood island with only transient proliferative Palis and colleagues on embryonic erythropoiesis in the YS potential. Because these HSC-independent definitive progeni - described the parallel emergence of multiple myeloid lineage tors were observed to produce definitive erythroid and myeloid Frontiers in Immunology | www.frontiersin.org 3 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny FiGURe 2 | Primitive hematopoiesis and yolk sac macrophage ontogeny. Early EMPs emerge in the YS around E7.5 before establishment of the blood circulation. They express CD41 and CSF-1R and are independent of the transcription factor C-Myb. Upon establishment of the blood circulation around E8.5, EMPs differentiate into primitive macrophages as well as primitive erythrocytes and granulocytes. Primitive macrophages seed all fetal tissues, in particular the head where they will give rise to future brain microglia that are able to continuously self-renew throughout adulthood. EMPs seeding the fetal liver briefly expand to generate a local macrophage population, likely important for sustaining enucleation of primitive erythrocytes passing through the sinusoid prior to the establishment of definitive hematopoiesis and the generation of fetal monocyte-derived macrophages in the fetal liver. cell types, but not to colonize the zebrafish thymus (implying FL with T cell, B cell, and macrophage potential (63), although that they are devoid of lymphoid potential), this population was the precise origin of these progenitors was not addressed. At the termed EMPs (57). Interestingly, EMPs can also emerge from same time, the team of Jacobssen identified Flt3 lympho-myeloid the hemogenic endothelium (HE) located in the placenta and progenitors (LMPs) devoid of erythrocyte and megakaryocyte umbilical cord (58) and colonize the FL from E9.5 (55) to par- capacity (64). Later, cells with myelo-erythroid and lymphoid ticipate in definitive hematopoiesis. Further studies advanced lineage potential, such as B-1 cells present in the adult spleen, the field significantly by identifying CD41 as an early marker were associated with E9.5 YS progenitors expressing AA4.1 and (pre-CD45) for defining hematopoietic progenitors, including CD19 (65). A year later, the same team also identified T cell EMPs, emerging from the YS (59, 60). Altogether, these impor- potential within the E9.5 YS progenitors (66). Using a Rag-1-Cre tant studies provided phenotypic and functional analyses of the fate-mapping model, Boiers et  al. confirmed that these LMPs first hematopoietic progenitors and demonstrated that defini- emerged at approximately E9.5 in the YS, seeding the FL by E11.5 tive hematopoiesis proceeds through two distinct waves during to give rise to T and B cells, as well as granulocytes and monocytes embryonic development (Figure 3). in the E14.5 FL, prior to HSCs (67). Finally, lympho-myeloid In parallel, several groups have also identified other multipo - progenitors isolated from the dorsal aorta at E9.0 were shown tential progenitors with lymphoid- or myelo-lymphoid-restricted to acquire long-term reconstitution capacity aer a f ft ew days of potential in the YS and the developing para-aortic splanchno- in  vitro culture with stromal cells, and were called immature pleura (P-Sp) prior to HSCs (61, 62). Lacaud et al. also described HSCs (68). Without preculture, these multipotential progenitors + −/− AA4.1 (CD93) multipotential progenitors present in the E14.5 can only engraft natural killer (NK)-deficient Rag2 γc mice. Frontiers in Immunology | www.frontiersin.org 4 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny FiGURe 3 | Fetal monopoiesis and macrophage ontogeny. At E8.5 as the blood circulation is established, the YS HE, possibly in conjunction with other hemogenic sites, generates EMPs expressing CD41 and c-Myb. These late EMPs seed the fetal liver around E9.5 where they expand rapidly to give rise to CSF-1R myeloid progenitors that are able to generate fetal monocytes through cMoP intermediates at E12.5. Fetal monocytes then spread via the blood circulation to all tissues, with the exception of the brain, which is isolated by the establishment of the blood–brain barrier at approximately E13.5. In the tissues, fetal monocytes begin to differentiate into macrophages, progressively outnumbering the previously established primitive macrophages. These fetal monocyte-derived macrophages maintain the capacity to self-renew throughout adulthood in certain tissues, such as the liver or the lung, where they will not be replaced by adult BM-derived monocytes. Whether these immature HSCs arise from LMPs or represent To conclude, commitment to hematopoietic fates begins dur- a distinct wave of progenitors remains to be clarified. However, ing gastrulation in the YS, which represents the only site of primi- these seminal studies provided strong evidence that lymphoid tive erythropoiesis and also serves as the first source of transient potential can emerge from the YS, prior to HSC-budding from definitive hematopoietic progenitors. HE develops from the YS the AGM (69). to various intra-embryonic sites, and acquires myeloid and then Because the emergence of EMPs and LMPs overlaps in time lymphoid lineage potentials in overlapping waves, highlighting and space, they could not be