Review

Astrocytes: Key Regulators of

Neuroinflammation
Emanuela Colombo1 and Cinthia Farina1,*
Astrocytes are crucial regulators of innate and adaptive immune responses in
Trends
the injured central nervous system. Depending on timing and context, astrocyte
Astrocytes are active players in neu-
activity may exacerbate inflammatory reactions and tissue damage, or promote roinflammation, and their response
immunosuppression and tissue repair. Recent literature has unveiled key fac- may be beneficial or detrimental for
tissue repair, depending on the kind
tors and intracellular signaling pathways that govern astrocyte behavior during of stimuli offered by the inflamed milieu.
neuroinflammation. Here we have re-visited in vivo studies on astrocyte signal-
ing in neuroinflammatory models focusing on evidences obtained from the The regulatory function of the astrocyte
in vivo is regulated by specific signaling
analysis of transgenic mice where distinct genes involved in ligand binding, pathways. Transgenic animals carrying
transcriptional regulation and cell communication have been manipulated in mutations in astrocyte proteins
astrocytes. The integration of in vivo observations with in vitro data clarifies involved in ligand binding, transcrip-
tional regulation, and cell communica-
precise signaling steps, highlights the crosstalk among pathways and identifies tion have been instrumental to dissect
shared effector mechanisms in neuroinflammation. astrocyte signaling in distinct neuroin-
flammatory models.

A Controversial Role for Astrocytes in Neuroinflammation TGFb, IFNg, gp130, estrogen, STAT3,
BDNF, and FasL support the protective
Astrocytes are the most abundant glia cell type of the central nervous system (CNS) and are
phenotype of the astrocyte, whereas
essential for brain homeostasis, as they provide metabolites and growth factors to neurons, IL17, sphingolipids, TrkB, SOCS3,
support synapse formation and plasticity, and regulate the extracellular balance of ions, fluid and NFkB, chemokines and VEGF trigger
neurotransmitters [1]. Thanks to their strategic location in close contact with CNS-resident cells damaging pathways.
(neurons, microglia, oligodendrocytes and other astrocytes) and blood vessels, astrocytes partici-
During neuroinflammation, astrocytes
pate in blood brain barrier (BBB) maintenance and permeability. They also play a role in the control are exposed simultaneously to a
of immune cell trafficking and activation. Astrocytes are immune-competent cells able to detect plethora of stimuli leading to a complex
danger signals, respond via secretion of cytokines and chemokines, and activate adaptive network of intracellular events. How-
ever, distinct activation modes may
immune defense [2,3]. CNS injury triggers a process leading to scar formation, whose impact
share signaling steps and effector
on tissue homeostasis is ambivalent, as inflammatory and neurotoxic mediators are produced at mechanisms.
injury site but remain confined to that area thanks to the glial scar [4]. Expression of the cytoskeletal
glial fibrillary acidic protein (GFAP) is widely used for the identification of astroglia in vivo and in vitro,
and upregulation of this marker in astrocytes is a typical hallmark for CNS pathologies [1]. GFAP is
also widely expressed by progenitor cells of neurons, oligodendrocytes and astrocytes [5], and
therefore its loss during development in constitutive GFAP knockout (KO) mice may have a long-
term impact on cell types other than astrocytes. Pioneering studies in such mice have shown no
gross alterations in brain architecture and BBB tasks in young animals, but white matter pathology
in old mice, indicating a physiological relevance for those intermediate filaments only during aging
[6,7]. Yet, GFAP plays a role during CNS infection and autoimmunity, as young GFAP KO mice
show more severe clinical expression of Toxoplasma encephalitis (TE, see Glossary), Staphy-
lococcus aureus-induced brain abscess and experimental autoimmune encephalomyelitis
(EAE) than wild type (wt) animals [8,9] (Table 1). To better evaluate the role of astrogliosis during 1
Institute of Experimental Neurology
CNS injury in adult animals, Sofroniew and colleagues have generated inducible transgenic mice (INSpe), Division of Neuroscience, San
where selective ablation of proliferating astrocytes is achieved in adult mice by ganciclovir Raffaele Scientific Institute, Milan, Italy

administration (GFAP-TK mice) [10] (Table 1). In vivo experiments in distinct disease models
(brain injury (BI), spinal cord injury (SCI) or EAE) consistently show that the loss of reactive *Correspondence: farina.cinthia@hsr.it
astrocytes during the early phases of injury results in exacerbation of clinical signs and motor (C. Farina).

