015 The immune system as a novel regulator of sex differences in brain and behavioral development

Prévia do material em texto

Review
The Immune System as a Novel Regulator
of Sex Differences in Brain and Behavioral
Development
Lars H. Nelson1,2 and Kathryn M. Lenz2,3,4*
1Program in Neuroscience, The Ohio State University, Columbus, Ohio
2Group in Behavioral Neuroendocrinology, The Ohio State University, Columbus, Ohio
3Department of Psychology, The Ohio State University, Columbus, Ohio
4Department of Neuroscience, The Ohio State University, Columbus, Ohio
Sexual differentiation of the brain occurs early in life as a
result of sex-typical hormone action and sex chromo-
some effects. Immunocompetent cells are being recog-
nized as underappreciated regulators of sex differences
in brain and behavioral development, including microglia,
astrocytes, and possibly other less well studied cell
types, including T cells and mast cells. Immunocompe-
tent cells in the brain are responsive to steroid hormones,
but their role in sex-specific brain development is an
emerging field of interest. This Review presents a sum-
mary of what is currently known about sex differences in
the number, morphology, and signaling profile of immune
cells in the developing brain and their role in the early-life
programming of sex differences in brain and behavior. We
review what is currently known about sex differences in
the response to early-life perturbations, including stress,
inflammation, diet, and environmental pollutants. We also
discuss how and why understanding sex differences in
the developing neuroimmune environment may provide
insight into understanding the etiology of several neuro-
developmental disorders. This Review also highlights
what remains to be discovered in this emerging field of
developmental neuroimmunology and underscores the
importance of filling in these knowledge gaps. VC 2016
Wiley Periodicals, Inc.
Key words: development; sex differences; sex; hor-
mone; microglia; astrocytes; brain; behavior; immune
response; inflammation
Recent research has demonstrated prominent sex differ-
ences in the number, morphology, and signaling of
immunocompetent cells within the brain, particularly
during development. This Review presents a summary of
the current knowledge about sex differences in immuno-
competent cells and their signaling during ontogeny
under baseline conditions as well as following early-life
perturbations. This Review also discusses how under-
standing sex differences in the developing neuroimmune
environment may provide insight into several neurodeve-
lopmental disorders. Throughout the Review, we
emphasize what remains to be discovered in this emerging
field of developmental neuroimmunology.
SEXUAL DIFFERENTIATION OF THE BRAIN
Sexual differentiation is the process by which the body is
made male or female typical during ontogeny. Sexual dif-
ferentiation is driven largely by hormones during the pre-
natal period. The male testes secrete sex steroid
hormones, particularly the androgens testosterone and
dihydrotestosterone. This androgen surge is responsible
for inducing male typical development of both the brain
and the peripheral organs. Androgens induce two parallel
developmental processes, masculinization, which is the
SIGNIFICANCE
Many differences between males and females in brain development
and behavior may be attributed to prominent sex differences in
immune cells in the immature brain. This Review discusses the rele-
vance of sex differences in the brain’s immune cells to sex differences
in the response to early-life infection, stress, or diet as well as how
the immune system may contribute to sex differences in the incidence
of developmental psychiatric disorders.
Contract grant sponsor: National Institutes of Health; Contract grant
number: R21MH105826; Contract grant sponsor: Brain and Behavior
Research Foundation; Contract grant sponsor: The Ohio State University
(to K.M.L.).
*Correspondence to: Kathryn M. Lenz, Group in Behavioral Neuroen-
docrinology, The Ohio State University, Columbus, OH 43210. E-mail:
lenz.56@osu.edu
Received 15 April 2016; Revised 9 June 2016; Accepted 13 June 2016
Published online 7 November 2016 in Wiley Online Library
(wileyonlinelibrary.com). DOI: 10.1002/jnr.23821
VC 2016 Wiley Periodicals, Inc.
Journal of Neuroscience Research 95:447–461 (2017)
active induction of male-typical features, and defeminiza-
tion, which is the removal of female-typical features. The
lack of androgen exposure in the female brain leads to
two corresponding processes, feminization, which is the
induction of female-typical brain development, and
demasculinization, which is the prevention of male-
typical development. In rodents, androgens are converted
into estrogens in the brain by the enzyme P450 aroma-
tase. Thus, masculinization and defeminization of the
brain are both dependent on estrogen action on estrogen
receptors (for review see McCarthy, 2008). Male-typical
hormone exposure during this developmental period pro-
grams gross size of several brain regions; the number of
cells, synapses, dendritic spines, functional connectivity,
signaling among brain regions and gene expression
(McCarthy, 2008). All of these developmental differences
are relatively permanent and are collectively referred to as
the organizational effects of hormones (Phoenix et al., 1959;
Arnold, 2009). This early organization programs lifelong
sex-specific behavioral repertoires that are essential for
reproductive and nonreproductive behaviors, including
sex differences in copulatory solicitation and reflexes,
parental behavior, territorial and maternal aggression,
emotionality, cognition, cognitive strategy, and sensory
processing (for reviews see Balthazart and Ball, 1995;
Morris et al., 2004; McCarthy and Arnold, 2011). In
humans and nonhuman primates, the organizational peri-
od for sexual differentiation begins prenatally and extends
into the early postnatal period (Forest et al., 1973; Reyes
et al., 1973; Gendrel et al., 1980). However, unlike the
case in rodents, masculinization of the primate brain relies
on androgens acting on androgen receptors (for reviews
see Morris et al., 2004). Many research studies have been
devoted to understanding which developmental processes
hormones regulate to produce sex differences in the brain,
for example, cell genesis vs. programmed cell death or
synaptogenesis vs. synaptic pruning (for review see Lenz
et al., 2012). Sex steroid hormones bind to their nuclear
receptors, which home to hormone response elements on
DNA and regulate gene transcription (for review see
McCarthy, 2008). However, only in a select few cases
have the downstream gene expression profiles and signal-
ing molecules regulating sexual differentiation of the brain
been elucidated (see, e.g., Nugent et al., 2015).
Sex chromosome complement also plays a role in
sex-specific development of brain and behavior. This role
for sex chromosome has been elegantly demonstrated
with the “four core genotypes” mouse model (De Vries
et al., 2002). In this model, the gene responsible for male
gonadal development (the sex-determining region of the
Y chromosome, or SRY) has been relocated from the Y
chromosome to an autosome. This model allows for dis-
sociation of gonadal/hormone vs. sex chromosome con-
tributions to sex differences in brain and behavior (Arnold
and Chen, 2009). These studies reveal that reproductive
behaviors are programmed primarily by gonadal and hor-
monal sex, whereas addiction, habit learning, obesity, and
progression of autoimmune neuropathology show a sig-
nificant hormone-independent role for sex chromosome
complement (De Vries et al., 2002; Gatewood et al.,
2006; Quinn et al., 2007; Arnold and Chen, 2009; Chen
et al., 2009; Kopsida et al., 2013).
Y chromosome genes have been detected in many
tissues during ontogeny, including the rodent brain (for
review see Sekido, 2014), and throughout life in humanbrain (Mayer et al., 1998; Dewing et al., 2006; Czech
et al., 2012). The role that Y chromosome genes play in
governing the process of brain sexual differentiation is
unknown. A recent analysis of Y chromosome evolution
suggests that Y chromosome genes duplicate the function
of the X chromosome genes that are expressed more
highly in females (Bellott et al., 2014). An important
future research question asks how X–Y pair genes might
serve to influence sex-specific brain development. It is
also unknown whether individual X chromosome genes
contribute to sexual differentiation of the brain. Given
the context of this Review, it is fascinating that the X
chromosome has the highest concentration of immune
genes of the entire genome (Bianchi et al., 2012). The
high concentration of immune genes on the X chromo-
some has been proposed as a possible reason that females
have a higher incidence of several autoimmune conditions
that occur postpuberty (Bianchi et al., 2012). However,
only a few studies have investigated whether X-linked
immune genes are expressed in the brain during develop-
ment (see, e.g., Xu et al., 2006) or whether these genes
influence sex-specific brain development. Future research
should employ a combination of the four core genotypes
mouse model and specific manipulations of sex chromo-
some genes to add substantial insight into the genetic
mechanisms that govern sexual differentiation of the
brain.
SEX DIFFERENCES IN IMMUNOCOMPETENT
CELLS AND INFLAMMATORY SIGNALING
DURING DEVELOPMENT
Immunocompetent cells reside in the central nervous sys-
tem (CNS) under healthy conditions. Brain-resident cell
types include microglia, dendritic cells, and mast cells, all
of which are cells of peripheral origin. The same immune
cells reside within the meninges along with immune cells
of the adaptive immune system, such as T cells and B
cells. Astrocytes are derived from neural stem cells, but
they also secrete and respond to inflammatory mediators,
so astrocytes are also included in this Review of sex dif-
ferences in immunocompetent cells. Sex differences in
immunocompetent cells in the brain have been found
during the critical period for sexual differentiation, with
accompanying sex differences in cytokine or chemokine
levels. This section summarizes those findings and high-
lights important questions that remain with regard to sex
differences in neuroimmunology during development.
