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Multilayered ancestry of arterial macrophages
Kay Klapproth, Felix Lasitschka & Hans-Reimer Rodewald
Arteries are colonized by macrophages of multiple origins, derived prenatally from the yolk sac and during an early
postnatal wave from the bone marrow. During sepsis, blood monocyte-derived macrophages transiently contribute to,
but do not replace, resident arterial macrophages that largely self-renew in situ.
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© 2016 Nature America, Inc. All rights reserved.
C
ardiovascular diseases can be tightly linked
to immunology. As a prominent example,
occlusions of arteries in atherosclerosis are
accompanied, if not driven, by inflammation
in the arterial wall. Macrophages seem to be
key participants during the initiation and
maintenance of arterial inflammation, as well
as plaque formation via their transformation
into foam cells1,2. Many studies have characterized the properties of arterial macrophage
populations in health and disease, yet the origins, heterogeneity, residence times, degree
of self-renewal at steady state and contribution of monocyte-derived macrophages infiltrating arterial walls after perturbation have
remained unclear. Now many of these issues
can be resolved by in vivo fate mapping, which
has been applied to macrophages for many
tissues and developmental stages3–7. In this
issue of Nature Immunology, Ensan et al.
address the origin of arterial macrophage populations in development, during maintenance
and after challenge by septic inflammation8.
By using a variety of genetic lineagetracing experiments in mice, the authors show
that resident arterial wall macrophages are
of multiple origins, first arising from yolk sac–
derived CX3CR1+ progenitor cells or primitive
macrophages, followed by a minor contribution from Flt3+ fetal liver progenitor cells and,
finally, from Flt3+ perinatal or postnatal progenitor cells (Fig. 1). Once sessile in the arterial wall, macrophages persist for long periods
of time, indicative of maintenance mostly via
local proliferation, a situation akin to that of
other tissue-resident macrophage populations.
While self-renewal seems to sustain this compartment, it is not the exclusive mechanism,
and over time a contribution from adult bone
marrow hematopoiesis is measurable. Under
inflammatory conditions (as shown in sepsis
models), many monocyte-derived macro­phages
enter the arterial wall. These inflammatory
Kay Klapproth and Hans-Reimer Rodewald
are in the Division of Cellular Immunology,
German Cancer Research Center, Heidelberg,
Germany. Felix Lasitschka is in the Department
of Pathology, University Hospital Heidelberg,
Heidelberg, Germany.
e-mail: [email protected]
macrophages are more phagocytic than are
their resident counterparts, but their presence
is limited to ongoing inflammation.
In healthy arteries, macrophages are located
mostly in the outer connective tissue layer of
the arterial wall, called the ‘adventitia’ (Fig. 1).
The inner endothelial and the outer adventitia
layers are well separated by smooth muscle cells
and elastic fibers (called the ‘media layer’) that
provide the mechanical structure of arteries and
are key for regulation of blood pressure. This
layer seems to be impermeable to macrophages
and prevents their direct extravasation from
the lumen into the adventitia. Instead, the vast
majority of macrophages present in normal
arteries enter the adventitia via small vessels
that supply the arteries ‘from the back’ (the
vasa vasorum (‘vessels of vessels’)).
It has not been clear which and how many
hematopoietic pathways feed the macrophage
compartment in the adventitia during normal
development. In addition to artery-resident
macrophage population(s), blood-derived
monocytes can infiltrate inflamed arteries,
where they can acquire macrophage phenotypes, which makes it difficult to distinguish
the resident cells from the newly colonizing
cells. In this scenario, cells may enter via the
endothelium (the intima layer), reside in the
intima and undergo pathogenic transformation
into the aforementioned foam cells. It appears,
however, that the main route in the arterial wall,
under inflammatory conditions as well, is into
the adventitia (Fig. 1). Because distinct ancestry
probably also bears functional implications for
the mature macrophages, delineating the developmental sources in a quantitative manner is
of central interest. It is this question that has
now been elegantly addressed by Ensan et al.8.
