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Inflammation and epilepsy: the contribution of astrocytes

Zurolo, E.

Publication date2013

Link to publication

Citation for published version (APA):Zurolo, E. (2013). Inflammation and epilepsy: the contribution of astrocytes.

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Download date:20 Apr 2021

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1.2Astrocyteimmuneresponsesinepilepsy

Eleonora Aronica1,2 Teresa Ravizza3, Emanuele Zurolo1, Annamaria Vezzani3

1Department (Neuro) Pathology, Academisch Medisch Centrum, Amsterdam, and 2 Epilepsy Institute in The Netherlands Foundation

(Stichting Epilepsie Instellingen Nederland, SEIN), Heemstede, The Netherlands.3Mario Negri Institute for Pharmacological Research, Via La Masa 19, 20156 Milano; Italy

Glia.2012 Aug;60(8):1258-68.

AbstractAstrocytes, the major glial cell type of the central nervous system (CNS), are known to play

a major role in the regulation of the immune/inflammatory response in several human CNS

diseases. In epilepsy-associated pathologies, the presence of astrogliosis has stimulated ex-

tensive research focused on the role of reactive astrocytes in the pathophysiological pro-

cesses that underlie the development of epilepsy. In brain tissue from patients with epi-

lepsy, astrocytes undergo significant changes in their physiological properties, including the

activation of inflammatory pathways. Accumulating experimental evidence suggests that

proinflammatory molecules can alter glio-neuronal communications contributing to the

generation of seizures and seizure-related neuronal damage. In particular, both in vitro and

in vivo data point to the role of astrocytes as both major source and target of epileptogenic

inflammatory signaling. In this context, understanding the astroglial inflammatory response

occurring in epileptic brain tissue may provide new strategies for targeting astrocyte-medi-

ated epileptogenesis.

This article reviews current evidence regarding the role of astrocytes in the regulation of the

innate immune responses in epilepsy. Both clinical observations in drug-resistant human ep-

ilepsies and experimental findings in clinically relevant models will be discussed and elabo-

rated, highlighting specific inflammatory pathways (such as interleukin-1β/toll-like receptor

4) that could be potential targets for antiepileptic, disease-modifying therapeutic strategies.

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Astrocytesaskeyplayersinbraininflammation The central nervous system (CNS) has commonly been considered an immune-privileged

site in the presence of an intact blood brain barrier (BBB). However, this concept is gradually

changing as a result of recent developments in the field of innate immunity supporting the

role of CNS-resident cells acting as innate-immune-competent cells; [for reviews see (Amor

et al. 2010; McNaull et al. 2010; Schwartz and Kipnis 2011)]. Both innate and adaptive in-

flammatory responses occur in the CNS. The innate immune response is a non-specific,

acute defense against external agents or local injuries, and cell types chiefly involved in this

response include monocytes/macrophages and microglia. The adaptive immune response

is antigen-specific, and B and T lymphocytes are the key players in adaptive immunity. Com-

munication between the innate and adaptive responses involves cell-cell interactions, as

well as soluble factors such as cytokines and chemokines. In earlier studies, a considerable

amount of attention was focused on the immune function of microglial cells, which are gen-

erally considered to be the “CNS-based resident macrophages” (Aloisi 2001; Graeber and

Streit 1990). However, over the past decade there has been accumulating evidence support-

ing the central role of astrocytes in the CNS innate immune response induced by a variety

of insults. Astrocytes have been shown to initiate, regulate and amplify immune-mediated

mechanisms involved in different human CNS diseases, including epilepsy [for reviews see

(Farina et al. 2007; Seifert et al. 2010)]. In this review we will largely focus on protoplas-

mic astrocytes that predominate in gray matter, and fibrous astrocytes that predominate

in white matter. Astrocytes undergo complex morphological and functional changes. These

changes may vary with the severity and the type of insult and a continuum of progressive

morphological alterations can be observed in human brain tissue under different pathologi-

cal conditions [for review see (Sofroniew and Vinters 2010)].

Astrocytesasinnate-immune-competentcellsAstrocytesasantigen-presentingcellsIn contrast to the well-established role of microglia as antigen presenting cells (APCs) the role

of astrocytes in antigen presentation is still a matter of debate. Previous studies have shown

the induction of major histocompatibility (MHC) class I and II molecules on interferon-γ

(IFN-γ) exposure in vitro [for reviews see (Aloisi et al. 2000a; Dong and Benveniste 2001)].