608 Trends in Immunology, September 2016, Vol. 37, No. 9 http://dx.doi.org/10.1016/j.it.2016.06.006

© 2016 Elsevier Ltd. All rights reserved.
Table 1. Impact of Astrocyte Depletion in Neuroinflammatory Modelsd. Glossary
Transgenic Disease model Effects of deletion Refs Brain abscess: focal brain infection
model induced by stereotactic injection of
Staphylococcus aureus.
Disease Inflammation Demyelination Neuronal Brain injury (BI): contusion or stab
severity damage injury performed with a steel-tipped
EAE " = = N.A. [8] piston or a blade respectively.
Experimental autoimmune
GFAP -/- TE " " N.A. N.A. [9] encephalomyelistis (EAE): T cell-
Brain abscess " " N.A. N.A. [9] mediated autoimmune disease of the
central nervous system with clinical
Forebrain Stab injury a
" " N.A. " [10] and neuropathological similarities to
SCIa " " " " [11,16] multiple sclerosis. It is induced by
active immunization with myelin
Cortical contusion Injury a
" " N.A. " [12] extracts, purified myelin proteins, or
GFAP-TK EAEa " " N.A. N.A. [15] immunogenic myelin peptides; or by
adoptive transfer of myelin-reactive T
EAE a
" " " " [13] lymphocytes.
EAEb " " N.A. N.A. [14] Floxed mice: transgenic mice where
two LoxP sites are positioned in
EAE c
# # N.A. N.A. [15] intronic regions flanking one or
a several essential exons of the target
Ganciclovir (GCV) administration pre- or immediately post-injury.
b gene. Recognition of LoxP sites by
GCV administration after disease onset.
c
GCV administration 30 days post immunization. Cre recombinase leads to excision of
d
Key. ", worsening in specific disease outcomes; #, improvement in specific disease outcomes; = no difference; N.A., not LoxP-flanked DNA. Thus, tissue-
assessed. specific gene deletion is achieved by
breeding floxed mice with transgenic
mice expressing Cre recombinase
under the control of a tissue-specific
deficits, scar disorganization, spreading and persistence of inflammatory cells, BBB alterations, promoter.
and, when analyzed, demyelination and neuronal death [10–16] (Table 1). By contrast, astrocyte Spinal cord injury (SCI): contusion,
depletion during the chronic phase of EAE ameliorates disease expression and reduces leukocyte compression, distraction, dislocation,
infiltration into the CNS [15] (Table 1). These data indicate that the outcome of astrogliosis is transection or chemical damage of
the spinal cord.
regulated in a time- and context-specific manner, and that stimuli from the microenvironment Stroke: induced by moderate to
during neuroinflammation may shift astrocyte action from beneficial to detrimental for the neural severe middle cerebral artery
tissue. The direct impact of the loss of a structural protein on glial inflammatory signaling remains, occlusion.
Toxic demyelination: acute
however, to be elucidated. Thus, the development of novel therapies for neuroinflammatory
demyelination induced by diet
disorders relies on the identification and selective targeting of specific astrocytic functions. To enriched with Cuprizone (a copper-
this end, the GFAP promoter has been instrumental to drive or abolish gene expression in astroglia chelating agent toxic to
in vivo and study astrocyte function in distinct neuroinflammatory models. Table 2 details the oligodendrocytes).
Toxoplasma encephalitis (TE):
impact of single molecular perturbations in the astrocyte on disease outcomes. In the next
systemic parasitic infection followed
paragraphs we discuss how the protective versus detrimental behavior of glia cells is regulated by a chronic stage with cyst
by surface or cytoplasmic proteins, transcription factors and released mediators, and integrate the formation in the brain. It is induced
information derived from in vivo studies with in vitro findings to highlight the crosstalk among by oral or intraperitoneal
administration of Toxoplasma gondii.
distinct pathways and identify shared effector mechanisms in neuroinflammation.

Protective Astrocyte Signaling Pathways Regulated by Cytokines, Growth

Factors and Hormones
In vivo studies have provided compelling evidence that astrocyte responses to certain cytokines,
growth factors and hormones are protective, as their absence worsens CNS injury (Table 2 and
Figure 1).

The first protective pathway is mediated by the glycoprotein gp130, an essential signal trans-
ducer for members of the IL6 cytokine family. The investigation of TE and EAE in mice lacking
gp130 in GFAP-positive cells (GFAPcre-gp130fl/fl or floxed mice) has demonstrated that
astrocytic gp130 signaling is crucial for glia cell survival and control of disease expression
(Table 2 and Figure 1) [17,18]. In fact GFAPcre-gp130fl/fl mice display astrocyte apoptosis, larger
areas of parasite-induced tissue necrosis, higher mortality after TE infection [17] and

Trends in Immunology, September 2016, Vol. 37, No. 9 609

Table 2. Impact of Defined Astrocyte Perturbations in Neuroinflammatory Modelsa.
Deleted or Disease Effects of deletion Refs
inactivated model
target

Disease Inflammation Glia Demyelination Neuronal

severity activation damage

gp130 TE " = " N.A. N.A. [17]

EAE " " N.A. " N.A. [18]

TGFbR Stroke " " " N.A. N.A. [22]

TE = " " N.A. " [23]

IFNgR EAE " " " " " [30]

ER/ EAE " " " " " [32,34]

Protective
astrocyte A20 EAE " " N.A. " N.A. [58]
pathways STAT3 SCI " " " " " [16,61,
62,65]

