ReviewEpilepsy and brain inflammation
Introduction
Accumulating clinical evidence strongly supports the relevance of inflammation in the pathophysiology of human epilepsy (Vezzani et al., 2011a, Vezzani et al., 2011b). Different common infectious or autoimmune diseases can cause recurrent seizures (Bien et al., 2007, Choi and Koh, 2008). The prototype of inflammatory epileptic encephalopathy is represented by the Rasmussen's encephalitis, a severe epileptic encephalopathy of childhood, characterized by intractable focal seizures, hemiparesis and progressive deterioration of cognitive function (Bauer and Bien, 2009, Bien et al., 2002, Pardo et al., 2004). Neuropathological evaluation of tissue of individuals affected by Rasmussen's encephalitis provides evidence of a progressive immune-mediated process of neuronal damage, involving both glial and lymphocytic responses (Pardo et al., 2004). Recent studies suggest as key pathogenetic mechanism a cytotoxic CD8 T cells-mediated attack on both neurons and astrocytes (Bauer et al., 2007, Schwab et al., 2009). The autoimmune nature of Rasmussen's encephalitis is supported by the discovery of antibodies against glutamate-receptor subunit 3 (GluR3) (Rogers et al., 1994). However, GluR3 antibodies are not present in all cases (Watson et al., 2004) and have been also detected in other epilepsy patients with severe intractable seizures (Mantegazza et al., 2002). In addition, autoantibodies against the presynaptic protein Munc18-1 have been identified in a subgroup of these patients (Alvarez-Baron et al., 2008).
A growing number of specific antibodies are being detected in patients with new onset epilepsy and immuno-mediated seizure disorders (Vincent et al., 2010). These antibodies are directed to intracellular targets (glutamic acid decarboxylase), or to cell-surface membrane proteins, such as voltage-gated potassium channels (VGKC-complex proteins) or N-methyl-d-aspartate receptors (Irani et al., 2010, Vincent et al., 2010). Increasing evidence shows that these antibodies are biomarkers for underlying immunopathology of limbic encephalitis which represents a precipitating event in adult-onset temporal lobe epilepsy with hippocampal sclerosis (TLE-MTS) (Bien et al., 2007, Niehusmann et al., 2009).
Clinical and neuropathological evidence suggest that inflammation could play also a central role in seizure disorders without infectious or immune-mediated etiology. Several clinical studies demonstrated increased levels of inflammatory mediators (e.g. cytokines such as Interleukin (IL)-6, Tumor Necrosis Factor (TNF)-α and IL-1β and the IL-1 receptor antagonist(ra)) in serum or CSF (Aronica and Crino, 2011). The activation of the cytokine network in patients with refractory epilepsy (e.g. cell types contributing to inflammation, extent of inflammation in brain tissue) may vary not only depending on seizure severity or duration but also on the epilepsy syndromes (Alapirtti et al., 2009, Bauer et al., 2009). Currently there are no inflammatory biomarkers, detectable in CSF and/or serum, with proven clinical utility for patients with chronic refractory focal epilepsy. A major challenge for the future is to define specific biomarkers which would allow the recognition of appropriate patient populations who might benefit from antiinflammatory or immunomodulatory therapies.
Neuroimaging approaches represent another interesting challenge to study the inflammatory processes in chronic epilepsy. Iron oxide contrast-enhanced MRI has been used to visualize cellular inflammation in patients with multiple sclerosis and stroke (Stoll and Bendszus, 2009). However, the possible application of this approach in patients with focal epilepsy and more subtle inflammatory processes deserves further investigation. Positron emission tomography (PET) with 11C-PK11195 has been also used to show activated astrocytes and microglia in epileptic patients with encephalitis (Banati, 2002, Kumar et al., 2008) or focal cortical dysplasia (Butler et al., 2011). However, this tracer may be not sensitive enough to detect inflammatory changes associated with microglia activation in patients with TLE-MTS (Banati, 2002, Butler et al., 2009). Recently, new promising, more sensitive, PET tracers have been developed to visualize activated glial cells in the epileptogenic zone of MTS patients (Hirvonen et al., 2010). Additional studies are, however, needed to correlate imaging with the neuropathological finding in patients undergoing epilepsy surgery.