distinguished clearly until recently. the complexity of the hematopoietic output. Whether some of Previous reports had suggested that lymphoid potential was these progenitors arise from independent sources or represent restricted to the CD41-negative cell fraction (59, 65). However, different maturation stages of a shared hematopoietic wave, cul- CD41 is also expressed in a sub-fraction of FL HSCs, and so this minating with the generation of HSCs, needs to be further clari- phenotypical distinction spread some confusion (47). Finally, fied. However, it is tempting to speculate that the clear contrasts a recent report from the group of Palis clarified this point by in differentiation/lineage potential do not reside in their intrinsic showing that co-expression of c-kit, CD41, and CD16/32 defines potential, but rather in the extrinsic signals provided by the local EMPs and allows their separation from other progenitors with environment. lymphoid potential, such as those giving rise to the B-1 cell (70). McGrath et  al. extended the notion of EMPs by showing their Definitive Hematopoiesis potential to generate neutrophils, megakaryocytes, macrophages, e co Th mplex hierarchy of stem and progenitor cells in the BM and erythrocytes. Finally, transplantation of EMPs in immune- is first established during embryonic development starting compromised adult mice can also provide transient adult red with the emergence of small numbers of HSCs from the AGM blood cell reconstitution (70). at E10.5 in murine embryos or at 5  weeks in human embryos Frontiers in Immunology | www.frontiersin.org 5 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny (71, 72). Aer E9.5 in t ft he mouse, with the determination of the e co Th ntribution of HSCs to FL hematopoiesis is complex intra-embryonic mesoderm toward a hematopoietic lineage, new to evaluate, partly because of the lack of specific fate-mapping waves of hematopoietic progenitors emerge within the HE of the models, and also the relatively limited knowledge regarding embryo proper (Figure 4), first in the P-Sp region and the umbili - embryonic HSC maintenance and homeostasis in this environ- cal and vitelline arterial regions of the embryo, then in the AGM ment. The capacity for long-term reconstitution, which defines region and the placenta (55, 73, 74). e h Th ematopoietic activities functional HSCs, is present in the AGM by E10.5 (76). However, of the P-Sp and AGM first generate immature HSCs and then lineage-specific commitment may not occur in  vivo imme- mature HSCs, which are defined by their capacity to reconstitute diately aer r ft eaching the FL environment. A number of other adult conventional mice (long-term reconstitution; LTR). Both progenitors generated during transient definitive hematopoiesis, immature and mature HSCs seed the FL at approximately E10.5 as discussed above, are already present and able to give rise to (68, 71, 75, 76) to establish definitive hematopoiesis ( 40, 77, 78). almost all cell lineages, which could prevent HSC consumption A maturation step seems necessary for immature HSCs to express and differentiation ( Figure  5). Evaluation of HSC contribution their LTR activity in full, which is then maintained until adult- has long been based on the assumption that all hematopoietic hood (68). However, further investigations using a fate-mapping cells in the FL were derived from HSCs as is the case in the BM system would be necessary to confirm this model. (81). Many multipotential progenitors share the same phenotype e FL b Th ecomes the major hematopoietic organ aer E11.5, ft with pre-HSC and HSCs, such as the expression of CD41 and generating all hematopoietic lineages. Importantly, the FL AA4.1 (60), adding to this confusion. e Th combination of the itself does not produce progenitors de novo, but rather recruits marker Sca-1 and new markers such as those from the SLAM progenitors derived from the YS and other hemogenic sites, to family (82) have greatly helped to clarify the characterization of − + + + − − ckit Sca-1 CD150 CD48 CD244 . initiate definitive hematopoiesis (79) in parallel with the expan- HSCs, defined now as Lin sion of the definitive HSC population before their migration to However, no specific fate-mapping model exists to characterize embryonic HSC progeny with the exception of the Flt3-Cre the spleen and BM (80). FiGURe 4 | Transition between fetal and adult hematopoiesis. Hemogenic endothelial cells from extra and intra-embryonic hematopoietic tissues generate C-Myb-dependent multipotential progenitors, such as LMPs and pre-HSCs, between E9.0 and E10.5, culminating with the emergence of mature HSCs with long-term reconstitution-bearing potential. CD93 (AA4.1) expression is associated with the emergence of lymphoid potential, whereas Sca-1 is the hallmark of HSCs. These progenitors seed the fetal liver around E10/E11, expanding and giving rise to the various lineages of the hematopoietic system, including fetal monocytes. These late fetal monocytes continue to participate in the tissue-resident macrophage network until hematopoiesis switches completely from the fetal liver to the bone marrow. Once adult hematopoiesis begins to take place in the bone marrow generating monocytes, certain tissues, such as the dermis, heart peritoneum, and the gut, continue to