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Review Article
Involvement of MicroRNA in Microglia-Mediated
Immune Response
J. Guedes,1,2 A. L. C. Cardoso,3andM.C.PedrosodeLima
3,4
1PhD Programme in Experimental Biology and Biomedicine (PDBEB), CNC - Center for Neuroscience and Cell Biology,
University of Coimbra, 3004-517 Coimbra, Portugal
2Institute for Interdisciplinary Research (IIIUC), University of Coimbra, 3030-789 Coimbra, Portugal
3CNC - Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal
4Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, 3001-401 Coimbra, Portugal
Correspondence should be addressed to M. C. Pedroso de Lima; mdelima@ci.uc.pt
Received March ; Accepted May
Academic Editor: Anirban Basu
Copyright © J. Guedes et al. is is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
MicroRNAs (miRNAs) are an abundant class of small noncoding RNA molecules that play an important role in the regulation
of gene expression at the posttranscriptional level. Due to their ability to simultaneously modulate the fate of dierent genes,
these molecules are particularly well suited to act as key regulators during immune cell dierentiation and activation, and their
dysfunction can contribute to pathological conditions associated with neuroinammation. Recent studies have addressed the role
of miRNAs in the dierentiation of progenitor cells into microglia and in the activation process, aiming at clarifying the origin
of adult microglia cells and the contribution of the central nervous system (CNS) environment to microglia phenotype, in health
and disease. Altered expression of several miRNAs has been associated with Alzheimer’s disease, multiple sclerosis, and ischemic
injury, hence strongly advocating the use of these small molecules as disease markers and new therapeutic targets. is review
summarizes the recent advances in the eld of miRNA-mediated regulation of microglia development and activation. We discuss
the role of specic miRNAs in the maintenance and switching of microglia activation states and illustrate the potential of this class
of nucleic acids both as biomarkers of inammation and new therapeutic tools for the modulation of microglia behavior in the
CNS.
1. Introduction
Microglia cells are crucial for the development and main-
tenance of the central nervous system (CNS). In addition
to acting as sensors of environmental changes that precede
pathological events, these cells have been shown to support
neuronal function by monitoring synaptic activity, control-
ling synaptogenesis, and promoting neuronal apoptosis dur-
ing development [–]. Although they are considered one of
the four major cellular types of the CNS, they do not originate
from the same precursor cells as astrocytes, oligodendrocytes,
or neurons. Instead, they derive from myeloid progenitor
cells and share several markers with peripheral monocytes,
macrophages, and dendritic cells, such as CDb, F/, and
CD []. e rst resident parenchymal microglia cells are
believed to originate from yolk sac immature macrophages
in early stages of fetal development. In humans, microglia
precursor cells arrive at the brain in two waves during the
rst and second trimester of gestation, while in rodents this
migration occurs shortly before and aer birth.
e sudden increase in CDb+and F/+cells observed
in the early postnatal period in rodents was until recently
attributed to the recruitment of bone marrow derived cells,
suggesting that myeloid precursors could also contribute to
the initial pool of microglia cells in the CNS. However, most
of the studies supporting these ndings used irradiation of
the recipient animals to allow bone marrow engrament
of genetically-labeled cells [], which was later found to
strongly inuence the observed results []. In , Ginhoux
andcolleaguesshedlightontheoriginofmicroglia.e
authors performed in vivo lineage tracing studies using Cre
recombinase activity, which was induced into pregnant mice
between days and of fetal development, when embryonic
hematopoiesis is restricted to the yolk sac. e results from
Clinical and Developmental Immunology
this study clearly demonstrated that postnatal hematopoietic
progenitors do not contribute signicantly to microglia post-
natal numbers and that the cellular expansion observed in
this period is mainly dependent on the proliferation of the
resident yolk sac-derived microglia population [].
e question remains whether this is also true in the
adultbrain,especiallyfollowinganeurologicalinsultorin
the case of a neurodegenerative disease, wherein the integrity
of the blood-brain barrier (BBB) may be compromised.
Several studies have shown that the inltration of bone
marrow-derived cells into the brain is possible under those
circumstances and may even play a central role in disease
modulation. Nevertheless, the exact nature of the contri-
bution of parenchymal and blood-derived microglia to the
neuroimmune response, in the context of neuronal disease,
remainstobeclaried[–].
Following their migration to the neuronal tissue, micro-
glia cells assume a surveying phenotype, usually referred as
“resting microglia,” characterized by a small and static cell
body,alargenumberofhighlymotileramications,andlow
expression of macrophage-related surface markers, such as
the major histocompatibility complex II (MHC II) and CD
[]. e low levels of these markers distinguish parenchymal
“resting” microglia from peripheral macrophages. However,
following a neuronal insult, such as ischemia, infection,
and trauma or in the presence of inammatory mediators
(IFN-𝛾), microglia cells assume an amoeboid form, losing
their ramications, and overexpressing the above-mentioned
markers. is process is referred to as microglia “activation”
and is known to induce profound phenotypical changes,
making parenchymal microglia to become almost indistin-
guishable from peripheral macrophages [].