However, there are discrepancies in the literature concerning the ability of astrocytes to act

as fully competent APCs (Aloisi et al. 2000a; Becher et al. 2000; Cornet et al. 2000; Cross and

Ku 2000; Dong and Benveniste 2001; Girvin et al. 2002; Weber et al. 1994). Moreover, the

demonstration of MHC-positive astrocytes in pathological human brain remains a contro-

versial topic, even in inflammatory neurological disorders such as multiple sclerosis (MS) (De

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Keyser et al. 2003; Gobin et al. 2001; Traugott and Raine 1985). In Rasmussen’s encephalitis

(RE), a severe inflammatory epileptic encephalopathy of childhood, expression of MHC class

I molecules has been reported in astrocytes (Bauer et al. 2007; Farrell et al. 1995), suggest-

ing an MHC class I–restricted T-cell response as a possible mechanism for the occurrence

of the astrocytic degeneration observed in RE (Bauer et al. 2007). However, the final effect

of astrocyte-T-lymphocyte interactions is complex and depends on the type of responding

T cells (Th1 or Th2 cells). Accordingly, whereas microglia may activate both Th1 and Th2

cells, astrocytes have been shown to stimulate mainly Th2 responses, providing homeostat-

ic mechanisms which may limit brain inflammation (Aloisi et al. 1998; Aloisi et al. 2000b).

The physiological role of MHC molecules (MHC class I), as well as of other immunologi-

cal molecules/receptors expressed by astrocytes during brain development, is another im-

portant area for future research. Recent studies suggest that immune molecules critically

modulate the development and function of the CNS [for review see (Glynn et al. 2011)].

AstrocytesassourceandtargetofinflammatorymoleculesAstrocytes represent an important source of immunologically relevant cytokines and

chemokines. In vitro studies document the ability of astrocytes (particularly reactive astro-

cytes) to produce cytokines such as interleukin(IL) -1β, IL-6, tumor necrosis factor (TNF)-α,

transforming growth factor beta (TGF)-β and chemokines, such as monocyte chemoattract-

ant protein-1 (MCP-1; chemokine, C-C motif, ligand 2; CCL2), which are highly expressed in

both experimental and human epileptogenic brain tissue [for reviews see (Aronica and Crino

2011; Vezzani et al. 2008b) Fig. 1]. The expression pattern and the role of astrocyte-derived

inflammatory molecules in seizure generation and progression will be discussed in the fol-

lowing sections.

Astrocytes are also the target of inflammatory molecules which, through the activation of

specific receptors (including pattern-recognition receptors (PRRs) and related intracellular

signaling pathways), may aggravate astrogliosis and amplify the pro-epileptogenic inflam-

matory signaling [for reviews see (Aronica and Crino 2011; Farina et al. 2007; Sofroniew and

Vinters 2010); Fig. 1].

Particular attention has recently been focused on the role of the IL-1 receptor/toll-like re-

ceptor superfamily (IL-1R/TLR) in epilepsy (Aronica and Crino 2011; Maroso et al. 2011;

Vezzani et al. 2010; Vezzani et al. 2011d; Vezzani et al. 2008b). The IL-1R/TLR superfamily

comprises cell surface PRRs sharing a conserved region termed the Toll/IL-1R (TIR) domain

(O’Neill 2008; O’Neill and Dinarello 2000). Several TLRs are expressed in human astrocytes

in vitro, including TLR2, TLR3 and TLR4 (Farina et al. 2007). However, whereas TLR3 shows

consistent expression in the resting state, studies examining TLR2 and TLR4 expression in

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astrocytes have produced conflicting results, which may reflect differences in cell source

and culture conditions (Crack and Bray 2007; Farina et al. 2007; Kielian 2006). Expression

of IL-1R1 or TLRs in astrocytes has also been demonstrated in human brain, with low levels

in resting astrocytes and upregulation in reactive astrocytes under different pathological

conditions, including epilepsy [(Maroso et al. 2010; Maroso et al. 2011; Ravizza et al. 2006a;

Ravizza et al. 2008a; Vezzani et al. 2011d; Zurolo et al. 2011); Fig.1].

TLRs have a key role in pathogen recognition (Kawai and Akira 2007), but in the absence

of pathogens, TLR can be activated by endogenous molecules, named damage-associated

molecular patterns (DAMPs), released from injured or activated cells. One of these mol-

ecules is the high mobility group box 1 (HMGB1) (Bianchi and Manfredi 2009), a ubiquitous

chromatin component that can be actively secreted by immuno-competent cells in response

to immune challenges (Muller et al. 2004). Both in vitro and in vivo findings suggest that

astrocytes are a source of extracellular HMGB1 (Maroso et al. 2010 ; Zurolo et al. 2011). In

particular, HMGB1 release has been shown to be induced in both rat (Hayakawa et al. 2010)

and human astrocytes in culture (Zurolo et al. 2011) in response to the pro-inflammatory

Figure1.Astrocytesaskeyplayersinneuroinflammationintheepilepticbrain. Schematic represen-tation of the hypothetical cascade of inflammatory processes in which astrocytes are involved. Knowl-edge of the astrocyte immune-inflammatory function in epilepsy may create the basis for developing effective therapeutic strategies to control seizures.

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cytokine IL-1β, and nuclear to cytoplasmic translocation has been observed in human and

experimental epileptic tissue (Maroso et al. 2010). In addition, astrocytes have also been

shown to respond to HMGB1 stimulation with induction of several inflammatory mediators

(Pedrazzi et al. 2007).