recruit adult monocytes to generate resident macrophages and replace with time the embryonic-derived macrophages. Frontiers in Immunology | www.frontiersin.org 6 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny FiGURe 5 | Timeline of fetal and adult hematopoiesis. The primitive hematopoiesis is initiated in the yolk sac independently of C-Myb activity, and generates early CSF-1R EMPs that give rise to YS macrophages without monocytic intermediates during a short time window and will establish the brain microglia. The transient definitive hematopoiesis and then the definitive hematopoiesis are both dependent on C-Myb activity and generate progenitors that differentiate in the fetal liver. The transient definitive wave, which include EMPs and then LMPs, give rise in particular to fetal monocytes that seed the tissues prior to birth to establish the self-renewing tissue-resident macrophage network. Although only HSCs, which result from the definitive hematopoiesis, seem to be maintained in the bone marrow in adults, the relative contribution of the transient definitive wave to the adult immune system remains unclear. model (83), which was used until now with the assumption that colonize various tissues (52, 93–95). Primitive macrophages may embryonic and adult HSCs follow similar differentiation path - contribute to many fundamental processes during mid and late ways. Our recent report suggests that the Flt3-Cre model can embryogenesis, such as clearance of dead cells or tissue matura- also be used to follow the progeny of LMPs (15). Furthermore, tion. In this regard, the developmental process of interdigital in the nascent BM, the long-term repopulation (LTR) capacity cell death removal during the mouse footplate remodeling that that characterizes functional HSCs is only observed at around occurs between E12.5 and E14.5 is of interest as the interdigit E17.5 (84). Considering the time required to initiate full HSC regions become heavily populated by macrophages and most of differentiation, these data suggest that proper adult HSC-derived the dead cells were shown to be rapidly engulfed by macrophages hematopoiesis does not take place in the BM until a few days (96). However, mouse models devoid of primitive macrophages aer b ft irth. Characterization of the functional specificities and such as the colony-stimulating factor 1 receptor (CSF-1R) KO regulatory pathways of HE that give rise to HSCs versus those (Florent Ginhoux, unpublished data,) and PU.1 KO (97) appears that generate EMPs and other multipotential progenitors could to exhibit a normal interdigit web tissue. Wood et  al. observed aid the development of new fate-mapping models and improve that interdigit web tissue in PU.1 KO was only slightly retarded, our understanding of this process (85). Use of other fate-mapping suggesting that other cell type such as neighboring mesenchymal models such as the Runx1-Mer-Cre-Mer (Runx1-iCre) (86), cells were compensating (97). In addition, we recently showed Tie2-Mer-Cre-Mer mice (14), and the c-kit-Mer-Cre-Mer mice that depletion of primitive macrophages and hence of embryonic (87) provided complementary results, although a careful analysis microglia, ae ff cted the progression of dopaminergic axons in the of the targeted cells in time and space is not yet fully available forebrain and the laminar positioning of subsets of neocortical for the last two models. We present here our best interpretation interneurons, likely through phagocytic mechanisms (98). of the data provided in these two recent studies that have used Schulz et al. highlighted further differences between primitive these models in light of the literature and our own results and and definitive hematopoiesis, showing that the latter relies on experience using the Runx1-iCre model (Figure 6). the transcription factor Myb, while YS-derived macrophages are Myb-independent, and are instead dependent on PU.1 (12). This e mbryonic and Adult Precursors of Adult again reinforces the view that YS-derived macrophages constitute an independent lineage, distinct from the progeny of definitive Tissue-Resident Macrophages HSCs. Schulz et  al. exploited the differential dependence of Yolk Sac Macrophages primitive versus definitive hematopoiesis on the transcription Yolk sac macrophages first appear in the YS blood islands at E9 factor c-Myb and reported that E16.5 tissue macrophage popula- (albeit in small numbers) with a unique pattern of differentiation tions were not ae ff cted by the loss of c-Myb. Using a CSF-1R-iCre that bypasses the monocytic intermediate stage seen in adult mac- fate-mapping model of YS macrophages, they also reported the rophages (50, 52). YS-derived primitive macrophages spread into persistence of YS macrophages progeny in adult tissue-resident the embryo proper through the blood as soon as the circulatory macrophage populations (lung, liver, and pancreas, as well as in system is fully established (from E8.5 to E10) (56), and migrate the brain and skin), although the level of labeling was minimal to various tissues, including the brain. Importantly, this occurs (below 3–5%) and decreased with time. The authors concluded before the onset of fetal monocyte production by the FL, which that tissue-resident macrophages were therefore derived from starts around