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Acta Neuropathol (2014) 128:319–331
DOI 10.1007/s00401-014-1267-1
REVIEW
Microglia: unique and common features with other tissue
macrophages
Marco Prinz · Tuan Leng Tay · Yochai Wolf ·
Steffen Jung
Received: 7 February 2014 / Revised: 28 February 2014 / Accepted: 6 March 2014 / Published online: 21 March 2014
© Springer-Verlag Berlin Heidelberg 2014
uniqueness among other tissue macrophages will be pivotal
for potential manipulation of immune cell function in the
CNS, thereby reducing disease burden. Here, we discuss
new aspects of myeloid cell biology in general with special
emphasis on the brain-resident macrophages and microglia.
Keywords Microglia · Yolk sac · Bone marrow ·
Inflammation · Monocytes · Neurodegeneration ·
CX3CR1 · CX3CR1Cre
The origin of microglia compared to other tissue
macrophages
Tissue macrophages are part of the mononuclear phago-
cytes and were classically thought to derive from blood
monocytes [30]. This assumption was based on the fact
that monocytes are leukocytes with a very short circula-
tion half-live and they were considered to act mainly as
precursors of peripheral mononuclear phagocytes. Sec-
ondly, monocytes recruited to sites of inflammation, such
as for instance a challenged peritoneum, were shown to
give rise to macrophages [64]. Finally, also in vitro cul-
tured monocytes were shown to differentiate into cells with
macrophage features [125]. However, the recent past has
seen a major revision of this established dogma. As a tis-
sue macrophage archetype defined by its seclusion behind
the blood–brain barrier (BBB), microglia played a major
role in these discoveries. Early studies involving chick-
quail chimeras had already suggested that microglia are
established during embryonic development [63]. However,
it took the advent of novel fate mapping methods to firmly
establish the origins of adult microglia in mammals. Spe-
cifically, these novel findings are based on a combination of
transgenic mice that harbor cell-type restricted constitutive
Abstract Microglia are highly specialized tissue mac-
rophages of the brain with dedicated functions in neuronal
development, homeostasis and recovery from pathology
Despite their unique localization in the central nervous sys-
tem (CNS), microglia are ontogenetically and functionally
related to their peripheral counterparts of the mononuclear
phagocytic system in the body, namely tissue macrophages
and circulating myeloid cells. Recent developments pro-
vided new insights into the myeloid system in the body with
microglia emerging as intriguing unique archetypes. Similar
to other tissue macrophages, microglia develop early dur-
ing embryogenesis from immature yolk sac progenitors. But
in contrast to most of their tissue relatives microglia persist
throughout the entire life of the organism without any sig-
nificant input from circulating blood cells due to their lon-
gevity and their capacity of self-renewal. Notably, micro-
glia share some features with short-lived blood monocytes
to limit CNS tissue damage in pathologies, but only bone
marrow-derived cells display the ability to become perma-
nently integrated in the parenchyma. This emphasizes the
therapeutic potential of bone marrow-derived microglia-
like cells. Further understanding of both fate and function
of microglia during CNS pathologies and considering their
M. Prinz (*) · T. L. Tay
Institute of Neuropathology, University of Freiburg, Breisacher
Str. 64, 79106 Freiburg, Germany
e-mail: marco.prinz@uniklinik-freiburg.de
M. Prinz
BIOSS Centre for Biological Signalling Studies, University
of Freiburg, Freiburg, Germany
Y. Wolf · S. Jung (*)
Department of Immunology, The Weizmann Institute of Science,
234 Herzl St, 76100 Rehovot, Israel
e-mail: s.jung@weizmann.ac.il
320 Acta Neuropathol (2014) 128:319–331
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or conditional Cre recombinase activity with mice harbor-
ing reporter gene alleles that are activated upon excision of
loxP-flanked (‘floxed’) STOP cassettes. As the Cre-medi-
ated DNA rearrangements in the given cells are permanent,
this approach reveals information about the ontogeny and
history of the studied cells. Applying this approach to the
study of microglia, Ginhoux and colleagues discovered
that the microglia compartment is established well before
birth from primitive macrophages that are generated dur-
ing an early ‘primitive’ wave of hematopoiesis in the yolk
sac [35]. Moreover, the authors showed that after birth,
microglia remain independent from input from bone mar-
row-derived monocytes and adult hematopoiesis, but rather
maintain itself by longevity and limited self-renewal. These
findings were subsequently corroborated and extended to
establish the critical role of Irf8 in the generation of yolk
sac-derived microglia [58]. Moreover, additional studies by
us and others showed that the embryonic origin is a com-
mon feature of most tissue macrophages [47, 116, 141]. Of
note, recent work on microglia development in mouse was
preceded by live time-lapse recordings that fate-mapped
early hematopoietic precursors from the yolk sac to the
mesenchyme of the head and the depths of the brain in
transparent embryonic zebrafish [49].
As opposed to the microglia that seems almost exclu-
sively yolk sac-derived, other tissue macrophage compart-
ments display, however, various contributions from cells
derived at a later time point from the fetal liver (Ginhoux and
Jung, in press) (Fig. 1). Interestingly, Geissmann and col-
leagues showed that their unique yolk sac origin is reflected
in the independence of microglia from the transcription
factor Myb [116]. It is currently unclear to what extent the
generation of fetal monocytes is dependent or independent
of Myb and its link to primitive or definitive hematopoiesis.