Neuropathological examination of surgical epilepsy specimens provided evidence of a complex and sustained inflammatory phenomenon, and the associated production of proinflammatory molecules. In patients with TLE-MTS, besides astrogliosis, which is a major histopathological feature of hippocampal sclerosis, prominent activation of cells of the microglia/macrophages lineage has been shown in the hippocampus (Aronica and Gorter, 2007, Ravizza et al., 2008a); in contrast, only few cells of adaptive immunity (CD3/CD8 positive T-lymphocytes) are observed in these specimens, mainly associated with microvessels (Marchi et al., 2010, Ravizza et al., 2008a, Ravizza et al., 2008b).
The activation of both astrocytes and microglial cells is associated with the induction of major proinflammatory pathways in TLE-MTS (Vezzani et al., 2011a, Vezzani et al., 2011b), and gene expression profile analysis studies confirmed the prominent upregulation of genes associated with the immune/inflammatory pathways, including several chemokines and pro-inflammatory cytokines (Aronica and Gorter, 2007). Activation the IL-1β system in glial as well as neuronal cells (expressing both IL-1β and its receptor, IL-1R1), has been shown in TLE-MTS specimens, confirming the findings reported in chronic epileptic rats (Ravizza and Vezzani, 2006, Ravizza et al., 2008a). Both the complement pathway and the plasminogen system are also activated within the sclerotic hippocampus in TLE patients (Aronica et al., 2007, Iyer et al., 2010a, Iyer et al., 2010b). Since IL-1β, complement components and plasminogen activators can affect the permeability properties of the blood brain barrier (BBB) (Ballabh et al., 2004, Lucas et al., 2006), we can speculate the existence of a reinforcing feedback loop between these pathways, which may contribute the BBB breakdown observed in hippocampal sclerosis (Ravizza et al., 2008a, van Vliet et al., 2007).
Recent studies support the activation of Toll-like receptor (TLR) signaling pathways in epilepsy (Maroso et al., 2010, Riazi et al., 2010). This prototypical inflammatory pathway is activated in response either to pathogens or endogenous ligands released by damaged or stressor-activated cells, named danger signals (see later for more details). Interestingly, overexpression of TLR4 in neurons and astrocytes, and its endogenous ligand high mobility group box 1 (HMGB1) has been demonstrated in microglia and astrocytes in TLE-MTS and focal cortical dysplasia (FCD), confirming the findings reported in chronic epileptic mice (Maroso et al., 2010, Zurolo et al., 2011). Since microglia and astrocytes respond to HMGB1 stimulation with the production of several inflammatory mediators (Andersson et al., 2008, Pedrazzi et al., 2007), and IL-1β can induce the release of HMGB1 in human (Zurolo et al., 2011) and rat (Hayakawa et al., 2010) cultured astrocytes, these cells are likely to play a crucial role in perpetuating the inflammatory response in epilepsy.
Attention has been recently focused on the role of microRNAs (miRNA) in the regulation of the innate and adaptive immune responses. In particular, miR-146a, which can be induced by different pro-inflammatory stimuli such as IL-1β and TNF-α, has been shown to critically modulate innate immunity through regulation of TLR signaling and cytokine responses (Sheedy and O'Neill, 2008, Taganov et al., 2006). Interestingly, this miRNA is upregulated in TLE as well as in experimental models of epilepsy (Aronica et al., 2010, Song et al., 2011). These observations suggest miRNA as potential targets to modulate inflammatory pathways.