E11.5/E12.5 (92). These primitive macrophages a c-Myb-independent lineage via YS macrophages (12), data retain the high proliferative potential observed in the YS as they supporting the initial report showing that microglia arise from Frontiers in Immunology | www.frontiersin.org 7 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny FiGURe 6 | Fate-mapping systems. (A) The Runx-iCre fate-mapping model (86) used in our study targets the hemogenic transition (88), hence, labeling specifically pr ogenitors in the process of budding out from the hemogenic endothelium. The Runx1 expression decreases in progenitors once they start to express Vav thus reducing the chances of tagging released progenitors from precedent waves (88). As a consequence, Runx-iCre tagging is restricted to a short time window in the lifespan of a given progenitor and allows a sharp definition of each hematopoietic wave. However, this model also restricts the tagging to only a small fraction of the targeted progenitor wave. (B) Tie2 is expressed in all endothelial cells that constitute the hemogenic endothelia even before the hemogenic transition (89). Thus, all endothelial cells and their progeny (non-hematopoietic and hematopoietic cells) will be labeled after tamoxifen injection using the Tie2-iCre model. As a consequence, an early tamoxifen injection (such as at E7.5) will result in the tagging of all hematopoietic cells emerging before the time of analysis. This will include progenitors from the primitive, the transient definitive, and the definitive waves if, for example, the analysis is done at E11.5. A late injection (such as at E10.5) will restrict the tagging to only the latest hematopoietic stem cells wave as they are just budding from HEs (90). Thus, this model might not be suitable to clearly separate the primitive from the transient definitive waves of hematopoiesis. However, this model could be important to study late HSC progeny as no other progenitors than HSCs emerge from HE after E10.5 (91). (C) C-kit is expressed by all hematopoietic progenitors and does not label endothelial cells that constitute the HEs (89). An early tamoxifen injection (such as at E7.5) will restrict the labeling to early progenitors making suitable the c-kit-iCre model to study the primitive hematopoiesis. However, the FL recruits progenitors of each hematopoietic wave from E8.5 until E11 (79). These progenitors still express c-kit and coexist after seeding the FL during the time necessary for their differentiation (47, 55). A later tamoxifen injection (such as at E9.5) might thus result in the cumulative labeling of undifferentiated primitive and definitive progenitors, including the transient wave of EMPs and LMPs. Thus, such model may not be suitable to resolve the complexity of the different embryonic hematopoietic waves characterized by short time windows of emergence and strong overlapping tendencies. Primitive hematopoietic progenitors are rapidly consumed and the engagement of EMPs and LMPs in FL hematopoiesis reduces the expression of c-kit on their surface. Thus, later tamoxifen injection (such as at E11.5) could restrict the labeling to newly derived HSCs expressing high level of c-kit without labeling precedent progenitor waves (87). Such model might be interesting to study the progeny of late HSCs although the risk of tagging the progeny of EMPs and LMPs or later committed progenitors derived from HSCs remains high and difficult to exclude. Further analysis would be necessary to clarify the potential of such model. YS macrophages (9). Embryonic origin of macrophages was although the exact nature of this precursor was not elucidated. In further supported by the work of Yona et al. and Hashimoto et al. fact, both YS macrophages and fetal monocytes express CX3CR1 showing that adult monocytes do not substantially contribute (9, 11, 15) and could therefore correspond to the unidentified to tissue macrophages under steady-state conditions (13, 33). precursors suggested by Yona et  al. However, using a CSF-1R- Furthermore, Yona et al. suggested the existence of a CX3CR1 iCre fate-mapping model, also used by Schulz et al. (12), another precursor for some of the monocyte-independent macrophages, study noted that the YS macrophage contribution in the brain, Frontiers in Immunology | www.frontiersin.org 8 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny the adult liver, and the heart was maintained although at a mini- stage, direct differentiation of fetal monocytes into macrophages mal level that decreased with time (99). Interestingly, the level of in vivo had not been demonstrated (100). labeling was always higher in microglia than that in the liver or To investigate the developmental event leading to the emer- the heart, suggesting that the level of YS macrophage contribu- gence of tissue-specific macrophages, we initially focused on tion may differ between tissues and that YS macrophages may the LC, the specialized myeloid population of the epidermis. be differently replaced over time by later waves of progenitors, While YS macrophages seed the embryonic skin before E13.5, we which follow tissue-distinct kinetics (discussed below). Our discovered that the major fraction of adult LCs is in fact derived own report using the Runx1-iCre fate-mapping model (86) from