Similarly to what has been described in macrophages,
microglia activation can also originate dierent subsets of
cells, depending on the nature of the activating stimulus
and surrounding environment. ese dierent activation
phenotypes express distinct molecular markers and exert
dierent functions in the neuronal tissue []. e denition
of the dierent activation states of macrophages was initially
based on the expression of proinammatory receptors and
cytokines (M phenotype—classical activation) or on the
expression of anti-inammatory receptors and cytokines (M
phenotype—alternative activation). Further studies revealed
the existence of several intermediate activation states, which
led to the conclusion that these two basic phenotypes can
overlap and that macrophages are able to assume a broad
spectrum of phenotypes that cannot be oversimplied and
separatedintodiscretecategories.
Contrarily to macrophages, the mechanisms responsible
for microglia phenotype regulation in the CNS are poorly
understood. M activation, which is characterized by an
increase in the production of IL-𝛽,IL-,TNF-𝛼,andnitric
oxide (NO), has been identied following acute brain injury
caused by trauma or stroke [,]. However, in the context of
neurodegenerative disorders, the distinction between the M
and M phenotypes has proven to be more challenging. While
several studies have identied the presence of M markers,
such as TGF-𝛽and IL-, in the brain of Alzheimer’s disease
(AD) animal models, as well as an increase in the expression
of M genes AG (arginase ) and CHIL/CHIL (chitinase
-like /) in AD patients [], inammation in the human
AD brain has also been associated with upregulation of IL-
, IL-𝛽,andTNF-𝛼,allmarkersoftheMstate,inthe
vicinity of amyloid deposits [,]. Most authors believe that
the M-like phenotypes are less aggressive to the neuronal
tissue, promoting tissue repair and phagocytosis of protein
aggregatesandcelldebris,whiletheM-likephenotypes
are more prone to induce neuronal toxicity by themselves,
due to the high expression of inammatory mediators and
NO.WhetherhumanmicrogliacanswitchfromanMto
an M phenotype with a detrimental eect to the brain is
still not clear. However, several studies have pointed to the
possibility of microglia “priming,” a phenomenon associated
with age and chronic inammation, in which exposure to
low levels of systemic signaling molecules can exacerbate
microglia response to a second local stimulus, such as the
presence of A𝛽aggregates, potentiating the development
of tissue damaging phenotypes []. Although some of the
molecular intervenients and exogenous stressors underlying
microglia activation in vitro have been identied [–],
in more complex environments, such as the diseased brain,
there is still a lack of answers concerning the molecular
mechanisms responsible for microglia phenotypic changes
[].isledseveralscientiststoproposearoleforcertain
key transcription factors and microRNAs (miRNAs) in these
processes [].
2. MiRNA Biogenesis and Activity
MiRNAs are transcribed from intragenic or intergenic
regions by RNA polymerase II or RNA polymerase III,
originating large stem-loop hairpin structures, designated
pri-miRNAs []. ese structures, which are asymmetrically
cleaved by an enzymatic complex containing Drosha, a
RNAse III endonuclease, originate hairpin-structured pre-
cursors designated pre-miRNA [,]. Alternatively, non-
canonical pathways for pre-miRNA biogenesis can occur,
such as the production of mirtrons, which consist in pre-
miRNAs generated through the direct splicing of introns [].
Pre-miRNAs are exported to the cytoplasm by the com-
plex Exportin-/RanGTP, which recognizes and binds the
characteristic overhangs of pre-miRNAs. ese precursors
arethenincorporatedintoaprocessingcomplexcontaining
another RNAse III endonuclease (Dicer), which is responsi-
ble for removing the stem-loop of the precursor, originating a
double-stranded miRNA molecule with overhangs (mature
miRNA). is duplex molecule is then incorporated into
the RNA-induced silencing complex (RISC) or the microri-
bonucleoprotein complex (miRNP), where the strand with
the least thermodynamically stable endisusedtoactivate
the complex and guide it to complementary mRNA targets
[]. During miRNA-mediated posttranscriptional gene reg-
ulation, the RISC complex, containing the single stranded
miRNA template, binds mainly to the untranslated region
(UTR) of the target mRNA, in one or more locations. e
complementarity between the mRNA and the nucleotides –
onthe
region of the miRNA (seed region) is responsible
for this binding and allows the recognition of multiple mRNA
Clinical and Developmental Immunology
targets by a single miRNA molecule []. MiRNA-mediated
regulation of gene expression is achieved either by translation
repression or degradation of the mRNA target molecule []
and has been associated with many important biological
processes, including inammation, apoptosis, angiogenesis,
development, proliferation, patterning, and dierentiation.
3. MiRNA Role in Microglia Development
e mechanism of miRNA-mediated posttranscriptional reg-
ulation is very well suited to the control of fate-changing
cellular events, such as dierentiation and activation. Since
these processes usually involve changes in several proteins
and dierent signaling pathways, such control can be easily
achieved through the expression of a single miRNA or a set
of specic miRNAs. In addition, miRNAs can directly target
transcription factors, the master regulators of most cellular
events. A number of studies have revealed that miRNAs
may provide a genetic switch to inactivate a set of target
genes through the regulation of a specic transcription factor,
while miRNA expression can be regulated by transcription
factors that bind upstream of pre-miRNA genes [,].
erefore, these two classes of regulators can work together
to orchestrate complex cellular events, such as monocyte-
macrophage dierentiation and microglia development from
primitive macrophages in the yolk sac or bone marrow
precursors.