Activation of cells of the microglia/macrophage lineage and astrocytes, and concomitant induction of various inflammatory pathways, have been also observed in cortical tubers of patients with tuberous sclerosis complex (TSC) and in FCD, which represent major causes of pediatric epilepsy (Blumcke et al., 2010). Both the innate and the adaptive immune responses are activated in these lesions (Boer et al., 2010, Choi et al., 2009, Iyer et al., 2010a, Iyer et al., 2010b, Ravizza et al., 2006a). Activation of plasminogen and focal BBB damage were also observed (Boer et al., 2008, Iyer et al., 2010a, Iyer et al., 2010b) as in TLE.
In a cohort of patients with FCD type II, the density of activated Human Leukocyte Antigen (DR)-positive microglial cells and the endogenous levels of IL-1β and its receptor IL-1R1 correlated with the duration of epilepsy, as well as with the frequency of seizures prior to surgical resection (Boer et al., 2006, Ravizza et al., 2006a). The number of HLA-DR-positive microglial cells is significantly higher in FCD type II than in specimens from patients with FCD type I, despite of lack of significant differences in seizure frequency and duration (Iyer et al., 2010a). In both FCD and TSC, CD3/CD8 positive T-cells are detected in close apposition with malformed cells (Boer et al., 2008, Iyer et al., 2010a, Iyer et al., 2010b); moreover, the number of T lymphocytes is greater in FCD type II specimens than in FCD type I (Iyer et al., 2010a). More prominent activation of complement, IL-1β and chemokines signaling pathways is also observed in FCD type II (Iyer et al., 2010a, Iyer et al., 2010b). These observations suggest that both the recurrent seizures and the underlying neuropathology are important determinants of the extent and the type of inflammatory molecules and cells in epileptic tissue. It could be speculated that the activation of the mammalian target of rapamycin (mTOR) pathway observed in TSC and within the cellular components of FCD type II (but not in FCD type I) could contribute to the inflammatory response. Accordingly, the mTOR pathway has been shown to influence both innate and adaptive immune responses (Schmitz et al., 2008, Weichhart and Saemann, 2009).
Whether the presence of a prominent population of inflammatory cells may contribute to progressive cognitive dysfunction in patients with malformations of cortical development, and more in general in epilepsy, deserves further investigation.
The link between brain inflammation and epilepsy, which is supported by the clinical evidence discussed above, fostered experimental studies aimed at elucidating the major sources and inducers of inflammatory molecules in the brain, and the functional consequences of the activation of specific proinflammatory signals. These studies demonstrated several crucial aspects of the inflammatory process (reviewed in Vezzani et al., 2011a, Vezzani et al., 2011b): 1. Inflammation is induced by recurrent seizures; 2. Neuronal cell loss which can be caused by seizures is not a prerequisite for inflammation to occur; rather, the release of proinflammatory cytokines can contribute to cell loss (Allan and Rothwell, 2001), and dying cells may perpetuate inflammation; 3. Seizure-induced brain inflammation is long-lasting and can persist for days after the termination of seizures (De Simoni et al., 2000, Dhote et al., 2007, Ravizza et al., 2008a, Voutsinos-Porche et al., 2004), denoting a failure of endogenous anti-inflammatory control mechanisms. Indeed, measurements of endogenous anti-inflammatory molecules such as IL-1ra demonstrated an inefficient control of the inflammatory response to seizures (De Simoni et al., 2000, Eriksson et al., 2000); 4. In models of epilepsy induced by status epilepticus, traumatic brain injury or prolonged febrile seizures, inflammation precedes the onset of spontaneous seizures suggesting that uncontrolled inflammation may contribute to the development of the epileptic process (De Simoni et al., 2000, Marcon et al., 2009, Ravizza et al., 2008a, Ravizza et al., 2011). Indeed, microarray studies of altered gene transcription in rodent models of TLE, showed that the inflammatory response is among the biological processes mostly upregulated during the epileptogenesis phase (i.e. occurring between the initial brain injury and the onset of epilepsy) (Gorter et al., 2006, Lukasiuk et al., 2006, Majores et al., 2004); 5. Pharmacological blockade of specific pro-inflammatory pathways (e.g. IL-1R/TLR signaling, COX-2) reduces experimental seizures, and transgenic mouse models with altered inflammatory molecules (e.g. cytokines and their receptors or inflammasome components) show changes in seizure threshold (reviewed in Ravizza et al., 2011, Riazi et al., 2010, Vezzani et al., 2008). This evidence indicates that brain inflammation contributes to seizures, therefore it should not be considered a mere epiphenomenon of the pathology.