Of note, the number of tissue macrophages that have been
studied in sufficient detail using the fate mapping approach
is still limited. However, for epidermal Langerhans cells, as
well as the majority of heart macrophages and lung mac-
rophages, it has been established that they are mostly fetal
liver-derived [27, 43, 52]. Microglia therefore clearly repre-
sent an exception in that they are homogeneously yolk sac-
derived (Fig. 1). Of note, the spectrum of tissue macrophage
origins also includes the other extreme, that is, a population
that is entirely monocyte-derived during steady state. Mac-
rophages that reside in the intestinal lamina propria, the con-
nective tissue underlying the gut epithelium, were shown
to have a rather short-half life and rely on constant renewal
from Ly6C+ blood monocytes [142]. Monocyte-dependent
macrophage subpopulations have also been noticed in other
steady-state tissues, such as the skin and heart [27, 129], but
it remains to be shown whether these cells differ in function.
Moreover, monocytes are well known to be prominently
recruited to sites of inflammation and during pathology. The
monocyte-derived macrophages that arise from these inflam-
matory monocyte infiltrates are however probably mostly
short lived. This has most convincingly been shown in an
elegant study involving EAE and the use of parabionts [3].
Again, exceptions seem however to exist, as it was recently
demonstrated that monocyte-derived macrophages that
reside in atherosclerotic plaques can persist and undergo
considerable proliferative expansion [109].
At least in the mouse, microglia are established before the
formation of the BBB and are independent from hematopoie-
sis in adulthood. However, it has been noted that following
irradiation and bone marrow transplantation, the brain can be
seeded by hematopoietic stem cell (HSC)-derived cells that
persist and constitute a progressively increasing and consid-
erable fraction of the brain macrophage compartment (Vol-
asky and Jung, unpublished results). These cells originate
clearly from cells that are distinct from monocytes, but might
be earlier myeloid precursors that entered the damaged brain
following the irradiation. Importantly, this finding indicates
that the adult bone marrow hosts cells that can give rise to
long-lived radio-resistant microglia. Interestingly, it has
been shown that this population can reconstitute microglia
defects [16, 23] and thus has therapeutic potential. However,
it remains to be established whether these brain macrophages
have all activities of bonafide microglia, both in homeostasis
and pathological settings.
Microglial functions within the non‑inflamed CNS
Since the early studies of Ilya (Eli) Metchnikov mac-
rophages are well appreciated to have dual functions, both
in inflammatory and pathological settings, as well as in
‘housekeeping’ that is critical to maintain tissue homeo-
stasis under non-inflammatory conditions. Though early
studies have emphasized the contributions of microglia to
pathology, more recent studies demonstrate that microglia
Fig. 1 Microglia origin compared to other tissue macrophages.
Developmental scale of macrophage development from embryonic
stages that contain yolk sac (YS)-derived macrophages (MF), fetal
liver (FL) monocytes until postnatal (adult) bone marrow (BM)-
derived macrophages. Dotted line indicates the time point of birth
321Acta Neuropathol (2014) 128:319–331
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are hardly ‘quiescent’ in steady state, but in fact actively
participate both in the shaping of the developing and young
CNS, and its maintenance, regardless of immune chal-
lenges [131, 137]. Microglia function is therefore similar to
reports describing macrophages involved in tissue remod-
eling [101] and wound healing [89].
Once considered ‘immune-privileged’ and excluded
from ongoing immune surveillance, the brain is now recog-
nized to host not only resident microglia as the representa-
tive macrophage population, but also other immune cells in
steady state [105]. Since neuronal damage is largely irre-
versible once it has occurred, the CNS parenchyma requires
tightly regulated and orchestrated inflammatory reactions
that aim at restoration, in which microglia participate in
substantially. Genome-wide expression profiling of mul-
tiple immune cell subsets by the Immunological Genome
Consortium has established that microglia stand out with
a unique transcriptome among other tissue macrophages,
such as the ones in the splenic red pulp, peritoneum or lung
alveolar space [32]. Similar analysis has emphasized the
differences of gene expression profiles of microglia and
Ly6C+ monocytes [11, 12] (Fig. 2). Moreover, these recent
studies have highlighted the role of TGFβ in imprinting
this unique microglia signature, which includes known
macrophage genes such as cx3cr1, itgam1 and aif1 and
also microglia-specific genes p2ry12, fcrls and tmem119.
These data support the notion that to perform within their
unique and sensitive microenvironment, microglia require
a characteristic gene expression distinct from other Myb-
dependent or independent, embryo-derived macrophages.
This probably includes the ability to phagocytose and
secrete soluble factors in a regulated manner, with mini-
mum inflammatory output.
During development, microglia can interact with neu-
rons either by direct contact resulting in phagocytosis or
by secretion of soluble factors. The capability of microglia
to phagocytose apoptotic neurons [73, 99, 122], synaptic
material [97, 115] and cellular debris [23] is well estab-
lished. Moreover, in the developing cerebellum, microglia
were reported not only to phagocytose, but also to actively
induce apoptosis of developmental Purkinje cells by means
of respiratory burst [73]. In adolescent mice, in which
many redundant neurons undergo elimination, microglia
actively phagocytose apoptotic neurons and thus participate
in early postnatal neurogenesis [122] and modulate synap-
tic pruning during development [97].