Recently, a new class of regulators of the immune responses has been recognised in the

form of microRNAs (miRNA), acting as post-transcriptional regulators of gene expression

(Gantier 2010; Quinn and O’Neill 2011; Sonkoly et al. 2008). In particular, the miRNA-146a

has been specifically associated with the regulation of TLR signaling (Cui et al. 2010; Quinn

and O’Neill 2011; Sheedy and O’Neill 2008; Taganov et al. 2006). miRNA-146a is expressed

in human brain, and astrocytes have been shown to be key players in the regulation of this

miRNA in response to inflammatory molecules, such as IL-1β (Aronica et al. 2010; Cui et al.

2010).

Another important component of the innate immune response is the complement system;

this consists of a variety of soluble and surface proteins which, when activated, result in

a complex cascade of processes contributing to the amplification of the inflammatory re-

sponse [for review see (Bonifati and Kishore 2007)]. Reactive astrocytes are a source of

complement components and also express complement-regulatory proteins, as well as

complement receptors [for review see (Farina et al. 2007); Fig. 1]. Complement activation

products, such as C3, regulate cytokine synthesis, and cytokines (such as IL-1β) may also in-

duce complement factor expression in human astrocytes (Barnum and Jones 1995; Bonifati

and Kishore 2007; Veerhuis et al. 1999). Astrocytes can also contribute to regulation of this

inflammatory pathway by induction of inhibitory factors, such as the complement factor H

(CFH) (Aronica et al. 2007; Boon et al. 2009; Griffiths et al. 2009). In addition, recent evi-

dence suggests an extensive and complex cross-regulation between complement and the

TLRs, which deserves further investigation in astrocytes (Hajishengallis and Lambris 2010).

Astrocyteimmune-inflammatoryfunctionandneurotrasmitterreceptorsSignaling via neurotransmitter receptors provides an additional mechanism by which astro-

cytes can sense and respond to changes in the extracellular environment, influencing the

inflammatory and immune response under pathological conditions associated with astro-

gliosis. An example is provided by the activation of astroglial G protein-coupled glutamate

and purinergic receptors, the expression of which is deregulated in epileptogenic brain tis-

sue (Byrnes et al. 2009; D’Antoni et al. 2008; Gomes et al. 2011; Hasko et al. 2005; Matute

and Cavaliere 2011).

Both in vitro and in vivo studies suggest an up-regulation of group I and II metabotropic glu-

tamate receptor (mGluR) subtypes (mGluR5 and mGluR3) in reactive astrocytes [(Aronica et

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al. 2003; Aronica et al. 2005a,b; Aronica et al. 2000); for review see (D’Antoni et al. 2008)].

Activation of mGluR3 in human astrocytes in culture modulates the release of IL-6 in the

presence of IL-1β, supporting the role of this receptor subtype in regulating the capacity of

activated astrocytes to produce inflammatory cytokines (Aronica et al. 2005a).

Increasing evidence points towards a critical role of purinergic receptors in neuron–glia

communication and neuroinflammation (Boison 2010; Gomes et al. 2011). Stimulation of

the adenosine receptor (P1 receptor) A2B induces the release of IL-6 from astrocytes and

the activation of the A3 receptor induces the synthesis of the chemokine MCP-1 [for reviews

see (Abbracchio and Ceruti 2007; Gomes et al. 2011; Hasko et al. 2005)]. The P2 purinergic

receptors modulate the cytokine-mediated signal transduction in human astrocytes in cul-

ture (Liu et al. 2000). Although the expression of both P2X (4,6,7) and P2Y(1,2) receptor-

subtypes has been reported in cultured astrocytes, P2Y rather than P2X receptor-subtypes

have been suggested as being involved in the modulation of intracellular Ca2+ (Fischer et al.

2009). In human fetal astrocytes, the blockade of P2Y receptors affects both IL-1β and TNFa

signaling (Liu et al. 2000), whereas the P2X7 receptor has been implicated in the regulation

of chemokine synthesis in astrocytes (Panenka et al. 2001). In addition, inflammatory mol-

ecules, such as IL-1β, may modulate the expression of adenosine kinase (ADK), providing a

potential modulatory crosstalk between the astrocyte-based adenosine cycle and inflamma-

tion (Aronica et al. 2011).

Finally, it has also been suggested that cannabinoid (CB) receptors, as mediators of endocan-

nabinoid signaling, exert an immunomodulatory function on astrocytes (Sheng et al. 2005).

AstroglialinflammatoryresponseinepilepsyIncreasing evidence supports the concept of activation of innate immune responses in both

experimental and human epilepsy and the critical involvement of inflammatory processes

in the etiopathogenesis of seizures (Aronica and Crino 2011; Vezzani et al. 2010; Vezzani et

al. 2011d). In particular, recent clinical-neuropathological and experimental observations

support the notion that dysregulation of the astrocyte immune-inflammatory function (dis-

cussed above) is a common factor, which may predispose or directly contribute to the gen-

eration of seizures and to seizure-related neuronal damage in epilepsy of various etiologies

(Fig. 1). Current knowledge concerning common inflammatory signaling pathways involving

astrocytes that may alter neuronal excitability is discussed below.