Microglia
Microglia are the tissue-resident macrophages of the
brain. Microglia are derived from the embryonic yolk sac
before either the fetal liver or the bone marrow begins
448 Nelson and Lenz
Journal of Neuroscience Research
producing immune cells (Alliot et al., 1999; Ginhoux
et al., 2010). Microglial progenitors initially infiltrate the
rodent CNS on embryonic days 8–9.5 (Alliot et al., 1999;
Ginhoux et al., 2010). Microglia rapidly proliferate
(Ajami et al., 2007; Nikodemova et al., 2015) and reach
maximal numbers approximately 4–5 weeks later (Niko-
demova et al., 2015). Microglia subsequently undergo a
cull that mirrors the overproduction and programmed loss
of neurons in the developing brain (Nikodemova et al.,
2015). In the developing human brain, microglia are first
detectable at 4.5 weeks of gestation, ostensibly entering
the brain via meninges, choroid plexus, and ventricles
(Verney et al., 2010). Later in gestation, microglia contin-
ue to infiltrate the brain via a transvascular route and then
proliferate (Verney et al., 2010). Microglia then sustain
their numbers locally throughout adulthood without
being replenished by peripheral macrophages under base-
line conditions (Hickey et al., 1992; Alliot et al., 1999;
Ajami et al., 2007). If depleted in adulthood, microglia
can be replenished from latent nestin-positive progenitor
cells within days (Elmore et al., 2014). Thus, the brain
somehow senses microglial loss and stimulates microglial
repopulation, which suggests that maintaining microglial
homeostasis is adaptive in the brain.
In the healthy adult brain, most microglia are in a
“ramified” or “surveying” state. Ramified/surveying
microglia have small cell bodies and many long, thin pro-
cesses that are used to sample the local microenviron-
ment dynamically for pathogens, debris, or dying cells
(Nimmerjahn et al., 2005; Hanisch and Kettenmann,
2007). When ramified/surveying microglia encounter
such detritus, they engulf and digest it in a process called
phagocytosis (Kettenmann et al., 2011). Under patholog-
ical conditions (e.g., brain injury, infection, chronic
stress, or neurodegeneration), microglial branches often
thicken or may even retract to take on an enlarged,
round, ameboid shape. These so-called “activated”
microglia then rapidly mobilize toward sites of injury/
damage (Kettenmann et al., 2011). After having been
activated, microglia engage in increased phagocytosis to
control or mitigate damage and prolonged stress to the
nearby tissue. Activated microglia secrete both proin-
flammatory cytokines and chemokines to recruit other
immune cells to sites of damage, and they also secrete
anti-inflammatory cytokines and growth factors to stimu-
late regrowth and repair within the CNS (Kreutzberg,
1996; Lenzlinger et al., 2001). Immature microglia in the
developing brain more closely resemble these activated
microglia that are also seen in the injured adult brain.
During development, microglia secrete high levels of
inflammatory mediators under healthy conditions
(Schwarz et al., 2012; Lenz et al., 2013; Schafer and Ste-
vens, 2013; Nikodemova et al., 2015). These mediators
are implicated in many normal processes of development,
including cell proliferation, differentiation, migration,
cell death, and synaptic patterning (for review see
Kettenmann et al., 2011; Schafer et al., 2013; Lenz and
McCarthy, 2015). In the context of sexual differentiation
of the brain, however, fairly little is known about the
contribution of microglia. The following section summa-
rizes what is currently known with regard to sex differ-
ences in microglia during development and how
microglia might impact sex-specific brain development.
Sex Differences in Microglia
There are many sex differences in microglial num-
ber, phenotype, and gene expression profiles during early
ontogeny. The data presented in this section are summa-
rized in Figure 1 (lane 1, with blue boxes indicating a
male bias in the stated outcome, red boxes indicating a
female bias, and purple boxes indicating that no sex differ-
ence was observed).
Male and female rats do not show sex differences
in microglial number in the brain prior to the onset of
testicular androgen secretion (Schwarz et al., 2012).
During the organizational period for sexual differentia-
tion, sex differences in microglia begin to emerge. On
postnatal day (PN) 0, females have a transient increase
in microglial number in the paraventricular nucleus of
the hypothalamus, hippocampus, and amygdala relative
to males (Schwarz et al., 2012). By PN4, males show
greater numbers of ameboid microglia in the hippocam-
pus, amygdala, and cortex relative to females (Schwarz
et al., 2012). These sex differences diminish by the
adolescent period (Schwarz et al., 2012). The timing of
these dynamic changes in microglia across the perinatal
period suggests that gonadal hormones regulate sex dif-
ferences in microglia. In the neonatal medial preoptic
area (POA), males have 30% more microglia overall
and twofold more ameboid microglia at PN0–2 than
females (Lenz et al., 2013). Treating females with a
male-typical dose of estradiol induces increased micro-
glial number and ameboid microglia (Lenz et al., 2013).
More subtle sex differences in the number of ramified
microglia have been reported in the cerebellum, with
females having more than males, but no overall differ-
ences in ameboid microglia or total microglia during
thelate neonatal period were found (Perez-Pouchoulen
et al., 2015). We have demonstrated that sex differences
in microglial number in the POA have consequences
for sex-typical behavioral development (see below), but
the functional role of sex differences in microglia in
other brain regions (e.g., Schwarz et al., 2012) has not
yet been identified. Thus, future studies should attempt
to connect these sex differences in microglia with nor-
mal developmental processes in the brain or sex-specific
behavioral outcomes.
How do these sex differences in microglia in the
developing brain occur? Several mechanisms are possi-
ble. Sex differences in microglial number may result
from sex differences in chemotactic signals that differen-
tially recruit the cells in males and females (Schwarz
et al., 2012; see below for discussion). Alternatively,
microglia may proliferate at different rates in males and
females after they have colonized the brain. Microglial
numbers in the developing mouse brain decrease
dramatically in the third postnatal week because of
Immune System, Sex Differences, and Brain and Behavioral Development 449
Journal of Neuroscience Research
apoptosis (Nikodemova et al., 2015), so it is possible that
microglia in the male vs. the female brain may undergo
apoptosis at different rates or across different develop-
mental trajectories. Sex differences in microglial coloni-
zation, proliferation, and survival likely converge to
induce greater microglial numbers in the developing
male brain. Future studies are required to tease out the
relative contribution of these mechanisms across differ-
ent brain regions and time points.
Inflammatory Signaling
Several groups have documented basal sex differ-
ences in cytokine, chemokine, and other inflammatory
mediators in the developing brain. From PN0 to PN4,
males have higher levels of the chemokines CCL20 and
CCL4 in the hippocampus and cortex (Schwarz et al.,
2012). In contrast, females have higher levels of the
proinflammatory cytokine interleukin (IL)-1b, its recep-
tors IL-1 receptor antagonist (IL-1Ra) and IL-1 receptor
Fig. 1. Sex differences in innate immune cells, signaling, and the
response to early-life perturbations. Summary of known sex differ-
ences in microglia (lane 1), inflammatory signaling (lane 2), and other
immunocompetent cells (lane 3) in the developing brain. Lane 4 sum-
marizes the known sex-specific behavioral responses to early-life per-
turbations as well as sex differences in neurodevelopmental disorders
or disorder risk following inflammation. Blue shading indicates an
effect that is more pronounced in males than in females; red shading
indicates an effect that is more pronounced in females than in males;
purple shading indicates an effect that is equally pronounced in males
and in females. Amyg, amygdala; BG, basal ganglia; CB, cerebellum;
Ctx, cortex; HPC, hippocampus; Hypo, hypothalamus; TLR4, Toll-
like receptor 4.
450 Nelson and Lenz
Journal of Neuroscience Research
(IL-1R), and the chemokine CCL22 and its receptor,
CCR4 (Schwarz et al., 2012). Within the amygdala,
females have much higher expression of the chemokine
CXCL9 (Schwarz et al., 2012). With regard to the differ-
entially expressed chemokines, Schwarz et al. (2012)
hypothesize that these chemokines are responsible for sex
differences in the recruitment of microglia to particular
brain regions and critical periods of sex-specific develop-
ment. However, chemokines have not been studied in
great detail within the healthy brain, especially during
development. CCL20 has been implicated in the recruit-
ment of immune cells into the CNS in disorders such as
multiple sclerosis (Reboldi et al., 2009) and after brain
injury (Das et al., 2011). Microglia, astrocytes, and neu-
rons express myriad chemokine receptors, which are G-
protein-coupled receptors that activate various second
messenger cascades (for review see Cartier et al., 2005).
The fact that chemokines and their receptors are detect-
able within the healthy brain suggests that they have func-
tional roles in brain development that have not been
investigated. Additional studies are required to elucidate
how chemokines regulate brain development in both
sexes.
For the POA, we found that microglia are a signifi-
cant source of the inflammatory lipid prostaglandin E2
(PGE2). PGE2 secretion is induced by male-typical expo-
sure to estradiol early in life (Amateau and McCarthy,
2002a). This PGE2 surge induces a twofold increase in
dendritic spines on POA neurons in males and resultant
male-typical sexual behavior in adulthood (Amateau
and McCarthy, 2002a, 2004; Wright and McCarthy,
2009). Inhibiting microglial activation with the anti-
inflammatory drug minocycline prevents estradiol from
inducing male-typical PGE2 levels and increasing dendrit-
ic spine density in the POA (Lenz et al., 2013). Further-
more, minocycline blocks male-typical copulatory
behavior in adulthood (Lenz et al., 2013). Thus, in the
POA, microglia are crucial to sex-typical organization of
the brain and resultant reproductive behavior later in life.
Currently, this is the only data set that directly links sex-
specific microglial properties with a sex-specific behavior-
al outcome in adulthood.