Microglial–neuronal crosstalk can involve several sign-
aling pathways, such as the CD200-CD200R [53], the
Fig. 2 Neuronal–microglial dialog during CNS homeostasis. Micro-
glia participate in synaptic pruning during development via the
CX3CR1/CX3CL1 axis, mediated either by transmembranal or solu-
ble neuronal CX3CL1, which may be influenced by TGFβ-mediated
expression of CX3CR1 via SMAD2/3 phosphorylation. Microglia can
influence neuronal fate by inflammatory output, such as IL-6, which
inhibits the differentiation of neuronal precursor cells (NPCs) into
neurons during early postnatal days, or by IL-4-mediated activation,
which may drive NPCs into newborn neurons. Microglia phagocy-
tose weak synapses by the CR3 complement pathway (CD11b → C3/
C1q), which is regulated by decreased neuronal activity or via TGFβ
signaling. Neurons can also express TGFβ under inflammatory con-
ditions, which may enhance microglial phagocytosis. Finally, ATP
signaling can give rise to the secretion of microglial BDNF, which
drive TrKB phosphorylation and thus contribute to the formation of
glutamatergic synapses
322 Acta Neuropathol (2014) 128:319–331
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CD47-CD172a [36] and the CX3CR1-CX3CL1 axis [60, 61,
107, 140] (Fig. 2). In the above cases, the ligand is expressed
by neurons or other glial cells, with the receptor located on
the microglia. It is generally thought that many of these sign-
aling pathways maintain microglia in a quiescent state [10].
In support of this notion, disruption of such communication,
such as for instance the CX3CL1-CX3CR1 axis, impairs the
maturation of dendritic spines during development, probably
by excessive synaptic pruning [97]. Importantly, the classi-
cal complement proteins, such as C1q, are present within the
CNS [126], and microglia were shown to use the comple-
ment receptor 3 pathway to engulf presynaptic inputs dur-
ing the pruning peak of retinal ganglion cells (RGCs) in an
activity-dependent manner [115]. Several other reports have
shown that microglia phagocytosis is dependent on neu-
ronal activity, such as visual experience [130] or antagonist-
mediated neuronal silencing, which decreases the contact
frequency between microglia and synapses [136, 137]. It is
therefore intriguing to postulate that neurons might be able
to fine tune the inhibitory ‘off’ signal to microglia according
to their activity levels or the strength of their synapses [10].
Direct microglial impact on neurons by secreted fac-
tors is less well established, although several such factors
were proposed. The majority of the literature is focused
on the interplay between adult neurogenesis and micro-
glia-derived cytokines. Adult neurogenesis is confined to
two locations in the brain, the subventricular zone (SVZ)
in which the newborn olfactory neurons are generated and
migrate into the olfactory bulb [85] and the subgranular
zone of the hippocampus (SGZ) [122]. Microglia seem to
be the key players for neurogenesis [34] as they phago-
cytose apoptotic neurons in the SGZ during early postna-
tal development without being activated by inflammation
[122]. Importance of the robust microglia silence is high-
lighted by the fact that SGZ neurogenesis is inversely cor-
related with pro-inflammatory microglia activation [26].
Moreover, neuronal in vitro cultures supplemented with
media from cultured LPS-activated microglia also display
less DCX+ immature neurons and this effect could be par-
tially reversed using antibody-mediated IL-6 blockade [87].
Interestingly, IL-6 also blocks the differentiation of GFAP+
neuronal stem cells into mature neurons [6], and IL-6 over-
expressing transgenic mice have reduced neurogenesis in
the hippocampus [135]. A cytokine which is considered to
promote neuronal differentiation is IL-4, which polarizes
microglia to acquire M2-like, non-inflammatory features
[117] and may contribute to the preservation of cognitive
functions [31]. Accordingly, cultured neurons in the pres-
ence of conditioned medium of IL-4 activated microglia
display more immature βIII tubulin+ neurons [17].
One cytokine, which recently gained particular focus
with respect to microglia in the steady-state CNS, is the
transforming growth factor (TGF) β (Fig. 2). TGFβ was
reported to promote the development of bonafide microglia
in vitro via the SMAD2/3 pathway and to regulate micro-
glial CX3CR1 expression [1]. TGFβ imprints a microglia
gene expression signature and mice which lack TGFβ seem
to have reduced numbers of microglia [12]. Neurons can,
at least under inflammatory conditions, secrete TGFβ [72].
However, neurons also require TGFβ to fully express the
complement protein C1q and thus initiate the CR3 pathway
to induce microglial phagocytosis. As a result TGFβ-R2−/−
mice exhibit an impaired segregation of contra- and ipsilat-
eral retinal input in the RGC [9]. Thus, TGFβ is required
both directly for microglia development and maturation, and
also indirectly for their proper homeostatic functions.