Two transcription factors, namely, CEBP𝛼and PU.,
have been shown to be critical for monocyte/macrophage
dierentiation and microglia development, CEBP𝛼being
considered the master regulator of hematopoietic stem cell
dierentiation. Several miRNAs are controlled directly by
CEBP𝛼, including miR-, which is also regulated by the
transcription factor NFI-A. However, while CEBP𝛼promotes
miR- expression, NFI-A inhibits the expression of this
miRNA [], which represses NFI-A through a feedback loop.
erefore, when CEBP𝛼levels are high, miR- expression
is enhanced, and NFI-A levels decrease, promoting granulo-
cyte dierentiation, but when NFI-A levels are relatively high
and miR- levels are low, other pathways are favored, such
as monocyte dierentiation.
On the other hand, PU. is required to promote the skew-
ing of granulocyte-macrophage progenitors to the monocyte
lineage. PU. levels, although directly controlled by CEBP𝛼,
have to be relatively high when compared to CEBP𝛼lev-
els, to avoid favoring the granulocyte lineage. MiR- is
upregulatedbyPU.and,throughthetargetingofNFI-A,
upregulates the expression of the M-CSF receptor, skewing
dierentiation towards the monocyte/macrophage lineage in
thebonemarrow[]. Forrest and coworkers have demon-
strated that, in addition to miR-, miR-, miR-, and
miR- play an important role in monocyte dierentiation
through combinatorial regulation []. ese authors have
shown that when overexpressed, these miRNAs are able to
cause cell-cycle arrest and partial dierentiation in THP-
cells (leukemia model). MiR- and miR- caused G
arrest and apoptosis, while miR- and miR- caused G
arrest and the downregulation of miR-, an antidierentiative
miRNA, by targeting a site in its primary transcript.
In another recent study, the oncogenic miR-- cluster
(which carries six miRNAs: −, −a, −a, −a, −b-
, and −a) has been directly associated with the process
of monocyte to macrophage dierentiation, in which PU.
and Egr are also involved []. Upon dierentiation into
macrophages, the transcription factor PU. was found to
induce the secondary determinant Egr which, in turn,
directly represses miR-- expression by promoting histone
H demethylation within the CpG island of the miR--
promoter. Conversely, Egr itself is targeted by miR--,
indicating the existence of a mutual regulatory relationship
between miR-- and Egr []. Given the similarities
betweenmacrophagesandmicroglia,itisreasonableto
assume that some of these miRNAs and transcription factors
also play an important role in microglia dierentiation in
the brain, and it would be interesting to ascertain whether
the same regulatory loops might exist in yolk sac-derived
microglia.
So far, the only study addressing miRNA contribution to
microglia development was performed by Ponomarev and
colleagues [,].eseauthorsshowedthatmiR-,one
ofthemostabundantmiRNAsinthebrain,isrequiredto
maintain the quiescent state of microglia in the brain. By
targeting the transcription factor CEBP𝛼and the cyclins
CDKandCDK,miR-isabletoreducetheexpressionof
PU. and its downstream target, the M-CSF receptor, restrict-
ing cellular proliferation and potentiating the dierentiation
of primitive macrophages to adult microglia in the brain
[]. While during the rst two weeks aer birth, microglia
isolated from the brain presented low levels of miR- and
aCD
high/MHC class IIhigh phenotype, characteristic of
active and proliferating cells, adult microglia presented the
opposite phenotype, CDlow/MHC class IIlow/miR-high
[,]. e authors hypothesized that the high levels of miR-
observed in adult microglia are a specic consequence of
the CNS environment. is idea was based on their observa-
tion that sublethally irradiated mice, transplanted with bone
marrow GFP+progenitor cells exhibiting a CDhigh/MHC
class IIhigh/miR-low phenotype, presented GFP+CDb+
positive cells in the brain with a CDlow/MHC class
IIlow/miR-high phenotype. To conrm this hypothesis,
Ponomarev and colleagues cocultured bone-marrow-derived
macrophages with astroglial or neuronal cell lines, and they
observed, in both cases, a downregulation of MHC class
IIandCDlevelsaswellasanupregulationofmiR-
expression. Several suggestions were made concerning the
mechanisms underlying miR- upregulation in microglia,
including the direct transfer of miR- from neuronal cells
to microglia through exosomal shuttle vesicles, direct cell-to-
cell contact between these two cell types and the release of
anti-inammatory factors, such as CXCL and TGF-𝛽,by
neuronal cells [,].
4. MiRNA Role in Microglia Activation
In addition to being involved in the regulation of the dieren-
tiation process of microglia, several studies suggest a role for
miRNAs as modulators of M and M polarization in both
Clinical and Developmental Immunology
microglia and macrophages. MiR-, broadly considered a
proinammatory miRNA, was one of the rst miRNAs to be
directly linked to the M phenotype (Figure ). is miRNA
was shown to be upregulated in macrophages, monocytes,
and microglia in response to several proinammatory stim-
uli, such as LPS, IFN-𝛾,andTNF-𝛼[,,]. In this
regard, we have recently shown that miR- targets anti-
inammatory proteins in microglia, such as the suppressor of
cytokine signaling (SOCS-), leading to the upregulation of
several inammatory mediators characteristic of the M phe-
notype, including the inducible nitrogen synthase (iNOS), IL-
, and TNF-𝛼[], as described in Figure . In addition, miR-
upregulation increases the expression of IFN-𝛽,which
is probably related with a feedback mechanism to control
theimmuneresponse,sinceIFN-𝛽isknowntoupregulate
the expression of SOCS- and IL-, two important anti-
inammatory mediators [,]. Our results conrm that
miR- upregulation contributes to a microglia-mediated
neurotoxic response, which has been largely associated with
the M phenotype. Furthermore, recent studies have shown
that miR- is able to target M-associated genes, such as
that encoding SMAD, a protein involved in the TGF-𝛽
pathway [], and CEBP𝛽,atranscriptionfactorimportant
fortheexpressionofIL-,arginase,andCD[], further
supporting the hypothesis that miR- is required to skew
microglia activation to M-like phenotypes.
