Han et al. Journal of Neuroinflammation(2021) 18:74
https://doi.org/10.1186/s12974-021-02124-z

REVIEW

Open Access

Uncovering sex differences of rodent
microglia
Jinming Han1*, Yueshan Fan1,2,3,4, Kai Zhou5,6, Klas Blomgren5,7 and Robert A. Harris1*

Abstract
There are inherent structural and functional differences in the central nervous systems (CNS) of females and males.
It has been gradually established that these sex-specific differences are due to a spectrum of genetic, epigenetic,
and hormonal factors which actively contribute to the differential incidences, disease courses, and even outcomesof CNS diseases between sexes. Microglia, as principle resident macrophages in the CNS, play a crucial role in both
CNS physiology and pathology. However, sex differences of microglia have been relatively unexplored until
recently. Emerging data has convincingly demonstrated the existence of sex-dependent structural and functional
differences of rodent microglia, consequently changing our current understanding of these versatile cells. In thisreview, we attempt to comprehensively outline the current advances revealing microglial sex differences in rodent
and their potential implications for specific CNS diseases with a stark sex difference. A detailed understanding ofmolecular processes underlying microglial sex differences is of major importance in design of translational sex- andmicroglia-specific therapeutic approaches.
Keywords: Microglia, Sex differences, Epigenetics, Disease

Exploring the sex differences of microglia: adeveloping story
Every cell has a sex [1]. Neurons and astrocytes in the
CNS exhibit obvious sex differences in terms of volume,
cell number, and even functional features [2]. However,
little attention has been paid to biological sex differencesof microglia and associated functions in the neuroimmune
system. Microglia have critical effects on CNS development such as contributing to sex differentiation of thebrain [3, 4]. They have emerged as central players andpromising targets in neurological diseases [5–7], since a
spectrum of brain pathologies secondary to neuroinflammation are mainly induced by activated microglia [8, 9].

* Correspondence: jinming.han@ki.se; hanjinming1202@126.com;
robert.harris@ki.se
1
Applied Immunology and Immunotherapy, Department of Clinical
Neuroscience, Karolinska Institutet, Center for Molecular Medicine, Karolinska
University Hospital, CMM L8:04, Karolinska Sjukhuset, S-171 76 Stockholm,
SwedenFull list of author information is available at the end of the article

Males and females may even experience adverse treatment
outcomes following specific microglial intervention [10].
Microglia can be reprogrammed during early life andthus exert crucial effects on behavior and disease in later
life [11–13]. We reach the realization that CNS diseases
developing early in life may be more common in males,
while females are most likely to suffer from CNS disordersthat emerge later in life [14]. Despite these marked differences, sex has been considered sparsely when making
treatment decisions in clinical practice, which might affectthe variability of clinical outcomes [15]. Multiple lines ofevidence point to dysfunctional glial and neuronal functions as one possible factor that explains sex differences in
CNS disease [16]. Sex steroid hormone exposure during
critical periods may also exert crucial effects on the functions of several immune cell types [17, 18]. In an attempt
to understand such sex differences of microglia, the critical question is when and how microglia become different
between males and females?

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Han et al. Journal of Neuroinflammation(2021) 18:74

In this review manuscript, we first summarize recent
novel evidence showing the sex differences of rodent
microglia. We then discuss potential differences of
microglial numbers and phenotypes between sexes.
More importantly, microglial endogenous functionality
and response to exogenous stimuli also differ between
males and females. Epigenetic mechanisms including
DNA methylation, histone modifications, and long noncoding RNAs that may partly explain these sex differences of rodent microglia are discussed.