White " N.A. # " N.A. [64]

matter
injury

FasL EAE " " N.A. " N.A. [89]

BDNF EAE " = = = " [90]

Act1 EAE # # # N.A. N.A. [38,39]

S1P1 EAE # N.A. N.A. # # [42]

B4GALT6 EAE # # # N.A. N.A. [15]

TrkB EAE # # # N.A. # [45]

NFkB SCI # # # # # [47,56]

EAE # # # # # [48–50]

BI N.A. # = N.A. N.A. [52,53]

Detrimental
Retinal # # # N.A. # [54,55]
astrocyte
ischemia
pathways
Nerve # # N.A. N.A. N.A. [51]
Injury

Cuprizone # # # # N.A. [57]

SOCS3 SCI # # " # N.A. [61]

CCL2 EAE # # # # # [82–84]

CXCL10 EAE # # = # # [85]

VEGF EAE # # = # N.A. [86]

Key. ", worsening in specific disease outcomes; #, improvement in specific disease outcomes; = no difference; N.A., not
a

assessed.

exacerbated EAE scores associated with enhanced demyelination and T cell recruitment [18]
compared to control animals. Ligand binding to the gp130 receptor activates signal transducer
and activator of transcription (STAT) 1/3 and mitogen-activated protein kinase (MAPK) (SHP2/
Ras/ERK) signaling cascades, which negatively control each other [19] (Figure 2). By crossing
the GFAPcre-gp130fl/fl mice with animals harboring either gp130DSTAT mutation (resulting in
the lack of STAT1/3 and excessive SHP2/Ras/ERK activation) or gp130Y757F allele (resulting in
the lack of SHP2/Ras/ERK and excessive STAT1/3 activation), Haroon et al. have observed
enhanced EAE severity in mice with defective SHP2/Ras/ERK but not STAT1/3 cascades,

610 Trends in Immunology, September 2016, Vol. 37, No. 9

 Protecve Detrimental
pathways pathways

TRKB-T1
IL17R

TGFβR

gp130-IL6R

Act1 S1P1
IFNγR

A20 SOCS3
LacCer
B4GALT6

ERα

STAT3 NFκB

CCL2
BDNF
STAT3 NFκB
VEGF Cytokine/growth factor Outside cell
FasL receptors
CXCL10
γ IκK
SOCS3
α β complex
JAK
JAK P

STAT3
P Proteasomal
Iκb
STAT3 P Cytoplasm degradaon
STAT3 P p50 p65

STAT3
Nucleus p50 p65
STAT3
Clinical improvement Clinical deterioraon
Reduced astrogliosis Enhanced astrogliosis
Immunosuppression Immune cell recruitment
An-inflammatory cytokine release Cytokine/chemokine release
Neuronal survival Neuronal death
Preserved myelin Oxidave stress
Blood brain barrier damage
Demyelinaon

Figure 1. Impact of Astrocyte Perturbations on Neuroinflammation. The figure depicts transmembrane receptors, cytoplasmic proteins, transcription factors or
released mediators involved in astrocyte activation and their effects on disease outcome. Inset highlights details in STAT3 and NFkB signaling pathways. Abbreviations:
B4GALT6, b-1,4-galactosyltransferase 6; BDNF, brain-derived neurotrophic factor; CCL2, C-C motif chemokine ligand 2; CXCL10, C-X-C motif chemokine ligand 10;
ER/, estrogen receptor /; IFNgR, interferon g receptor; IkB, inhibitor of nuclear factor kB; IkK, IkB kinase complex; IL17R, interleukin 17 receptor; JAK, Janus kinase;
NFkB, nuclear factor kB; S1P1, sphingosine -1-phosphate receptor 1; SOCS3, suppressor of cytokine signaling 3; STAT3, signal transducer and activator of
transcription 3; TGFbR, transforming growth factor b receptor; TRKB-T1, truncated tropomyosin receptor kinase B; VEGF, vascular endothelial growth factor.

indicating that gp130-mediated SHP2/Ras/ERK activation limits neuroinflammation [18]

(Figure 2).

TGFb signaling in astrocytes is of particular interest because the ligand has important immuno-
suppressive properties, is produced by glia cells under physiological conditions and strongly
upregulated after brain injury [20,21]. Mice with depletion of TGFb signaling in astrocytes (Ast-
Tbr2DN mice) display impaired upregulation of TGFb in the peri-infarct cortex, defective motor
functions, diffused inflammation and enhanced myeloid cell activation after stroke (Table 2 and
Figure 1) [22]. After TE infection, Ast-Tbr2DN mice are characterized by enhanced gliosis, T cell
infiltration, and proinflammatory cytokine and chemokine production, and neuronal damage,
despite the lack of TGFb signaling does not affect Toxoplasma burden or distribution in the brain