Several other miRNAs have been directly related to
the M phenotype, including miR- and miR-b. Zhu
and coworkers observed an upregulation in miR- levels
in response to several TLR ligands in macrophages. e
overexpression of this miRNA resulted in the downreg-
ulation of MAPK phosphatase (MPK-), promoting the
activation of MAPK and the expression of M-associated
proinammatory cytokines, such as IL-, TNF-𝛼,andIL-
. On the other hand, miR- inhibition enhanced MPK-
expression and decreased p and JNK activation [].
Regarding miR-b, Chaudhuri and colleagues reported an
increase in M macrophage activation, with upregulation of
MHC class II, CD, CD, and CD, upon overexpression
of this miRNA. e authors related these eects to miR-
targeting of interferon regulatory factor (IRF) and
also observed that, upon forced expression of miR-b,
macrophages adopted an M cytotoxic phenotype, presented
elevated responsiveness to IFN-𝛾, and were more eective in
killing EL T-lymphoma tumor cells in vitro and in vivo [].
In contrast to miR- and miR-b, miR-a was
recentlyshowntobedownregulatedinresponsetothe
activation of dierent TLRs, this decrease being neces-
sary to enhance TLR-triggered production of inammatory
cytokines in macrophages []. On the other hand, miR-
a overexpression inhibited the activation of the JNK/C-
JUN pathway through the targeting of mitogen-activated
protein kinase kinase (MKK), which activates JNK/stress-
activated protein kinase and a direct target of miR-a.
erefore, miR-a downregulation can be considered as an
additional requirement for M macrophage activation [].
Finally, as illustrated in Figure ,miR-hasbeen
reported to contribute to the M phenotype of macrophages
and microglia, since its forced overexpression led to the
downregulation of M-associated markers, such as IL-,
TNF-𝛼,andiNOS,andtoanincreaseofproteinsassociated
with the M phenotype, as is the case of TGF-𝛽, arginase-,
and FIZZ [].Itwasconcludedthat,inmicroglia,aninverse
correlation exists between the expression of M activation
markers and that of miR- (Figure ). Indeed, the highest
levels of miR- were detected in CDlow/MHC class
IIlow nonactivated resident microglia, but miR- expression
decreased dramatically upon cell treatment with IFN-𝛾and
GM-CSF, two stimulus known to upregulate CD and MHC
class II expression and to potentiate the M phenotype [].
Table lists several miRNAs involved in microglia activation,
indicating the phenotype (M or M) favored by their up- or
downregulation.
Taken together, the above-mentioned results highlight
the prominent role of miRNAs in the regulation of the
activation of both microglia and macrophages and open new
possibilities in the eld of anti-inammatory therapies. Using
appropriate gene therapy tools, miRNA modulation could
be an interesting and promising strategy to ne-tune the
immune response, skewing cell activation to the M or M
phenotypes according to the specic requirements of each
disease setting.
5. MiRNA Dysregulation in
Neurodegeneration and Neuroinflammation
Neurodegeneration is characterized by neuronal loss of spe-
cic neuronal circuits associated with cognitive and motor
functions. In this context, neuronal death is considered both
cause and consequence of neuroinammation, a process
involving microglia and astrocyte proliferation and activa-
tion. e excessive production of inammatory mediators
by these cells propagates inammation in the brain and
contributes to the triggering of a local and systemic immune
response, which is characterized by changes in miRNA levels
in the nervous tissue and in the periphery. Although the
trac of peripheral mononuclear cells from the blood stream
to the CNS is tightly regulated at the level of the BBB, it is
knownthattheintegrityofthisbarrierishighlycompromised
in severe brain diseases, such as stroke, brain trauma [],
and AD [,,]. e local disruption of the BBB in
a disease context facilitates the passage of blood-derived
cells to the nervous tissue. erefore, both microglia and
peripheral mononuclear phagocytes are able to impact the
pathoetiology of neurodegenerative diseases, and miRNAs
presenting altered expression levels in both of these cell
types can be used as new potential biomarkers and targets of
neurodegeneration.
rough the cellular crosstalk between the brain and
the periphery, the nervous system can inuence peripheral
immune functions, and, conversely, the immune system can
aect neuronal activity []. A specic subset of miRNAs
is able to regulate both cognitive and immune features, but
the neuroimmune impact of these molecules is far from
being fully understood. However, the role of miRNAs in
neuroimmune pathologies has recently started to be unveiled.