One hundred years of microglial biology
First described a century ago, microglia are specialized
resident macrophages within the central nervous system(CNS) [19–22]. The relatively long-lived microglia are
uniquely positioned within the brain parenchyma, colonized from early embryogenesis [23], self-renewed byproliferation throughout adult life [24, 25], underwent
four distinct phases of differentiation during development [26], are identified by unique markers [27–29], andare epigenetically primed by early stimulations [30]. Importantly, they are quite distinct from other tissue
macrophage populations.
Reports that describe the substantial plasticity and complexity of microglia through a spatiotemporal manner are
readily accumulating [31–33]. They are the architects for
CNS development and are crucially involved in engulfing
cell debris, modulating synaptic elimination by complement [34], axonal migration, and remodeling neuralcircuits [35, 36]. Apart from their well-appreciated immunological roles, newly emerging microglial functions
have been discovered including assisting in oligodendrocyte progenitor maintenance and inducing adult hippocampal neurogenesis [37, 38]. In addition, microglia andastrocytes coordinate their functions via secreted mediators and complement activation, which is fundamental toneuronal functions [39–41].
It is gratifying that most of Hortega’s original postulates of microglial biology have now been proven quite
correct, despite that this has required the most recent
technical innovations to unequivocally prove his foresight. The most recent findings of spatiotemporal differences between resident microglia subpopulations open
up a new era of scientific discovery in which the individual contributions of microglia subsets in both driving orprotecting from disease will be possible to define.
Sex bias in neurological diseases
Sex should be considered as a biological variable when designing both preclinical experiments and clinical trials [42,43]. Indeed, many neurological disorders demonstrate a
sex bias, with neurodevelopmental diseases exhibiting amale bias in incidence [44], while neuroinflammatory diseases such as multiple sclerosis (MS) are female

Page 2 of 11

dominated [45]. Taking MS as an example, a recent Danish study demonstrated that the incidence of late onset
MS has increased profoundly in women over the previous
60 years but has only slightly increased in men [46]. When
compared with male MS patients, female patients have ahigher relapse rate, more radiological contrast-enhancing
lesions, and stronger adaptive immune activity [45]. However, male MS patients may have more obvious evidenceof neurodegeneration, supported by faster brain atrophy
and more severe cognitive decline than is experienced byfemale patients [45].
Colony-stimulating factor 1 receptor (CSF1R)-related leukoencephalopathy, mainly caused by CSF1R gene mutations
and regarded as the most common type of adult-onset
leukoencephalopathy, currently without a cure, typically presents with progressive neuropsychiatric and motor symptoms [47, 48]. CSF1R is predominately expressed on
microglia within the CNS and thus CSF1R-related leukoencephalopathy is considered as a primary CNS microgliopathy
with dystrophic microglia playing a pivotal role in disease
pathogenesis [47, 49]. Although there are no sex differencesof CSF1R-related leukoencephalopathy in prevalence and disease duration, male patients develop disease significantly later
than do females [50].
Sexual dimorphism of microglial function in other disease conditions was reviewed elsewhere [4, 51]. Given
the commonality of the concept, further investigation ofother neurological disease settings is warranted.

Sex differences of microglial morphology andnumbers through development
The current evidence obtained from preclinical models
indicates that the number and phenotype of microglia
differ between females and males in a region- and agespecific manner (Fig. 1) [52]. The cortex, amygdala,hippocampus, and preoptic area (POA) are wellaccepted regions in which obvious sex differences of
microglial numbers have been noted [52–55] (Table 1).
During normal neurodevelopment, microglia undergo
sex-specific and distinct maturation processes over time[57]. There are no significant differences in brain regionspecific volume and microglial numbers between maleand female rats at early embryonic day 17 (E17) [55].
The unique microglial developmental index based ontranscriptomic profiling of purified mouse microglia
throughout development was also similar between males
and females at E18 [57]. Sex differentiation may start
after E18 in rodents and extends into the early postnatal
period [58]. During postnatal days 2–3 (P2-3), phagocytic hippocampal Iba1+ microglia (the presence of atleast one phagocytic cup) are significantly more numerous in female rats than in males [53]. In general, exogenous estradiol enhances humoral immunity while
testosterone has the opposite effect [15]. In research