Trends in Immunology, September 2016, Vol. 37, No. 9 611

 TGFβR
gp130-IL6R

Act1

IFNγR IL17R

SOCS3

S1P1
JAK

STAT3

BDNF STAT3
P
MAPK
IL1R
P
STAT3

IκB

NFκB
A20
TRKB-T1

STAT3
STAT3 NFκB

ERα
LacCer

B4GALT6
ERα

Nitric
oxide CXCL10 CCL2

VEGF

Figure 2. Crosstalk among Distinct Intracellular Signaling Pathways in the Astrocyte. In vivo and in vitro observations highlight MAPK, NFkB and/or STAT3
activation as critical convergence events among distinct astrocyte responses. Abbreviations: NFkB, nuclear factor kB; STAT3, signal transducer and activator of
transcription 3;. B4GALT6, b-1,4-galactosyltransferase 6; BDNF, brain-derived neurotrophic factor; CCL2, C-C motif chemokine ligand 2; CXCL10, C-X-C motif
chemokine ligand 10; ER/, estrogen receptor /; IFNgR, interferon g receptor; IkB, inhibitor of nuclear factor kB; IL17R, interleukin 17 receptor; JAK, Janus kinase; MAPK,
mitogen-activated protein kinase; NFkB, nuclear factor kB; S1P1, sphingosine 1-phosphate receptor 1; SOCS3, suppressor of cytokine signaling 3; STAT3, signal
transducer and activator of transcription 3; TGFbR, transforming growth factor b receptor; TRkB-T1 truncated tropomyosin receptor kinase B-T1,; VEGF, vascular
endothelial growth factor.

[23] (Table 2 and Figure 1). It is known that TGFb pathway inhibits the activation and nuclear
translocation of the proinflammatory transcription factor nuclear factor kB (NFkB) [24]. Accord-
ingly, Cekanaviciute et al. observe a significantly higher proportion of astrocytes with nuclear
NFkB expression in Ast-Tbr2DN mice compared with control animals after Toxoplasma gondii
infection, suggesting that astrocytic TGFb signaling may reduce neuroinflammation and neuro-
nal damage via downregulation of NFkB signaling [23] (Figure 2).

612 Trends in Immunology, September 2016, Vol. 37, No. 9

Interferon (IFN)-g is a typical pro-inflammatory cytokine primarily produced by activated T
lymphocytes and natural killer cells. During CNS inflammation IFN-g acts in multifaceted ways,
as it may initiate oligodendrocyte death [25] and contribute to macrophage and microglia
activation [26] or support neuroprotection by negative regulation of neutrophil and T cell
accumulation [27–29]. Given the dual role of this mediator in neuroinflammation, Stohlman
et al. have analyzed the impact of IFN-g pathway in astrocytes during EAE [30] (Table 2). Despite
normal onset of clinical symptoms, mice with defective IFN-g signaling in astrocytes (GFAPgR1D)
exhibit aggravated EAE leading to enhanced mortality [30]. Escalating disease in GFAPgR1D
animals is paralleled by accentuated demyelination during acute and chronic phases of EAE,
enhanced leukocyte infiltration during late stage of the disease, upregulation of transcripts of
inflammatory genes [C-C motif chemokine ligand 2 (CCL2), CCL5, C-X-C motif chemokine
ligand 10 (CXCL10), iNOS, and tumor necrosis factor (TNF)] and, conversely, reduction of the
anti-inflammatory cytokines IL-10 and IL-27 when compared to control mice [30] (Figure 1).
Although the sequence of events and the cellular players regulated by IFN-g signaling in
astrocytes remain to be elucidated, these data indicate that the pathway provides important
braking signals to neuroinflammation via the astrocyte.

Finally, protective effects are achieved upon astrocyte activation in response to estrogens
(Figure 1). Several lines of evidence support the therapeutic potential of estrogens in human
and experimental CNS disorders, including multiple sclerosis (MS) and EAE [31,32]. Treatment
with ligands for estrogen receptor (ER) / or b ameliorates EAE and reduces inflammation,
demyelination and axonal loss [33]. Importantly, the anti-inflammatory and neuroprotective
effects of systemic treatment with ER/ ligand are completely prevented by conditional deletion
of ER/ in astrocytes but not neurons [34] (Figure 1). In fact estrogen administration to transgenic
mice defective for ER/ signaling in astrocytes during EAE does not prevent reactive astrogliosis,
demyelination and axonal damage, and does not reduce chemokine expression in activated
astrocytes [32]. On the contrary, treatment with ERb ligand ameliorates EAE independently from
ERb expression in astrocytes [32]. Together, these findings indicate that ER/ but not ERb
signaling in astrocytes is necessary to limit neuroinflammation (Table 2 and Figure 1).