Another microglial-derived secreted factor that has been
proposed to be critical in microglia–neuron crosstalk is
brain-derived neurotrophic factor (BDNF), which acts on
the receptor tyrosine kinase TrkB (also known as NTRK2)
expressed on dendritic spines and is involved in neuronal
plasticity and synapse remodeling [83]. ATP stimulation
induces microglia secretion of BDNF, which is involved
in pain sensation [18]. By exploiting mice that harbor an
insertion of a tamoxifen-inducible Cre recombinase under
the control of the CX3CR1 promoter, and achieving micro-
glia-specific BDNF gene ablation, it was recently sug-
gested that microglia participate in the learning-dependent
synapse formation and elimination in the motor cortex [98].
Moreover, the authors suggested that microglial BDNF
might be critical for plasticity and neurotransmission, as
protein levels of glutamate receptor subunits GluN2B and
GluA1 were reduced in the absence of microglial BDNF.
In the developing barrel cortex, CX3CR1-deficient mice
exhibit delayed developmental switch of the glutamate
receptor subunits GluN2B to GluNA, one of the main fea-
tures of barrel cortex development [54]. The mechanism
suggested was the elimination by a soluble factor. However,
given the data of Parkhurst et al. [98], it is possible that
BDNF is involved in the mechanism. However, a molecular
link between CX3CR1 and BDNF production remains to be
shown. The detachment of afferent axonal endings from the
surface membrane of regenerating motoneurons and their
subsequent displacement by microglia in a process called
“synaptic stripping” has been described in rodent models
and patients of neurodegenerative conditions [88].
Bone marrow‑derived microglia‑like cells and tissue
macrophages
Do bone marrow-derived microglia-like cells (BMDM)
exist in nature and if so, are they functionally similar com-
pared to yolk sac-derived bonafide microglia? The answer
to this question could have tremendous clinical implications
for the treatment of many diseases of the human CNS, such
323Acta Neuropathol (2014) 128:319–331
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as brain tumors, amyotrophic lateral sclerosis (ALS), Alz-
heimer’s (AD), and Parkinson’s disease (PD). Specifically,
microglia precursors in the bone marrow could be used as
Trojan horses to deliver neuroprotective or immune-relevant
genes to the diseased CNS to modulate pathology. Upon
injury or infection, BM-derived inflammatory or patrol-
ling monocytes are summoned from the bloodstream to the
inflammatory lesion, followed by terminal differentiation
into macrophages [70]. The availability of the CX3CR1GFP
mouse [56] enabled the identification of a short-lived blood
monocyte subset characterized as CX3CR1loCCR2+Gr1+
cells that are actively attracted to inflamed tissue, while an
alternate CX3CR1hiCCR2+Gr1+ class of monocytes was
shown to reside in the vascular lumen and regulate endothe-
lial integrity [5, 33]. Davies et al. recently showed that both
resident macrophages and recruited inflammatory mono-
cytes expand their populations with different kinetics at the
onset and recovery of the tissue insult to regain homeosta-
sis [21, 22]. Does this also apply to the brain parenchymal
microglia of the immune-privileged CNS?
Several approaches were taken to resolve this enigma.
Also using incorporation of [3H]-thymidine, in combina-
tion with F4/80 immunohistochemistry to identify resident
microglia in the adult murine brain, researchers came to
the conclusion that these tissue macrophages are a self-
renewing population, which nevertheless derive in addition
from circulating monocytes that continuously engraft the
brain via an intact BBB and subsequently differentiate into
resident microglia [68]. However, other reports involving
BM chimeras with alternatively marked donor cells, such
as MHC class I in rat [76] or Y chromosome containing
cells in female BM transplant hosts [134], were not able to
demonstrate engraftment of donor BM-derived microglia in
the brain parenchyma. Studies indicated that perivascular
macrophages in mouse [7], rat [50] and human [134] brains
were donor-derived from circulating myeloid cells. In a
meningitis infection model, we showed that GFP-labeled
circulating cells entered the brain of a reconstituted host
and differentiated into microglia with multiple processes
reminiscent of a typical microglia cell [24]. Later studies in
non-human primates [124] and a comparative investigation
involving radiation chimeric mice and rats [65] demon-
strated differential recruitment of donor BM-derived mac-
rophages to the periphery of the CNS (perivascular space
and choroid plexus) and engraftment into the brain (neo-
cortex and cerebellum).
All previous reports, however, did not directly address
the question of whether BMDM are recruited to the
immune-privileged brain parenchyma under physiologi-
cal conditions. In fact, the use of irradiation followed by
BM transplantation leads to non-physiological permanent
alterations of the BBB, induces a chemokine storm among
other changes in the brain [59, 79], and elevates HSC count
in blood, all of which promote entry of circulating myeloid
cells into the CNS [104, 106] (Fig. 3).
Two groups further investigated the engraftment of
BMDM while avoiding the induction of non-physiological
conditions via head protection of irradiated hosts [79], or
using parabiosis, where the circulation of both host and
recipient is surgically conjoined to allow mixing of the
blood elements [2]. In the head protection model, engraft-
ment by peripheral GFP-labeled BM cells was absent even
upon cuprizone-induced demyelination or transection of
the facial motor nerve [79]. Both studies came to the con-
clusion that in the presence of an intact BBB, native micro-
glia undergo local proliferation during homeostasis. The
recruitment of BM cells into the brain was only possible
with conditioning of the host via irradiation [79] (Fig. 3).