Classically, tissue macrophages have been
studied by histology or by flow cytometry,
which provides an informative yet static picture of healthy and diseased tissues. To gain
insight into the ancestry and heterogeneity of
arterial macrophages, Ensan et al. make use of
inducible and constitutive genetic fate mapping8. They use mice that express Cre recombinase under control of specific promoters,
such as the promoter of the gene encoding the
chemokine receptor CX3CR1 (Cx3cr1CreER).
After exposure to tamoxifen, Cre activity turns
nature immunology volume 17 number 2 February 2016
on a gene encoding a fluorescent reporter. In
these experiments, cells can be labeled in situ
in a time-controlled manner (at least for the
inducible systems) without a requirement for
cell isolation or transplantation. The inheritable marker allows visualization of all progeny
that arise in vivo from the initially labeled cells.
After introduction of the label in Cx3cr1CreER
mice on embryonic day 8.5, and thus at a stage
of yolk sac hematopoiesis, the authors find a
substantial contribution of labeled cells to the
adult arterial macrophage pool. The labeling
frequency of these arterial macro­phages is lower
than that of microglia in the brain but greater
than that of Kupffer cells in the liver, consistent
with the chronological order of organ development. In contrast, cells arising from fetal liver
progenitor cells marked by expression of the
receptor tyrosine kinase Flt3 contribute only
at a low frequency to arterial macro­phages at
birth. In summary, the yolk sac is initially the
main source of arterial macrophages.
The induction of Cre activity in the
Cx3cr1CreER mice at embryonic day 8.5 leads to
labeling of the vast majority of arterial macro­
phages at 2 days after birth. Interestingly, the labeling frequency decreases rapidly thereafter and
stabilizes by 2 weeks of age at around only 50%
of the initial labeling frequency. This dilution by
cells not initially labeled via injection of tamoxifen into Cx3cr1CreER mice at embryonic day 8.5
strongly suggests that the arterial wall continues
to be colonized by precursors of macrophages.
This second influx of cells into the arterial macrophage pool is completed by about 2 weeks. The
authors do not determine its source, but eventually over half of arterial macrophages are labeled
via Cre recombinase expressed under control of
the promoter of the gene encoding Flt3, which
marks hematopoietic stem cell–dependent lineages originating in the fetal liver and in the bone
marrow. Combined, these data indicate two main
origins of resident arterial macrophages: an initial
yolk sac–derived wave, followed by Flt3-marked,
mostly postnatal hematopoiesis.
While tissue macrophage populations are
considered long-lived, ultimately they depend
on either local proliferation (self-renewal) or
replacement by new precursor cells from the
circulation. On the basis of cell-cycle analysis
and dilution of labeling of green fluorescent
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news and v iews
Fetal liver
CMP
Yolk sac
2 weeks
postnatally
HSC
Bone marrow
Monocytes
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© 2016 Nature America, Inc. All rights reserved.
Marina Corral Spence/Nature Publishing Group
CX3CR1+
precursor
Development and
homeostasis
Flt3+
precursor
Inflammation
Inflammation
Resolution
Intima
Media
Adventitia
Resident macrophages
(CD11b+F4/80+CD115+Lyve-1+)
Inflammatory macrophages
(CD11b+F4/80+CD115-Lyve-1-)
Figure 1 Origins of arterial macrophages in development, at steady state and during arterial
inflammation. The arterial wall consists of three layers: the intima (yellow), composed of endothelial cells
surrounding the lumen; the media (dark red), a smooth-muscle-cell layer; and the adventitia (light red),
connective tissue that contains resident macrophage populations under homeostatic conditions. Resident
macrophages (CD11b+F4/80+CD115+Lyve-1+) arise prenatally, first from yolk sac–derived CX3CR1+
precursor cells and subsequently from Flt3+ fetal liver precursor cells. In the first 2 weeks after birth, the
immigration of cells derived from Flt3+ bone marrow cells completes the resident pool of macrophages
in the adventitia. Thereafter, this population is maintained mainly through local proliferation (left),
with little input from the bone marrow (homeostasis). During sepsis (right), phenotypically distinct
macrophages (CD11b+F4/80+CD115–Lyve-1–) emerge in the arterial wall (inflammation). These
inflammatory macrophages are derived from bone marrow hematopoiesis, and the cells of this phenotype
disappear with resolution of the inflammation. Moreover, they do not differentiate into resident-type
arterial macrophages. CMP, common myeloid progenitor; HSC, hematopoietic stem cell.