Detrimental Astrocyte Signaling Pathways Induced by Cytokines,

Sphingolipids and Neurotrophins
During neuroinflammation, astrocytes upregulate the receptor for IL17 [35], an important
inflammatory cytokine released by effector T lymphocytes. IL17 binding to heteromeric trans-
membrane receptors results in the recruitment of NFkB activator 1 (Act1) and formation of a
signaling complex which triggers the production of pro-inflammatory cytokines, chemokines and
metalloproteinases [36]. Mice lacking Act1 in the neuroectodermal lineage (neurons, oligoden-
drocytes, and astrocytes) are less susceptible to EAE induction [37], and this phenotype can be
reproduced in mice lacking Act1 in astrocytes (GFAPcre-Act1fl/fl) [38] (Table 2 and Figure 1).
Consistent with the reduced clinical score, mononuclear cell infiltration and induction of IL17-
regulated inflammatory genes are attenuated in the spinal cord of GFAPcre-Act1fl/fl EAE mice
[37,38]. To test the therapeutic effects of IL17 pathway blockade on ongoing neuroinflammation,
Zhang and colleagues have injected vectors expressing Act1-specific short hairpin RNA (shAct1)
or control lentivirus (shVec) under the control of GFAP promoter intracerebroventricularly during
EAE. While shVec does not affect typical development of EAE, shAct1 significantly reduces EAE
severity [39], indicating that targeting of IL17 pathway in astrocytes is effective in treating
established neuroinflammation (Table 2 and Figure 1).

Another important detrimental signaling pathway in astrocytes is regulated by sphingolipids,

including sphingosine 1-phosphate (S1P) and LacCer (Table 2 and Figure 1). S1P regulates
cellular processes such as growth, survival and differentiation via binding to specific G-protein-
coupled receptors (S1P1-5) [40]. It is well recognized that in both homeostatic and pathological

Trends in Immunology, September 2016, Vol. 37, No. 9 613

settings the S1P–S1P1 axis controls leukocyte trafficking from the lymphoid tissues to blood
circulation [40]. Importantly, S1P receptors are strongly upregulated on activated glia cells in MS
and EAE lesions [35,41]. Conditional mice lacking S1P1 in astrocytes display ameliorated EAE
expression, reduced demyelination and axonal loss [42], indicating that S1P signaling in
astrocytes is pathogenic (Table 2 and Figure 1). In vitro studies have dissected the effects of
S1P on astrocyte–neuron interaction and shown that, while physiological S1P concentrations do
not induce changes when given directly to neurons, they do promote neurodegeneration when
given to astrocytes, demonstrating that astrocytes are crucial in driving S1P-induced neuro-
degeneration [35].

LacCer is another lipid mediator triggering inflammation and astrogliosis. Its synthesis is cata-
lyzed by b-1,4-galactosyltransferase 5 (B4GALT5) and B4GALT6, two members of the
b4-galactosyltransferase family, both highly expressed by astrocytes during chronic EAE and
MS [43]. Interestingly, the knockdown of B4GALT6 but not of B4GALT5 by intracerebroven-
tricular injection of shRNA-encoding lentiviruses under the control of GFAP promoter decreases
the concentration of LacCer in the CNS during the progressive phase of EAE, suppresses
disease progression, and reduces recruitment of inflammatory monocytes [15] (Table 2 and
Figure 1). In vitro experiments indicate that LacCer leads to the activation of interferon regulatory
factor 1 (IRF-1) and NFkB transcription factors and to their recruitment to Cfs2 (GM-CSF)
promoter in astrocytes (Figure 2). Further, B4GALT6 knockdown in astrocytes during EAE
lowers Nos2 expression in microglia, suggesting that GM-CSF produced by astrocytes in a
B4GALT6-LacCer-dependent manner modulates microglia activation [15] (Table 2).

Finally, glia cells may be a target of neurotrophins, growth factors essential for neuron survival
and axonal growth [44]. While occasionally present on astrocytes in control white matter, the
TrkB receptor for the brain-derived neurotrophic factor (BDNF) is strongly upregulated on glia
cells in MS and EAE lesions [45]. Surprisingly, conditional mice lacking TrkB in GFAP-positive
cells (GFAPcre-TrkBfl/fl) show milder EAE susceptibility and severity than control mice (Table 2
and Figure 1). Accordingly, the number of myeloid cells, T and B lymphocytes, and degenerating
neurons are reduced in the spinal cord of GFAPcre-TrkBfl/fl EAE mice compared to controls [45].
In vitro and in vivo evidences indicate that TrkB signaling regulates nitric oxide release from
astrocytes [45] (Figure 2). Thus, in contrast to the well-established neurotrophic functions of
TrkB ligands, we have demonstrated that astrocyte activation via TrkB supports expression of
neuroinflammation [45].