In another study involving whole BM and HSC transplanta-
tion and parabiosis models, it was striking that nearly no
GFP+ donor cells were detected, even with injury to the
hippocampus induced by intraperitoneal application of
kainate acid, in the parabiosis model where the BBB was
left intact [74]. Of note, peripheral chimerism was rather
low at 0.5–10.7 % even in transplanted mice [74]. Simi-
larly, a recent report comparing brain conditioning by lethal
dose irradiation and application of the alkylating agent
busulfan indicated that damage to the BBB is required for
engraftment of BMDM in both steady and challenged con-
ditions [59]. Treatment with busulfan did not disrupt the
BBB as drastically as by irradiation, which led to several-
fold higher myeloid cell entry into certain brain regions
of radiation chimeras [59]. Others also showed using only
busulfan-based chemotherapy regime that the introduction
of BMDM into the brain parenchyma required pathological
conditions, such as in hypoxic–ischemic stroke and APP/
PS1 AD models [66], or stress [139]. In the latter case,
CD45hi cells entered the brain parenchyma in a CCR2-
independent, social stress-driven manner, and eventually
adopted endogenous microglia morphology in the dentate
gyrus [139].
Interestingly, BM-derived myeloid cells and yolk sac-
born microglia contribute differently to inflammatory
response of the CNS [80] and neurodegenerative diseases
as shown in mouse models of AD [82, 123] and multiple
sclerosis (MS) [3, 81], as well as in a spinal cord injury
model [120]. For example, CCR2+ BMDM were suggested
to be superior in plaque clearance and reduction of disease
burden in AD models [82, 123]. Furthermore, recruitment
of BM-derived Ly6ChiCCR2+ monocytes has been reported
in the experimental autoimmune encephalomyelitis (EAE)
animal model of autoimmune CNS disease mimicking MS
and was suggested to exacerbate disease progression [81].
Notably, these infiltrating monocytes were short-lived and
vanished several days after engraftment, thereby excluding
their stable integration into the endogenous CNS microglial
324 Acta Neuropathol (2014) 128:319–331
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network [3]. Using a CCR2-deficient mouse model, Serbina
and Pamer demonstrated the necessity of this chemokine
signaling for the extravasation of Ly6Chi monocytes from
the BM into the blood stream, but that it was not required
for translocation to the tissue [118].
BMDM are characterized by the expression of
Ly6ChiCCR2+ surface markers, in contrast to Ly6C−CCR2−
microglia [79, 81, 82, 86]. To date, however, it is still not
completely clear whether BMDM are able to fulfill the
whole range of functions of endogenous microglia. If the
development of sophisticated microglial phenotypes is a
consequence of long term co-evolution with other cell popu-
lations and networks in the CNS, could newly immigrated
BMDM simply replace native microglia cells [92]? Inter-
estingly, the route of entry of BMDM into the conditioned
brain parenchyma was described to be via the choroid
plexus [121]. Other studies hinted a preferential engraftment
of BMDM in the olfactory bulb and cerebellum in contrast
to the cortex, striatum and hippocampus [103].
Microglia as disease inducing cells
Despite the low number of microglia compared to other
resident brain cell types, these small cells play significant
roles in the developing early postnatal brain [97, 115, 133]
as well as maintain homeostasis in the healthy adult brain
[46, 131]. During challenges such as injury [20, 93], infec-
tion or disease [19, 24, 113], microglia nimbly navigate
the altered brain physiological landscape via changes in
their morphology, presentation of antigens and release of
cytokines. Whether microglia contribute as beneficial or
harmful mediators in autoimmune diseases [37] or neuro-
degenerative disorders [104, 127] is open to deeper investi-
gation. Recent studies have linked microglia-related genetic
mutations to neurological or behavioral abnormalities
in humans and mouse models [14, 16, 23, 29, 62, 94, 96,
119]. On the contrary, some genetic depletions in microglia
revealed neuroprotective outcomes in disease background
[13, 48, 104, 106].