In the nearby anteroventral periventricular (AVPV)
nucleus of the POA, sex differences in immune signaling
instead serve the processes of feminization in females and
defeminization in males. The AVPV is larger in females
than in males because of estradiol-mediated cell death in
males (Sumida et al., 1993). Higher levels of cell death in
males are driven by the tumor necrosis factor (TNF) fami-
ly member repressor protein TRIP (Krishnan et al.,
2009). The higher expression of TRIP in males prevents
the induction of a cell survival cascade that is induced by
TNFa in females. The result of this differential signaling
is that GABAergic cells of the AVPV undergo more cell
death in males than in females (Krishnan et al., 2009). In
addition to the sex differences in TRIP that regulate sex-
specific cell death in the AVPV, several other immune
genes related to TNF signaling showed significant sex dif-
ferences in the Krishnan et al. (2009) study, including
TRAIL (TNFsf10) and Traf family member nuclear
factor-jB activator. The functional roles of these other
immune genes in the AVPV have not yet been identified.
Two points are worth noting with regard to these studies
on the mechanisms governing sexual differentiation of the
POA. The first is that two nearby regions of the POA
both rely on immune mediators to produce sex differ-
ences in neuronal number and morphology following
exposure to sex-specific hormonal signals, suggesting that
immune mediators may govern sexual differentiation of
other brain regions. The second is that the AVPV (studied
by Krishnan et al., 2009) and medial POA (studied by
Lenz et al., 2013) are very close together anatomically and
yet rely on different immune mediators to produce sexual
dimorphism. This suggests that very regionally specific
cues orchestrate inflammatory signaling to induce sex dif-
ferences in the brain.
Primary microglia exhibit sex differences in gene
expression profiles as well. In mice, microglia from PN3
males have significantly lower expression of the puriner-
gic receptors P2X5 and P2Y4 than microglia from females
and higher expression of the P2X4 receptor at PN21
(Crain et al., 2009). The functional significance of these
sex differences is unknown; however, purinergic signaling
is important for microglial transition from a quiescent to
an activated/phagocytic phenotype (for review see Koizu-
mi et al., 2013). Purinergic receptors are also important
for neuronal–glial cross-talk during normal brain develop-
ment (for review see Del Puerto et al., 2013). Thus, these
sex differences may be important for sex differences in
cell genesis,axonal outgrowth, or synaptic connectivity
(Del Puerto et al., 2013).
Primary microglia from whole mouse brain on PN3
show higher expression of the proinflammatory cytokines
TNFa, IL-1b, and IL-6 in females relative to males as
well as higher levels of the anti-inflammatory cytokine
IL-10 (Crain et al., 2013). These sex differences disappear
by PN21, again suggesting an organizational role for ste-
roid hormones in producing the effects (Crain et al.,
2013). A difficulty in interpreting these data is that
whole-brain microglia were assessed and regional differ-
ences were not documented. Thus, the same group ana-
lyzed microglia in different brain regions and found that
microglia in cerebral cortex of female mice on PN21 had
significantly higher levels of the inducible nitric oxide
synthase gene expression than those of males, and brain-
stem microglia showed no such sex difference (Crain and
Watters, 2015). At PN21, microglia from the male cere-
bellum showed greater expression of purinergic receptors
P2X4 and P2X7 than microglia from other brain regions
or any brain region in females (Crain and Watters, 2015).
Nevertheless, the biological significance of most docu-
mented sex differences in immune molecules in brain
development is unknown. Overall, most studies that have
examined males and females have shown more substantial
sex differences in the early postnatal period than in later
development, and studies have also tended to find more
proinflammatory tone in the male brain. These sex differ-
ences in the neuroimmune system may prove particularly
Immune System, Sex Differences, and Brain and Behavioral Development 451
Journal of Neuroscience Research
relevant for sex-specific brain development. The data
presented in this section are summarized in Figure 1
(lanes 1, 2).
Other Innate Immune Cells
There are other immunocompetent cells in the
CNS, including dendritic cells, mast cells, and neutro-
phils. In the periphery, dendritic cells engage in antigen
presentation and activate T cells (D’Agostino et al., 2012).
Dendritic cells preferentially secrete the cytokines IL-12
and interferons to activate T cells and recruit macro-
phages, respectively (Mildner and Jung, 2014). In the
rodent CNS, dendritic cells are also present under normal
conditions. With a CD11c-yellow fluorescent protein
reporter mouse, dendritic cells have been detected in the
male brain parenchyma beginning in the embryonic peri-
od and extending into adulthood (Bulloch et al., 2008).
In adulthood, dendritic cells are found throughout the
forebrain, primarily in the hippocampus, ventricular
zones, extended amygdala, and white matter tracts (Bul-
loch et al., 2008). These dendritic cells closely resemble
microglia in morphology and can coexpress putative
microglial markers (Bulloch et al., 2008). Thus, studies of
microglia using these markers (such as CD11b or ionized
calcium-binding adaptor molecule 1) may in fact be
studying microglia and dendritic cells. Brain-resident den-
dritic cells respond to seizure and medial cerebral artery
occlusion both with morphological change and by hom-
ing to site of injury (Bulloch et al., 2008; Felger et al.,
2010). Studies on brain dendritic cells have been per-
formed only in males thus far, so it remains to be deter-
mined whether there are any sex differences in brain-
resident dendritic cell numbers or phenotype and whether
they participate in healthy brain development.
Another type of innate immune cell, the mast cell,
also resides within the brain parenchyma. Mast cells are
granulated innate immune cells of hematopoetic origin
and are responsible for type I hypersensitivity conditions,
such as food allergy, atopy, and asthma as well as the
response to pathogens (Abraham and St. John, 2010).
Although most mast cells are found in peripheral tissues,
including the skin, colon, and airway, mast cells also
locate to the normal, healthy brain. Mast cells colonize
the rodent brain prenatally, localizing near brain micro-
vessels inside the blood–brain barrier (BBB; Khalil et al.,
2007). In male mice, mast cell numbers in the entire brain
increase until PN21, and the proportion of those mast
cells located in or near the hippocampus peaks during the
first postnatal week (Nautiyal et al., 2012). In humans,
mast cells are found in the brain during infancy and are
most commonly found in brains of individuals less than
19 years old (Dropp, 1979; Ma�sli�niska et al., 2001). Under
baseline conditions, brain mast cells undergo a process
called degranulation. Mast cell granules contain primarily
serotonin, histamine, and several proteases, and these
granules are capable of traveling to sites hundreds of
micrometers away from the putative site of release (for
review see Silver and Curley, 2013). Pharmacological
activation of mast cells in the brain leads to increased
microglial activation and a spike in proinflammatory cyto-
kines (Dong et al., 2016), suggesting that cross-talk
between mast cells and microglia occurs within the brain.
Mast-cell–deficient (male) mice have altered anxiety
behavior, learning and memory, and stress reactivity in
adulthood (Nautiyal et al., 2008, 2012), but it is unknown
whether these effects of mast cell loss are due to develop-
mental effects or whether they occur in both males and
females. Mast cells release the hormone gonadotropin-
releasing hormone (Khalil et al., 2003), and mast cell
numbers increase in the brain following hormonal manip-
ulations and during sexual behavior (Zhuang et al., 1993,
1997), so sex differences in mast cells may be relevant for
sex-specific physiology. In the developing POA of rats,
the mast-cell–derived enzyme mast cell protease 2 is
detectable only in males (Nugent et al., 2015). We are
currently investigating whether this sex difference in mast
cell signaling is relevant for sexual differentiation of the
brain and behavior or sex differences in the response to
early-life inflammatory events. The fact that mast cells are
prevalent within the brain during the perinatal period and
signal to microglia suggests that they are likely unappreci-
ated contributors to normal brain development.
Astrocytes
Astrocytes are not embryologically related to micro-
glia but are instead derived from neural stem cells (Sofro-
niew and Vinters, 2010). However, astrocytes are
considered immunocompetent cells because they are
capable of antigen presentation, express a variety of toll-
like receptors and cytokine receptors, and secrete inflam-
matory molecules (Dong and Benveniste, 2001). Devel-
opmental sex differences in astrocyte number and
morphology have been documented in the POA (Ama-
teau and McCarthy, 2002b) and hypothalamus (Mong
et al., 1999). Astrocytes in the neonatal male hypothala-
mus have a more complex, stellate morphology, whereas
astrocytes in females are bipolar in appearance (Mong and
McCarthy, 2002). These sex differences in astrocyte mor-
phology are organized by early-life exposure to male-
typical hormones (Mong and McCarthy, 2002). Glial
fibrillary acidic protein (GFAP) is an intermediate fila-
ment protein that is preferentially expressed in astrocytes
(for review see Sofroniew and Vinters, 2010; Middeldorp
and Hol, 2011). GFAP is often used as an astrocytic mark-
er, although it does not always stain every astrocyte with-
in the brain (Sofroniew and Vinters, 2010; Middeldorp
and Hol, 2011). There are sex differences in GFAP
immunostaining in the adult hippocampus, globus pal-
lidus, and hypothalamus but not in the number of GFAP-
positive cells (Garcia-Segura et al., 1988). Steroid hor-
mone exposure in neonatal females increases GFAP stain-
ing to male-typical levels, and castration of neonatal males
reduces GFAP staining in these regions (Garcia-Segura
et al., 1988). Although astrocytes appear to be sexually
differentiated in many brain regions, the functional signif-icance of these sex differences for normal brain function
452 Nelson and Lenz
Journal of Neuroscience Research
or the response to early-life perturbations remain open
questions. Astrocytes are important mediators of synaptic
activity and neurotransmitter levels within the brain, and
astrocytes also can cross-talk with microglia by releasing
inflammatory mediators, so determining the role of sex-
specific astrocyte function in the developing brain is an
important area of future inquiry.
