Abstract
Epilepsy is a chronic disorder characterized by spontaneous recurrent seizures. Several lines of evidence demonstrate that inflammatory processes within the brain parenchyma contribute to recurrence and precipitation of seizures. In both epileptic patients and animal models, seizures upregulate inflammatory mediators, which in turn may enhance brain excitability. We recently showed that the C-C motif ligand 2 (CCL2) chemokine (also known as monocyte chemoattractant protein-1 [MCP-1]) mediates the seizure-promoting effects of inflammation. Systemic inflammatory challenge in chronically epileptic mice markedly enhanced seizure frequency and upregulated CCL2 expression in the brain. Selective pharmacological blockade of CCL2 synthesis or C-C chemokine receptor type 2 (CCR2) significantly suppressed inflammation-induced seizures. These results have important implications for the development of novel anticonvulsant therapies: drugs interfering with CCL2 signaling are used clinically for several human disorders and might be redirected for use in pharmacoresistant epilepsy. Here we review the role of CCL2/CCR2 signaling in linking systemic inflammation with seizure susceptibility and discuss some open questions that arise from our recent studies.
Introduction
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Systemic Inflammation Enhances Spontaneous Seizures and Upregulates CCL2
In a recent study, we carried out a molecular screening to identify novel inflammatory mediators involved in seizure precipitation in a chronic epileptic condition. As a model of chronic epilepsy, we used the injection of kainic acid (KA) in the adult mouse hippocampus, which induces spontaneous recurrent seizures and extensive hippocampal damage (Riban et al., 2002; Antonucci et al., 2008). In agreement with previous studies showing proepileptic effects of systemic inflammation (Heida et al., 2005; Friedman and Dingledine, 2011; Marchi et al., 2014), we found that systemic inflammatory challenge by lipopolysaccharide (LPS) injection was able to enhance seizure frequency in chronically epileptic mice. This LPS-induced increase of seizure frequency was accompanied by a marked upregulation of inflammatory proteins in both blood and brain parenchyma (Cerri et al., 2016). Among these proteins, CCL2 emerged as a key mediator of systemic inflammation, being upregulated at the time of LPS-induced seizure enhancement (4 h) and decreasing at the time seizure frequency returned to baseline (24 h) (Fig. 1A, B). In keeping with previous findings (Vezzani et al., 2013), IL-1β also showed a similar behavior in our epilepsy model (Cerri et al., 2016).
Schematic summary of the role of CCL2 signaling in mediating the proepileptic effects of systemic inflammation.
Expression of CCL2 and Its Receptor in the Epileptic Brain
CCL2, also known as monocyte chemoattractant protein-1 (MCP-1), was the first human chemokine to be characterized. Chemokines, or chemotactic cytokines, are defined by their ability to induce directional migration and activation of leukocyte subsets into inflammatory sites. In the brain, CCL2 specifically activates and attracts cells of the monocyte lineage (including macrophages, monocytes, and microglia), as well as T lymphocytes, basophils, natural killer cells, and astrocytes (Semple et al., 2010). In the brain, CCL2 is mainly produced by astrocytes, resident microglia, and endothelial cells. Infiltrating macrophages also release CCL2 upon their migration into the brain parenchyma, and neurons can produce detectable levels of CCL2 after damage or inflammation (Semple et al., 2010; Réaux-Le Goazigo et al., 2013; Stuart et al., 2015). CCL2 binds as a dimer to the G-protein-coupled receptor C-C chemokine receptor type 2 (CCR2), which is then internalized and removed from the cell surface. Downstream targets of CCR2 include several signaling cascades, including mitogen-activated protein kinase and NF-κB pathways (Semple et al., 2010; Mora et al., 2012). CCR2 expression has been reported on various brain cell types, including neurons, astrocytes, microglia, neural progenitor cells, and microvascular endothelial cells (Semple et al., 2010; Stuart et al., 2015).
CCL2/CCR2 signaling has been implicated in many different neuropathologies, including epilepsy. Increased levels of CCL2 have been detected in the brain of patients with intractable epilepsy (Choi et al., 2009). In rodent models of temporal lobe epilepsy, seizures have been shown to transiently increase CCL2 and CCR2 expression in the hippocampus. In dentate gyrus and CA1/CA3 pyramidal layers, CCL2 upregulation after seizures has been detected in both neurons and glial cells, whereas CCR2 induction has been observed in astrocytes, microglia, and ectopic progenitors of hilar neurons (Manley et al., 2007; Foresti et al., 2009; Hung et al., 2013; Arisi, 2014; Arisi et al., 2015; Cerri et al., 2016) (Fig. 1B).