Immunohistochemical studies in experimental models of epilepsy showed that various inflammatory molecules are rapidly induced by seizures, or by brain injury, in locally activated astrocytes and microglia, thus demonstrating that the first wave of inflammation induced by a brain damaging event, occurs in parenchymal brain cells (reviewed in Vezzani et al., 2008, Vezzani et al., 2011a, Vezzani et al., 2011b). Inflammatory mediators are also induced in endothelial cells of the BBB, indicating that inflammation propagates from glial cells to the brain microvasculature. Moreover, inflammatory mediators could be also released by macrophages and granulocytes entering the brain from the blood during the epileptogenesis phase (Fabene et al., 2008, Ravizza et al., 2008a, Ravizza et al., 2008b, Zattoni et al., 2011). The interaction of inflammatory molecules produced by perivascular glia, and the extravasation of leukocytes may cause BBB damage and the subsequent leakage of serum proteins into the brain (Oby and Janigro, 2006, Shlosberg et al., 2010, Vezzani et al., 2011a, Vezzani et al., 2011b). Serum protein extravasation appears to contribute to neuronal network hyperexcitability (see later). Inflammatory mediators are also measured in regions of seizure generalization. Notably, these molecules are over-expressed not only after the acute pro-epileptogenic injury and during the epileptogenesis phase, but also in chronic epileptic tissue in the cell populations where they are detected in human TLE specimens (reviewed in Vezzani et al., 2011a, Vezzani et al., 2011b).
Recently, increased IL-1β has been measured in the somatosensory cortex of Genetic Absence Epilepsy Rat from Strasbourg (GAERS), a rat model of absence seizures (Akin et al., 2011), indicating that brain inflammation can be triggered by different types of recurrent seizure activity.
The presence of brain inflammation in epilepsy raised the crucial question about the functional consequences of this phenomenon, thus prompting pharmacological studies in seizure models, and investigations on seizure susceptibility in transgenic mice with altered inflammatory pathways. The initial studies showed that blockade of IL-1β signaling in the brain using IL-1ra drastically reduced seizures in various animal models (De Simoni et al., 2000, Vezzani et al., 2000, Vezzani et al., 2002); moreover, mice overexpressing the soluble form of human IL-1ra in astrocytes were intrinsically resistant to seizures (Vezzani et al., 2000). These findings, and the evidence that IL-1β is increased in brain after a pro-convulsant challenge (De Simoni et al., 2000, Minami et al., 1990, Vezzani et al., 1999), indicate that this cytokine contributes to the precipitation and recurrence of seizures. Subsequent studies showed that blockade of IL-1β biosynthesis using specific Interleukin Converting Enzyme (ICE)/Caspase-1 inhibitors (Maroso et al., 2011a, Maroso et al., 2011b, Ravizza et al., 2006b, Ravizza et al., 2008b) caused powerful anticonvulsant effects on acute and chronic seizures. Additional investigations showed the role of other cytokines, specific prostaglandins and complement factors in seizures (reviewed in Kulkarni and Dhir, 2009, Riazi et al., 2010, Vezzani et al., 2011a, Vezzani et al., 2011b). More recently, HMGB1 a danger signal with pro-inflammatory properties (Bianchi and Manfredi, 2007) which is released from activated or damaged neurons and glia, was identified as a molecular event crucial for lowering the threshold to seizures (Maroso et al., 2010), in association with IL-1β (Maroso et al., 2011a, Maroso et al., 2011b). HMGB1 effects were mediated by the activation of TLR4 overexpressed by neurons undergoing hyperexcitability induced by proconvulsant drugs (Maroso et al., 2010, Maroso et al., 2011a, Maroso et al., 2011b). In line with these pharmacological data, mice lacking the ICE/Caspase-1 gene, thus unable to produce and release IL-1β, or lacking the IL-1R1 gene thus being unable to activate the IL-1β signaling cascade, or with a defective TLR4 signaling due to a spontaneous loss-of-function receptor mutation, showed a significant delay in the onset of seizures and were intrinsically resistant to seizure activity (Maroso et al., 2010, Ravizza et al., 2006b, Vezzani et al., 2000). Notably, both the IL-1β and TLR signaling are pivotal for the activation of innate immunity and inflammation following pathogen recognition, thus highlighting convergent molecular pathways possibly underlying the proposed causal link between CNS infection and epilepsy (Singh et al., 2008).