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Astrocytes and inflammatory processes in patients withmedicallyrefractoryepilepsyReactive astrogliosis is a pathological hallmark of various types of medically refractory focal

epilepsy, including epilepsy that develops following ischemic, traumatic, or infectious brain

injury (Sofroniew and Vinters 2010). Reactive astrogliosis is also the pathological hallmark of

two major epilepsy-associated pathologies [hippocampal sclerosis and focal malformations

of cortical development (MCD; FCD and cortical tubers in tuberous sclerosis complex TSC)].

Hippocampal sclerosis (HS) is the most common neuropathological finding in patients un-

dergoing surgery for intractable temporal lobe epilepsy [TLE; (Wieser 2004)]. Although

specimens obtained from patients undergoing surgery for intractable TLE often represent

the end-stage of the pathological cascade that leads to HS, histopathological and molecular

analysis of this tissue is essential to confirm the relevance and cellular sources of inflamma-

tory molecules and related signaling. Large-scale analysis of gene expression profiles sug-

gests a prominent upregulation of genes related to astroglial activation and innate immune/

inflammatory response in human TLE [for review see (Aronica and Gorter 2007 )] This evi-

dence, at gene expression level, has been confirmed by histopathological studies demon-

strating the association between activated astrocytes and microglial cells and the induction

of major proinflammatory pathways in human TLE (Aronica and Gorter 2007; Ravizza et al.

2008a; Vezzani et al. 2010).

The transcription factor nuclear factor-kappa B (NFkB) plays a central role in regulating im-

mune and inflammatory responses, including the IL-1R/TLR signaling pathways (Oecking-

haus et al. 2011). Activation of IL-1R1-mediated signaling in cells targeted by the released

IL-1β induces, via an NFkB-dependent mechanism, the transcription of other genes encod-

ing downstream mediators of inflammation, including IL-6, TNF-α, cyclooxygenase-2 (Cox-

2) or CCL2 (i.e. monocyte chemotactic protein-1) (Andjelkovic et al. 2000; Dinarello 2004;

Meeuwsen et al. 2003). Crespel et al. (Crespel et al. 2002) reported NFkB over-expression

in reactive astrocytes in human HS specimens. The activation of the NFkB signaling pathway

in astrocytes has been confirmed by subsequent studies demonstrating prominent expres-

sion in astrocytes of both L-1β and its functional receptor, IL-1R1, providing evidence of a

persistent activation of this specific inflammatory pathway in human tissue from people

with chronic epilepsy (Ravizza et al. 2008a). In addition, up-regulation of astroglial Cox-2 and

CCL2 has been reported in human TLE (Desjardins et al. 2003; Holtman et al. 2009; Wu et

al. 2008) suggesting the activation of a complex, highly interconnected, cytokine network.

Human studies in TLE also support the involvement of the complement system. Expression

of various complement components, such as C1q, C3c and C3d, has been observed in reac-

tive astrocytes within the sclerotic hippocampus of people with TLE (Aronica et al. 2007).

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Complement activation in astrocytes may regulate cytokine synthesis thus critically contrib-

uting to the propagation and persistence of the inflammatory response.

The activation of the plasminogen system in astrocytes in human TLE may contribute to the

regulation of the immune responses and related inflammation within the epileptic lesion

(Benarroch 2007). Accordingly, in addition to neurons and microglial cells, expression of

both tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) has been

observed in reactive astrocytes (Iyer et al. 2010). Although PAs may contribute to the activa-

tion of astrocytes (Gravanis and Tsirka 2005), astrocytes are also involved in the neutraliza-

tion of tPA toxicity via an endocytic tPA receptor (Fernandez-Monreal et al. 2004).

Induction of the pathways discussed above (IL-1β, complement and PA) in perivascular as-

trocytes suggests that the alterations of BBB permeability described in human TLE (Rigau et

al. 2007; van Vliet et al. 2007) probably result from the convergence of the actions of dif-

ferent inflammatory molecules released from parenchymal brain cells, then acting on blood

vessels. In this context, the vascular endothelial growth factor A (VEGFA)-signaling pathway

may also affect the integrity of the BBB (Schoch et al. 2002). VEGFA is upregulated in reac-

tive astrocytes (including perivascular astrocytes) in human epileptogenic tissue [HS and

FCD; (Boer et al. 2008; Morin-Brureau et al. 2011; Rigau et al. 2007)], and increased VEGFA

expression has recently been reported within cortical tubers of people with TSC (Parker et

al. 2011).

