REVIEW MACROPHAGES

The development and maintenance of

resident macrophages
Elisa Gomez Perdiguero1 & Frederic Geissmann2

The molecular and cellular mechanisms that underlie the many roles of macrophages in health and disease states in vivo remain
poorly understood. The purpose of this Review is to present and discuss current knowledge on the developmental biology of
macrophages, as it underlies the concept of a layered myeloid system composed of ‘resident’ macrophages that originate mainly
© 2016 Nature America, Inc. All rights reserved.

from progenitor cells generated in the yolk sac and of ‘passenger’ or ‘transitory’ myeloid cells that originate and renew from bone
marrow hematopoietic stem cells, and to provide a framework for investigating the functions of macrophages in vivo.

The current ‘mononuclear phagocyte system’ model1 has guided many
investigators studying the functions of macrophages over the past 30 years.
It postulates that tissue macrophages are maintained from a constant sup-
ply of bone marrow–derived circulating blood monocytes that extrava-
sate into tissues2. This model was initially based on the observation that
blood leukocytes recruited into the inflamed peritoneum differentiate
into macrophages. Subsequent experiments of transplantation into irra-
diated recipients demonstrated that bone marrow hematopoietic stem cells
(HSCs) and HSC-derived myeloid progenitors can indeed give rise to tissue
macrophages or dendritic cells in mice and humans3–7. Therefore, investi-
gations designed to study the molecular and cellular basis of macrophage
phenotypic diversity and function have focused largely on the activation
or polarization of monocyte-derived macrophages by external cues, such
as cytokines and microbe-associated signals8–10. Nevertheless, it remains
unclear whether the polarization stages of monocytes are stable or represent
npg
include spleen red-pulp macrophages, lung alveolar macrophages, epider-
mal Langerhans cells, brain microglia, liver Kupffer cells, large peritoneal
macrophages, and F4/80bright pancreatic, kidney and cardiac macrophages.
Many tissue-resident macrophages are long lived in mice and can prolifer-
ate within their tissue of residence, a mechanism involved in their mainte-
nance in adults20–27. Nevertheless, bone marrow–derived progenitor cells
also contribute to subsets that reside in the lamina propria, spleen, brain,
skin, heart, liver and kidneys in a proportion that varies by tissue, mouse
age and pathological processes12–18,28,29.
Therefore, the purpose of this Review is to present and discuss current
knowledge of the developmental biology of resident macrophages, as it
underlies the concept of a layered myeloid system composed of tissue-
resident macrophages distinct from passenger macrophages and myeloid
cells and provides a new framework and experimental tools with which to
characterize the functions of macrophages in vivo, in health and in disease

transient states of activation, whether they account for the phenotypic and
functional heterogeneity of macrophages in vivo in health or disease states,
and how many such polarized states exist10,11.
A new model of macrophage development is emerging that is based on
many independent observations that two distinct populations of macro-
phages, which can be distinguished by their progenitors, developmental
history, turnover and mechanisms of maintenance, coexist within the tis-
sues of an adult mouse. The macrophage system of a mouse can now be
described as a ‘layered’ system in which ‘resident’ macrophages that develop
in embryos independently of HSCs12 persist in adult tissues independently
of adult HSCs12–18 and coexist with ‘passenger’ leukocytes, such as mono-
cytes and classic dendritic cells, that originate and renew from bone mar-
row HSCs and myeloid progenitors4,6,19. Tissue-resident macrophages
1Macrophages and Endothelial Cells group, Department of Developmental
and Stem Cell Biology, CNRS URA 2578, Institut Pasteur, Paris, France.
2Immunology Program, Memorial Sloan Kettering Cancer Center, New York,
New York, USA. Correspondence should be addressed to F.G. (geissmaf@
mskcc.org).
Received 24 September; accepted 3 November; published online
17 December 2015; doi:10.1038/ni.3341
settings.
The original concept of ‘primitive macrophages’
During ontogeny, macrophages are found in the mouse yolk sac and the
embryo before the initiation of HSC-derived hematopoiesis and before
monocytes can be detected in the fetal blood. These ‘primitive macro-
phages’ were described in a series of seminal works20,30,31 that reported
the presence of large cells with macrophage morphology in blood islands
of the yolk sac at embryonic day 9 (E9) and in the fetal liver at E10. These
were described as ‘immature’ because of their lack of detectable phagocytic
activity and weak immunoreactivity for the macrophage marker F4/80,
as detected by electron microscopy20,30. In addition to reporting those
‘immature macrophages’, these early studies also reported the existence
of ‘fetal macrophages’ from E10 to E17 as slightly smaller cells with pseu-
dopodia, phagocytic activity and frequent mitotic figures31. At E17, the
‘fetal macrophages’ were reported as more differentiated, as they acquired
ultrastructural features similar to those of adult tissue macrophages30,32.
These early studies noted that the remaining difference between fetal tissue
macrophages and adult tissue macrophages was the presence of numer-
ous mitotic figures in fetal macrophages throughout development. It was
also noted that poorly differentiated myeloid cells were detected in the
blood vessels of the yolk sac at E10 (ref. 20), and immature stages of the