Adaptive Immune Cells
T cells and B cells are cells of the adaptive immune
system prevalent in the meninges and cerebrospinal fluid
(for review see Hickey, 2001; Filiano et al., 2015). These
adaptive immune cells have been implicated in normal
brain function. Severe combined immunodeficiency mice
lack B cells and T cells and display significant deficits in
learning and memory tasks (Brynskikh et al., 2008). These
effects are attributed to CD41 T cells in the meninges,
which may signal to microglia, astrocytes, or other brain
cells across the blood brain barrier (BBB) (Radjavi et al.,
2014a,b; Filiano et al., 2015). The role that adaptive
immune cells play in brain development and specifically
in brain sexual differentiation has been recently investigat-
ed. Mice that congenitally lack T cells show a loss of vol-
umetric sex differences in several brain regions (Rilett
et al., 2015). The most dramatically affected brain region
is the bed nucleus of the stria terminalis (BNST), which is
a highly sexually dimorphic brain structure that regulates
mood and reproductive behavior (for review see De Vries
and Forger, 2015). Female T-cell–deficient mice show
increased BNST volume relative to wild-type females,
which represents a masculinization of the brain region
(Rilett et al., 2015). T-cell deficiency also diminishes sex
differences in anxiety-like behavior and exploration on
the open-field test (Rilett et al., 2015), behaviors that
strongly depend on the BNST. These studies implicate T
cells in normal sexual differentiation of the brain. Because
T cells do not reside in the brain, future research should
seek to determine whether peripheral immune cells influ-
ence the CNS via mediating effects on humoral signaling,
the vagus nerve, or BBB function. The data presented in
the preceding three sections are summarized in Figure 1
(lanes 2, 3).
HORMONAL REGULATION OF BRAIN-
RESIDENT IMMUNOCOMPETENT CELLS
AND INFLAMMATORY SIGNALING
Immunocompetent cells of the brain express steroid hor-
mone receptors and are regulated by hormones. For the
POA, we did not detect any colocalization between
immunostaining for estrogen receptor a (ERa) and the
microglial marker isolectin (Lenz et al., 2013), so estro-
gens may affect microglia in the POA via other estrogen
receptor isoforms or indirectly via either neurons or astro-
cytes. Microglia isolated from whole mouse brain on PN3
express ERa, and transcript levels increase across devel-
opment in both males and females (Crain et al., 2013).
No sex differences in levels of ERa were detected at any
age (Crain et al., 2013). In a followup study, microglia
isolated from different brain regions were compared, and
cortical microglia demonstrated higher ERa gene expres-
sion than brainstem/spinal cord microglia (Crain and
Watters, 2015). At 3 and 7 weeks of age but not at older
ages, cortical microglia from males showed higher ERa
expression than those from females (Crain and Watters,
2015). In contrast, ERb was undetectable in mouse
microglia throughout life (Crain et al., 2013). Several
research groups have reported detectable ERb in micro-
glial cell lines, primary cultured microglia, or adults fol-
lowing immune activation or stroke (Vegeto et al., 2001;
Takahashi et al., 2004; Liu et al., 2005; Baker et al.,
2011). Thus, microglial ERb may play a preferential role
in the adult brain’s response to stressors. In contrast, the
effects of estrogens on normal microglial function may
depend more on ERa or nonnuclear estrogen receptors,
such as the G-protein–coupled estrogen receptor
GPER1. Microglia also highly express the glucocorticoid
receptor, suggesting that microglia may be more sensitive
to stress hormones than to sex hormones (Sierra et al.,
2008). Few studies have examined androgen receptor
function in microglia, but low levels have been detected
at baseline in rodent microglia (Garcia-Ovejero et al.,
2002; Brown et al., 2007). Under Gestational/early-life
stress below, we discuss more fully the effects of early-life
stress on microglia and other neuroimmune endpoints.
Estrogens exert anti-inflammatory effects in the
adult brain following injury or under neurodegenerative
or autoimmune conditions. These effects are mediated by
both microglia and astrocytes (Vegeto et al., 2008). Sex
hormones also regulate the response of both microglia
and astrocytes to immune challenge. In culture, microglia
from neonatal males showed a greater IL-1b response to a
high-dose challenge with the bacterial endotoxin lipo-
polysaccharide (LPS) than those from females (Loram
et al., 2012). Estradiol treatment led to a sexually diver-
gent response in IL-1b levels, with male microglia show-
ing suppression of IL-1b in response to LPS following
estradiol treatment but female microglia showing
enhanced IL-1b response (Loram et al., 2012). This
divergent response to the same hormone in male and
female microglia may be attributed to sex differences in
estrogen receptor density, second messenger cascades, or
differential expression of sex chromosome or autosomal
genes. Although they have been studied considerably less
than estrogens, androgens also exert anti-inflammatory
effects on microglia following brain injury and in Alz-
heimer’s disease models (see, e.g., Garcia-Ovejero et al.,
2002; Barreto et al., 2007; Brown et al., 2007). Injury
increases androgen receptor expression in microglia (Bar-
reto et al., 2007), and the anti-inflammatory effects of
androgens are mediated both by direct effects on andro-
gen receptors and by conversion of androgens into estro-
gens (Garcia-Ovejero et al., 2002).
Immune challenge also induces sex-specific
responses in astrocytes of the neonatal rodent brain. Cul-
tured astrocytes from neonatal males produce greater IL-
1b to LPS challenge than those from females (Loram
et al., 2012). Unlike estradiol effects on microglia,
Immune System, Sex Differences, and Brain and Behavioral Development 453
Journal of Neuroscience Research
estradiol has no effect on IL-1b release from astrocytes
(Loram et al., 2012), which suggests that the effects of
estradiol on the brain’s inflammatory response in neonates
may preferentially depend on microglia. Another study of
primary cultured astrocytes derived from PN1 mouse
brains showed no baseline sex differences in mRNA for
IL-6, TNFa, IL-1b, or the inflammatory chemokine
CXCL10; moreover, treatment with male-typical hor-
mones did not alter female levels of these transcripts (San-
tos-Galindo et al., 2011). However, in response to
immune challenge with LPS, male astrocytes showed a sig-
nificantly larger increase in IL-6, TNFa, and IL-1b and
decrease in CXCL10 than female astrocytes (Santos-
Galindo et al., 2011). Treating females with estradiol mas-
culinizes astrocyte gene expression patterns to male levels
(Santos-Galindo et al., 2011). Direct comparisons between
microglia and astrocytes have not been made in these cul-
ture experiments, so conclusions are hard to draw with
regard to the relative contribution of these two cell types
to the brain’s inflammatory response during development.
In addition, culture experiments that include only one
immunocompetent cell type cannot account for any cross-
talk between the two cell types that is likely to occur in
vivo. Future studies should employ coculture experiments
to tease out how microglia and astrocytes regulate eachother’s inflammatory function within the developing brain.
SEX DIFFERENCES IN RESPONSE TO
EARLY-LIFE PERTURBATIONS
Perinatal Infection/Inflammatory Challenge
Early-life immune challenge has been used in hun-
dreds of animal studies to perturb the developing immune
system and assess resultant changes in brain development
and lifelong function. Immune activation can be induced
with the bacterial endotoxin LPS, the viral mimetic poly-
I:C, exogenous cytokines (e.g., IL-6, TNFa), live bacte-
ria (Escherichia coli), or intact viruses. Although many
early-life immune activation studies have been performed,
relatively few have directly compared outcomes in males
and females. Reviewing the body of early-life immune
challenge studies is beyond the scope of this Review, but
we direct readers to other excellent reviews on the topic
(Meyer et al., 2009; Bilbo and Schwarz, 2012; Knuesel
et al., 2014). Here we briefly review the data on sex dif-
ferences in the response to early-life immune activation.
Microglial activation has been documented within
days after prenatal immune challenge (Cunningham et al.,
2013; Smith et al., 2014). Early-life immune challenge
induces increases in overall density and numbers of reac-
tive microglia throughout the adult brain in some studies
(Bland et al., 2010; Juckel et al., 2011; Sominsky et al.,
2012; Van den Eynde et al., 2014; Zhu et al., 2014; Man-
itz et al., 2016). In contrast, other groups have shown no
change in microglial gene expression and morphology in
response to prenatal immune challenge (Smolders et al.,
2015; Giovanoli et al., 2016). Sex differences in either
microglial density or expression of inflammatory markers
have not been observed in the adult brain following
perinatal immune challenge (Ratnayake et al., 2014; Van
den Eynde et al., 2014; Zhu et al., 2014). One study
assessed expression of CD11b, the complement receptor 3
known to be essential for microglial synaptic pruning, and
CD45, a marker for activated microglia following prenatal
immune activation with poly-I:C. In that study, microglia
from the male and female offspring of poly-I:C–treated
dams showed decreased CD11b but showed decreased
CD45 only in males (Manitz et al., 2016). These data
indicate that microglial signaling and phagocytic activity
may be differentially impacted in males and females.
Although microglial changes may not be dramatic follow-
ing early-life inflammatory events, these early-life pertur-
bations have been shown to “prime” microglia, such that
latent changes in microglial function or signaling may be
apparent only after a “second hit” of inflammation later in
life (Williamson et al., 2011). Thus, studying only changes
in baseline morphology and gene expression of microglia
may not be the best indicator that microgilal properties
have been impacted by early-life inflammation.