As mentioned above, the activation of the TLR signaling pathway in astrocytes plays a key

role in the regulation of the innate immune response. Interestingly overexpression of TLR4

and its endogenous ligand HMGB1 has been demonstrated in human TLE in reactive as-

trocyes, confirming the role of these cells as both source and target of HMGB1 (Maroso et

al. 2010). The functional consequences of the astrocyte-mediated HMGB1-TLR signaling in

ictogenesis and epileptogenesis in experimental models are discussed below (see Fig. 2).

Reactive astrogliosis is also a major feature of focal MCD, such as FCD and cortical tubers in

TSC (Blumcke et al. 2011; Blümcke et al. 2009; Orlova and Crino ; Sosunov et al. 2008; Wong

2008).

The association between astrogliosis and activation of inflammatory pathways is supported

by gene expression analysis of cortical tubers in TSC; high expression levels have been ob-

served for genes encoding complement components, chemokines, MHC elements and com-

ponents of the IL-1R/TLR signaling pathways (Boer et al. 2010).

Activation of various proinflammatory molecules and related pathways has been confirmed

at the cellular level in both TSC and FCD specimens, pointing to the central role of reactive

astrocytes in the immune/inflammatory response of these developmental epileptogenic le-

sions as well [for review see (Aronica and Crino 2011)].

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A recent study (Zurolo et al. 2011) demonstrates the activation of the TLR signaling pathway

in reactive astrocyes both in FCD type and TSC specimens, as shown by overexpression of

TLR4 and RAGE (receptor for advanced glycation end products) and cytoplasmic transloca-

tion of HMGB1 [as reported in human HS specimens; (Maroso et al. 2010)].

The transforming growth factor (TGF)-β mediated pathway is also upregulated in focal MCD

(Boer et al. 2010; Kim et al. 2003). TGF-β is a multifunctional cytokine which, acting through

its specific receptors, modulates the astrocyte immune responses and which has recently

been suggested as playing a critical role in neocortical epileptogenesis acting through its

specific receptors (Ivens et al. 2007).

A controversial issue is whether the dysregulation of astrocyte immune-inflammatory re-

sponses is related to the underlying cellular pathology in epileptic tissue or whether it oc-

curs as a consequence of recurrent seizures. The observation of prenatal TLRs and HMGB1

expression in giant cells within the tuber in TSC (Aronica et al. 2008; Samadani et al. 2007;

Zurolo et al. 2011) suggests that the induction of these signaling pathways could be intrinsic

to the developmental lesion per se, and that the development of seizures later in life could

contribute to perpetuation of their activation.

These observations provide evidence of the potential role of astrocytes in the chronic in-

Figure 2. Functional consequences of the production of inflammatorymediators by astrocytes. Proepileptogenic brain injuries trigger brain inflammation, involving the induction/overexpression of cytokines (e.g. IL-1β), danger signals (e.g. HMGB1) and TLRs (e.g. TLR4) in parenchymal/perivascular astrocytes. This event leads to changes in brain physiology such as neuronal hyperexcitability, BBB dys-function and cell damage that can contribute to lower seizure threshold and trigger epileptogenesis.

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flammatory state observed in focal MCD. Whether the dysregulation of astrocyte immune-

inflammatory responses could contribute to progressive cognitive dysfunction in children

deserves further investigation (Chew et al. 2006; Cohly and Panja 2005).

Finally, both gene expression and immunocytochemical studies suggest prominent activa-

tion of major proinflammatory pathways within the astroglial component of highly epilepto-

genic tumors (glioneuronal tumors, such as gangliogliomas) (Aronica et al. 2008; Samadani

et al. 2007; Zurolo et al. 2011).

AstrocyteandbraininflammationinexperimentalmodelsofseizuresandepilepsyExperimental studies provided the first evidence showing a significant contribution of reac-

tive astrocytes to the inflammatory processes developing after seizures or induced by an ep-

ileptogenic brain injury. Acute seizures induced in rodents (following intracerebral applica-

tion of kainate or bicuculline, or electrically induced status epilepticus) were shown rapidly

to upregulate prototypical inflammatory cytokines in microglia and astrocytes in the brain

areas where seizures originate and spread; as a consequence of this event, a downstream

cascade of inflammatory mediators is transcriptionally upregulated in brain tissue similar to

what has been shown in human epilepsy [(De Simoni et al. 2000; Gorter et al. 2006; Vezzani

et al. 1999; Vezzani et al. 2000); reviewed by (Kulkarni and Dhir 2009; Vezzani et al. 2008a;