Han et al. Journal of Neuroinflammation(2021) 18:74

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Fig. 1 Sex-specific differences of microglia during development and whole life span. Microglia initially colonize the brain during embryogenesis
when obvious sex differences of microglia are not apparent. Estradiol and progesterone fluctuations are noted. During the postnatal period, sex
hormones such as testosterone can actively influence both the number and function of microglia between males and females. Sex differences of
microglia may still exist even later in life. Male microglia (blue) and female microglia (pink)

and function of microglia in the developing brain, since
microglia expressed related receptors for steroid hormones[58, 59]. In support of this, in the developing POA, a brain
region associated with expression of sex behavior and highproduction of prostaglandin in males, male rat pups havemore numerous activated/amoeboid microglia while female
rat pups had fewer Iba1+-stained microglia on P2 [56].

settings, the number of female rat phagocytic microglia
can be reduced to that of male levels following treatment
with estradiol during the early postnatal period, indicating a hormonal role for microglial sex differences during
rodent brain development [53].
Around the time of birth, a testosterone surge has been
noted in male rodents that sensitively influences the number

Table 1 Overview of studies comparing the microglial numbers between males and females in diverse brain regions
Species

Age

Brain region

Main differences

References

Rat

P4

CortexMales have more amoeboid microglia

[55]

Rat

P4

Hippocampus

Males have more amoeboid microglia

[55]

Rat

P4

Amygdala

Males have more amoeboid microglia

[55]

Rat

P30

CortexFemales have more activated microglia

[55]

Rat

P30

Hippocampus

Females have more activated microglia

[55]

Rat

P30

Amygdala

Females have more activated microglia

[55]

Rat

P60

CortexFemales have more activated microglia

[55]

Rat

P60

Hippocampus

Females have more activated microglia

[55]

Rat

P60

Amygdala

Females have more activated microglia

[55]

Rat

P2

Preoptic area

Males have more amoeboid-shaped microglia

[56]

Rat

P3

Hippocampus

Females have more phagocytic microglia

[53]

Mice

13 weeks

Hippocampus

Males have more Iba1+ microglial cell density

[52]

Mice

13 weeks

CortexMales have more Iba1+ microglial cell density

[52]

Mice

13 weeks

Amygdala

Males have more Iba1+ microglial cell density

[52]

Mice

3 weeks

Hippocampus

Males have more Iba1+ microglial cell density

[52]

Mice

3 weeks

Amygdala

Females have more Iba1+ microglial cell density[52]

Abbreviations: P2 postnatal day 2, Iba1 Ionized calcium binding adaptor molecule 1

Han et al. Journal of Neuroinflammation(2021) 18:74

The sex differences of microglia described above could
be mediated by sex hormones, as evidenced by an increased number of amoeboid microglia in the POA in
females treated neonatally with estradiol [56, 58]. Furthermore, apparent microglial differences in microglia
between males and females have also been noted on P4,
shortly after the testosterone surge, when male rats havea more amoeboid microglia than do females in the cortex, hippocampus, and amygdala which diminishes
quickly at P30 [55].
As measured using deep single-cell RNA sequencing
across different developmental periods and brain regions,adult microglia do not seem as complex and heterogeneous as do postnatal microglia [60]. Nonetheless, microglial sex differences may also be evident in adult rodents(Table 1). Microglia can be influenced by sex identity
postnatally and maintained until adulthood independentlyof circulating sex hormones. Specifically, Iba1+ microglial
cell density is significantly higher in the hippocampus,
cortex, and amygdala of adult male mice when comparedwith adult female mice [52]. Microglia actually undergo
four sequential distinct phases across development basedon gene expression [26]. In this case, the microglial
morphology, number, and cytokine expression can be
modified dramatically throughout neurodevelopment. Indeed, on P30 and P60, rat microglial numbers had opposite trends between males and females in diverse brain
regions [55]. It is currently unclear if more microglial progenitor cells may be recruited into the male brain during
early development (E8 to E9) and whether more microglial
proliferation or less microglial apoptosis occurs in malerodents within a given brain region throughout their life
span than in females [4, 24, 61].
We can conclude that microglial sex differences inboth space and time are significant in developmental
processes and that greater understanding of the underlying molecular mechanisms and environmental cues involved could be vital to the design of tailored therapies.