In the past few years, a large number of studies have emerged
Clinical and Developmental Immunology
M2 phenotype
Resting microglia
M1 phenotype
CD45 MHC II CD206
↑miR-124
↓miR-155
↑miR-124
↓miR-155
↑miR-155
↓miR-124
TGF-𝛽
IL-10
IFN-𝛾
GM-CSF
LPS
↑iNOS ↑FIZZ-1
A𝛽??
↑IL-6, TNF-𝛼and IFN-𝛽
↓SOCS-1, CEBP𝛽and SMAD2
↑IL-10, Arg-1 and TGF-𝛽
↓c-Jun, NF-𝜅B
F : MiRNA contribution to microglia activation phenotypes. Resting microglia cells are characterized by low expression levels of
miR- and a relatively high expression of miR-. In the presence of strong inammatory stimuli, such as IFN-𝛾,GM-CSF,andLPS,
microglia assume a classical activation phenotype (M), characterized by the upregulation of both CD and MHC II, the expression of several
inammatory mediators, such as iNOS, the inammatory cytokines IL- and TNF-𝛼, the type I interferon IFN-𝛽, and the downregulation
of miR-. MiR- upregulation is thought to be crucial for the establishment of this phenotype, since this miRNA directly targets several
anti-inammatory molecules, including SOCS-. Alternatively, in the presence of TGF-𝛽or the anti-inammatory cytokine IL-, a dierent
activation phenotype is observed (M). In this case, CD is upregulated at the cell surface, and anti-inammatory molecules involved
in tissue repair and angiogenesis are expressed. Moreover, most proinammatory pathways, including those regulated by the transcription
factors c-Jun and NF-𝜅B, are inactivated, and miR- upregulation is not observed. Certain endogenous danger signals associated with
neurodegenerative disorders, such as A𝛽brils present in senile plaques of AD patients, can also cause microglia activation, although the
exact nature of the observed phenotypic changes is yet to be fully characterized.
T : MiRNAs in microglia and macrophage activation.
Role Phenotype Ref
↑miR-
Tar g e t s S O CS - c ausing ↑of iNOS, TNF-𝛼,IL-,
and IFN-𝛽
Tar g e t s S M A D a n d C E B P 𝛽M [,,]
↑miR- Lead to ↓IL-, iNOS, and TNF-𝛼and ↑TGF-𝛽,
arginase-, and FIZZ M [,]
↑miR- ↓MPK- promoting MAPK activation and IL-,
TNF-𝛼, and IL- expression M []
↑miR-b ↑MHC class II, CD, CD, and CD through
targeting of IFR
Elevates responsiveness to IFN-𝛾M []
↓miR-a
Inhibited activation of JNK/c-Jun through
targeting of MKK
Its downregulation is necessary to promote the
M phenotype
M []
reporting the disruption of miRNA expression during neu-
roinammation and neurodegeneration processes []. For
example, miR-, which has been directly related with AD
[] and epilepsy [], has also been shown to be highly
overexpressed in A𝛽and TNF-𝛼stressed human microglia
cells, and this eect was inversely correlated with the levels of
inammation-related proteins, such as CFH and IRAK- [].
Another brain enriched miRNA, miR-, which has been
directly related with the maintenance of the quiescent state of
microglia [], has also been shown to inhibit the neuronal
transcription regulator complex REST, which is involved in
Rett syndrome [] and neuronal changes during chronic
cocaine intake [].
MiRNA dysregulation in the context of neurodegenera-
tion can be a consequence of genetic or sporadic anomalies.
In addition, the miRNA-related machinery can be impaired
Clinical and Developmental Immunology
in neurological disorders []. Accordingly, altered miRNA
proles have been identied in several neurodegenerative
diseases. Striatal miR-, miR-c, miR-, miR-, miR-
, miR-, miR-, miR-, and miR--p, as well as
the cellular levels of Drosha were shown to be downregulated
in both YAC and R/ transgenic mouse models of
Huntington’s disease (HD) []. In the frontal cortex and
striatum of human HD brains, several miRNAs were found
to be altered with respect to the brains of healthy subjects,
andchangesintheprimarynucleotidestructureofthe
terminus of certain miRNAs were also reported []. MiRNA
proling studies in Parkinson’s disease (PD) revealed an early
downregulation of miR-b/c in human brain areas, with
variable neuropathological eects at clinical (motor) stages
[],whileinALSmousemodelsdeciencyinmiR-
accelerated disease progression [].
A large number of studies using cellular and mouse mod-
els, human hippocampus, human cortex, and whole brain
samples have revealed altered miRNA expression proles
in AD. MiR-b was found to be aberrantly expressed in
the APPswe/PSΔE AD mouse model, and its levels were
correlated with the dysregulated expression of the TGF-𝛽
type II receptor []. Wang and coworkers have shown that
the exposure of SH-SYY cells to A𝛽1−42 oligomers leads
to the increase of miR-b expression and to the conse-
quent impairment in TGF-𝛽signaling, providing a possible
explanation for the observed neurodegeneration []. As
mentioned before, TGF-𝛽signaling is a marker of the M
microglia phenotype, and, as such, miR-b overexpression
could be considered an additional cause of microglia skewing
to the M phenotype following A𝛽exposure.