Astrocyte Transcription Factors and Neuroinflammation

A crucial transcription factor in any inflammatory reaction is NFkB (Figure 1 and Box 1). NFkB
activation and NFkB-dependent transcription of pro-inflammatory factors are relevant for the
amplification of the inflammatory and neurodegenerative processes [46]. To dissect the contri-
bution of astroglial NFkB to CNS neuroinflammation, Bethea et al. have generated a transgenic
mouse overexpressing a dominant negative form of IkB/ in glia cells, which blocks nuclear
translocation of NFkB [47]. NFkB inactivation in astrocytes improves the clinical outcome in
distinct inflammatory models including SCI [47], EAE [48–50], nerve injury [51], brain injury
[52,53] and retinal ischemia [54,55] (Table 2). A reproducible observation is the decreased level
of chemokines and oxidative stress within the injured nervous system in absence of astrocyte
NFkB [47–56], thus supporting the hypothesis that astrocyte NFkB regulation of proinflamma-
tory and oxidative pathways is central to neuroinflammation and neurotoxicity (Table 2 and
Figure 1). In harmony with these findings, the spinal cord of GFAP-IkB/-DN EAE animals shows
a clear reduction in the frequency of infiltrating immune cells, including T helper 1 (Th1) and Th17
CD4 T cells, B cells and macrophages compared to that of control EAE mice [48,50]. Further,
when exposed to the compound cuprizone, which induces toxic demyelination in the absence
of immune cell infiltration, GFAP-IkB/-DN mice show more limited gliosis and myelin

614 Trends in Immunology, September 2016, Vol. 37, No. 9

 Box 1. Transcription factors in neuroinflammation.
NFkB. In addition to the well-known role in immune responses and inflammation, emerging evidences indicate that NFkB
is expressed in all cell types in the CNS and involved in processes as neuronal development, synaptic plasticity and
neurodegeneration [92]. NFkB dimers are commonly composed of p50 and p65 subunits in the CNS, and are trapped in
the cytoplasm by the inhibitor IkB/ [93]. Cell activation involves phosphorylation of IkB/ by IkB kinase (IkK) complex,
ubiquitination and proteasomal degradation of IkB/, and NFkB translocation to the nucleus, where it modulates the
expression of a complex array of genes encoding cytokines and chemokines, antiapoptotic proteins, cell adhesion
molecules, glial fibrillary acidic protein, inducible nitric oxide synthase, and neurotrophic factors (for review [92,93]).
Experimental models of neuroinflammation and in vitro studies have revealed complex and, sometimes, opposite
functions of NFkB in the CNS. For example, in the EAE model, NFkB inhibition in all neuroectodermal cells of the
CNS is protective for disease development, because NFkB activation in CNS-resident cells is necessary to establish and
support the inflammatory milieu [94]. On the contrary, NFkB inactivation specifically in neuronal cells results in more
severe EAE clinical symptoms and increased axonal loss [95]. Mice with inactivation of microglia NFkB show a ten-times
reduction in infarct size after experimental stroke, suggesting that activation of NFkB in microglia is pathogenic [96].
Accordingly, in vitro studies have shown that NFkB protects neurons against excitotoxic and metabolic insults [97], but it
induces production of neurotoxic agents by other cell types such as microglia and astrocytes, thus leading to neuronal
apoptosis indirectly [35,98].

STAT3. STAT3 is a member of the STAT family of cytoplasmic transcription factors. The phosphorylation of a specific
tyrosine residue by Janus kinases (JAK) promotes the formation of STAT dimers, which are transported into the nucleus.
The modulation of this pathway involves different mechanisms including direct inhibition of JAK activity by SOCS3
(suppressor of cytokine signaling 3) [99]. Whereas STAT3 is not activated in healthy CNS, its levels and activity increase
during CNS inflammation [59,60,100]. In vivo evidences suggest that STAT3 is implicated in axon regeneration after
injury, as STAT3 overexpression or SOCS3 deletion promote regeneration of optic nerve [101,102].

preservation compared to control mice, indicating that blockade of NFkB pathway in astrocytes
is sufficient to reduce the inflammatory burden sustained by CNS-resident cells and associated
with myelin damage [57] (Table 2 and Figure 1). Additional evidence for a role of astrocyte NFkB
in neuroinflammation comes from the analysis of another transgenic mouse where hyper-
activation of NFkB is achieved in astrocytes by selective deletion of the ubiquitin-modifying
protein A20 [58] (Table 2 and Figures 1 and 2). GFAPcre-A20fl/fl mice are more susceptible to
EAE and display pronounced demyelination and leukocyte infiltration, accompanied by a
proinflammatory gene transcription profile [58]. Taken together, these findings indicate that
the NFkB pathway in astrocytes fosters neuroinflammation and that NFkB inhibition in glia cells
may be beneficial in inflammatory neurological disorders.

Another transcription factor activated in response to extracellular signals like cytokines and
growth factors is signal transducer and activator of transcription 3 (STAT3) (Box 1 and Figure 1).
STAT3 is expressed in the adult CNS by resident cells such as astrocytes and neurons, and its
phosphorylation markedly increases after injury [59]. Activation of the STAT3 pathway has been
reproducibly observed in reactive astrocytes under acute injury [60–63] (Table 2 and Figures 1
and 2). Conditional deletion of STAT3 in astrocytes exacerbates neonatal white matter injury
[64] and adult SCI [61,62] (Table 2). In the last disease model the spinal cords of transgenic mice
are characterized by widespread infiltration of CD11b+ inflammatory cells, pronounced demy-
elination and neuronal loss due to disorganized scar formation [61,62,65]. By contrast, SCI in
mice with selective ablation of suppressor of cytokine signaling 3 (SOCS3, the negative
regulator of STAT3) in neuroectodermal cells is characterized by improved motor performance
associated with prolonged STAT3 phosphorylation, rapid scar formation, and limited spreading
of inflammatory cells. Together these findings indicate that STAT3 signaling in astrocytes
regulates those aspects of astrogliosis essential for restricting inflammation and lesion size
[61] (Figures 1 and 2).