Several molecular modulators of microglia homeosta-
sis that may subsequently mediate or induce CNS diseases
[60] include transcription factors and other nuclear proteins
(Fig. 4). The development of yolk sac-derived microglia
hinges on two important transcription factors PU.1 (also
known as SFPI1) and interferon regulatory factor (IRF) 8
[8, 58]. PU.1 is exclusively expressed in the hematopoietic
cell lineage and required for normal myeloid cell devel-
opment among members of the ets family of transcrip-
tion factors [110]. Mice deficient in PU.1 lack circulating
monocytes and tissue macrophages including microglia
[8, 77]. The regulation of the receptor of the macrophage
colony-stimulating factor CSF-1, which is required for
macrophage survival and proliferation, was shown in vitro
Fig. 3 Conditions for recruitment of bone marrow-derived microglia
(BMDM). Postnatal BMDM form only under defined host condi-
tions in the CNS. BM cells (purple round cells) are released into the
bloodstream in a chemokine receptor (CCR) 2-dependent fashion and
can enter the conditioned CNS. Local conditioning of the CNS can
occur via irradiation or neurodegeneration, which lead to subsequent
disruption of the blood–brain barrier and concomitant induction of
chemokines such as CCL2, thus allowing engraftment of BM-derived
macrophages (purple elongated cells). In conditioned CNS, BMDM
(purple ramified cells) undergo hyperplasia in challenged or disease
states (indicated by purple arrow). Yolk sac-derived microglia (green)
are radio-resistant and perform self-renewal by undergoing homeo-
static proliferation (indicated by green arrow)
325Acta Neuropathol (2014) 128:319–331
1 3
to be dependent on PU.1 [15]. In a mouse model of ALS,
microglia from wild-type BM-derived cells transferred to
PU.1−/− neonates with a point mutation for ALS-caus-
ing gene were able to partially but significantly rescue the
defects seen in knockouts [8]. IRF8 is a weak DNA-binding
protein on its own and requires interaction with other tran-
scription factors, such as PU.1 [84]. Interestingly, IRF8-
deficient mice developed altered distribution of microglia
with reduced cell surface area and different expression of
microglia markers including Iba-1, CD45, CD11b, F4/80
and CX3CR1 [84]. Thus, IRF8 was proposed as a key fac-
tor of microglia development and activation [58, 84]. In
a peripheral nerve injury model, Masuda et al. showed
that IRF8 is up-regulated and claimed that its expression
shifts microglia towards a reactive phenotype via promo-
tion of gene expression of innate responses such as TLR2
and TLR4, chemotaxis, CX3CR1 and inflammatory factors
IL-1β and P2X4R [75]. However, it is still open whether
IRF8 is critical in regulating homeostasis of mature
microglia. Point mutations in IRF8 were reported in three
patients of immunodeficiency syndromes, characterized by
the absence or abnormality of peripheral myeloid cells such
as monocytes and dendritic cells, but skin samples revealed
normal LCs, while brain biopsies were unavailable [44].
Fig. 4 Factors for microglia homeostasis and disease induction. Up-
regulation of IRF8 expression promotes a reactive microglia (MG)
phenotype via TLR2, TLR4, P2X4R, and inflammatory factors. IRF8
DNA-binding activity is dependent on other transcription factors,
such as PU.1. PU.1 regulates the receptor for macrophage colony-
stimulating factor CSF-1 (CSF-1R), which is required for microglia
survival and proliferation. In the CNS, the ligand interleukin (IL)-
34 is more prominent than CSF-1 in microglia–neuron interaction.
Microglia-specific CSF-1R signaling requires the adaptor DNAX-
activating protein of 12 kDa (DAP12). DAP12 maintains synaptic
signaling via the brain-derived neurotrophic factor (BDNF)—tyrosine
kinase receptor B (TrKB). Functional loss of DAP12 and triggering
receptor expressed on myeloid cells 2 (TREM2) has been implicated
in the pathogenesis of neurodegenerative diseases such as Alzhei-
mer’s, Parkinson’s, Nasu–Hakola disease, and multiple sclerosis.
TREM-2 suppresses the expression of pro-inflammatory factors and
promotes phagocytosis of cellular debris. The ABCD1 protein, an
adenosine triphosphate-binding cassette transporter located in the
peroxisomal membrane, together with the myelin-associated gly-
coprotein (MAG) expressed in Schwann cells or oligodendrocytes is
important for maintaining myelin integrity and modulating microglia
activation state. In AD, the microglial inflammasome, comprising
caspase 1 (Casp 1), apoptosis-associated speck-like protein contain-
ing a caspase recruitment domain (Asc), and nucleotide-binding and
oligomerization domain-like receptor family pyrin domain-containing
3 (NLRP3), contributes to increased amyloid-β (Aβ) load, shift of
microglia towards pro-inflammatory-activated phenotype, neurode-
generation, and cognitive defects. The orphan nuclear receptor Nurr1
may be neuroprotective via NF-κB-p65 signaling. Loss of Hox-B8
in microglia leads to excessive grooming. Deficiency in the methyl-
CpG-binding protein (MECP) 2 leads to the X-linked autism spec-
trum disorder Rett syndrome. MicroRNA miR-146a could maintain
microglia phenotype via suppression of neuron-specific genes. The
resting state of microglia may be maintained by miR-124 via the tran-
scription factors PU.1 and C/EBPα. Cell membrane, cytoplasm and
nucleus of a microglia cell are depicted in different shades of green
326 Acta Neuropathol (2014) 128:319–331
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The immune-related role of another microglia-related
transcription factor, Nurr1, was investigated in the context
of inflammation-mediated neuronal cell death [112]. Nurr1
is an orphan nuclear receptor known to be required for the
maintenance of post-mitotic midbrain dopaminergic neu-
rons and their neuronal phenotype [4]. Human point muta-
tions in the Nurr1-coding gene NR4A2 were identified in
patients of late onset familial, but not sporadic, PD [69]. In
these affected patients, the levels of NR4A2 mRNA were
decreased in lymphocytes, together with altered expression
of tyrosine hydroxylase, which is the rate-limiting enzyme
in the biosynthesis of dopamine [69]. By analyzing the
responses to LPS-induced inflammation in primary mouse
and human microglia in which Nurr1 was knocked down
by silencing RNA or lentivirus small-hairpin RNA, it was
proposed that Nurr1 played a neuroprotective role via the
NF-κB-p65 signaling pathway in glia [112]. However, this
putative function of microglial Nurr1 in vivo has not been
directly addressed so far.