Maternal immune activation also induces sex-
specific changes in astrocyte markers in the brain. Prena-
tally challenged pups show increased expression of the gli-
al marker S100B in the frontal cortex and hippocampus
(de Souza et al., 2015). de Souza and colleagues (2015)
also found that GFAP levels were increased in prenatally
challenged animals at PN30 in the frontal cortex of males
and females, but only males had increased hippocampal
GFAP expression. Thus, astrocytes may show more pro-
nounced changes in morphology and gene expression
than microglia following prenatal immune challenge, and
males are more affected than females.
Even in the absence of dramatic changes in micro-
glial morphology, early-life immune activation induces
behavioral deficits in various tasks later in life. Deficits in
prepulse inhibition, latent inhibition, memory tasks,
motor tasks, and adolescent social interaction have been
observed in both males and females (Smith et al., 2007;
Meyer et al., 2008; Lin et al., 2012; Mattei et al., 2014;
Ratnayake et al., 2014; Van den Eynde et al., 2014; Zhu
et al., 2014; Aavani et al., 2015; Choi et al., 2016). Prena-
tal exposure to LPS has been shown to induce male-
specific deficits in juvenile social play behavior, with
females seemingly resilient to this immune perturbation
(Taylor et al., 2012). Additionally, male neonates chal-
lenged with TNFa show larger increases in anxiety and
despair-like behaviors later in life compared with females
(Babri et al., 2014).
Interpreting the body of early-life immune challenge
studies is complicated because the timing, type of chal-
lenge, and route of administration can influence the
maternal immune response and consequent fetal immune
response (Meyer et al., 2006a,b; Fortier et al., 2007; Mis-
sault et al., 2014). Moreover, a single immune challenge
alters different cytokines in the offspring brain across dif-
ferent time courses (Garay et al., 2013). In addition, many
of the studies that have included male and female subjects
have not been statistically powered to detect sex differ-
ences, for example, including only three or four males
454 Nelson and Lenz
Journal of Neuroscience Research
and females per condition, so future studies that are statis-
tically powered to detect sex differences following
immune challenge are essential to determine whether
there are sex-specific responses to early-life inflammation.
Gestational/Early-Life Stress
Prenatal stress is another early-life perturbation that
activates the neuroimmune system and induces lifelong
changes in brain and behavior. In humans, prenatal stress
increases circulating levels of cytokines in humans (see,
e.g., Coussons-Read et al., 2007). In rodents, prenatal
stress late in gestation can alter long-term microglial func-
tion, increasing proinflammatory signaling and shifting
microglia to a less ramified morphology (G�omez-Gonz�alez
and Escobar, 2010; Diz-Chaves et al., 2012, 2013). Prena-
tal stress also increases microglia reactivity to subsequent
immune challenge during adulthood in both males and
females (Diz-Chaves et al., 2012, 2013). Prenatal stress ear-
ly in gestation induces behavioral impairments, including
hyperactivity, increased behavioral despair and anhedonia,
and stress system dysregulation in males but not in females
(Mueller and Bale, 2008; Bronson and Bale, 2014). Male-
specific effects during early prenatal stress may result from a
larger inflammatory response in the male placenta (Mueller
and Bale, 2008; Bronson and Bale, 2014). Another prenatal
stress paradigm, bedding material restriction, leads to
increased glucocorticoid levels specifically in males (Bolton
et al., 2013). Early prenatal stress disrupts the masculiniza-
tion process in the hypothalamus via mediating effects on
micro-RNA expression in the brain (Morgan and Bale,
2011). Other work unrelated to stress has shown that
micro-RNAs, particularly mir-124, regulate the progres-
sion of microglia from an activated, phagocytic phenotype
to a mature, surveying phenotype in the developing brain
(Ponomarev et al., 2011; Svahn et al., 2016). Thus, sex dif-
ferences in microglia in the developing brain may depend
on sex differences in micro-RNAs, or sex differences in
the response to stress may in turn depend on differential
micro-RNA regulation of microglia.
Prenatal stress induces prolonged inflammation in
the brain. Microglia continue to release elevated levels of
inflammatory molecules into the early postnatal period
following prenatal stress (Slusarczyk et al., 2015). Mater-
nal or early-life stress can also interact with other inflam-
mogenic experiences to alter the CNS immune response.
A double hit of mild early-life stress and prenatal exposure
to diesel exhaust leads to increased brain levels of IL-1b,
Toll-like receptor 4, and caspase-1 and decreased levels of
anti-inflammatory cytokine IL-10, but only in male off-
spring. In fact, exposure to diesel exhaust increases IL-10
in the female brain (Bolton et al., 2013), suggestingthat
females have a more protective inflammatory response to
air pollution. In this same study, both males and females
showed increased anxiety-like behavior following this
double hit of early-life inflammation, but only males
showed cognitive deficits on contextual fear conditioning
(Bolton et al., 2013). Early-life maternal deprivation indu-
ces increased astrocyte immunostaining in the neonatal
hippocampus of both males and females, but only in the
cerebellar cortex of males (L�opez-Gallardo et al., 2008;
Llorente et al., 2009). These data once again suggest that
the male brain mounts a larger immune response to stress
during development. Overall, these data indicate that the
immune system is a major player in the brain’s response
to stress; thus, neuroimmune signaling may be responsible
for the early-life programming effects of stress on brain
and behavior. In addition, these data suggest that males
mount a larger inflammatory response in the brain and
periphery following early-life stress and also show more
behavioral disruptions following stress. It would be valu-
able to conduct future studies to determine why males
mount a larger immune response and appear more vulner-
able to early-life stress or, conversely, to determine what
endogenous factors protect females from a similar inflam-
matory response to early-life stress. In humans and rodent
models, females are more susceptible to stress-related
behavioral abnormalities and pathology in adulthood (for
a recent review see, e.g., Gobinath et al., 2015), so the
responses of males and females may be highly dependent
on developmental cues or differential effects of hormones
and sex chromosome genes across the life span.
Diet and Nutrition
Perinatal diet exposure also impacts neuroimmune
functioning in a sex-specific manner. Exposure to a high-
fat diet leads to male-specific alterations in development,
and prenatal exposure to diesel exhaust also leads to sex-
specific sensitivity to a high-fat diet in adulthood. Males
fed a high-fat diet following prenatal air pollution exposure
show dramatic weight gain and insulin resistance, whereas
females are resistant to these prenatal effects (Bolton et al.,
2014). Exposure to diesel exhaust in early life and adult
high-fat diet increase microglial activation markers within
the hippocampus of males but not females (Bolton et al.,
2014). Maternal high-fat diet influences fetal gene expres-
sion in the placenta differently in males and females, with
female placentae showing more genes to be affected and a
greater magnitude of altered expression (Mao et al., 2010).
Females also show greater changes in immune genes and
immune cell numbers following exposure to the inflamma-
tory experiences of preeclampsia or asthma in utero (Sood
et al., 2006; Scott et al., 2009). Overall female placentae
respond more dramatically following perturbations, and
females also are less affected behaviorally by these perturba-
tions. This suggests that the placental gene expression
changes in females may protect against the detrimental
effects of gestational perturbations. This provocative
hypothesis warrants further investigation.
High-fat diet or overnutrition during the perinatal
period also induces substantial neuroinflammation, and
possible sex differences in sensitivity to overnutrition have
been documented. In humans, male children of diabetic
women show greater metabolic dysregulation and
increased preterm birth and birth defects than female chil-
dren (Tundidor et al., 2012). In rodents, maternal high-
fat diet during pregnancy leads to increased microglial
Immune System, Sex Differences, and Brain and Behavioral Development 455
Journal of Neuroscience Research
densitometry and proinflammatory cytokine levels in the
brains of males and females (Bilbo and Tsang, 2010).
However, only male offspring exhibit increased body
weight, increased anxiety-like behavior, and increased
spatial learning acquisition in adulthood (Bilbo and Tsang,
2010). In contrast to prenatal exposure to a high-fat diet,
early postnatal overnutrition increases the stress axis
response to immune challenge in both male and female
offspring (Clarke et al., 2012). Together, these data sug-
gest that males are somewhat more susceptible to early-
life inflammogenic perturbations, particularly during the
prenatal period. Nevertheless, subtleties in the data are
numerous, and it is clear that the particular perturbation
experienced and outcome measured are important deter-
minants of whether males or females are more affected by
inflammogenic experiences during ontogeny. The data
presented under Sex differences in response to early-life
perturbations are summarized in Figure 1 (lane 4).
IMPLICATIONS: SEX DIFFERENCES IN
NEURODEVELOPMENTAL DISORDERS
There are sex differences in the prevalence of neurodeve-
lopmental disorders. Autism spectrum disorder, early onset
or severe schizophrenia, Tourette’s syndrome, and atten-
tion deficit hyperactivity disorder (ADHD) are all more
common in males than in females (Grossman et al., 2008;
Robertson, 2008; Xiang et al., 2010; Goldstein et al.,
2013; Wingate et al., 2014). Little is known about the sex-
specific mechanisms that result in higher rates of these neu-
rodevelopmental disorders. However, recent research has
linked altered steroid hormone levels during ontogeny
with an increased risk for autism in children. Males with
autism-spectrum disorder have higher amniotic fluid levels
of several steroid hormones during gestation, including
progesterone and testosterone (Baron-Cohen et al., 2015).