Interfering with CCL2/CCR2 Signaling Blocks Inflammation-Induced Seizures
To test the functional role of CCL2 in inflammation-induced seizures, we first used bindarit, an anti-inflammatory compound that inhibits transcription of monocyte chemoattractant subfamily of CC chemokines (Mirolo et al., 2008). In chronically epileptic mice, systemic administration of bindarit reduced blood and hippocampal levels of CCL2, resulting in a significantly lower frequency of LPS-induced seizures. The same results were obtained by blocking CCR2 by systemic administration of the selective antagonist RS102895, or by delivering CCL2-blocking antibodies directly into the seizure focus (Cerri et al., 2016) (Fig. 1C, D).
Open Questions
In light of previous studies (some of which are already quoted in this brief review), our study leaves several questions open, which would deserve further investigation.
(1) Does CCL2 mediate the proconvulsant effects of inflammation also in other models of epilepsy? To answer this question, the role of CCL2 should be tested in other models of generalized as well as focal epilepsy, and other inflammatory stimuli (different from LPS) should be used.
(2) Is the proconvulsant role of CCL2 peripheral or central? Our data suggest that centrally released CCL2 in chronically epileptic mice treated with LPS can increase seizure frequency (Fig. 1C, D). However, we cannot exclude that peripheral (circulating) CCL2 is also involved. Peripherally produced CCL2 may cross the blood–brain barrier (BBB), possibly by interacting with specific carriers such as caveolin-1; in addition, CCL2 itself seems to affect BBB permeability (Yao and Tsirka, 2014), which is altered in the KA-lesioned hippocampus (Zattoni et al., 2011). Thus, we cannot rule out the possibility that LPS-induced CCL2 upregulation is a consequence of BBB disruption. However, TNFα, interferon-γ, and IL-10 were upregulated by LPS only in the blood but not in the hippocampus of chronically epileptic mice (Cerri et al., 2016), suggesting that CCL2 (but not other inflammatory mediators) is selectively upregulated within the epileptic focus. This question could be addressed in different ways, for example, by testing seizure propensity after an inflammatory stimulus in mice with conditional overexpression or deletion of CCL2 in the brain.
(3) Which are the cellular and molecular mechanisms of action of CCL2 in the epileptic brain? Different mechanisms may underlie the seizure-promoting effect of CCL2. CCL2 might directly enhance neuronal excitability by altering Ca2+ signaling (van Gassen et al., 2005), reducing inhibition (Gosselin et al., 2005) or potentiating excitatory postsynaptic currents (Zhou et al., 2011). CCL2 might also act indirectly, inducing the synthesis of other proinflammatory factors such as IL-1β, whose seizure-promoting effects have been largely described (Vezzani et al., 2011). Indeed, we observed a reduced expression of IL-1β in the hippocampus of LPS-treated epileptic mice after systemic administration of the CCL2 synthesis inhibitor bindarit (Cerri et al., 2016). This is in agreement with previous studies showing a reduced synthesis of IL-1β after LPS treatment in CCL2−/− mice (Rankine et al., 2006). CCR2 is expressed in activated microglia/macrophages within the epileptic hippocampus (Fig. 1B) (Cerri et al., 2016 and references already mentioned). Since IL-1β is mainly produced and released by activated microglia in the epileptic brain (Vezzani et al., 1999), it is likely that CCL2 increases seizures through IL-1β. Finally, CCL2 might also be involved in neuropathological changes that accompany seizures, such as ectopic migration of neuronal progenitors in the dentate gyrus (Hung et al., 2013). Thus, CCL2/CCR2 signaling may act as a key proinflammatory factor in the epileptic brain by both directly increasing neuronal excitability and inducing downstream inflammatory effectors.
(4) Is CCL2 implicated in human epilepsy? Some studies demonstrated that CCL2 expression is increased in brain tissue of patients with intractable epilepsy (Wu et al., 2008; de Vries et al., 2016). Moreover, a genetic polymorphism located in the CCL2 promoter (resulting in increased CCL2 gene transcription and higher levels of circulating CCL2) has been associated with drug-resistant epilepsy in Chinese pediatric patients (He et al., 2013). Further studies are needed to confirm and strengthen the idea that CCL2-mediated inflammation is implicated in human epilepsy.
Concluding Remarks
In chronically epileptic mice, systemic inflammation induces a significant aggravation of seizures, accompanied by upregulation of CCL2 in the brain. Systemic and local interference with CCL2/CCR2 signaling significantly reduces seizures after systemic inflammation. These data show a previously unrecognized role for CCL2 in mediating the seizure-promoting effects of systemic inflammation. Drugs blocking CCL2/CCR2 signaling are currently under development for several inflammatory brain disorders (Semple et al., 2010) and could also be tested in drug-resistant epilepsy.
Footnotes
Acknowledgments
This work was funded by the Italian Ministry of Health (grant RF-TAA-2008-1141282 to Y.B. and M.C.), Fondazione Pisa (grant 158/2011 to M.C.), and University of Trento (start-up grant to Y.B.).
Disclosure Statement
No competing financial interests exist.