Interestingly, Schonrock and collaborators have shown
that half of the tested miRNAs in their study were downregu-
lated in hippocampal neuronal cultures in response to A𝛽1−42
peptides and that the dysregulated miRNAs were likely to
aect target genes belonging to signaling pathways known
to be disrupted in AD. ese results were further validated
in the hippocampus of APP mice and human AD brains
[]. Curiously, many of those miRNAs, as miR-, miR-
a, let-i, and miR-b, had already been associated with
inammation.
Although the majority of miRNA proling studies in
neurodegenerative diseases has been performed in brain
samples, miRNA dysregulation has been found in other
biological sources, such as plasma, peripheral blood, and
cerebral spinal uid (CSF). For example, miR-a was shown
to be signicantly increased in plasma from HD gene carriers
prior to symptom onset, suggesting that plasma miRNAs
canbeusedasbiomarkersinHD[]. In another study,
eighteen miRNAs were found to be dierentially expressed in
peripheral blood mononuclear cells of nineteen PD patients
with respect to miRNA levels in thirteen control subjects
[]. In blood serum from AD patients, miR-, miR-c,
miR-, and miR-a/b were found to be downregulated,
and although the ability of these miRNAs to conclusively
diagnose AD is currently unknown, these blood-circulating
miRNAs have potential to be used as additional biomarkers
of the disease []. Although more dicult to obtain, CSF
has also been considered a source of biomarkers for many
neurological diseases, since this uid constantly exchanges
material with the brain parenchyma and is less prone to the
inuence of peripheral organs. Alexandrov and collaborators
analyzed miRNA abundance in the CSF of AD patients and
observed a signicant increase in the levels of miR-, miR-
b, miR-a, and miR-, with respect to age-matched
controls. Interestingly, these miRNAs are NF-𝜅B-sensitive
proinammatory miRNAs, also known to be upregulated
in AD brains and have been associated with progressive
spreading of neuroinammation []. Taken together, these
results indicate that the eective application of miRNAs as
biomarkers for neurodegenerative diseases should include
miRNAproling,notonlyinthebloodbutalsoinserum,
plasma, and dierent cellular subtypes, as well as the parallel
correlation of the obtained results with brain morphology
overtime, in order to overcome clinical issues related with
disease staging and progression.
MiRNA deregulation has also been associated with viral-
induced neuroinammation and neurodegenerative pro-
cesses. Mishra and coworkers showed that human microglia
cells treated with HIV- Tat-C protein, a molecule involved
in neuroinammation in HIV-infected patients, present
increased miR- expression with consequent changes in
the levels of the downstream target TRAF, which, in turn,
was found to control IRF and IRF expression []. It was
also demonstrated that miR-a is upregulated in human
microglia cells under HIV infection, regulating the inam-
matory response by targeting the CCL [] chemokine.
erefore, it seems that miRNA expression is altered aer
inammation in immune and glial cells in order to support
the ne-tuning of immune functions essential to main-
tainbrainhomeostasis.AninterestingstudybyDaveand
Khalilishowedthatmorphine-inducedinammationand
oxidative stress in human monocyte-derived macrophages
contribute to the expansion of the HIV- viral reservoir in
the CNS and HIV-associated dementia []. e authors
provided evidence that this process is regulated by dier-
entially expressed miRNAs, including miR-b and miR-
b, both linked to several targets in the proinammatory
pathways.
MiRNAsarealsobelievedtomodulatemicrogliainam-
mation aer hypoxia/ischemia, which may contribute to
neuronal damage. Hypoxia causes upregulation of the Fas
ligand (FasL) and simultaneously downregulation of miR-
in microglia, inuencing neuronal apoptosis. Interest-
ingly, according to the work of Zhang and colleagues, the
ectopic expression of miR- partially protects neurons from
cell death caused by hypoxia-activated microglia []. e
same authors reported a potential role for miR-c in
the regulation of TNF-𝛼expression aer hypoxia/ischemia
and microglia-mediated neuronal injury. ey showed that
oxygen-glucose deprivation (OGD) induces microglia acti-
vation in vitro and in the hippocampus of Wistar rats
(four-vessel occlusion—-VO—rat model of ischemia), as
concluded by observation of the overproduction of TNF-𝛼,
IL-𝛽, and NO and the downregulation of miR-c. On the
other hand, the heterologous expression of this miRNA was
found to protect neurons from cell death caused by OGD-
activated microglia [].
Clinical and Developmental Immunology
Regarding neurodegenerative pathologies with a known
inammatory component, such as multiple sclerosis (MS),
miRNAshavealsobeenshowntoplayacentralrolein
theregulationofmicroglia-mediatedimmuneresponses.
Ponomarev and coworkers have demonstrated that miR-
is able to switch microglia from an inammatory to a
quiescent state, and this phenomena was considered essential
to successfully inhibit the onset of EAE []. Also in MS, miR-
−/− knockdown mice were shown to present signicantly
reduced numbers of encephalogenic CD+ cells, an
inammatoryT-cellsubsetwithanimportantroleinthis
disease []. In a very recent study, Butovsky et al. showed
that the modulation of the cytokine prole of inammatory
monocytes using an anti-LyC mAb reduced monocyte
recruitment to the spinal cord, decreased neuronal loss, and
extended survival in a mouse model of ALS. Interestingly, in
humans with ALS, the analogous CD+CD−monocytes
were shown to exhibit an ALS-specic miRNA inammatory
signature, which can be used as a biomarker of this disease
and reveal novel therapeutic targets [].