Polycystic ovary syndrome (PCOS) is a disorder that indu-
ces increased testosterone levels in women, and women
with PCOS are more likely to have a child with autism
(Kosidou et al., 2016). Higher maternal levels of the
hormone-sequestering protein a-fetoprotein as well as
lower levels of the estrogen estriol have also been associat-
ed with increased autism risk in offspring (Windham et al.,
2015). Overall these data suggest that changes in the steroid
hormone environment of developing fetuses may alter
brain development to produce autism.
High levels of inflammation during pregnancy, pos-
sibly because of infection, another immune-activating
agent, or autoimmune disorder, also increase the risk for
autism, ADHD, and schizophrenia (Brown et al., 2004,
2014; Brown, 2012; Canetta et al., 2014; Instanes et al.,
2015; Lee et al., 2015). Although these neurodevelop-
mental disorders are more common in males, immune
activation during gestation increases the risk for both
sexes (Brown et al., 2014; Canetta et al., 2014; Instanes
et al., 2015). Immune abnormalities have been demon-
strated in patients with autism-spectrum disorder, schizo-
phrenia, and Tourette’s syndrome. Post-mortem brains of
autistic and schizophrenic patients exhibit an activated
microglial phenotype, increased microglial density and
abnormal spacing of microglia and neurons (Vargas et al.,
2005; Morgan et al., 2010, 2012; Fillman et al., 2013).
Autistic and schizophrenic patients as well as those with a
high risk of psychosis have been shown to have higher
levels of microglial activation by using microglial-specific
ligands for positron emission tomography imaging (Suzuki
et al., 2013; Bloomfield et al., 2015). Post-mortem brain
tissue from Tourette’s syndrome patients also exhibits
increased microglial activation, and transcriptome data
from this tissue show increased expression of immune and
glial genes (Lennington et al., 2016). Collectively, these
data suggest that there may be microglial involvement in
these developmental disorders, but whether the increased
microglial activity is a cause or consequence of the disor-
der remains to be determined. It should be noted that few
human studies of neurodevelopmental disorders have
explicitly compared males and females. This may be
because the malepreponderance of autism, ADHD, and
Tourette’s often leads to underrepresentation of females
within human studies. Future research that adequately
represents females and directly compares the sexes would
be extremely valuable to our understanding of the sex-
specific mechanisms involved in these disorders.
Little is known about immune cells and inflammato-
ry mechanisms in the human brain during development.
Thus, a main question remaining is how might the
immune system and microglia shape development of the
brain in these neurodevelopmental disorders. In a recent
study, human male brains were shown to have higher
expression levels of genes within astrocyte and microglial
modules; people diagnosed with autism also showed
higher levels of the same immune and glial gene modules
(Werling et al., 2016). Werling et al. also showed that
autism risk genes in the cortices of developing humans
were equally expressed in males and females, so it is not
male overexpression of autism risk genes that accounts for
the male bias in autism. Instead, Werling and colleagues
(2016) hypothesize that autism risk genes could interact
downstream with normally sexually dimorphic processes
involving astrocyte and microglia functions to disrupt
brain development. Thus, higher expression of glial and
immune genes in males could increase male susceptibility
to developing autism if autism risk genes are also present.
This is a hypothesis that warrants experimental inquiry in
animal models and use of human brain tissue. It is vital to
gain knowledge of how hormones, the immune system,
and the nervous system interact during development to
understand fully the mechanisms that induce the occur-
rence of neurodevelopmental disorders. The data pre-
sented under Sex differences in response to early-life
perturbations are summarized in Figure 1 (lane 4).
CONCLUSIONS AND FUTURE DIRECTIONS
This Review presents a summary of that which is current-
ly known about sex differences in neuroimmune cells in
the developing brain and highlights what remains
unknown in the field. Currently, what is unknown
456 Nelson and Lenz
Journal of Neuroscience Research
greatly trumps what is known with regard to both sex dif-
ferences in the brain’s innate immune system and sex-
specific effects of early-life exposure to inflammogenic
experiences. It is crucial to characterize sex differences in
gene expression in innate immune cells across the prenatal
and postnatal periods further to add valuable insight with
regard to how innate immune cells influence sex-specific
brain development. Documenting a role for less well
studied immune cells may shed light on latent immune
factors that contribute to normal brain development, sex-
ual differentiation, and early-life programming of behav-
ior. Perinatal inflammation is a risk factor for many
neurodevelopmental disorders, and many of these same
disorders are more common in males than in females.
Therefore, we may increase our understanding of the eti-
ology of these disorders by gaining knowledge of the sex-
specific biology of the brain’s immune system.
ACKNOWLEDGMENTS
The authors thank Aarohi Joshi and our two expert
reviewers for their helpful comments on the Review.
CONFLICT OF INTEREST STATEMENT
The authors have no conflicts of interest to declare.
ROLE OF AUTHORS
KML designed the figure. LHN and KML planned and
wrote the Review.
REFERENCES
Aavani T, Rana SA, Hawkes R, Pittman QJ. 2015. Maternal immune
activation produces cerebellar hyperplasia and alterations in motor and
social behaviors in male and female mice. Cerebellum 14:491–505.
Abraham SN, St. John AL. 2010. Mast cell-orchestrated immunity to
pathogens. Nat Rev Immunol 10:440–452.
Ajami B, Bennett JL, Krieger C, Tetzlaff W, Rossi FM V. 2007. Local
self-renewal can sustain CNS microglia maintenance and function
throughout adult life. Nat Neurosci 10:1538–1543.
Alliot F, Godin I, Pessac B. 1999. Microglia derive from progenitors,
originating from the yolk sac, and which proliferate in the brain. Brain
Res Dev Brain Res 117:145–152.
Amateau SK, McCarthy MM. 2002a. A novel mechanism of dendritic
spine plasticity involving estradiol induction of prostaglandin-E2.
J Neurosci 22:8586–8596.
Amateau SK, McCarthy MM. 2002b. Sexual differentiation of astrocyte
morphology in the developing rat preoptic area. J Neuroendocrinol 14:
904–910.
Amateau SK, McCarthy MM. 2004. Induction of PGE2 by estradiol
mediates developmental masculinization of sex behavior. Nat Neurosci
7:643–650.
Arnold AP. 2009. The organizational-activational hypothesis as the foun-
dation for a unified theory of sexual differentiation of all mammalian
tissues. Horm Behav 55:570–578.
Arnold AP, Chen X. 2009. What does the “four core genotypes” mouse
model tell us about sex differences in the brain and other tissues? Front
Neuroendocrinol 30:1–9.
Babri S, Doosti MH, Salari AA. 2014. Tumor necrosis factor-alpha
during neonatal brain development affects anxiety- and depression-
related behaviors in adult male and female mice. Behav Brain Res 261:
305–314.
Baker AE, Brautigam VM, Watters JJ. 2011. Estrogen modulates micro-
glial inflammatory mediator production via interactions with estrogen
receptor b. Endocrinology 145:5021–5032.
Balthazart J, Ball GF. 1995. Sexual differentiation of brain and behavior
in birds. Trends Endocrinol Metab 6:21–29.
Baron-Cohen S, Auyeung B, Nørgaard-Pedersen B, Hougaard DM,
Abdallah MW, Melgaard L, Cohen AS, Chakrabarti B, Ruta L,
Lombardo MV. 2015. Elevated fetal steroidogenic activity in autism.
Mol Psychiatry 20:369–376.
Barreto G, Veiga S, Azcoitia I, Garcia-Segura LM, Garcia-Ovejero D.
2007. Testosterone decreases reactive astroglia and reactive microglia
after brain injury in male rats: role of its metabolites, oestradiol, and
dihydrotestosterone. Eur J Neurosci 25:3039–3046.
Bellott DW, Hughes JF, Skaletsky H, Brown LG, Pyntikova T, Cho T-
J, Koutseva N, Zaghlul S, Graves T, Rock S, Kremitzki C, Fulton RS,
Dugan S, Ding Y, Morton D, Khan Z, Lewis L, Buhay C, Wang Q,
Watt J, Holder M, Lee S, Nazareth L, Alfoldi J, Rozen S, Muzny DM,
Warren WC, Gibbs RA, Wilson RK, Page DC. 2014. Mammalian Y
chromosomes retain widely expressed dosage-sensitive regulators.
Nature 508:494–499.
Bianchi I, Lleo A, Gershwin ME, Invernizzi P. 2012. The X chromo-
some and immune associated genes. J Autoimmun 38:J187–J192.
Bilbo SD, Schwarz JM. 2012. The immune system and developmental
programming of brain and behavior. Front Neuroendocrinol 33:267–
286.
Bilbo SD, Tsang V. 2010. Enduring consequences of maternal obesity for
brain inflammation and behavior of offspring. FASEB J 24:2104–2115.
Bland ST, Beckley JT, Young S, Tsang V, Watkins LR, Maier SF, Bilbo
SD. 2010. Enduring consequences of early-life infection on glial and
neural cell genesis within cognitive regions of the brain. Brain Behav
Immun 24:329–338.
Bloomfield PS, Selvaraj S, Veronese M, Rizzo G, Bertoldo A, Owen
DR, Bloomfield MA, Bonoldi I, Kalk N, Turkheimer F, McGuire P,
de Paola V, Howes OD. 2015. Microglial activity in people at ultra
high risk of psychosis and in schizophrenia: an [11C]PBR28 PET brain
imaging study. Am J Psychiatry 173:44–52.
Bolton JL, Huff NC, Smith SH, Mason SN, Foster WM, Auten RL,
Bilbo SD. 2013. Maternal stress and effects of prenatal air pollution on
offspring mental health outcomes in mice. Environ Health Perspect
121:1075–1082.