fetal monocytes that are generated in the FL from E12.5 and indicated that only microglia, specialized macrophages of the are then recruited into fetal skin at E14.5 (11). These cells share central nervous system, were derived solely from primitive a similar phenotype to their adult counterparts; however, they macrophages while all other tissue macrophages derived from are generated independently of CSF-1R expression (9, 11). They definitive hematopoiesis (9 ). possess high proliferative potential, and, in contrast to their adult To understand whether YS macrophages might be the sole counterparts, express few genes related to pathogen recognition progenitors of every other adult macrophages, we asked what and immune activation (15). Further studies should clarify impact their in  utero depletion would have on the subsequent whether such differences reflect monocyte immaturity imposed generation of fetal tissue macrophages. CSF-1R is expressed on by a sterile fetal environment, or rather dedicated functional YS macrophages and fetal monocytes, but only the development specializations that have yet to be unraveled. In  utero adoptive of the former is actively dependent on CSF-1R (9, 11). Thus, we transfers combined with fate-mapping studies unequivocally attempted to deplete YS macrophages by transiently inhibiting confirmed in situ differentiation of fetal monocytes into adult LCs the CSF-1R signaling pathway using a blocking anti-CSF-1R (11). Fetal monocytes were then demonstrated to be the precur antibody, as recently described (98). Importantly, aer ft complete sor of adult macrophages in lung alveoli by intranasal injection (10, 101). Fetal monocytes were also shown to be involved in depletion of primitive YS macrophages in E10.5 embryos and thus of most macrophages in treated embryos at E14.5, tissue mac- the generation of adult macrophages of the heart (99). In fact, fetal monocytes become the major leukocyte within the blood rophages (including microglia) were able to repopulate to normal levels before birth. These data suggest that YS macrophages are circulation aer E13.5, s ft preading to all tissues. This occurred independently of the CCL2/CCR2 axis (15), suggesting an dispensable for the generation of tissue-resident macrophages in the embryo, and that another CSF-1R-independent embryonic alternative mechanism of exit from the FL and/or recruitment precursor can functionally replace YS macrophages during by fetal tissues. Moreover, we were able to fate-map, from before development (15, 98). Using a combination of both the CSF-1R- birth to adulthood, the local differentiation of fetal monocytes iCre and the Runx1-iCre fate-mapping models, we noted that into resident macrophages, by taking advantage of the specific although YS macrophages infiltrate all tissues (including lung, expression of S100a4 in fetal monocytes compared to YS mac- liver, kidney, skin, gut, heart, pancreas, and stomach) until E13.5, rophages (15). Only the brain remained free from fetal monocyte a second wave of precursors, with a monocytic morphology and infiltration, possibly resulting from the isolation of the brain by phenotype, supersedes them aer E14.5 w ft ith the exception of the nascent blood–brain barrier as early as E13.5 (15, 102). Thus, the brain where YS macrophages are maintained until adulthood these data now reveal that fetal monocytes are the major circulat- (15). A fuller understanding of this process may help to resolve ing embryonic precursor for all macrophages, with the exception some of the earlier discrepancies regarding the contribution of of the brain. The absence of monocyte precursor contribution to YS macrophages. the microglial pool could result from a lack of intrinsic potential or a lack of access to the developing brain due to the nascent Fetal Monocytes blood–brain barrier. Interestingly, we observed a major influx of Fetal monocytes were described by Naito et  al. (92). Focusing monocytes in the brain at E14.5 in our YS macrophage depletion their study on liver Kuper ce ff lls (the resident macrophages of the model, and preliminary data using our fetal monocyte S100a4- liver) during embryonic development, they exploited the endog- Cre/WT fate-mapping model combined with in  utero depletion enous peroxidase activity of monocytes and pro-monocytes of YS macrophages suggest that fetal monocytes are capable of granules described earlier by van Furth et  al. (18, 92). Naito giving rise to microglia under certain conditions (Hoeffel & et al. observed the transient appearance of peroxidase activity, a Ginhoux, personal communication). Whether this atypical fetal signature for monocyte and pro-monocyte granule activity, dur- monocyte infiltration reflects a compensatory mechanism to ing the in  vitro generation of macrophages from a preparation fulfill an empty niche in the brain or results from a disruption of of FL-dissociated cells (92). In the YS and at early stages of FL the blood–brain barrier remains to be investigated. development, no peroxidase activity was observed, suggesting Adult Monocytes that primitive macrophages first seed the FL. At a later stage, the peroxidase activity increased, suggesting the presence of BM-derived circulating monocytes were considered the only precursors for all tissue-resident macrophages