Sex differences of microglial functions
Despite the presence of conflicting evidence, differences
between rodent male and female comprehensive microglial functionality throughout the life span have also
been recently discerned (Table 2), indicating that microglia may not respond similarly between sexes in disease
conditions including ischemic stroke and obesity [62–64]. From a functional perspective, microglial phagocytosis is an integral aspect of synaptogenesis and synaptic
pruning during fetal development and in adults is an active process necessary to remove dead and dying neurons. Conversely, dysfunctional microglial phagocytosis
may contribute to CNS disorders [69]. Interestingly, theupregulation of autophagy via several routes has beenshown to promote microglial polarization toward an

Page 4 of 11anti-inflammatory phenotype, while Atg5 siRNA inhibited autophagy and aggravated proinflammatorypolarization [70]. In addition, activation of microglia canalso be tightly regulated by the cholinergic pathway
through α7 nicotinic receptors [71]. Sex differences of
microglial endogenous functions, microglial responses
following radiotherapy, and exogenous stimuli will now
be discussed.

Sex differences of microglial endogenous
functionsMale and female microglia have distinct fundamental
endogenous behaviors such as antigen presentation.
Some microglial sex differences are noted at both RNA
and protein levels with few differentially expressed genes
and proteins overlapping [52]. Male microglia exhibit
higher potential antigen presentation ability in the cortex, as evidenced by higher expression of major histocompatibility complex (MHC) I and MHC II than doage-matched female microglia [52]. Kinetical observations have demonstrated that microglia are the first local
antigen-presenting cells in the CNS to respond and takeup myelin antigen, while dendritic cells are more important later in the context of EAE [72]. A possible differential role of microglia during early disease periods
may thus be enacted in males and females. This hypothesis is further supported by a recent study elegantly
showing that microglia exert a sex-specific effect on thelong-term absence of the microbiome, with males being
significantly affected during early development while females showed profound changes at adult periods instead[26].
Sex differences of microglia in response toradiotherapy
Radiotherapy is an effective tool in the treatment of highgrade brain tumors, but it is associated with adverse sideeffects, where the long-term, so-called late effects are more
pronounced in children [73, 74]. One common such late effect after cranial radiotherapy is cognitive impairment, andthis is, at least partly, thought to be caused by reduced hippocampal neurogenesis, where neuroinflammation and a perturbed microenvironment are thought to be involved [75].
We have previously shown that irradiation to the brains of
P14 mice can cause in females a more marked upregulationof several cytokines and chemokines in the hippocampus 6 hafter irradiation [76]. During recovery, microglial density wasincreased in females, but not in males, indicating long-lasting
effects of irradiation on the hippocampal microenvironment[76]. The subsequent loss of neurogenic capacity and performance in cognitive tests was more pronounced in females[76, 77]. Interestingly, exposure to lipopolysaccharide (LPS)
prior to irradiation aggravated the loss of neurogenesis more
so in males than in females [76]. Further immuno-

Han et al. Journal of Neuroinflammation(2021) 18:74

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Table 2 Overview of studies comparing the functional differences between male and female microglia
Species

Age

Main functional differences

References

C57BL/6 mice and
NF-κB-luc2 mice

12 weeks

Male microglia express more NF-κB regulated genes and are more prone to inflammatory activation than are females. These differences can also be maintained in vitro and thus do nottotally depend on sex steroids. Females can even maintain sex-specific microglial differences
when transplanted into the opposite sex brain, and this protects the males from ischemic
stroke.