Vezzani et al. 2011b)]. Activation of astrocytes in the absence of neuronal degeneration has

been reported in a kindling model (Khurgel et al. 1995) and induction of GFAP has been

observed even after a single electroconvulsive seizure (Steward et al. 1992). A detailed time-

course analysis of these inflammatory processes occurring after status epilepticus in rats

was instrumental in demonstrating that the mRNA of inflammatory mediators are induced

within 30 minutes of seizure onset (De Simoni et al. 2000). The immunohistochemical analy-

sis of IL-1β in status epilepticus models showed that the expression of this cytokine in micro-

glia is time-locked to the occurrence of seizures and the extent of expression depends on the

recurrence of seizures, while astrocytes appear to be involved in perpetuating inflammation

even in the long-term after the initial injury (Ravizza et al., 2008a). Moreover, as in human

epileptic tissue, astrocytes often express both the inflammatory mediator and the cognate

cell signaling receptors, thus highlighting that these cells serve as sources and targets of in-

flammatory molecules (reviewed in (Maroso et al. 2011; Vezzani et al. 2011c). Experimental

findings clearly show that both parenchymal and perivascular astrocytes are activated and

express inflammatory molecules in epilepsy models with functional consequences on BBB

function (Fig. 2) (Bauer et al. 2008; Friedman et al. 2009; Ravizza et al. 2008a; van Vliet et al.

2009; Vezzani et al. 2011c). Notably, specific anti-inflammatory molecules, such as the IL-1

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receptor antagonist (IL-1ra) (De Simoni et al. 2000 ) or the C59 inhibitor of the complement

system (Aronica et al. 2007), are induced to a limited extent by seizures or following brain

injury, suggesting that the mechanisms involved in the resolution of brain inflammation are

not very efficient, and possibly explaining why inflammation is detrimental for tissue excit-

ability and cell survival.

Although induction of various inflammatory molecules has been demonstrated in astrocytes

in seizure models [reviewed in (Friedman and Dingledine 2011; Vezzani et al. 2008a; Weth-

erington et al. 2008)], the IL-1/TLR is the first inflammatory signalling to be induced during

innate immunity activation, either by a pathogen or a danger signal serving as endogenous

ligand (Maroso et al. 2011; Vezzani et al. 2011c). IL-1/TLR signalling is rapidly induced by tis-

sue injury or seizures in neurons, microglia and astrocytes resulting in transcriptional activa-

tion of inflammatory genes, and other genes potentially involved in synaptic and molecular

changes underlying epileptogenesis [reviewed in (Vezzani et al. 2011c)]. Two endogenous li-

gands, IL-1β and HMGB1 (Fig. 2) are released by glial cells; IL-1β activates IL-1R1 and HMGB1

activates TLR4 in neurons with significant consequences for ictogenesis, mainly mediated by

post-translational effects (see later).

Functional consequences of astrocyte-mediated brain in-flammationonneuronalexcitabilityThe activation of inflammatory pathways and the consequent release of inflammatory mol-

ecules by astrocytes alter neural network excitability via induction of various mechanisms,

with either direct or indirect impact on neuronal functions. Here we focus on the IL-1R1/

TLR4 signaling because of its prominent involvement in seizures and epileptogenesis [(Ma-

roso et al. 2011; Vezzani et al. 2011c); Fig. 2].

IL-1β, by acting on IL-1R type 1, can inhibit the astrocytic reuptake of glutamate (Hu et al.

2000; Ye and Sontheimer 1996) and increases its glial release likely via induction of TNFa

(Bezzi et al. 2001). These effects result in elevated extracellular glutamate levels, which in

turn can promote tissue excitability. IL-1β can also increase neuronal glutamate release via

the activation of inducible nitric oxide synthase in glial cells (Casamenti et al. 1999; Hewett

et al. 1994). Astrocytic glutamate release may have a role in the genesis or strength of sei-

zure-like events (Carmignoto and Fellin 2006; Tian et al. 2005).

In hippocampal neurons, IL-1R1 co-localizes with NMDA receptors, a subtype of glutamate

receptor involved in the onset and spread of seizures. IL-1β potentiates NMDA receptor

function in cultured hippocampal neurons (Lai et al. 2006; Viviani et al. 2003) by enhanc-

ing N-Methyl-D-aspartate (NMDA)-mediated Ca2+ influx via IL-1R1 dependent activation of

Src kinases and subsequent NMDA receptor subunit 2 (NR2B) phosphorylation. This rapid

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mechanism (within minutes) involves ceramide-mediated activation of Src kinases (Viviani

et al. 2003), and contributes to seizure generation and recurrence (Balosso, 2008). IL-1β also

down-regulates AMPA receptor expression and phosphorylation in hippocampal neurons in

a Ca2+- and NMDA-dependent manner (Lai et al. 2006).

Interactions of IL-1β with GABA-mediated inhibitory neurotransmission have also been re-

ported. However, the results obtained are not consistent. Thus IL-1β can either decrease

or increase GABA inhibition depending on the brain area (Alam, 2004; Wilkinson, 1993),

the cytokine concentration (Wang, 2000; Zeise, 1997; Serantes, 2006) and the functional

properties of the cells (Hori, 1988), highlighting a dual role of IL-1β in affecting GABAergic

inhibitory system.