Distinct Astrocyte Stimuli but Shared Intracellular Pathways

In the previous sections we have seen which individual stimuli and signaling proteins confer on
astrocytes a protective or detrimental phenotype in vivo during neuroinflammation (Figure 1).

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Importantly, distinct astrocyte perturbations may converge towards the same signaling pro-
teins and effectors (Figure 2). When implemented with mechanistic observations obtained by in
vitro studies, the intracellular scenario highlights three critical events shared among several
distinct astrocyte responses: MAPK, NFkB and/or STAT3 activation (Figure 2). These key
nodes are differently regulated by inflammatory, lipid and hormonal mediators. Thus, S1P and
TGFb activate ERK to induce astrocyte proliferation or migration respectively [66–68], while
estradiol decreases ERK1/2 phosphorylation and hampers glia cell proliferation in vitro [69].
ER/ agonists also inhibits TNF/ -induced NFkB-dependent transcription, thereby exerting an
anti-inflammatory action [70]. Interestingly, blockade of S1P signaling is sufficient to inhibit
NFkB activation and nitric oxide release evoked by IL1 and IL17, indicating that cytokine
pathways depend on S1P signaling to trigger activation in astrocytes [35]. This is relevant
in vivo, as astrocytes upregulate the receptors for IL1, IL17 and S1P in demyelinated MS and
EAE lesions [35], suggesting concomitant activation of distinct signaling pathways in glia cells.
In vitro dissection of the signaling events induced by IL17 in astrocytes has shown that
activation of ERK1/2, p38 and JNK MAPKs leads to the downstream nuclear translocation
of NFkB, thus triggering MIP-1/ expression, a b-chemokine important for the migration of
monocytes, T lymphocytes, and eosinophils during EAE [71,72]. Similarly, IL6 and TGF-b
pathways activate MAPK and mediate phosphorylation of IkB/ in astrocytes [71,73]. Further,
IL17 acts in a synergistic manner with IL6, to increase the recruitment of phosphorylated NFkB
to the C-C motif chemokine ligand 20 (CCL20) promoter and induce STAT3-dependent IL6
expression in astrocytes [74]. Also the inflammatory cytokine IFN-g activates glial STAT3 in vitro
[75,76]. Importantly, STAT3 activation induces expression of SOCS3, which in turn blocks the
JAK/STAT signaling cascade in a negative feedback loop [77]. Interestingly, SOCS3-deficient
astrocytes display enhanced activation of MAPK and NFkB in response to IL17 and IL6,
indicating that SOCS3 may hamper other signaling steps in addition to the well-defined
negative regulation of JAK kinase activity [71].

The Contribution of Mediators Released by Astrocytes to Neuroinflammation

Upon CNS injury activated astrocytes produce and secrete a variety of bioactive molecules,
including chemokines, growth factors and neurotrophins, which may enhance or reduce CNS
inflammation. The EAE model has proven to be the ideal system to study the role of factors
released by astrocytes (Table 2 and Figures 1 and 2). Interestingly, three of these mediators
[CCL2, CXCL10 and vascular endothelial growth factor (VEGF)] are regulated by NFkB
[47,48,78], while another one (BDNF) can enhance STAT3 activation [79,80] (Figures 1 and
2). The chemokine CCL2 appears to be critical for EAE onset, since CCL2-deficient mice are
protected from disease [81]. Interestingly, in vivo studies show that astrocyte CCL2 supports
EAE development by enhancing leukocyte recruitment into the CNS parenchyma. In fact mice
with specific depletion for CCL2 in astrocytes (GFAPcre-CCL2fl/fl mice) display reduced EAE
clinical scores associated with attenuated immune cell infiltration, demyelination, and axonal loss
[82–84] (Table 2 and Figure 1). Similar observations have been made in mice with conditional
deletion of CXCL10 in astrocytes [85], though the impact on clinical and neuropathological
measures in GFAPcre-CXCL10fl/fl mice is not as strong as in GFAPcre-CCL2fl/fl mice. Together
these data demonstrate a role for astrocytic chemokines in the recruitment of infiltrating immune
cells into CNS lesions during neuroinflammation.

The contribution of astrocytes to the formation and maintenance of the BBB has significant
implications during inflammation as astrocyte dysfunction may favor the entry of molecules and
immune cells into the CNS. Astrocyte VEGF has been recently identified as an important
mediator supporting vascular permeability and CNS damage in acute inflammatory lesions.
In fact mice with selective ablation of VEGF in GFAP-positive cells develop a milder EAE course
associated with limited astrogliosis, BBB damage, immune cell infiltration and demyelination
than wt mice [86] (Table 2 and Figure 1).