[62]

Csf1R-EGFP mice

13 weeks

Sex differences of electrophysiological responses to ATP which measured as membranecurrents were noted in isolated microglia. Male microglia have a higher antigen-presenting
capacity compared to females. The expression of MHCI was higher in male microglia in boththe cortex and hippocampus, while MHCII expression was higher in male cortical microglia.

[52]

C57BL/6 mice

13 weeks

Transcriptional profile differences were found between isolated male and female microglia inboth the hippocampus and cortex. Sex differences of microglial steady-state protein levels
were also noted from the whole brain.

[52]

CD1 mice

E3,5; adolescent Treatment of CSF1R inhibitor to deplete embryonic microglia cause sex-specific effects on
and adult
mice, evidenced by adolescent female mice showing hyperactive development and adult female mice showing anxiolytic-like behavior. However, these phenomena were not noted inmale adolescent and adult mice respectively.

[10]

C57BL6/J mice
CX3CR1gfp/gfp knockin mice

18 weeks

There are sex differences of hypothalamic microglial CX3CR1 signaling activation which
contribute to obesity susceptibility between male and female mice. Female mice are moreresistant to diet-induced obesity than are males. In contrast, female mice become susceptible
to diet-induced obesity in the absence of CX3CR1 signaling.

[63]

Sprague Dawley rats

60–90 days

Rat female microglia in the periaqueductal gray area exhibited a more activated phenotypeat baseline, produced higher transcription levels of IL-1β, and could be more responsive toimmune challenges such as LPS than were male rats, without overall microglial gender density differences in this region.

[64]

Wistar rats

Newborn (P0)
to P2

Cultured female rat newborn microglia had higher phagocytic activity than in males asmeasured by in vitro bead intake assays at both baseline and following by IFNγ stimulation.
In contrast, female rat newborn microglia had less basal and stimulated microglial migration
than the males as measured by in vitro Transwell assay.

[16]

Sprague Dawley rats

Neonatal

During early postnatal development, phagocytic female microglia in the hippocampus had
significantly more highly expressed phagocytic pathway genes and phagocytic associatedfunctions when compared to the males.

[53]

C57BL/6 SPF and GF
mice

E18.5
P60

Microglia exert a sex-specific effect on long-term absence of the microbiome, with males being significantly affected during early development while females exhibited profoundchanges in adulthood instead.

[26]

Sprague Dawley rats

Around 70 days
old

Acute or chronic behavioral stress has distinct direct effects on corticolimbic microglial
morphology and immune factor transcriptional expression such as CD40, CX3CR1, andCD200R in a number of brain regions which is mediated by microglia in a sex-dependent
manner.

[65]

Sprague Dawley rats

Around 70 days
old

Differential effects of stress on microglial cell activation in male and female medial prefrontal
cortex[66]

Sprague Dawley rats

P1 and P21

Cultured rat male microglia had increased migration compared to females after single IL-4
stimulation. The mRNA level of K+ channel (Kcna3) and Kv current were also higher in male
P21 microglia than in females after inflammatory stimulation, while unstimulated microglia
had similar levels in males and females.

[67]

C57BI/6J mice

17–18 months

Female aged microglia exhibited higher expression of translocator protein and higher
expression of M1 markers following peripheral LPS challenge than did male aged microglia.

[68]

Abbreviations: NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells, Csf1R colony-stimulating factor 1 receptor, ATP adenosine triphosphate, MHC
major histocompatibility complex, E3,5 embryonic day 3.5, CX3CR1 CX3C chemokine receptor 1, LPS lipopolysaccharide, IL-1β interleukin-1beta, IFNγ interferon-γ,
GF germ-free, SPF specific-pathogen free

radiobiological investigations are needed to understand why
girls develop more pronounced cognitive late effects than doboys after cranial radiotherapy [78–81].