2 VOLUME 17 NUMBER 1 JANUARY 2016 NATURE IMMUNOLOGY

 REVIEW

monocyte and neutrophil differentiation series could be detected from E12
onward, and finally true neutrophils and monocytes, identified on the basis
of ultrastructural features and endogenous peroxidase-cytochemistry, were
present in the fetal liver at E15 and E16, respectively30. These researchers
therefore proposed that fetal macrophages develop without a monocytic
intermediate and in parallel with the monocyte and granulocyte lineage.
In subsequent literature, that early work was sometimes reported as evi-
dence of the early colonization of the fetus by ‘yolk-sac macrophages’ before
their replacement by monocyte-derived macrophages. However, this body
of work described the identification of proliferating non-phagocytic pre-
cursors of macrophages (described as ‘immature macrophages’) that origi-
nate in the yolk sac, colonize the embryo and finish their differentiation in
situ within the fetal tissues20,30,31. This description did not correspond to
a particular wave of hematopoietic progenitors (‘primitive’ or ‘definitive’),
as discovered subsequently. However, the description of erythro-myeloid
progenitors (EMPs)33–36 (discussed below) and additional fate-mapping
data are in accordance with the original interpretation of the morphological
data. Indeed, genetic pulse labeling of Csf1r-positive c-Kit+CD45loAA4.1+
EMPs present in the yolk sac at E8.5 is followed by the detection of labeled
CD11b+ cells with negative or faint immunoreactivity for F4/80 at E9.5 in
both the yolk sac and embryo and then by the detection of F4/80bright cells
© 2016 Nature America, Inc. All rights reserved.
region) in which they are generated, expand and differentiate, their differ-
entiation potential and their dependence on transcription factors (Table 1
and Fig. 1). Here we will focus on the myeloid lineages; erythroid lineages
have already been reviewed37.
Primitive hematopoiesis as a source of macrophages in embryos
Primitive hematopoiesis is the source of the first hematopoietic cells
detected in the developing embryo. It is characterized by the emergence of
progenitor cells with limited potential for development into erythrocytes
and macrophages and that can be found in the extra-embryonic yolk-sac
blood islands in mice36. These progenitor cells are thought to arise directly
from the posterior plate mesoderm, and clonal analysis of the blood islands
has revealed that the endothelial cells lining the blood islands and the
hematopoietic cells they contain do not share a common origin38. This
distinguishes them from the subsequent ‘definitive’ hematopoietic waves,
which are thought to arise from a hemogenic endothelium. Accordingly,
primitive progenitors and their daughter cells are not affected when tran-
scription factors required for the endothelial-hematopoietic transition,
such as Runx1, are absent (Table 1).
In zebrafish embryos, the ‘primitive’ wave can give rise to erythrocytes
and macrophages, as well as neutrophils, in the absence of the gene encod-

starting from E10.5 (ref. 18).
More recent work has identified three successive but overlapping waves
of hematopoietic progenitors during development, all of which have the
potential to give rise to fetal macrophages34. These can be distinguished
by the anatomical site (extra-embryonic yolk sac versus intra-embryonic
ing Runx1 (refs. 39–41). In the mouse embryo, in vivo characterization of
the macrophage progeny of the primitive wave is still hampered by experi-
mental constrains. However, (rare) progenitors with restricted macrophage
potential in vitro can be found in the yolk sac at the neural-plate stage
between E7.5 and E8 (refs. 34,36).