The loss of DNA-binding nuclear protein Hox-B8 in the
hematopoietic lineage revealed a striking excessive patho-
logic grooming behavior in the mutant mice [16]. This
behavior was reportedly similar to that of sufferers of the
obsessive–compulsive spectrum disorder trichotillomania.
Interestingly, the authors attributed this strong phenotype
to microglia, as they were unable to detect reporter expres-
sion in other CNS cell types based on their Hoxb8-IRES-
Cre-mediated reporting of Hox-B8 localization [16]. Fur-
thermore, they demonstrated that wild-type BMDM could
rescue the behavioral phenotype in their mutant model
[16].
In a similar vein, Derecki et al. were able to ameliorate
the pathology in a methyl-CpG-binding protein (MECP)
2-null mouse model of Rett syndrome, an X-linked autism
spectrum disorder, by transplantation of wild-type BM
[23]. Rett syndrome is severely debilitating in males, which
have only one X chromosome. However, CNS engraftment
of BMDM, which displayed properties of resting micro-
glia, largely alleviated the symptoms in MECP2-null males
and extended their life spans [23].
Microglial phenotype is also controlled by several
microRNAs (miRNA) [42]. In a microarray analysis of
miRNA expression in neurons, astrocytes, oligodendrocytes
and microglia, cell type-specific enrichment of miRNAs
such as miR-146a in microglia was described [55]. Bind-
ing of miR-146a to neuron-specific genes shown in lucif-
erase reporter assays led the authors to speculate that this
miRNA could be important for maintaining a non-neuronal
phenotype in microglia [55]. In another study, miR-124
was proposed to maintain the quiescent state of microglia,
where elevated levels of miR-124 kept activation mark-
ers MHC Class II and CD45 down-regulated through the
C/EBPα-PU.1 pathway [102]. Therefore, it is reasonable to
speculate that mutations in miRNA-coding regions could
contribute considerably to neurological dysfunction.
An important factor for the survival, maintenance
and proliferation of macrophages is CSF-1 that signals
through its receptor CSF-1R (also known as CD115) [41,
67]. Impaired auditory and visual processing of Csf1op/op
osteopetrotic mice, in which CSF-1 is deficient, has been
described [78]. A comparison of CSF-1R null mutant and
Csf1op/op survival rates 1-month after birth revealed com-
plete mortality of CSF-1R knockouts and a 40 % survival
of Csf1op/op [90]. CSF-1R mutants reportedly have smaller
brains and atrophic olfactory bulbs with obvious strong
defects in olfactory function [28, 90]. While embryonic
brains appeared to develop normally in CSF-1R knock-
outs, microglia are completely absent in these mutants [28].
It was reported that in postnatal mouse brain, CSF-1R is
only expressed on microglia but not in other neural cell
types [28]. These findings suggested that development of
microglia in the CNS is dependent on CSF-1R signaling
albeit via a different ligand compared to other tissue mac-
rophages. Indeed, the expression of CSF-1 was described to
be low throughout the brain, with slightly higher expression
in the cerebellum throughout development and in adult ani-
mals [78]. Subsequently, it was found that microglial phe-
notype in mutants of the cytokine IL-34, a recently discov-
ered alternative ligand of CSF-1R [71], is closer to that of
the CSF-1R null, as a severe reduction of microglia number
as well as skin LCs was observed here [138]. Furthermore,
a lack of alteration in blood monocytes, other tissue mac-
rophages and DCs, apart from a subset of DCs in the lung,
points to the tissue-restricted requirement of IL-34 for LC
development and microglia maintenance [138]. Compara-
tive analysis of both CSF-1R ligands revealed a broader
regional expression of IL-34 than CSF-1 in the brain, with
complementary expression of each gene in the neocor-
tex [90]. Using a mouse model of prion disease and from
analyses of clinical samples of variant Creutzfeldt–Jacob
disease and AD, PU.1 and CSF-1R were postulated to be
molecular factors regulating microglial proliferation in
these pathologies, suggesting that a delay in neuronal dam-
age and disease progression could be achieved by target-
ing CSF-1R signaling between microglia and neurons [40].
CSF-1R signaling-dependent survival and proliferation of
macrophages were shown to be inhibited in mutant mice
for the transmembrane tyrosine kinase-binding DNAX-
activating protein of 12 kDa (DAP12); in particular, fewer
microglia cells were found in certain brain regions [95].
Functional loss of the adaptor protein DAP12 (alter-
natively named KARAP or TYROBP) in conjunction
with mutations in the surface receptor, triggering receptor
expressed on myeloid cells 2 (TREM2), has been impli-
cated in the pathogenesis of several neurodegenerative dis-
eases, including AD, Nasu-Hakola disease (NHD), PD, and
327Acta Neuropathol (2014) 128:319–331
1 3
MS [29, 62, 91, 94, 96, 100, 108, 119, 128]. Expression of
DAP12 in the brain is detected in microglia only and its
mutation was linked to altered synaptic plasticity due to
a large decrease in signaling via the brain-derived neuro-
trophic factor—tyrosine kinase receptor B [111]. TREM-2
is a pattern recognition receptor located on the cell surface
of dendritic cells, bone osteoclasts and brain parenchymal
microglia [119]. It plays a role in suppressing the synthe-
sis of pro-inflammatory factors [45, 132] and promotes
protective phagocytosis of cellular debris, such as degen-
erated myelin, as demonstrated in an animal model of MS
[128]. In support of these findings, another study detected
enhanced expression of TREM-2 on microglia during EAE
and that blockade of the receptor exacerbated disease pro-
gression [100]. Just recently, the mutant allele p.R47H of
TREM-2 was identified in a genome-wide association
study on thousands of patients to be a risk factor for fronto-
temporal dementia and PD [108], in addition to the known
risk for AD [91].