In the hippocampus, IL-1β affects synaptic transmission, and inhibits long-term potentiation

(LTP) via activation of Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase

(MAPK) (Bellinger et al. 1993; O’Donnell et al. 2000; Schneider et al. 1998). This cytokine can

also modulate neurotransmitter release via inhibition of voltage-dependent Ca2+ channels,

an effect that involves pertussis-sensitive G proteins and protein kinase C ( PKC) (Plata-

Salaman and ffrench-Mullen 1994).

TNF-a is a cytokine released from activated astrocytes and microglia, and tightly associated

with IL-1β, since the two molecules reciprocally induce their respective release from glia

and mutually activate their gene transcription. TNF-a has been shown to increase the mean

frequency of AMPA-dependent miniature excitatory postsynaptic currents in hippocampal

neurons and to decrease GABAA-mediated inhibitory synaptic strength. These effects are

mediated by its ability to activate the recruitment of AMPA receptors lacking the GluR2

subunit at neuronal membranes (thus in a molecular conformation which favors Ca2+ influx

into neurons) and to induce endocytosis of GABAA receptors (Beattie et al. 2002; Stellwagen

et al. 2005).

TLR4 is the TLR subtype most extensively studied for its involvement in brain excitability.

Cortical rat exposure to lipopolysaccaride (LPS), a prototypical activator of TLR4, induces

rapid increases in neuronal excitability leading to seizures (Rodgers et al. 2009), which are

prevented by IL-1ra, implicating a role of released IL-1β.

Regarding IL-1β, LTP and long-term depression (LTD) impairment are induced by TLR4 stimu-

lation, compatible with cognitive deficits induced in rodents by brain inflammation (Galic

et al. 2009; Harre et al. 2008; Spencer et al. 2005). These modifications in brain physiology

are dependent on the release of TNF-a and IL-1β from activated glial cells (Riazi et al. 2010).

Cognitive deficits are associated with specific changes in NMDA receptor subunit expres-

sion in the cortex and hippocampus, predicting modifications in CNS excitability (Galic et al.

2009; Harre et al. 2008; Spencer et al. 2005).

PS Emanuele 10-12.indd 41 19-12-12 13:26

42

These fast post-translational effects of inflammatory cytokines are examples of novel path-

ways by which inflammatory molecules produced in diseased tissue by glia can modulate

neurotransmission and contribute to hyperexcitability and associated neuropathology.

In general, the amount and persistence of cytokines in brain tissue appears to be a crucial

factor which determines their consequences on neuronal excitability. Another important

aspect is the pattern of expression of cytokine receptors in the tissue exposed to inflamma-

tion. A typical example is represented by TNF-a, which may have inhibitory or permissive

effects on seizures depending both on its brain levels and on the receptor subtypes pre-

dominantly activated (Balosso et al. 2005). Moreover, transgenic mice with low to moderate

overexpression of TNF-a in astrocytes show decreased susceptibility to seizures (Balosso et

al. 2005), whereas mice with high overexpression of TNF-a in astrocytes develop signs of

neurologic dysfunction (Akassoglou et al. 1997; Probert et al. 1995).

Functional consequences of astrocyte-mediated braininflammationonseizuresandepileptogenesisThe role of cytokines released by astrocytes in seizures and epileptogenesis has been inves-

tigated at various levels, initially using genetically-modified mice with perturbed cytokine

systems and subsequently by pharmacological intervention using receptor antagonists or

cytokine synthesis inhibitors, or by injecting the cytokines themselves into rodent brain [re-

viewed in (Vezzani et al. 2008a; Vezzani et al. 2011b)].

IL-1β, TNF-a and HMGB1 are among the most studied cytokines for their permissive role

in seizures. Mice with upregulation of IL-1ra in astrocytes or lacking IL-1R1 are intrinsically

resistant to seizures (Vezzani et al. 2000) and the intracerebral injection of IL-1ra mediates

powerful anticonvulsant effects (Vezzani et al. 2000; Vezzani et al. 2002). Because the only

action ofIL-1ra is to inhibit the effects of IL-1β, these data demonstrate that an endogenous

increase in brain IL-1β contributes to seizures. Mice lacking caspase-1, the biosynthetic en-

zyme of IL-1β, and therefore unable to release the biologically active form of this cytokine,

show decreased seizure susceptibility (Ravizza et al. 2006b). Pharmacological data support

a proconvulsant role of IL-1β in several acute and chronic seizure models (Vezzani et al.

1999; Vezzani et al. 2000), as well as in the kindling model of epileptogenesis (Ravizza et

al. 2008b). Moreover, recent data showed that inhibition of IL-1β biosynthesis in astrocytes

reduces spike-and-wave discharges in rats with genetic absence epilepsy (GAERS) (Akin et

al. 2011). Increase in astrocytic IL-1β in the hippocampus due to fever/hyperthermia is in-

volved in decreasing the seizure threshold in the immature rodent (Dube et al. 2005; Heida

and Pittman 2005).