since the seminal monocytic intermediates. In  vitro clonal expansion assays con- firmed the existence of two types of colonies, those containing work of van Furth et  al. (17–18). Although this dogma was entirely revisited recently with the emergence of sophisticated fetal monocytes and those devoid of them. This provided early evidence for the existence of two distinct developmental path- fate-mapping tools as well as parabiotic models, the physi- ological contribution of circulating adult monocytes to the adult ways leading to the generation of Kuper ce ff lls, although at this Frontiers in Immunology | www.frontiersin.org 9 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny macrophage network remains valid at least in certain tissues. (108). The high level of foreign antigens passing through the LN e co Th ntinuous recruitment of circulating monocytes to the during the lifespan, support the model of a constant replenish- dermis has been shown to shape the adult dermal macrophage ment of the local macrophage pool by circulating adult mono- network (25). Although this study did not employ fate-mapping cytes. However, the work of Jakubzick et  al. suggests otherwise techniques, Tamoutounour’s data suggest the existence in the as tissue-patrolling monocytes at steady state seem to enter the dermis of both a prenatal pool of macrophages and a second LN without any sign of local differentiation to macrophages or pool derived from adult blood monocytes. The authors argue dendritic cells (34). Further studies using fate-mapping systems that the dermis, in contrast to the epidermis, continues to recruit should be addressed to clarify this point. Spleen macrophages are circulating monocytes in adulthood, most likely facilitated by generated prenatally (13, 33). However, red pulp macrophages its high level of vascularization. The macrophage network in the and marginal zone macrophages seem highly dependent, respec- intestine follows a similar model. Data from Bain et  al. suggest tively, on the transcription factor SPI-C (109) and on the nuclear that embryonic macrophages do not persist in adulthood in the receptor LXR (110), also expressed by circulating monocytes and gut, and are replaced constantly by circulating adult monocytes suggest again that embryonic-derived macrophages are replaced (23), convincingly showing that adult monocytes are the source over time by adult monocytes-derived macrophages. e Th use of of intestine-resident macrophages. e Th role of commensal the S100a4-Cre fate-mapping model in our hands supports these microbiota in this process is supported by the observation that observations and similar conclusions were obtained for BM and the use of germ-free animals or treatment with broad-spectrum peritoneal macrophages (15). Although tissue microenviron- antibiotics results in a significant reduction in the recruitment ment shapes certain macrophage functional specificities ( 111), of Ly6C monocytes to the colon (23). e Th macrophage network through an ontogenic point of view, the composition of each of the heart has also been shown to contain a component of YS tissue-resident macrophage pool evolves throughout life and the macrophages and fetal monocyte-derived macrophages, both of respective origins of each macrophage population may account which are maintained in adulthood (99). However, similar to the for some of their key functions and cellular behaviors in a given dermis and the gut, adult monocytes seem to replace embryonic tissue. Hence, a new challenge is to understand if an embryonic macrophages progressively over time (24). The decreasing capac - or adult origin matters for the function and the activation states ity for self-renewal of embryonic macrophages with age observed of tissue-resident macrophages. by Molawi et  al. may explain the requirement for continuous Origin and Development of YS recruitment of monocyte-derived macrophages to the heart in the absence of inflammation. It remains to be clarified whether Macrophages and Fetal Monocytes this phenomenon occurs in other tissues as a result of aging. In Origin of YS Macrophages agreement, proliferation of YS macrophages and fetal monocytes is very high during development (20–40% before E14.5) but Bertrand et  al., in line with the seminal work of Palis (45), described two sequential myeloid waves within the early YS decreases progressively to 10% few days aer b ft irth in most tis - sues and decreases to almost undetectable levels in adults (15). (42). Using an in  vitro culture reporter system, Bertrand et  al. observed a first wave of monopotent progenitors that gave rise Interestingly, macrophage turnover seems different from one tissue to another. Following BrdU incorporation at steady state, only to macrophages, followed by a second wave that gave rise to a mix of granulocytes, monocytes, and macrophages. More almost no proliferation was observed in adult gut macrophages (23), while 2–5% was measured in adult heart macrophages recently, Kierdorf et  al. revisited the work of Bertrand et  al. (24). Macrophage proliferation activity can also be mobilized exploiting organotypic embryonic brain slices to demonstrate upon inflammation. For example, peritoneal macrophages can that microglial cells derived from YS EMPs (112). Kierdorf et al. increase