Table 1 Primitive and definitive waves of hematopoiesis

Primitive Definitive
Progenitor name MP EMP HSC
Origin Posterior plate mesoderm Hemogenic endothelium Arterial hemogenic endothelium
Progenitor surface phenotype c-KitloCD41lo c-Kit+CD41+VE- Lin–c-Kit+Sca-1+CD41+VE-
Cad+CD16/32+AA4.1+Tie2+CD45loSca-1– Cad+Tie2+CD45+
Time window of generation E7.0–? E8.25–E10.5 E9.5–E12.5
Anatomical Generation YS (blood islands) YS AGM (and large arteries)
location of: Expansion YS FL FL
Differentiation-commitment YS YS and FL FL, BM
Differentiation potential Myeloid Erythroid Erythroid
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Myeloid Myeloid
Lymphoid
Macrophage lineage PU.1 dependent PU.1 dependent PU.1 dependent
Runx1 independent Runx1 dependent Runx1 dependent
c-Myb independent c-Myb independent c-Myb dependent
Notch1 independent Notch1 dependent
Macrophage self-renewal ? Yes ?
Progenitor long-term self-renewal No (transient) No (transient) Yes
TF expression Pu.1 (Spi1) NA + +
c-Myb NA + +
GATA-2 NA + +
GATA-3 NA – +
Lmo2 NA – +
TF requirement PU.1 (Spi1) Yes Yes No**
c-Myb No No* Yes
Runx1 No Yes Yes
CBFb No Yes Yes
Scl–Tal-1 Yes Yes Yes
Notch1 No No Yes
The surface phenotypes, ability to self-renew, anatomical sites of origin, expansion and differentiation, expression and dependence on transcription factors of various
progenitor cells. MP, myeloid progenitor; YS, yolk sac; FL, fetal liver; BM, bone marrow; TF, transcription factor; NA, not applicable.

*Red blood cell differentiation is compromised. **Macrophage differentiation is compromised.

NATURE IMMUNOLOGY VOLUME 17 NUMBER 1 JANUARY 2016 3

 REVIEW

Figure 1 Time scale for the development of Blood

hematopoietic progenitor cells in the mouse islands
Fetal liver
embryo. There are three successive but Hematopoietic Yolk
organs sac
overlapping waves of hematopoietic progenitor Bone marrow
cells during development, each of which has
the potential to give rise to fetal macrophages. “Primitive”
progenitors
These can be distinguished by the hematopoietic EMPs
niches in which the progenitor cells expand Hematopoietic
and differentiate during development (top): progenitors Fetal HSCs Adult HSCs
the yolk sac (primitive progenitors (green) and
EMPs (blue)), the fetal liver (EMPs and HSCs),
Erythrocytes and myeloid cells Resident macrophages
and, finally, the bone marrow (HSCs (orange)). Hematopoietic Erythrocytes
While primitive progenitor cells have been found cells (myeloid cells) Erythrocytes, passenger myeloid cells, lymphoid cells
only in a small time window (middle), definitive
progenitor cells (including EMPs and HSCs)
Timeline
co-exist during most fetal development in the
E7.5 E10.5 E12.5 E14.5 E16.5 E18.5 Birth Weaning Adulthood
fetal liver. Only HSC-derived hematopoiesis
then shifts to the bone marrow niche. The three
waves of progenitor cells can also be distinguished by their differentiation potential in vivo (bottom). Primitive progenitor cells are restricted to the erythroid
or myeloid lineage, while EMPs have both erythroid and myeloid potential. EMP-derived hematopoiesis gives rise to erythrocytes, macrophages, monocytes,
granulocytes and mast cells and is sufficient to support survival of HSC-deficient embryos until birth. EMP-derived macrophages persist after birth in most
tissues as resident macrophages, except in the intestinal lamina propria. HSCs are distinguished from other progenitor cells by their ability to perform long-
term repopulation of all leukocyte lineages when transplanted into a conventional irradiated recipient mouse.
© 2016 Nature America, Inc. All rights reserved.