protein–tagged histone H2B, the authors
estimate that nearly all arterial macrophages
undergo proliferation within 1 year, suggestive
of slow but eventually almost complete turnover in this compartment. Through the use of a
parabiosis model (gain of partner-derived macrophages in arteries over time) and inducible
CX3CR1-dependent labeling (dilution of label
over time), Ensan et al. find only a limited contribution to the aorta via recruitment of circulating precursor cells (below 20% over a period
of 8 months)8. This suggests that for the majority of arterial macrophages, self-renewal by proliferation dominates over de novo recruitment,
at least under healthy conditions. Whether the
observed low-level entry of new cells substitutes
for the yolk sac–derived macrophages and/or
the Flt3-marked macrophages is not clear.
Arterial walls can be acutely and chronically
inflamed in the course of many pathological
conditions, including atherosclerosis, hypertension and vascular remodeling, infection and
sepsis. An early response to local or systemic
inflammatory stimuli is leukocyte extravasation that occurs mainly in post-capillary venules.
However, leukocytes, including monocytes, also
enter arteries, where they are found in arterial
lesions in the intima and in the adventitia9,10.
Immigrating monocytes can differentiate further and can acquire an activated macrophage
118
phenotype, which can make it difficult to distinguish them from preexisting activated resident macrophages. Hence, the accumulation
of macrophages in inflamed arteries can be
brought about by the proliferation of resident
macrophages and/or by the influx of monocytederived macro­phages. Ensan et al. construct
parabiotic mice and subject them later to inflammatory conditions to distinguish resident macrophages from de novo invading macrophages
under these conditions8. They give these
mice injections of lipopolysaccharide or induce
microbial sepsis by cecal ligation and puncture,
each of which causes activation of macrophages.
In inflamed arteries, a second population of
macrophages appears that are different from
resident macro­phages in lacking expression of
CD115 (the receptor for the cytokine M-CSF)
and the lymphatic endothelial cell marker
Lyve-1. These inflammatory macrophages display the same level of parabiont chimerism as
circulating monocytes, which indicates that they
were derived from recruited cells. In contrast,
the endogenous resident compartment remains
free of cells from the parabiont partner, even
under inflammatory conditions. With resolution of inflammation, recruited blood-borne
macrophages vanish from the arterial walls.
Collectively, the genuine resident macro­phages
of embryonic and early postnatal origin are not
replaced during the course of inflammation;
instead, lipopolysaccharide or microbial sepsis
lead to a transient influx of monocyte-derived
macrophages (which remain phenotypically distinct from the former). Fate mapping is, in any
case, key to distinguishing these possibilities.
Currently, it seems difficult to build a coherent view across many different tissues on the
relationships between tissue-resident macrophages and infiltrating monocyte-derived
macrophages. An emerging common theme is
that inflammation-driven recruitment of new
macrophages seems to be mostly transient or
lasts at least until the inflammation has been
resolved, as shown here for arteries8 or elsewhere for microglia11. However, the relative
proportions of resident macrophages and
recruited macrophages may vary depending on
the specific tissue and pathological condition.
Very little is known about specific functions
that can be attributed to macrophages of either
origin. Interestingly, Ensan et al. find greater
phagocytic potential for immigrating macro­
phages than for resident macrophages8.
In summary, arterial macrophages have a
multilayered ancestry. There are at least three
origins: an embryonic source; an early postnatal
source; and, upon demand, a monocyte source.
This pattern is another interesting example of
distinct and successive layers in the development of the immune system. Monocyte-derived
macrophages seem to have classic immunological functions in resolving inflammation,
including phagocytosis. The adventitia has been
recognized as a site for the formation of tertiary
lymphoid organs, which might be an important
hub for sustained inflammation. Interestingly,
macrophages have been suggested to contribute
to this ectopic lymphoid tissue organization9.
Given the new findings of Ensan et al.8, additional work will be needed to link the ancestry
of macrophages to their roles under physiological and patho­logical conditions.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
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