Recently, we have shown that endogenous release of “danger signals” produced by stressed

PS Emanuele 10-12.indd 42 19-12-12 13:26

43

or injured neurons (i.e. HMGB1), promotes seizures by activation of neuronal TLR. Seizures,

in turn, induce an additional wave of HMGB1 release from activated astrocytes and micro-

glia, leading to a vicious positive feedback cycle of seizures and inflammation. This novel

pathway may be a crucial mechanism for recurrent seizures [(Maroso et al. 2010); reviewed

in (Maroso et al. 2011; Vezzani et al. 2011d); Fig. 2].

Cytokines and other inflammatory mediators have been shown to contribute to both ex-

citotoxic and apoptotic neuronal death (Allan et al. 2005), highlighting the possibility that

they contribute to seizure-mediated neuronal damage. The deleterious effects of cytokines

on neuronal survival involve the production of neurotoxic compounds via autocrine or par-

acrine mechanisms (Allan et al. 2005; Vezzani and Baram 2007). Importantly, although cy-

tokines can promote neurodegeneration (Fig.2), their effects on the threshold, frequency

and duration of seizures are not dependent on cell death (Vezzani et al. 2011c).

Notable examples exist of a dual role of cytokines on neuronal survival in diseased tissue

(Allan et al. 2005; Bernardino et al. 2005); it has been shown the ability of cytokines to

induce the synthesis of growth factors in astrocytes, to activate antioxidant pathways, man-

ganese superoxide dismutase, or calbindin which counteracts the elevation of intracellular

Ca2+ induced by cell injuries (Allan et al. 2005), thus promoting cell repair mechanisms. In

this respect, IL-1β and TNF-a can either reduce or exacerbate glutamate receptor–mediated

excitotoxicity in organotypic slice cultures, depending on their extracellular concentrations,

the length of time the tissue is exposed to these cytokines, and the receptor types activated

by these cytokines (Bernardino et al. 2005).

Finally, the possible involvement of inflammatory mediators in epileptogenesis has been

suggested by two main lines of evidence: the induction of an inflammatory state in the brain

by administering proinflammatory molecules in rodents, or the use of mice that overexpress

specific cytokines in astrocytes. This leads to decreased seizure threshold and induces long-

term neurological deficits, particularly if applied to immature rodents, thus suggesting long-

term effects of inflammation on brain functions [reviewed by (Ravizza et al. 2011; Riazi et

al. 2010)]. In this context, brain inflammation has been implicated in the pathophysiology of

several neuropsychiatric conditions (such as depression, memory impairments, and autism

spectrum disorder) which are comorbidities of epilepsy, (Vezzani et al. 2011a).

Pharmacological interference with specific inflammatory pathways activated during epi-

leptogenesis may reduce the severity and frequency of spontaneous seizures [reviewed in

(Ravizza et al. 2011)].

Additionally, cytokines such as IL-1β and HMGB1 released in diseased tissue by parenchy-

mal and, in particular, by perivascular astrocytes, can play a major role in BBB breakdown

associated with brain inflammation in human and experimental epileptic tissue [reviewed

PS Emanuele 10-12.indd 43 19-12-12 13:26

44

by (Friedman et al. 2009)]. The opening of the BBB rapidly activates the innate immune

response (Cacheaux, 2002) and the accumulation of albumin in the brain because of BBB

damage. Albumin triggers long-lasting hyperexcitability in surrounding tissue by impairing

astrocyte capacity to buffer extracellular K+ and glutamate via activation of the TGF-β path-

way [reviewed by (Friedman et al. 2009)].

Finally, it is increasingly recognized that pro-inflammatory molecules released by glia con-

tribute to some of the acquired channelopathies described in epilepsy by inducing altera-

tions in voltage- and receptor-gated ion channels via either post-translational or transcrip-

tional mechanisms [reviewed by (Vezzani et al. 2011b; Viviani et al. 2004)].

ConcludingremarksDuring the past decade, detailed molecular characterization of astrocytes, in particular re-

active astrocytes, demonstrates that these cells are active players in the development and

progression of the immune/inflammatory response that takes place in epileptic brain tis-

sue. Both human and experimental data suggest the activation of specific proinflammatory

pathways in astrocytes, which may also recruit neuronal cells and, in some cases, cells of

the adaptive immune system. The identification of “harmful” pro-inflammatory pathways

contributing to seizure onset and recurrence, as well as to comorbities often associated

with epilepsy, highlights the possibility of developing a therapeutic strategy targeting the

astrocyte-mediated inflammatory signalings. However, we need to wait for the outcome of

clinical studies before we can consider whether this approach is the right strategy. If this

is so, it may not only improve control of seizures, but may also act as disease-modifying

therapy in patients with epilepsy resistant to conventional antiepileptic drugs.

AcknowledgementsAV is supported by Fondazione Monzino, Fondazione Cariplo and PACE; EA is supported by

National Epilepsy Funds (NEF 09-05) and EU FP7 project NeuroGlia (Grant Agreement N°

202167).

PS Emanuele 10-12.indd 44 19-12-12 13:26

45

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