EMPs give rise to most tissue-resident macrophages
The first ‘definitive’ progenitor cells emerge in the yolk sac of mouse
embryos at E8.25 (refs. 34,36). These progenitors (EMPs) are phenotypi-
cally defined as c-Kit+, AA4.1+ (CD93+), CD41+, positive for the endothelial
marker VE-cadherin, positive for CD16/32 (FCgII and FCgIII recep-
tors), and CD45lo (refs. 33,35) (Table 1). Examination of their erythroid
progeny led to their classification as ‘definitive’ progenitors42. However,
they can be distinguished from HSCs by their lack of lymphoid poten-
tial, both in vitro and in vivo, their lack of long-term repopulating poten-
tial35 and lack of surface expression of the lineage marker Sca-1 (ref. 35).
EMP-derived hematopoiesis is sufficient to support survival of HSC-
deficient embryos until birth43. EMP emerge from the yolk-sac hemogenic
endothelium in a Runx1-dependant endothelial-to-hematopoietic transi-
tion44. The emergence of EMPs from the yolk sac does not require blood
flow, as indicated by results obtained by the study of embryos deficient
in the sodium-calcium exchanger NCX1 or VE-cadherin35,45,46. They are
generated from Tie2+ yolk-sac ancestors from E7.5 to E10.5 (ref. 18). The
number of EMPs in the yolk sac peaks between E9.5 and E10.5, and they
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has been described elsewhere52,53. HSCs and EMPs arise from distinct
hemogenic endothelial cells43. HSCs emerge from the hemogenic endothe-
lium of the aorto-gonado-mesonephros (AGM) region (starting at E10.5 in
mice)39,54,55 and of the umbilical and vitelline arteries56 and migrate to the
fetal liver, where they expand and differentiate from E12.5 until definitive
hematopoiesis begins to shift to the bone marrow. HSCs colonize the embry-
onic bone marrow at E15, and active hematopoiesis starts there at E17 (refs.
47,57). Fetal and adult HSCs require c-Myb for their self-renewal and main-
tenance, and loss of c-Myb expression leads to rapid HSC-derived hemato-
poiesis failure12,58–60. In addition, HSCs also require the transmembrane
receptor Notch1 for their emergence, in contrast to EMPs, as Notch1–/–
embryos have normal numbers of hematopoietic progenitor cells in the
yolk sac but very few in the body of the embryo61. Several groups have
attempted to pinpoint the specific site and time at which HSCs can be first
detected within the embryo proper. Pioneering work demonstrated that
the AGM region contains the progenitor cells endowed with long-term–
repopulating ability at E10.5 (ref. 62). However, immature HSCs have been
detected at E9–E9.5 in the para-aortic splanchnopleura, a structure that

seed the fetal liver as soon as E9 (ref. 47). Circulating EMPs are still detected
in peripheral blood at E14.5 (ref. 35), and EMP-derived c-Kit+ cells are
found in the fetal liver at 16.5 (ref. 18). EMPs expand in the fetal liver and
differentiate into erythrocytes, megakaryocytes, macrophages, monocytes,
granulocytes and mast cells18. EMPs isolated from the yolk sac at E8.5 and
the fetal liver at E12.5 have the same differentiation potential in vitro, but
erythroid, monocyte and granulocyte and mast-cell potential is observed
in vivo only after seeding of the fetal liver18. Thus, the fetal liver niche pro-
vides critical cues or an environment for EMPs to reach their full potential.
Yolk-sac EMPs express the gene encoding the transcription factor c-Myb
(Myb)48, but their commitment and differentiation into the myeloid fate is
unaltered in c-Myb-deficient embryos, although their erythroid potential
is blocked12,18,49,50 (Table 1). Therefore, c-Myb is required for the commit-
ment and differentiation of EMPs into the erythroid fate50 but is dispens-
able for myeloid differentiation and is possibly redundant for this, because
EMPs express the gene encoding b-Myb (Mybl2), a transcription factor that
can restore hematopoiesis in c-Myb-deficient Drosophila melanogaster51.
later evolves into the AGM region. Such immature HSCs migrate into the
fetal liver at E10, mature into HSCs and contribute to the adult HSC pool47.
Genetic models distinguish the three progenitor waves
As described above, the emergence of different waves of hematopoietic
precursor cells partially overlaps in a short time frame, which has made
fate-mapping difficult. However, these precursor cells can be distin-
guished genetically, and overall, the layering of the hematopoietic system
and in particular of macrophage development seems to stand on a robust
genetic basis. Primitive hematopoiesis occurs in the absence of Runx1 and
c-Myb63,64, while EMPs are dependent on Runx1 for their emergence64 but
are independent of c-Myb for their myeloid differentiation12,18,49 and are
Notch1 independent61. In contrast, fetal and adult HSCs require Runx1,
c-Myb and Notch1 (refs. 39,40,58,59,61) (Table 1). On the basis of these
data, several reports have investigated the lineage of origin of fetal (primi-
tive) and adult macrophages and the mechanisms that might account for
their persistence in adults.

HSCs are developmentally distinct from EMPs Resident macrophages originate mainly from yolk-sac EMPs
HSCs are distinguished from other progenitor cells by their ability to per- The development of fetal F4/80bright macrophages is unaltered in c-Myb-
form long-term repopulation of all leukocyte lineages when transplanted deficient mice, and adult F4/80bright macrophages are largely maintained
into a conventional irradiated recipient mouse. HSC-derived hematopoiesis in adult mice in the absence of c-Myb and independently of bone marrow