Sex differences of microglia in response toexogenous stimuli
Either acute or chronic behavioral stress has distinct direct
effects on corticolimbic microglial morphology and immune factor transcriptional expression, such as of CD40,

CX3CR1, and CD200R in a number of brain regions in a
microglial sex-specific manner [65, 66]. In addition, cultured rat male microglia had greater migration function
than did female microglia following IL-4 stimulation [67],implying that male microglia might have the ability to surround a lesion earlier after brain injury than can females[67, 82]. The mRNA level of K+ channel (Kcna3) and Kv
current were also higher in male P21 microglia than in females following inflammatory stimulation, while

Han et al. Journal of Neuroinflammation(2021) 18:74

unstimulated microglia had similar levels between males
and females [67]. It has been documented that estrogen
receptors contribute to mediating sex differences of
microglia, as evidenced by perinatal activation of estrogen
receptor-α being restricted to males during early development [83]. Overall, such differences may subsequently leadto sex-dependent changes and susceptibility to CNS
diseases.
Sex differences in brain innate immune responses toneonatal ischemia have been reported in P12 mice, withhigher microglia immunoreactivity, greater proinflammatory gene expression, and increased myeloid cells being noted in male mice [84]. In addition, male neonatal
microglia had a significantly greater IL-1β response to
LPS compared to neonatal females [85]. Aged mice
microglia also exhibited sex differences of immune responses in the context of disease, as evidenced by female
aged microglia having higher levels of microglial activation and higher expression of M1 markers following peripheral stimulation than did male aged microglia [68].
However, it is important to note that these microglial
sex differences may already exist at this time point before disease induction and may also have a role in differences in secondary damage between males and females.
Starting from P60, male microglial maturation is delayed
relative to females, and such notable sex differences in
microglial maturity may also contribute to disease susceptibility [57]. Specifically, following an acute stimulation such as LPS challenge, an obvious increased level ofmicroglial transcriptional maturation was only observedin males [57]. It has been claimed that male microglia
may be more vulnerable to inflammatory stimuli and tobe over-activated than in females during a particularly
sensitive time window when males have more microglia.
This could be a contributory factor to sex differences inboth behavior and disease [15, 57].
Male and female microglia may have distinct
mechanisms to resolve an inflammatory insult. Specifically, female rat neonatal microglia had both lessbasal and stimulated microglial migration than did
male microglia [16]. During early postnatal development, phagocytic microglia in the female rat hippocampus express significantly more phagocytic
pathway genes and phagocytic associated functions
when compared to males [53]. In support of this notion, cultured female rat newborn microglia hadhigher phagocytic activity than males as assessed byin vitro bead uptake assays at both baseline and following interferon (IFN)-γ stimulation [16]. The accumulated data strongly suggest that rat microglia may
exhibit sex differences of phagocytic capacity during
development [14]. One recent study inferred the opposite conclusion that microglia in male adult mice
had similar phagocytic ability as females as measured