Another interesting eect associated with miRNAs
derives from their capacity to activate receptors by them-
selves, aer being secreted by cells within the CNS, which
allows them to function as signaling molecules. For example,
let- is known to induce neurodegeneration by binding to
TLR in neurons and microglia. Let- is increased in the CSF
of AD patients, and accordingly, the injection of this miRNA
in the CSF of wild-type animals caused neurodegeneration,
which did not occur in mice lacking TLR []. Also in the
context of AD, the NF-𝜅B-dependent miR-a was reported
to be upregulated in the brain of AD patients and to enhance
inammation by targeting the complement factor H (CFH)
[]. MiR-a was also found to be overexpressed in prion-
infected mouse brain tissues, concurrent with the onset of
prion deposition and microglia activation, which suggests
that this miRNA plays a role in the proinammatory response
of microglia to prion replication [].
e above-mentioned studies stress the role of miRNAs
as modulators of both neuroinammation and neurodegen-
eration and illustrate their potential as biomarkers and novel
therapeutic targets in CNS diseases.
6. MiRNA-Based Therapeutic
Applications in Neuroimmune and
Neurodegenerative Diseases
Over the last few years, there has been a signicant progress
in the development of strategies to modulate the levels of
certain miRNAs and miRNA clusters, aiming at adjusting
cellular functions dysregulated in several pathologies. Mod-
ulation of mature miRNAs can be accomplished by oligonu-
cleotides complementary to miRNA sequences (miRNA
inhibitors), causing miRNA knockdown or, alternatively,
by miRNA precursor oligonucleotides (miRNA mimics),
which cause miRNA overexpression [–]. Usually, these
oligonucleotides present chemical modications, such as the
incorporation of locked nucleic acids (LNA) nucleotides,
which have a methylene bridge between the -oxygen and
the -carbon of the ribose moiety or the incorporation of
-O-methyl modied nucleotides (antagomirs) in certain
positions of the oligonucleotide sequence. e purpose of
these modications is to increase the chemical stability and
resistance of the miRNA inhibitors or mimics to serum nucle-
ases, thus potentiating their therapeutic potential. However,
in order to achieve a successful clinical application of miRNA-
based therapies it is crucial that these oligonucleotides are
properly delivered by vehicles that not only reliably and
eectively overcome cellular and physiological barriers but
are also highly target specic. e presence of the BBB, which
restricts entry of therapeutic molecules into the brain, as
well as the possible degradation of nucleic acids by nucleases
present in the blood, constitutes major obstacles associated
with nucleic acid delivery in vivo. Nonviral vectors have been
developed to ensure protection and improvement of nucleic
acid delivery into target cells and have been employed in
miRNA-based therapeutic approaches to modulate neuroin-
ammatory signaling pathways [].
Inourownwork,wehaveshownthattheuseofa
nonviral strategy to promote miR- silencing in microglia,
prior to their activation, is able to reduce the release of
NO and other inammatory mediators, such as the major
inammatory cytokines TNF-𝛼and IL- to the extracellular
environment, through an increase in the levels of SOCS-, a
direct target of miR- (Figure ). e modulation of this
miRNA in microglia cells, prior to their activation with LPS,
proved to be enough to improve cell viability in neuronal
cultures incubated with microglia conditioned medium [].
ese results stress the neuroprotective potential of miR-
silencing in the context of neuroinammation.
Other miRNAs have been related with the regulation of
the neuroimmune response. As discussed before, miR- is
one of the main miRNAs responsible for the maintenance of
the quiescent state of microglia/macrophages in the brain and
spinal cord, and therefore, miR- may constitute an impor-
tant target for the development of therapeutic approaches
towards the control of neuroinammation. Recently, Wille-
men and coworkers investigated the contribution of miR-
to the regulation of hyperalgesia and microglia/macrophage
activation in LysM-GRK+/− mice, in which the expression of
the G protein-coupled receptor kinase (GRK) is decreased
to about % in microglia/macrophages, with respect to wild-
type animals. ese mice develop inammatory hyperalgesia
caused by activation of microglia/macrophages in the spinal
cord. e authors showed that the pathological transition
from acute to persistent hyperalgesia is associated with
reduced levels of miR- in spinal cord microglia and
with a microglia M phenotype switch, leading to increased
production of proinammatory cytokines. e intrathecal
administration of miR- mimics prevented completely the
transition to persistent pain in LysM-GRK+/− mice and
normalized the expression of microglial M/M markers,
suggesting that miR- administration may be a promising
approach to prevent and treat persistent inammatory and
neuropathic pain [].