4 VOLUME 17 NUMBER 1 JANUARY 2016 NATURE IMMUNOLOGY

 REVIEW

progenitor cells12. In addition, adult tissue-resident F4/80bright macro- Mesoderm

phages have been traced back to c-Myb-independent precursor cells
expressing the cytokine receptor CSF1R and present in the early mouse
Runx1 independent
embryo at E8.5, as assessed through the use of Cre recombinase–mediated, YS hemogenic
E7.5
hydroxytamoxifen-induced pulse labeling65,66 of transgenic mice express- endothelium

ing a tamoxifen-inducible fusion protein of ‘improved’ Cre recombinase

(iCre) and the mouse estrogen receptor (Mer-iCre-Mer) under control of E8.5 Primitive YS-derived EMPs
hematopoiesis
the macrophage-specific mouse gene encoding CSF1R12 (discussed below). Runx1 c-Myb-
dependent independent
The data noted above would suggest that F4/80bright macrophages origi-
nate not from HSCs but from yolk-sac progenitor cells such as either primi- E9.5 Intraembyonic
tive progenitor cells or EMPs48. In accordance with that, brain microglia hemogenic
endothelium
develop from yolk-sac progenitor cells14 that have been functionally identi-
fied as EMPs48. Following pulse labeling of CSF1R-expressing cells with E10.5
c-Myb-dependent Fetal macrophages
hydroxytamoxifen at E8.5 (in the mouse model described above), the label-
ing efficiency of F4/80bright macrophages is 30% at day 10.5 and remains E12.5
HSCs
high (~20%) in microglia but progressively decreases from E12.5 onward
in other organs to reach a plateau at birth that is maintained even in 1-year- Erythrocytes,
old mice (5% for the liver Kupffer cells; 1% for epidermal Langerhans E16.5 Flt3+ progenitors megakaryocytes
cells)12,18,67 (data not shown). This might be explained by the observation granulocytes
monocytes
that pulse labeling with hydroxytamoxifen at E8.5 labels only the very first mast cells
Birth
EMPs that emerge in the yolk sac, while EMPs continue to emerge in the
© 2016 Nature America, Inc. All rights reserved.

yolk sac until at least E10.5 (ref. 18), and therefore the frequency with which
the macrophages are labeled in embryonic tissues might reflect the time
at which their colonization by fetal macrophages is completed. However,
it is also possible that this ‘dilution’ reflects a contribution by another pro-
genitor cells, such as fetal HSCs or HSC-derived monocytes, to several
fetal macrophage populations17,67–69. A contribution by fetal monocytes
is unlikely, as they also derive from EMP until E16.5 (refs. 18,35). However,
a contribution by fetal HSCs is still under debate17,68–70.
The main argument against the possibility of a substantial contribu-
tion by fetal HSCs has been provided by experiments with a fate-mapping
mouse model in which the expression of Cre recombinase (as the Mer-
iCre-Mer fusion) is inducible in cells expressing the tyrosine kinase Tie2
(encoded by Tek)18,71. Tie2 is expressed in the hemogenic endothelium,
endothelial cells and hematopoietic progenitor cells in the yolk sac72,
AGM region, fetal liver HSCs and adult HSCs52. Pulse labeling of these
mice with tamoxifen at E7.5, E8.5, E9.5 and E10.5 results in the labeling
of adult HSCs, lymphocytes, monocytes and neutrophils with the same
efficiency, as expected. In contrast, fetal macrophages and adult tissue-
npg
Tissue-resident
Erythrocytes,
Aging macrophages
megakaryocytes
(Kupffer cells,
lymphoid cells
myeloid cells
microglia,
Langerhans cells)
Figure 2 Myeloid development and maintenance in the mouse embryo
and adult mouse. Primitive progenitor cells do not share a common origin
with endothelial cells lining the blood islands in the yolk sac (YS), and they
emerge the absence of Runx1 and c-Myb. During definitive hematopoiesis,
HSCs and EMPs arise from distinct hemogenic endothelial cells through
a Runx1-dependent endothelial-to-hematopoietic transition. Both EMPs
and HSCs express c-Myb, and although no fetal or adult HSC-derived
hematopoiesis can occur in the absence of c-Myb, EMPs are c-Myb
independent for their myeloid differentiation. EMP-derived hematopoiesis
gives rises to erythrocytes and short-lived myeloid cells (monocytes,
granulocytes and mast cells) that are replaced by HSC-derived cells late
during gestation. EMP-derived macrophages colonize all tissues during