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by in situ phagocytosis assays in both hippocampal
and cortical slices [52].
A series of factors such as different ages, species, brain
regions, and methods in these studies may account forthis discrepancy [86]. In research settings, microglia haveto be dissociated from the CNS into a single-cell suspension using both mechanical dissociation and enzymatic
dissociation in order to facilitate culture or analyses.
Some may argue that mechanical dissociation and enzymatic dissociation may alter the microglial activation
status and mRNA expression profiles [87, 88]. Microglia
may lose their coordinated cell-to-cell communications
when isolated from other CNS cell types, complicating
appreciation of their precise roles in vivo [89]. For thesereasons, one must be careful in interpreting in vitro
studies regarding functional microglial sex differences
purported in vivo. Further investigations using advanced
methods such as live imaging techniques are warranted
to uncover related mechanisms, taking sex differences of
microglial phagocytosis into consideration.
Microchimerism is defined as the phenomenon of two
genetically distinct cell populations in an individual, withmale microchimerism occurring more frequently than in
the female brain [90]. Fetal microchimeric cells, genetically foreign maternal cells, persist throughout postnatal
development into adulthood, and these microchimericcells may play a role in responding to injury [90, 91].
Therefore, sexual microglial dimorphism mediated by
microchimeric cell effects should not be ignored.
All these observed functional differences may predispose as yet underestimated but marked sex-dependent
microglial activation patterns and signaling cascades in
the injured CNS [54, 92]. Emerging data has challenged
the classical viewpoint that sexual microglial dimorphism is solely due to hormones. The X chromosome inactivation is a main epigenetic feature in females that
equalizes dosage of gene products [93]. However, incomplete X chromosome inactivation may cause some genes
to be doubly expressed, which could explain specific sex
biases [94]. Furthermore, microglial functional balancecan be affected by a diversity of environmental factors
including stress, with females exhibiting increased stresssensitivity [95]. These non-hormonal mechanisms mightalso affect microglial development and function. It has
been proposed that chronic pregnancy stress and inflammation can be mediated via microglial innate immune
memory and that such memory may increase the susceptibility of neurodegenerative conditions later in life
[11].
Most of the evidence presented is derived from preclinical studies, showing dynamic sex differences of
microglia. Understanding how sex differences of microglia contribute to the occurrence and progression of human CNS disorders may explain sex differences in CNS

Han et al. Journal of Neuroinflammation(2021) 18:74

disease susceptibility and provide new optimism for developing sex-specific treatments.

Epigenetic control of microglia: novel potential
players for sex differences of microglia
Microglia can be pre-conditioned through epigeneticdriven innate immune memory that persists for several
months [30, 96]. Epigenetic alterations of microglia during early life may re-program the communication between microglia and other cells in the CNS and havesignificant later consequences for behavior and disease[11, 13, 61]. We now know that differential use of glycolytic or fatty acid metabolic pathways indices different
myeloid cell activation states [97] and so differential
microglial functionality between males and females
might be due to underlying metabolic differences. Epigenetic processes can integrate intrinsic signals and environmental variables into the genome and then regulate
microglial genes and functions that contribute to the sex
bias in autoimmune diseases [45, 98]. Microglial differentiation and response to stimuli can be governed by
epigenetic mechanisms including DNA methylation, histone modifications, and long non-coding RNAs [99].
DNA methylation is an epigenetic mechanism that occurs by the addition of methyl groups to DNA, modifying genetic functions without changing the sequence[96]. From a sex viewpoint, females have been reported
to have higher levels of DNA methyltransferase enzyme
activity, DNA methylation, and methylated CpG sites
than do males in the neonatal POA [100]. Inhibiting
DNA methyltransferase causes masculinized neuronalmarkers and male sexual behavior in female rats [100],
suggesting that DNA methylation serves as the basis forthe sex-specific transcriptomes in selective brain regions.
Histone modifications are epigenetic regulators that
control chromatin structure and gene expression [101].
Histone modifications associated with neural sexual differentiation were recorded [102]. Epigenetic regulationof microglia may vary across life span, prenatal depletionof the histone deacetylases Hadc1 and Hadc2 using
Cx3cr1CreHdac1fl/flHdac2fl/fl mice being harmful for
microglial development by inducing apoptosis and reducing survival [103, 104]. However, experimental depletion of the histone deacetylases Hadc1 and Hadc2
during adulthood did not impair microglial numbers andmorphology, indicating that epigenetic factors may not
be critical for adult steady-state microglia [103, 104].
While no sex differences in histone modifications were
evident in whole embryonic brains [102], regionally theacetylation and methylation of histone H3 in neonatal
male mice were increased in the cortex and hippocampus when compared with females, but not in the POA,
amygdala, and hypothalamus [102].