Human mutations in TREM-2/DAP12 are known in the
rare autosomal recessive NHD [29, 62, 94, 96], which is
characterized by pathological lesions in the cortex, thala-
mus and basal ganglia, progressive early-onset dementia
and formation of bone cysts, consistent with the known
tissue distribution of this receptor–adaptor complex [119].
Notably, peripheral myeloid cells appear to be unaffected
in loss of TREM-2/DAP12 functions [119]. An investiga-
tion on NHD-specific biomarkers expressed by microglia
confirmed the absence of DAP12 expression in NHD brains
while detecting DAP12 protein on quiescent microglia in
control samples [114]. Interestingly, this study included a
claim that human microglia do not express TREM-2, lead-
ing to the antithesis proposed by the authors that loss of
microglial TREM-2/DAP12 function in humans may not be
the primary cause of NHD phenotype [114].
Intracellular signaling pathways also influence microglia-
mediated innate immune responses. Analysis of brain lysates
from AD patients and controls clearly demonstrated elevated
levels of cleaved caspase-1 (Casp1) in the pathology, which
was reflected in the APP/PS1 transgenic mouse model for
familial AD [48]. Casp 1 is a component of the microglial
nucleotide-binding and oligomerization domain-like recep-
tor family pyrin domain-containing 3 (NLRP3) inflam-
masome, together with NLRP3 and the adaptor molecule
apoptosis-associated speck-like protein containing a caspase
recruitment domain [38]. NLRP3 inflammasomes have been
implicated in several chronic inflammatory diseases, because
they act as a sensor for inflammatory compounds such as
amyloid-β (Aβ) [38, 48]. To examine the contribution of
NLRP3 inflammasome to AD, the authors bred mutant
mice carrying a defective component of the inflammasome
with APP/PS1 mice [48]. The absence of either NLRP3 or
Casp1 in the AD model significantly improved clearance of
Aβ and led to overall neuroprotection, apparently via a shift
of microglia activation towards anti-inflammatory state (i.e.,
lower IL-1β and nitric oxide synthase 2 and higher argin-
ase-1, IL-4 and found in inflammatory zone 1, compared to
wild-type microglia in AD) [48].
Mutations in the ABCD1 gene encoding the ALD pro-
tein, an adenosine triphosphate‑binding cassette trans-
porter, cause the clinically heterogeneous disorder X-linked
adrenoleukodystrophy (ALD) [14]. In boys, ALD is a
severe brain inflammation and demyelination disease.
Transfusion of autologous HSCs that were ex vivo infected
with ABCD1-encoding lentivirus in two patients showed a
halt in disease progression in less than 2 years from onset
of gene therapy, even while reconstitution of myeloid and
lymphoid cell types was only 9–14 % [14]. This report
indicates that ABCD1-deficient microglia in the CNS could
be replaced by BMDM carrying the wild-type ABCD1
allele. It was also described in ABCD1-null and myelin-
associated glycoprotein-deficient mice models of ALD that
a combined lack of both functional genes in metabolic con-
trol and myelin integrity led to an additive effect on micro-
glia activation and axonal degeneration [25].
Outlook
Recent developments in investigative tools for immunol-
ogy, imaging and genetics have progressed our under-
standing of the unique nature of microglia in development,
homeostasis and disease. However, we are just beginning
to decipher the enigmatic nature of microglia. For future
experiments in mouse model systems, it would be vital to
take advantage of recently developed microglia-specific
genetic systems [39, 98, 141]. In particular, the use of these
inducible systems for gene depletion in microglia would
reduce the observation of artifacts arising from peripheral
myeloid cells or other brain cell types in generic knockout
animals [19]. These investigations may potentially reveal
new intercellular interactions modulated by microglia and
may recapitulate aberrant phenotypes observed in humans.
As multifaceted guardian of the CNS, microglia possess a
specialized transcriptomic signature that encodes proteins
for detecting endogenous ligands and infectious agents
[51], and myriad membrane channels and receptors [57].
It is thus vital to continue exploring the possibilities where
these cells could be harnessed for gene delivery to the CNS,
or act as direct targets of pharmacological interventions.
Acknowledgments This work was supported by the DFG funded
research unit (FOR) 1336 (to MP & SJ), the BMBF-funded Compe-
tence Network of Multiple Sclerosis (KKNMS to MP), the Compe-
tence Network of Neurodegenerative Disorders (KNDD to MP), the
Centre of Chronic Immunodeficiency (CCI to MP), and the DFG (PR
577/8-2 to MP and TA1029/1-1 to TLT).
328 Acta Neuropathol (2014) 128:319–331
1 3
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