Bolton JL, Auten RL, Bilbo SD. 2014. Prenatal air pollution exposure
induces sexually dimorphic fetal programming of metabolic and neuro-
inflammatory outcomes in adult offspring. Brain Behav Immun 37:30–
44.
Bronson SL, Bale TL. 2014. Prenatal stress-induced increases in placental
inflammation and offspring hyperactivity are male-specific and amelio-rated by maternal anti-inflammatory treatment. Endocrinology 155:
2635–2646.
Brown AS. 2012. Epidemiologic studies of exposure to prenatal infection
and risk of schizophrenia and autism. Dev Neurobiol. 72:1272–1276.
Brown AS, Begg MD, Gravenstein S, Schaefer CA, Wyatt RJ,
Bresnahan M, Babulas VP, Susser ES. 2004. Serologic evidence of pre-
natal influenza in the etiology of schizophrenia. Arch Gen Psychiatry
61:774–780.
Brown AS, Sourander A, Hinkka-Yli-Salom€aki S, McKeague IW,
Sundvall J, Surcel H-M. 2014. Elevated maternal C-reactive protein
and autism in a national birth cohort. Mol Psychiatry 19:259–264.
Brown CM, Xu Q, Okhubo N, Vitek MP, Colton CA. 2007. Andro-
gen-mediated immune function is altered by the apolipoprotein E gene.
Endocrinology 148:3383–3390.
Brynskikh A, Warren T, Zhu J, Kipnis J. 2008. Adaptive immunity
affects learning behavior in mice. Brain Behav Immun 22:861–869.
Bulloch K, Miller MM, Gal-Toth J, Milner TA, Gottfried-Blackmore A,
Waters EM, Kaunzner UW, Liu K, Lindquist R, Nussenzweig MC,
Immune System, Sex Differences, and Brain and Behavioral Development 457
Journal of Neuroscience Research
Steinman RM, McEwen BS. 2008. CD11c/EYFP transgene illuminates
a discrete network of dendritic cells within the embryonic, neonatal,
adult, and injured mouse brain. J Comp Neurol 508:687–710.
Canetta S, Sourander A, Hinkka-Yli-Salom€aki S, Leivisk€a J, Kellendonk
C, McKeague, IW, Brown AS. 2014. Elevated maternal C-reactive
protein and increased risk of schizophrenia in a national birth cohort.
Am J Psychiatry 17:960–968.
Cartier L, Hartley O, Dubois-Dauphin M, Krause K-H. 2005. Chemo-
kine receptors in the central nervous system: role in brain inflammation
and neurodegenerative diseases. Brain Res Rev 48:16–42.
Chen X, Grisham W, Arnold AP. 2009. X chromosome number causes
sex differences in gene expression in adult mouse striatum. Eur J Neu-
rosci 29:768–776.
Choi GB, Yim YS, Wong H, Kim S, Kim H, Kim SV, Hoeffer CA,
Littman DR, Huh JR. 2016. The maternal interleukin-17a pathway in
mice promotes autism-like phenotypes in offspring. Science 351:933–939.
Clarke MA, Stefanidis A, Spencer SJ. 2012. Postnatal overfeeding leads
to obesity and exacerbated febrile responses to lipopolysaccharide
throughout life. J Neuroendocrinol 24:511–524.
Coussons-Read ME, Okun ML, Nettles CD. 2007. Psychosocial stress
increases inflammatory markers and alters cytokine production across
pregnancy. Brain Behav Immun 21:343–350.
Crain JM, Watters JJ. 2015. Microglial P2 purinergic receptor and immu-
nomodulatory gene transcripts vary by region, sex, and age in the
healthy mouse CNS. Transcr Open Access 3:124.
Crain JM, Nikodemova M, Watters JJ. 2009. Expression of P2 nucleotide
receptors varies with age and sex in murine brain microglia.
J Neuroinflamm 6:24.
Crain JM, Nikodemova M, Watters JJ. 2013. Microglia express distinct
M1 and M2 phenotypic markers in the postnatal and adult central ner-
vous system in male and female mice. J Neurosci Res 91:1143–1151.
Cunningham C, Martinez-Cerdeno V, Noctor SC. 2013. Microglia reg-
ulate the number of neural precursor cells in the developing cerebral
cortex. J Neurosci 33:4216–4233.
Czech DP, Lee J, Sim H, Parish CL, Vilain E, Harley VR. 2012. The
human testis-determining factor SRY localizes in midbrain dopamine
neurons and regulates multiple components of catecholamine synthesis
and metabolism. J Neurochem 122:260–271.
D’Agostino PM, Gottfried-Blackmore A, Anandasabapathy N, Bulloch
K. 2012. Brain dendritic cells: biology and pathology. Acta Neuropa-
thol 124:599–614.
Das M, Leonardo CC, Rangooni S, Pennypacker KR, Mohapatra S,
Mohapatra SS. 2011. Lateral fluid percussion injury of the brain induces
CCL20 inflammatory chemokine expression in rats.
J Neuroinflammation. 8:148.
de Souza DF, Wartchow KM, Lunardi PS, Brolese G, Tortorelli LS,
Batassini C, Biasibetti R, Gonc¸alves C-A. 2015. Changes in astroglial
markers in a maternal immune activation model of schizophrenia in
Wistar rats are dependent on sex. Front Cell Neurosci 9:489.
De Vries GJ, Forger NG. 2015. Sex differences in the brain: a whole
body perspective. Biol Sex Differ 6:15.
De Vries GJ, Rissman EF, Simerly RB, Yang LY, Scordalakes EM,
Auger CJ, Swain A, Lovell-Badge R, Burgoyne PS, Arnold AP. 2002.
A model system for study of sex chromosome effects on sexually
dimorphic neural and behavioral traits. J Neurosci 22:9005–9014.
Del Puerto A, Wandosell F, Garrido JJ. 2013. Neuronal and glial puri-
nergic receptors functions in neuron development and brain disease.
Front Cell Neurosci 7:197.
Dewing P, Chiang CWK, Sinchak K, Sim H, Fernagut PO, KellyS,
Chesselet MF, Micevych PE, Albrecht KH, Harley VR, Vilain E.
2006. Direct regulation of adult brain function by the male-specific fac-
tor SRY. Curr Biol 16:415–420.
Diz-Chaves Y, Pern�ıa O, Carrero P, Garcia-Segura LM. 2012. Prenatal
stress causes alterations in the morphology of microglia and the
inflammatory response of the hippocampus of adult female mice.
J Neuroinflammation 9:2094–2099.
Diz-Chaves Y, Astiz M, Bellini MJ, Garcia-Segura LM. 2013. Prenatal
stress increases the expression of proinflammatory cytokines and exacer-
bates the inflammatory response to LPS in the hippocampal formation
of adult male mice. Brain Behav Immun 28:196–206.
Dong H, Zhang X, Wang Y, Zhou X, Qian Y, Zhang S. 2016. Suppres-
sion of brain mast cells degranulation inhibits microglial activation and
central nervous system inflammation. Mol Neurobiol [E-pub ahead of
print].
Dong Y, Benveniste EN. 2001. Immune function of astrocytes. Glia 36:
180–190.
Dropp JJ. 1979. Mast cells in the human brain. Acta Anat 105:505–513.
Elmore MRP, Najafi AR, Koike MA, Dagher NN, Spangenberg EE,
Rice RA, Kitazawa M, Matusow B, Nguyen H, West BL, Green KN.
2014. Colony-stimulating factor 1 receptor signaling is necessary for
microglia viability, unmasking a microglia progenitor cell in the adult
brain. Neuron 82:380–397.
Felger JC, Abe T, Kaunzner UW, Gottfried-Blackmore A, Gal-Toth J,
McEwen BS, Iadecola C, Bulloch K. 2010. Brain dendritic cells in
ischemic stroke: time course, activation state, and origin. Brain Behav
Immun 24:724–737.
Filiano AJ, Gadani SP, Kipnis J. 2015. Interactions of innate and adaptive
immunity in brain development and function. Brain Res 1617:18–27.
Fillman SG, Cloonan N, Catt VS, Miller LC, Wong J, McCrossin T,
Cairns M, Weickert CS. 2013. Increased inflammatory markers identi-
fied in the dorsolateral prefrontal cortex of individuals with schizophre-
nia. Mol Psychiatry 18:206–214.
Forest MG, Cathiard AM, Bertrand JA. 1973. Evidence of testicular
activity in early infancy. J Clin Endocrinol Metab 37:148–151.
Fortier M-E, Luheshi GN, Boksa P. 2007. Effects of prenatal infection
on prepulse inhibition in the rat depend on the nature of the infectious
agent and the stage of pregnancy. Behav Brain Res 181:270–277.
Garay PA, Hsiao EY, Patterson PH, McAllister AK. 2013. Maternal
immune activation causes age- and region-specific changes in brain
cytokines in offspring throughout development. Brain Behav Immun
31:54–68.
Garcia-Ovejero D, Veiga S, Garcia-Segura LM, Doncarlos LL. 2002. Gli-
al expression of estrogen and androgen receptors after brain injury.
J Comp Neurol 450:256–271.
Garcia-Segura LM, Suarez I, Segovia S, Tranque PA, Cal�es JM, Aguilera
P, Olmos G, Guillam�on A. 1988. The distribution of glial fibrillary
acidic protein in the adult rat brain is influenced by the neonatal levels
of sex steroids. Brain Res 456:357–363.