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MicroRNAs are small non-coding RNA molecules that act
as post-transcriptional negative regulators of gene expression.
Different expressions of isolated microglial microRNAs in
the adult mice brain between male and female at baseline
have also been uncovered using microRNA sequencing,which was also validated using qPCR [105]. Selective loss ofthese microRNAs by ablating the microRNA-processing enzyme Dicer lead to significant sex-dependent changes of
microglial transcriptomes with male adult microglia exhibiting a higher extent than the females [105]. Furthermore,male and female mice showed a similar Tau burden in
P301S tauopathy mice, while sex-related changes of microRNA profiles were evident in the condition of tau pathology,
suggesting that microglial microRNAs may serve as a potential player for mediating sex differences of microglia [105]. In
support of this, certain microRNA can modulate acetylcholine signaling by regulating cholinergic gene expression [106].
Of relevance, dysregulated acetylcholine signaling can lead to
CNS and peripheral dysfunctions via a sex-specific manner[106, 107], potentially shedding light onto other neuroinflammatory diseases.
Collectively, these data imply that epigenetic mechanisms such as DNA methylation, histone modifications,and microRNAs impact an array of transcriptional responses and, at least in part, represent a potential mechanism that might explain microglial sex-related functional
differences. We are optimistic that future research regarding epigenetic control of male and female microglia willbear exciting novel discoveries to better understand thesex differences of microglia.

Conclusions
Sex differences of microglia exist among diverse regions withrespect to both CNS physiology and pathology. Sexual microglial dimorphism may be in part responsible for sex differences in the incidence and pathology of a variety ofneurological diseases. Males may be more vulnerable toneurological insults during early development in part as a result of having more highly activated microglia in the developing brain compared to females. In contrast, increased
activation of microglia in females during adulthood may contribute to their increased susceptibility to several inflammatory brain diseases that occur late in life. Given our increasing
understanding of these fascinating cells in some degree ofboth targets and drivers of sex differentiation [108], future investigation will bear exciting novel discoveries about sex
microglia. Sex differences of microglia could be caused by intrinsic differences evidenced by X chromosome containing alarge density of immune-related genes and some epigenetic
modifiers, while the differences can also be mediated by hormonal or other environmental influences expressed over thelifespan. Therefore, more effort will be needed to explore how
genes located on the X or Y chromosomes, epigenetic mechanisms, endocrine factors, and microenvironmental signals

Han et al. Journal of Neuroinflammation(2021) 18:74

synergistically contribute to microglial sex differences andthen fully understand the molecular bias of CNS diseases with
sex vulnerability [108, 109].

Page 8 of 11

9.

Acknowledgements
Not applicable.
Authors’ contributions
JH wrote the manuscript draft. YF prepared the figures. ZK, KB, and RAH
edited and revised the manuscript. All authors read and approved the final
manuscript for publication.
Funding
This work was supported by grants from the China Scholarship Council, theSwedish Research Council, Neuroforbundet, HjärnFonden, AlzheimerFonden,
Alltid Litt Sterkere, the Swedish Cancer Foundation, and the SwedishChildhood Cancer Fund. Open Access funding provided by Karolinska
Institutet.

10.

11.

12.

Availability of data and materials
Not applicable.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.

13.

14.

15.

Competing interests
The authors declare that they have no competing interest.
Author details
1
Applied Immunology and Immunotherapy, Department of Clinical
Neuroscience, Karolinska Institutet, Center for Molecular Medicine, Karolinska
University Hospital, CMM L8:04, Karolinska Sjukhuset, S-171 76 Stockholm,
Sweden. 2Department of Neurosurgery, Tianjin Medical University General
Hospital, Tianjin, China. 3Key Laboratory of Injuries, Variations andRegeneration of Nervous System, Tianjin Neurological Institute, Tianjin, China.
4
Tianjin Medical University, Tianjin, China. 5Department of Women’s andChildren’s Health, Karolinska Institutet, Stockholm, Sweden. 6Department ofPediatrics, Children’s Hospital of Zhengzhou, Zhengzhou, China. 7PediatricOncology, Karolinska University Hospital, Stockholm, Sweden.

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Received: 2 December 2020 Accepted: 5 March 2021
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