fetal development, where they specialize to their tissue of residency after

resident F4/80bright macrophages in all organs are labeled with a decreasing
efficiency (high at E7.5 to very low at E10.5), which indicates that fetal and
adult F4/80bright macrophages originate mainly from a progenitor cell that
does not express Tie2 after E10.5, with a minor contribution of progenitor
cells (such as HSCs) that express Tie2 after E10.5.
Research with fate-mapping models in which the expression of Cre
recombinase is either inducible (as the Mer-iCre-Mer fusion) or constitu-
tive (iCre alone) in CSF1R-expressing cells has identified the macrophage
progenitor itself as a CD45loKit+AA4.1+ EMP that appears in the yolk sac
from E8.5 (ref. 18). These EMPs migrate into the fetal liver, where they
expand and persist as c-Kit+ progenitors until at least E16.5 (ref. 18). These
EMPs give rise to erythrocytes, monocytes, granulocytes and mast cells
until late in fetal development18,35, in accordance with the observation that
HSC-independent hematopoiesis is not only necessary but also sufficient to
support the survival of HSC-deficient embryos until birth43. These EMPs
also give rise to F4/80bright fetal macrophages in all embryonic tissues from
E10.5 onward18. An EMP origin of the majority of tissue-resident macro-
phages has also been favored in an independent report67.
Together, the data reported above support the proposal of a major
contribution of yolk-sac EMPs to fetal and adult resident macrophages
(Figs. 1 and 2). EMP-derived erythrocytes, monocytes, granulocytes and
mast cells are ultimately replaced by HSC-derived myeloid cells in the late
birth and can persist throughout adult life by local proliferation (circular
red arrows indicate self-renewal potential). Depending on the age of the
organism and environmental challenges, HSC-derived cells can contribute to
adult tissue-resident populations.
embryo18, while EMP-derived macrophages persist all through fetal and
post-natal development as tissue-resident macrophages12,18 (Fig. 2). It has
also been speculated that a first wave of c-Myb-independent EMPs gives
rise to microglia and a second wave of c-Myb-dependent EMPs gives rise
to other macrophages67. However, there are no data available to support
the proposal of the existence of two genetically distinct populations of
EMPs, and this hypothesis is not in accordance with reports that c-Myb
is expressed in early yolk-sac EMPs36,48 or with the persistence of fetal
resident macrophages in all c-Myb-deficient embryonic tissues12.
The contribution of ‘primitive’ hematopoiesis to microglia
Primitive hematopoiesis could also contribute to ‘primitive’ and fetal mac-
rophages. Early pulse labeling of progenitor cells (at E7.5) with several dif-
ferent reporters, such as Runx1, CSF1R, Tie2 and c-Kit12,14,18,70, results in
more efficient labeling of adult microglia than of other adult tissue-resident
macrophages. As mentioned above, this might relate either to earlier ter-
mination of colonization of the brain by macrophages than that of other