Gatewood JD, Wills A, Shetty S, Xu J, Arnold AP, Burgoyne PS,
Rissman EF. 2006. Sex chromosome complement and gonadal sex
influence aggressive and parental behaviorsin mice. J Neurosci 26:
2335–2342.
Gendrel D, Chaussain JL, Roger M, Job JC. 1980. Simultaneous postna-
tal rise of plasma LH and testosterone in male infants. J Pediatr 97:600–
602.
Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, Mehler
MF, Conway SJ, Ng LG, Stanley ER, Samokhvalov IM, Merad M.
2010. Fate mapping analysis reveals that adult microglia derive from
primitive macrophages. Science 330:841–845.
Giovanoli S, Weber-Stadlbauer U, Schedlowski M, Meyer U, Engler H.
2016. Prenatal immune activation causes hippocampal synaptic deficits
in the absence of overt microglia anomalies. Brain Behavior Immun 55:
25–38.
Gobinath AR, Mahmoud R, Galea LA. 2015. Influence of sex and stress
exposure across the lifespan on endophenotypes of depression: focus on
behavior, glucocorticoids, and hippocampus. Front Neurosci 8:420.
Goldstein, JM, Cherkerzian S, Tsuang MT, Petryshen TL. 2013. Sex dif-
ferences in the genetic risk for schizophrenia: history of the evidence
458 Nelson and Lenz
Journal of Neuroscience Research
for sex-specific and sex-dependent effects. Am J Med Genet B Neuro-
psychiatr Genet 162:698–710.
G�omez-Gonz�alez B, Escobar A. 2010. Prenatal stress alters microglial
development and distribution in postnatal rat brain. Acta Neuropathol
119:303–315.
Grossman LS, Harrow M, Rosen C, Faull R, Strauss GP. 2008. Sex
differences in schizophrenia and other psychotic disorders: a 20-year
longitudinal study of psychosis and recovery. Compr Psychiatry 49:
523–529.
Hanisch U-K, Kettenmann H. 2007. Microglia: active sensor and versa-
tile effector cells in the normal and pathologic brain. Nat Neurosci 10:
1387–1394.
Hickey WF. 2001. Basic principles of immunological surveillance of the
normal central nervous system. Glia 36:118–124.
Hickey WF, Vass K, Lassmann H. 1992. Bone marrow-derived elements
in the central nervous system: an immunohistochemical and ultrastruc-
tural survey of rat chimeras. J Neuropathol Exp Neurol 51:246–256.
Instanes JT, Halmøy A, Engeland A, Haavik J, Furu K, Klungsøyr K.
2015. Attention-deficit/hyperactivity disorder in offspring of mothers
with inflammatory and immune system diseases. Biol Psychiatry doi:
10.1016/j.biopsych.2015.11.024 [E-pub ahead of print].
Juckel G, Manitz MP, Br€une M, Friebe A, Heneka MT, Wolf RJ. 2011.
Microglial activation in a neuroinflammational animal model of schizo-
phrenia—a pilot study. Schizophr Res 131:96–100.
Kettenmann H, Hanisch U-KU, Noda M, Verkhratsky A. 2011. Physiol-
ogy of microglia. Physiol Rev 91:461–553.
Khalil MH, Silverman A-J, Silver R. 2003. Mast cells in the rat brain
synthesize gonadotropin-releasing hormone. J Neurobiol 56:113–124.
Khalil M, Ronda J, Weintraub M, Jain K, Silver R, Silverman A-J.
2007. Brain mast cell relationship to neurovasculature during develop-
ment. Brain Res 1171:18–29.
Knuesel I, Chicha L, Britschgi M, Schobel SA, Bodmer M, Hellings JA,
Toovey S, Prinssen EP. 2014. Maternal immune activation and abnormal
brain development across CNS disorders. Nat Rev Neurol 10:643–660.
Koizumi S, Ohsawa K, Inoue K, Kohsaka S. 2013. Purinergic receptors
in microglia: functional modal shifts of microglia mediated by P2 and
P1 receptors. Glia 61:47–54.
Kopsida E, Lynn PM, Humby T, Wilkinson LS, Davies W. 2013. Disso-
ciable effects of Sry and sex chromosome complement on activity, feed-
ing and anxiety-related behaviours in mice. PLoS One 8:e73699.
Kosidou K, Dalman C, Widman L, Arver S, Lee BK, Magnusson C,
Gardner RM. 2016. Maternal polycystic ovary syndrome and the risk
of autism spectrum disorders in the offspring: a population-based
nationwide study in Sweden. Mol Psychiatry 21:1441–1448.
Kreutzberg GW. 1996. Microglia: a sensor for pathological events in the
CNS. Trends Neurosci 19:312–318.
Krishnan S, Intlekofer KA, Aggison LK, Petersen SL. 2009. Central role
of TRAF-interacting protein in a new model of brain sexual differenti-
ation. Proc Natl Acad Sci U S A 106:16692–16697.
Lee BK, Magnusson C, Gardner RM, Blomstr€om A˚, Newschaffer CJ,
Burstyn I, Karlsson H, Dalman C. 2015. Maternal hospitalization with
infection during pregnancy and risk of autism spectrum disorders. Brain
Behav Immun 44:100–105.
Lennington JB, Coppola G, Kataoka-Sasaki Y, Fernandez TV, Palejev D,
Li Y, Huttner A, Pletikos M, Sestan N, Leckman JF, Vaccarino FM.
2016. Transcriptome analysis of the human striatum in Tourette syn-
drome. Biol Psychiatry 79:372–382.
Lenz KM, McCarthy MM. 2015. A starring role for microglia in brain
sex differences. Neuroscientist 21:306–321.
Lenz KM, Nugent BM, McCarthy MM. 2012. Sexual differentiation of
the rodent brain: dogma and beyond. Front Neurosci 6:26.
Lenz KM, Nugent BM, Haliyur R, McCarthy MM. 2013. Microglia are
essential to masculinization of brain and behavior. J Neurosci 33:2761–
2772.
Lenzlinger PM, Morganti-Kossmann MC, Laurer HL, McIntosh TK.
2001. The duality of the inflammatory response to traumatic brain inju-
ry. Mol Neurobiol 24:169–181.
Lin Y-L, Lin S-Y, Wang S. 2012. Prenatal lipopolysaccharide exposure
increases anxiety-like behaviors and enhances stress-induced corticoste-
rone responses in adult rats. Brain Behav Immun 26:459–468.
Liu X, Fan X-L, Zhao Y, Luo G-R, Li X-P, Li R, Le W-D. 2005.
Estrogen provides neuroprotection against activated microglia-induced
dopaminergic neuronal injury through both estrogen receptor-alpha and
estrogen receptor-beta in microglia. J Neurosci Res 81:653–665.
Llorente R, Gallardo ML, Berzal AL, Prada C, Garcia-Segura LM,
Viveros M-P. 2009. Early maternal deprivation in rats induces gender-
dependent effects on developing hippocampal and cerebellar cells. Int J
Dev Neurosci 27:233–241.
Loram LC, Sholar PW, Taylor FR, Wiesler JL, Babb JA, Strand KA,
Berkelhammer D, Day HE, Maier SF, Watkins LR. 2012. Sex and
estradiol influence glial proinflammatory responses to lipopolysaccharide
in rats. Psychoneuroendocrinology 37:1688–1699.
L�opez-Gallardo M, Llorente R, Llorente-Berzal A, Marco EM, Prada C,
Di Marzo V, Viveros MP. 2008. Neuronal and glial alterations in the
cerebellar cortex of maternally deprived rats: gender differences and
modulatory effects of two inhibitors of endocannabinoid inactivation.
Dev Neurobiol 68:1429–1440.
Manitz M, Pl€umper J, Demir S, Ahrens M, Eßlinger M, Wachholz S,
Eisenacher M, Juckel G, Friebe A. 2016. Flow cytometric characteriza-
tion of microglia in the offspring of PolyI:C treated mice. Brain Res
1636:172–182.
Mao J, Zhang X, Sieli PT, Falduto MT, Torres KE, Rosenfeld CS.
2010. Contrasting effects of different maternal diets on sexually dimor-
phic gene expression in the murine placenta. Proc Natl Acad Sci U S
A 107:5557–5562.
Ma�sli�niska D, Dambska M, Kaliszek A, Ma�sli�nski S. 2001. Accumulation,
distribution, and phenotype heterogeneity of mast cells (MC) in human
brains with neurocysticercosis. Folia Neuropathol 39:7–13.
Mattei D, Djodari-Irani A, Hadar R, Pelz A, de Coss�ıo LF, Goetz T,
Matyash M, Kettenmann H, Winter C, Wolf SA. 2014. Minocycline
rescues decrease in neurogenesis, increase in microglia cytokines. and
deficits in sensorimotor gating in an animal model of schizophrenia.
Brain Behav Immun 38:175–184.
Mayer A, Lahr G, Swaab DF, Pilgrim C, Reisert I. 1998. The Y-
chromosomal genes SRY and ZFY are transcribed in adult human
brain. Neurogenetics 1:281–288.
McCarthy MM. 2008. Estradiol and the developing brain. Physiol Rev
88:91–124.
McCarthy MM, Arnold AP. 2011. Reframing sexual differentiation of
the brain. Nat Neurosci 14:677–683.
Meyer U, Nyffeler M, Engler A, Urwyler A, Schedlowski M, Knuesel I,
Yee BK, Feldon J. 2006a. The time of prenatal immune challenge
determines the specificity of inflammation-mediated brain