Although the contribution of brain inflammation to seizure activity has been demonstrated in various experimental settings, its involvement in epileptogenesis is still hypothetical, although long-term effect of brain inflammation on neuronal excitability have been reported (Riazi et al., 2010). In favor of such pro-epileptogenic role there is evidence that CNS injuries such as trauma, stroke, viral infection, febrile seizures, status epilepticus occurring either in infancy or during a lifetime are considered common risk factors for developing epilepsy, and long lasting CNS inflammation develops rapidly after these events. Moreover, astrocytic overexpression of cytokines such as TNF-α or IL-6 results in age-dependent development of neurological dysfunctions, including increased seizure susceptibility and spontaneous seizures (Akassoglou et al., 1997, Stalder et al., 1998). Pharmacological attempts to interfere with brain inflammation induced by status epilepticus or by kindling using COX-2 inhibitors (Jung et al., 2006), antibodies against endothelial cell adhesion molecules (Fabene et al., 2008), mTOR pathway inhibitor (Zeng et al., 2009) and immunosuppressant drugs (reviewed Ravizza et al., 2011), provided some support to the involvement of inflammation in the development of epileptogenesis. These studies indeed reported a reduction of the frequency of spontaneous seizures in post-SE models (Fabene et al., 2008, Jung et al., 2006, Zeng et al., 2009) or a delay in kindling epileptogenesis in the absence of afterdischarge modifications (reviewed in Ravizza et al., 2011).
Section snippets
Molecular mechanisms by which inflammation contributes to seizures
The mechanisms underlying the proconvulsant effects of IL-1β and HMGB1 include rapid post-translational changes in N-methyl-d-aspartate (NMDA) receptor phosphorylation leading to increased receptor function (Balosso et al., 2008, Maroso et al., 2010, Viviani et al., 2003). Namely, the activation of IL-1R1/TLR4 axis by their endogenous ligands induces Src kinase-dependent phosphorylation of the NR2B subunit of the NMDA receptors (Balosso et al., 2008, Maroso et al., 2010), a pathway responsible
Conclusions
Activation of specific pro-inflammatory pathways has been demonstrated in human and experimental epileptic brain tissue. Various convulsant and pro-epileptogenic brain insults (i.e. neurotrauma, stroke, infection, perinatal injury, febrile seizures, status epilepticus) induce long-lasting inflammation in the brain by activating specific pro-inflammatory pathways inefficiently controlled by endogenous anti-inflammatory mechanisms. The initiation of an inflammatory response in the brain may be a
Acknowledgments
AV is supported by Fondazione Monzino, Fondazione Cariplo and Progetto Ministero della Salute; EA supported by National Epilepsy Funds (NEF 09-05); AM is supported by NIH research grants , and EU FP7 project NeuroGlia (Grant Agreement No. 202167); QJP is supported by CIHR and NSERC and is an AHFMR Scientist.
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