NATURE IMMUNOLOGY VOLUME 17 NUMBER 1 JANUARY 2016 5

 REVIEW
organs or to the existence of several subsets of EMPs. However, it might
also reflect the contribution of primitive hematopoiesis to microglia. Future
studies should determine whether primitive progenitor cells are labeled
in these models and should investigate whether macrophages produced
by primitive hematopoiesis can persist in adult mice. Independence of
c-Myb does not allow genetic distinction of primitive hematopoiesis from
EMP-derived hematopoiesis, and it is not yet known whether Runx1 is
required for the differentiation of macrophages in mice. Together, the
lack of genetic delineation and the low temporal resolution of classic fate-
mapping approaches in the mouse have not yet allowed independent track-
ing of the respective progeny of the two progenitor populations. Better
understanding of the molecular control of primitive progenitor cells and
the commitment and differentiation of EMPs is needed for the develop-
ment of better tools for characterization of the possible contribution of
‘primitive’ hematopoiesis to the development of fetal macrophages and,
possibly, adult macrophages in mice.
Contribution of HSCs depends on organ and time
Analysis of the contribution of HSCs to adult resident macrophages in
the absence of bone marrow irradiation has been performed by several
groups studying mice expressing Cre recombinase from the gene encod-
© 2016 Nature America, Inc. All rights reserved.
ing the receptor tyrosine kinase Flt3 (Flk2) (refs. 12,17,18,73), on the
basis of the observation that Flt3 is expressed by HSC-derived progeni-
tor cells71,74 but not by fetal macrophages12. The contribution of HSCs
has also been studied in several independent models of bone marrow
transplantation without irradiation, such as Mybfl/flMx1-Cre mice (which
undergo deletion of loxP-flanked Myb alleles (Mybfl/fl) via Cre expressed
from the interferon-inducible gene Mx1) and KitW/WvRag2–/–Il2rg–/–
mice (which lack mast cells and have hypoplastic bone marrow (KitW/Wv)
and lack lymphocytes (Rag2–/–Il2rg–/–))12,18 and in parabiotic mice15,75.
Overall, these analyses have revealed that the contribution of HSCs
to tissue-resident macrophages differs among organs and frequently
increases with age. The contribution of HSCs to adult tissue-resident
macrophages is minor (<5%) in the brain, liver and epidermis12,14,48,
is small but increases with age in the lungs, heart and spleen18,75, and
sometimes predominates after weaning in gut lamina propria28,76 (Figs.
1 and 2). It is not yet known whether the precursor(s) of the HSC-derived
resident macrophages is (are) the monocyte or earlier myeloid progeni-
tor, except in the gut, in which Ly6C+ monocytes are proposed to be the
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immediate precursor of gut lamina propria macrophages28,76.
Of note, a published report has investigated the origin of tissue-resident
macrophages through the use of a fate-mapping model in which the expres-
sion of Cre recombinase (as Mer-iCre-Mer) is inducible in cells expressing
the gene encoding the stem-cell-factor receptor c-Kit (CD117) (ref. 70).
Notably, this study represents independent confirmation of the finding that
most adult tissue-resident macrophages originate from embryonic and fetal
progenitor cells and persist in adults independently of adult HSCs, as c-Kit+
progenitor cells do not seem to contribute to tissue-resident macrophages
after E16.5 (ref. 70). The authors also conclude that most tissue-resident
macrophages, except microglia, are descendants of fetal HSCs, not EMPs,
on the basis of the observation that labeling of c-Kit+Sca-1+ EMPs at E7.5
results in the labeling of microglia only, while labeling of c-Kit+ progenitor
cells at E8.5 or E9.5, in contrast, results in the labeling of all adult resident
macrophages and HSC-derived cells70. However, EMPs do not express
Sca-1 (ref. 35) (Table 1), while EMPs and fetal HSCs both express c-Kit
and co-exist in the fetal liver until E16.5 (refs. 18,35). Thus, c-Kit expres-
sion and c-Kit-based fate mapping might not allow the distinction of EMPs
versus fetal HSCs.
Studies of chickens have suggested that in contrast to results obtained
for mice, yolk sac–derived macrophages are not retained in chickens after
hatching and, surprisingly, that tissue macrophages in the adult birds
6 VOLUME 17 NUMBER 1 JANUARY 2016 NATURE IMMUNOLOGY
originate from a macrophage-restricted, self-renewing progenitor cell in
bone marrow that does not give rise to any other lineage73. These find-
ings, if confirmed, would indicate that myelopoiesis in birds is markedly
different from its counterparts in mice, zebrafish and human, because a
macrophage-restricted, self-renewing progenitor cell in the bone marrow
or its equivalent has not been identified in these species.
Maintenance of macrophages by local proliferation or recruitment
As indicated above, a proportion of alveolar macrophages, kidney mac-
rophages and heart macrophages seem to be replaced during the normal
aging process12,18,75. Partial replacement of tissue-resident macrophages
is also observed following g-irradiation, bone marrow transplantation or
adoptive-transfer experiments3,4,77 and in macrophage-depletion studies,
such as intravenous injection of clodronate liposomes. Liver Kupffer cells
are also reported to be replaced by bone marrow–derived progenitor cells
following, for example, the death of Kupffer cells in severe experimental
infection with Listeria monocytogenes29. However, resident macrophages
such as Langerhans cells, Kupffer cells, microglia, alveolar macrophages,
peritoneal macrophages and splenic macrophages all have the potential
to proliferate15,22,23,27,78–82. In some cases, tissue-resident macrophages
can immediately replenish themselves following severe depletion15,23,82.
A growing body of evidence now clearly shows that fully differentiated
resident macrophages can re-enter the cell cycle and maintain their num-
bers by local proliferation23–27. Macrophage proliferation increases sub-
stantially during inflammatory conditions, such as skin inflammation,
during which up to 30% of Langerhans cells express the proliferation
marker Ki67 (ref. 22). During an acute inflammatory response, the tissue-
resident peritoneal macrophages that survive undergo a transient and
intense proliferative burst in situ to repopulate the tissue23. In response
to helminth infection, macrophages residing in the pleural and peritoneal
cavities expand by local proliferation81. In glucan-induced formation of
granulomas in monocytopenic mice, Kupffer cells proliferate actively
and contribute to granuloma formation78. After conditional ablation of
microglia in adult mice, the microglial compartment is reconstituted
within 1 week by cells residing in the central nervous system, indepen-
dently of bone marrow–derived precursor cells82. However, it remains
unclear whether the tissue-resident macrophage populations contain a
local ‘progenitor compartment’ or whether all macrophages have the
ability to undergo mitosis. The mechanisms that underlie this ability of
tissue-resident macrophages to self-maintain and to proliferate are still
unclear. An important role for the Maf family of transcription factors is
likely, as combined deficiency in MafB and c-Maf enables the KLF4- and
c-Myc–dependent proliferation of differentiated macrophages in culture
in response to the cytokine CSF1 (M-CSF)83. In epidermal Langerhans
cell, proliferation in vivo has been shown to be driven by an unidentified
keratinocyte-derived signal22. During nematode infection, macrophage
proliferation is impaired in the absence of interleukin 4 (refs. 81,84).
The cellular and molecular mechanisms that control macrophage main-
tenance and expansion in adult mice, and the consequences of aging on
these processes, are an area of active investigation, as their elucidation
will shed light on the pathophysiology of inflammation.
Conclusions and perspectives on macrophage function
A revised model for macrophage development and function is therefore
emerging that distinguishes the tissue-resident macrophages that develop
during embryogenesis from the passenger myeloid cells that originate
and undergo renewal from bone marrow HSCs. This distinction between
passenger macrophages and tissue-resident macrophages is in line with
early kinetic studies of the splenic-myeloid compartment in adult mice,
which proposed a dual origin for myeloid cells, with half of myeloid
cells renewing from blood and the other half being produced within the