their proliferation rate from 1 to 9% in response to also showed that these EMPs did not express the transcription parasite infection or in response to IL-4 stimulation (35), while factor c-Myb, associating them with the progenitors reported enhanced local proliferation of macrophages in atherosclerotic by Schulz et  al. (12), although a direct link with the generation lesions sustain disease progression (103). The characterization of of microglia in  vivo in adulthood was not conclusively demon- local signals regulating macrophage proliferation as well as the strated. More recently, Perdiguero et  al. used the CSF-1R-iCre presence of specialized tissue niches that sustain macrophage fate-mapping model to show that YS macrophages are derived EMPs (14). Hence, these two studies suggest survival, proliferation, or even “stemness” will be fundamental to from CSF-1R better understand their tissue homeostasis. that YS macrophages, and thus microglia, would originate from c-Myb-independent CSF-1R EMPs. Furthermore, Perdiguero e m Th acrophage network of the lymphoid system seems to follow a similar pattern than in the gut and dermis. Although et al. demonstrated that CSF-1R EMPs were able to seed the FL by E10.5, suggesting that these progenitors could later populate the lymph nodes (LN) start to develop very early in the embryo (104), they become functionally active only within the first week other tissue niches and produce YS-like macrophage later during development in others tissues. Nevertheless, these data do not aer b ft irth recruiting and organizing B and T cell areas when fol - licles start to shape with connections to aer ff ent lymphatics via explain the low percentage of labeled adult macrophages observed by Schulz et al. using the CSF-1R-iCre fate-mapping model (12). the subcapsular sinus (105). Although macrophages are known to participate in lymphangiogenesis during development, notably Later observations by Epelman et al. (99), and more recently by by the production of VEGF (106, 107), the precise origin of the our group using the same fate-mapping model (15), indicated different LN macrophage populations remain poorly understood that the ability of CSF-1R EMP to reach the FL could explain Frontiers in Immunology | www.frontiersin.org 10 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny the surprising maintenance of primitive macrophages until E16.5 derivation from the recently described YS-derived LMPs (67). in c-Myb null embryos, where primitive macrophages generated u Th s, LMPs may be important for the generation of a small but in the YS as well as in the FL would be able to fulfill the empty significant proportion of fetal monocytes prior to the expansion niche left by the absence of c-Myb-dependent myeloid cells, that of mature HSCs (Figure  4). Further investigations using more include fetal monocytes. However, this may not reflect the physi - specific fate-mapping models will be necessary to elucidate the ological situation and may instead result from a compensatory exact contribution of LMPs as well as the hematopoietic transi- mechanism to ensure the presence of macrophages in all tissues tion between the FL and the BM. in the absence of c-Myb activity and fetal monocytes. Using the Importantly, we observed that fetal monocytes were not same CSF-1R-iCre fate-mapping model (15), we were able to tagged with the CSF-1R-Cre model that label early CSF-1R EMPs, suggesting that fetal monopoiesis is not dependent on follow the maintenance of microglia in the brain by self-renewal + + from E10.5 until adulthood, linking them with CSF-1R EMPs CSF-1R EMPs, consistent with our previous data (9, 11) and and confirming the previous observations of Perdiguero et  al. with our YS macrophage depletion results (15, 98). Furthermore, (14). However, for all other macrophage populations, the reduc- the Runx1-iCre fate-mapping model allowed us to identify two tion of fate-mapping reporter labeling aer E13.5 co ft nfirmed the waves of EMPs that arise sequentially before LMPs in the YS. progressive replacement of YS macrophages by another unlabeled es Th e included an early wave, arising at E7.5 that differentiates precursor arising from a different hematopoietic wave. locally into YS macrophages; and a later wave tagged at E8.5, that We previously showed that Runx1 YS progenitors that migrates and seeds the FL following the establishment of the blood emerged at E7.5 give rise to YS macrophages and microglia (9, 11). circulation before E9.0. Early EMPs tagged at E7.5 were therefore Using both the Runx1-iCre and the CSF-1R-iCre fate-mapping related to those described previously by Kierdorf and Perdiguero models, we showed that these E7.5 Runx1 YS progenitors were (14, 112). The late EMPs tagged at E8.5, however, expressed in fact the same CSF-1R EMPs described by Perdiguero et  al. c-Myb, expanded more efficiently in the FL, and differentiated and Kierdorf et  al., which contributed to the generation of YS in vivo into fetal cMoPs, constituting the major component of the macrophages and, to a lesser extent, those seeding the FL (14, 15, fetal monocyte population as well as the fetal monocyte-derived 112). However, we also observed their disappearance from the FL macrophage population (Figure  3), which was able to maintain aer E11.5 