Another relevant study on miRNA therapeutics in the
context of neuroinammation was reported by Selvamani
Clinical and Developmental Immunology
SOCS-1 mRNA (fold change)
Control LPS
2
3
4
5
6
1.5
1
0.5
0
∗∗
Oligo scramble
+ LPS
Anti-miR-155
+ LPS
(a)
Cytokine levels (fold change)
70
60
50
40
30
20
10
3
2
1
0
TNF-𝛼IL-1 𝛽IL-6
Control
LPS
Anti-miRl55 + LPS
∗∗∗
∗∗∗
(b)
F : MiR- inhibition in microglia cells increases SOCS- levels and decreases the release of inammatory cytokines to the extracellular
medium. N mouse microglia cells were transfected with anti-miR- oligonucleotides (anti-miR) or with nontargeting oligonucleotides
(oligo scramble) complexed with cationic liposomes for h. Twenty-four hours aer transfection, cells were incubated with LPS at .𝜇g/mL
for h. e cell culture medium was then collected to determine cytokine protein levels, and total RNA was extracted from the cultured
cells. (a) SOCS- mRNA levels were quantied by qRT-PCR. (b) e levels of TNF-𝛼,IL-𝛽, and IL- secreted to the cultured medium were
determined by ELISA. Results are expressed as fold change of mRNA or protein levels with respect to control (untransfected and untreated
cells). ∗∗𝑃< 0.01 compared to cells transfected with the scramble oligonucleotide and ∗∗∗𝑃< 0.01 compared to LPS-treated cells in the
absence of transfection. Results in (a) and (b) are representative of independent experiments performed in triplicate.
andcoworkers.eauthorshypothesizedthatmiRNAs
able to target proteins from the insulin-like growth factor-
(IGF-) signaling family could be suppressed to promote
the neuroprotection provided by IGF- following stroke.
Using middle-age female rats in which the treatment with
estrogen is known to be neurotoxic, the authors administered
stereotactically LNA antisense oligonucleotides against miR-
or let-f, h aer stroke, and observed that miRNA
inhibition extended the neuroprotection aorded by IGF-.
Interestingly, although let-f is a proinammatory miRNA
preferentially expressed in microglia from the ischemic hemi-
sphere, where the IGF- expression is detectable, the levels
of IGF- increased even further in microglia aer anti-let-f
treatment []. Finally, a study from Lukiw and coworkers
showed that the expression of miR-a is increased in AD
brains, which was correlated with a decrease in the CFH
expression, a protein responsible for the repression of the
inammatory response in the brain. e inhibition of miR-
, achieved through delivery of a specic LNA-antisense
oligonucleotide in human neural cells subjected to oxidative
stress, was found to restore CFH expression levels, which
weredecreasedfollowingoxidativedamage[].
Taken together, these reports illustrate the vast neuropro-
tective potential of miRNA-based therapeutic strategies using
anti-miRNA oligonucleotides targeting neuroinammation
and conrm the important role of miRNAs in modulating
neuroimmune responses to acute and chronic brain damage.
Although less explored, the application of miRNA mimics,
in order to restore miRNA expression and decrease the
levels of potentially neurotoxic proteins, is also an interesting
possibility with high therapeutic potential. Nevertheless, it is
important to mention that, due to incomplete pairing, each
miRNA has the ability to target multiple genes simultane-
ously, which signicantly increases the risks of unspecic
eects on miRNA-based therapeutic approaches. To avoid
this drawback, it will be relevant to consider not only the
strength of the binding between a certain miRNA and all
its available targets in a specic tissue but also the relative
amounts of these targets and the thermodynamic stability of
each miRNA : mRNA duplex [].
7. Conclusion
Although the exact nature of the contribution of microglia
and peripheral mononuclear phagocytes to neurodegenera-
tion remains to be fully elucidated, several benets have been
suggested for the use of these immune cells in therapeutic
strategies designed to curb amyloidosis, decrease EAE pro-
gression, ght CNS viral infection, or assist in the reduction
of neuroinammation associated with neurodegenerative
diseases. On the other hand, several groups have identied
signs of chronic activation and functional impairment in
microglia cells isolated from dierent brain disease mod-
els. Given their important role in the regulation of gene
expression, we believe that miRNA-based therapies could
constitute an interesting and attractive strategy to improve
Clinical and Developmental Immunology
microglia activity, modulating signaling pathways linked with
neuroinammation. In addition, the compromised activity
of the BBB in most neurodegenerative disorders, the lower
activation threshold of peripheral mononuclear phagocytes
compared to brain-derived microglia, and their easy access in
a clinical context make these cells another excellent tool for
future gene therapy approaches for brain disorders. Overall,
understanding the contribution of brain- and blood-derived
immune cells to microglia pools in the CNS in a disease
context will be of great importance to improve the existing
immunotherapies and generate new and eective therapeutic
strategies for these diseases.
e ne-tuning activity of miRNAs has been proven
crucial in the regulation of dierentiation of microglia allow-
ing the maintenance of brain homeostasis. Since a single
miRNA has the capacity to target more than one protein
involved in the same signaling pathway, their modulation can
signicantly change cell phenotypes that depend on the levels
and activation of specic proteins. Such capacity reects a
molecular paradigm suitable for therapeutic intervention.
Due to the lack of minimally invasive diagnosis tools and
eective therapeutic options for most CNS diseases, we
believethattheuseofmiRNAs,bothasdiseasebiomarkers
andtherapeutictargetsassociatedwithcellsoftheimmune
lineage, although yet poorly explored, will tend to grow in the
near future.
Acknowledgments
e present work was supported by Grants from the
Portuguese Foundation for Science and Technology and
FEDER/COMPETE (PTDC/BIM-MEC// and Pest-
C/SAU/LA/). Joana Guedes and Ana Lu´
ısa Cardoso
are recipients of fellowships from the Portuguese Founda-
tion for Science and Technology (SFRH/BD// and
SFRH/BPD//, resp.).
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