616 Trends in Immunology, September 2016, Vol. 37, No. 9

Elimination of infiltrating T cells in the CNS is crucial for resolution of EAE. Fas ligand (FasL) plays Outstanding Questions
a major role in this process as it binds the death receptor Fas expressed by activated immune What are the key pathways that shift
cells to induce programmed cell death via caspase signaling [87]. Astrocyte endfeet in contact the phenotype of astrocytes from det-
rimental to protective?
with blood vessels strategically express FasL in vivo, suggesting a vital role for astrocyte FasL in
inducing apoptosis of infiltrating T lymphocytes [88]. Indeed, transgenic mice deficient for FasL in Can we reprogram glial activity in
astrocytes develop severe EAE with abundant demyelination and T cell infiltration [89] (Table 2 inflamed CNS to achieve clinical
and Figure 1). In vitro experiments have clearly demonstrated that FasL-deficient astrocytes are benefit?
less efficient in inducing T cell apoptosis than wt astrocytes, thus confirming that astrocytic FasL
Is targeting a single function of astro-
favors elimination of T cells in the CNS during neuroinflammation [89].
cytes sufficient for the development of
novel, effective therapies for neuroin-
Finally, a protective role for astrocyte BDNF in the inflamed nervous system is supported by a flammatory diseases?
study showing that depletion of BDNF production by astrocytes induces a more severe EAE
course associated with enhanced axonal damage [90]. As the extent of immune cell infiltration Do single astrocyte perturbations con-
and demyelination in GFAPcre-BDNFfl/fl mice is similar to that in wt mice (Table 2 and Figure 1), tribute to all pathological outcomes in
neuroinflammation?
the authors postulate a neuroprotective effect exerted by astrocyte BDNF during
neuroinflammation. Are these effects conserved in distinct
phases of neuroinflammation?
Concluding Remarks
Neuroinflammation relies on the complex integration of the activity of all cells present in the CNS, How does the severity of the inflamma-
tion influence the response potential of
including neurons, glia cells and, eventually, infiltrating immune cells. The evidence discussed in
the astrocyte?
this review implicates the astrocytes as critical regulators of tissue responses, as they may
promote or dampen neuronal damage, demyelination, and inflammation depending on the kind What is the net outcome of the com-
of stimuli present in the inflamed milieu. The recent in vivo approaches have provided vital plex reaction of the astrocyte to multi-
insights into the contribution of distinct astrocyte pathways to neuroinflammation and have ple stimuli?

highlighted some effector mechanisms. Overall, we have learnt the following:

Is there a hierarchy among activation
(i) Individual stimuli may influence the behavior of astrocytes and confer on them a tissue- modes for the definition of the final
protective or -detrimental phenotype in vivo. Still, the definition of the processes by which astrocyte phenotype?
each perturbation acts on distinct disease outcomes is only at the beginning (see Outstand-
ing Questions). Moreover, the known heterogeneity in astrocyte morphology and physiology
[91] may influence glial propensity to respond to specific inflammatory stimuli, an unexplored
issue in published studies.
(ii) Disease outcome may change depending on when astrocyte perturbation is introduced
during neuroinflammation. This has been clearly shown for GFAP-TK mice (Table 1), but
needs to be demonstrated for most of the single molecular perturbations.
(iii) In real life astrocytes are exposed simultaneously to a plethora of stimuli and activate distinct
intracellular signaling pathways during neuroinflammation. Thus, astrocyte responses are
not the reaction to a single event but the net result of a complex and variegated network of
intracellular activations. Additional studies are necessary to characterize the changes in the
extracellular milieu determining the establishment of a neuroinflammatory reaction and to
correlate them with the overall response potential of the astrocyte (see Outstanding
Questions).
(iv) A single stimulus may trigger multiple intracellular signaling pathways.
(v) Signaling pathways elicited by distinct stimuli may share intracellular mediators, raising the
possibility to modulate astrocyte behavior via targeting of key steps in the crosstalk. We have
seen that NFkB activation in response to the inflammatory cytokines IL1 and IL17 depends
on S1P signaling [35], yet future studies are warranted to verify whether all pathogenic
pathways involve NFkB or other more relevant signaling hotspots.
In conclusion, the systematic identification of key astrocyte pathways in neuroinflammation
together with the timely reconstruction of the inflamed milieu deserves further investigation. The
global understanding of the pathogenic and protective networks in the astrocyte has become a
primary need for the development of new therapeutic approaches in neuroinflammatory
disorders.

Trends in Immunology, September 2016, Vol. 37, No. 9 617

Acknowledgments
This work was supported by Italian Ministry for Health and Fondazione Italiana Sclerosi Multipla (grant 2012/R/7 to CF;
fellowship award 2013/B/3 to EC).

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