spleen85. It also explains the observations that Langerhans cells do not
undergo exchange between parabiotic mice13 and that fetal microglia
persist in adult mice14.
That distinction is relevant to disease pathogenesis, as it is apparent
that the functions of HSC-derived monocytes can be very different from
those of tissue-resident macrophages in the same inflammatory environ-
ment. The two cell types have distinct expansion mechanisms and distinct
functions in the brain during experimental autoimmune encephalitis86.
Infiltrating monocytes are recruited via extravasation from blood vessels
and produce inflammatory mediators important for disease progression
but do not persist after the resolution of inflammation, while, in contrast,
activated resident microglia proliferate locally, persist and return to quies-
cence following remission86. Following parasitic helminth infection, pleural
resident macrophages adopt anti-inflammatory phenotypes, while pas-
senger macrophages exhibit pro-inflammatory responses81. However, in
a model of chronic neurodegeneration, proliferating resident microglial
cells contribute to neuronal damage during development of the disease87.
Thus, it might be inaccurate to describe tissue-resident macrophages as
always being ‘anti-inflammatory’ in contrast to passenger macrophages
that are always ‘pro-inflammatory’. The respective roles of tissue-resident
macrophages and passenger macrophages remain to be studied in detail,
© 2016 Nature America, Inc. All rights reserved.
and most experimental models available at present do not allow easy elu-
cidation of the roles of resident and recruited macrophages in adult mouse
tissues. Future work should help characterize the roles of (EMP-derived)
tissue-resident macrophages, separate them from the functions of HSC-
derived passenger macrophages and characterize the underlying molecular
mechanisms involved.
It is also important to recognize that the distinct subsets of resident mac-
rophages associated with different tissues, such as microglia, Langerhans
cells, Kupffer cells, peritoneal macrophages, red-pulp macrophages and
alveolar macrophages, have different phenotypes, transcriptional profiles
and chromatin ‘landscapes’88,89, require specific growth factors, and rely on
distinct transcription factors for their differentiation or maintenance. For
example, CSF1 is essential for Kupffer cells90, the growth factor TGF-b1 and
interleukin 34 are essential for microglia and Langerhans cells88,91–93, CSF2
(GM-CSF) is essential for alveolar macrophages in mice and humans94–99,
and retinoic acid and the transpiration factor GATA-6 are essential for
large peritoneal macrophages88,100,101. The transcription factor Spi-C is
important for the development of red-pulp macrophages102, while mice
npg
deficient in the nuclear receptor LXR are defective in the generation of
marginal-zone and metallophilic macrophages103.
Tissue-specific environments are important for the maintenance of mac-
rophage identity88,89. Several studies also suggest that individual resident
macrophage subsets follow a differentiation program during embryogene-
sis and early post-natal life that results in the establishment of stable pheno-
typic and functional subsets22,48. Future work investigating the respective
contributions and interplay of lineage-specific ‘hard-wired’ differentiation
programs and of the local tissue micro-environment to the differentiation,
maintenance and activation of tissue-specific resident macrophages will
contribute to the understanding of their functions.
ACKNOWLEDGMENTS
Supported by the National Cancer Institute of the US National Institutes of Health
(P30CA008748 to F.G.), the Wellcome Trust (WT101853MA to F.G.), the European
Research Council (2010-StG-261299 to F.G.), the Institut Pasteur (E.G.P.) and
Agence Nationale de la Recherche (Laboratoire d'Excellence Revive, Investissement
d'Avenir; ANR-10-LABX-73 to E.G.P.).
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
NATURE IMMUNOLOGY VOLUME 17 NUMBER 1 JANUARY 2016 7
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