in ft dicative of a rapid local consumption/differentiation itself in all tissues tested (15). rather than long-term maintenance. Our results also suggest that e exi Th stence of two distinct EMP waves is in agreement these early CSF-1R EMPs are able to contribute to a short-term with Bertrand et al. who reported an early wave of macrophage maintenance of macrophages in the FL (Figure  2), but do not progenitors restricted to the YS, and a second wave that was able contribute to other tissue macrophages as evidenced by their rapid to reach the FL to participate in definitive hematopoiesis ( 42). disappearance from the blood circulation aer E14.5 ( ft 15). This e diff Th erential expression of c-Myb between early and late EMPs transient population in the FL may be due to a local immediate is in agreement with previous reports indicating that primitive requirement for macrophages, at least during the onset of FL hematopoiesis can occur in the absence of c-Myb, especially for hematopoiesis, to perform efficient enucleation of primitive eryth - the generation of monopotent macrophage progenitors (114), rocytes passing through the FL sinusoids (100, 113). Combining whereas EMPs from definitive hematopoiesis express and are historical evidences showing their direct lineage connection with dependent on c-Myb activity (45, 62, 115). the emergence of YS macrophages and recent findings showing Notably, a previous study showed that c-Myb ablation strongly their independence with c-Myb activity, we propose that CSF-1R compromises definitive hematopoiesis ( 116). Palis et al. observed EMPs should be designated as primitive EMPs. that c-Myb is expressed prior to and during the early develop- ment of definitive erythrocyte progenitors (45). u Th s, late EMPs Origin of Fetal Monocytes and LMPs, as well as HSCs, express c-Myb (15, 45, 61, 62), Because adult monocytes are derived from HSCs in the BM, it suggesting that the entire fetal monopoiesis machinery is reliant hi lo would be reasonable to assume that embryonic HSCs might also F480 on this transcription factor. In agreement, the CD11b give rise to fetal monocytes in the developing liver. In agreement population, which in our hands contains fetal monocytes, was with this hypothesis, we have identified a population in the FL completely absent in the c-Myb-deficient embryo ( 12, 116). As a similar to adult MDPs that have the potential to generate fetal consequence, the contribution of c-Myb-dependent progenitors cMoPs and monocytes following in vitro culture (15). Exploiting to tissue-resident macrophage populations could not be evalu- the Flt3-Cre tomato fate-mapping model (83), we then fol- ated in c-Myb-deficient embryos, where c-Myb-independent YS macrophages maintain themselves as a compensatory mecha- lowed the progeny of embryonic HSCs. However, the poor labeling observed between E14.5 and E17.5 in FL monocytes and nism due to the absence of c-Myb-dependent fetal monocytes macrophages contrasted with the strong labeling of FL MDPs, that normally outcompete them. Because c-Myb expression is suggesting that HSCs had limited involvement in the generation upregulated during the successive steps of fetal monopoiesis of fetal monocytes (15). Nonetheless, the limited but significant (15), the switch in EMP localization between the YS and the labeling in fetal monocytes and macrophages at birth suggested FL may indeed be orchestrated by c-Myb. As a consequence, an increasing derivation from fetal HSCs, assuming that fetal most tissue-resident macrophages derived from fetal monocytes HSCs follow a similar Flt3-dependent differentiation pathway as would therefore rely on c-Myb activity. Altogether we propose EMPs giving rise to the first circulating monocytes adult HSCs. In parallel, gene array analysis highlighted a strong that c-Myb lymphoid signature within fetal MDPs (15), indicative of their should be designated as definitive EMPs. Frontiers in Immunology | www.frontiersin.org 11 September 2015 | Volume 6 | Article 486 Hoeffel and Ginhoux Macrophage ontogeny understanding of the mechanisms that control the tissue-specific Conclusion functions of macrophages in the steady state, and thus may Recent reports have drastically changed the view of the develop- uncover new therapeutic opportunities in diverse pathological ment of the MPS and shed light on the multiple layers that define settings such as metabolic diseases, fibrosis, and carcinogenesis. fetal hematopoiesis. It is now evident that fetal monocytes form the major precursors of most adult tissue-resident macrophages, Funding and further investigations are now necessary to clarify how they shape macrophage heterogeneity. Examining how tissues This work was supported by the Singapore Immunology Network imprint specific fates in these circulating precursors will aid our (SIgN) core grant. 19. Virolainen M. 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Microsc Res Tech (1997) 39:350–64. doi:10.1002/ is cited, in accordance with accepted academic practice. No use, distribution or (SICI)1097-0029(19971115)39:4<350::AID-JEMT5>3.3.CO;2-V reproduction is permitted which does not comply with these terms. Frontiers in Immunology | www.frontiersin.org 14 September 2015 | Volume 6 | Article 486

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