The Role Of IL-17in Epilepsy With Malformations Of Cortical Development

Malformations of cortical development (MCDs) are increasingly recognized as importantcauses of epilepsy in humans. MCDs (e.g., Focal Cortical Dysplasia, Tuberous Sclerosis,hemimegalencephaly, polymicrogyria, agyria, and Sturge-Weber syndrome) are oftenassociated with seizures, and the frequency of seizures were positively correlated with thepathological changes in MCDs. It has been demonstrated that more than80percent ofpediatric with intractable epilepsy are associated with MCD. Thus, MCD represents awell-recognized cause of intractable epilepsy, and the pathogenesis of MCD has become acentral concern in the field of intractable epilepsy. The research on the factors andmechanisms of epileptogensis in MCDs will be helpful for the treatment of refractoryepilepsy.Innate and adaptive immunity have been demonstrated as a striking feature that occurs inFCDs and the inflammatory response may contribute to the generation and recurrence ofseizures by increasing permeability of the blood–brain barrier (BBB), neuron circuitexcitability and decreasing the seizure threshold. It has been shown that IL-17can play abridging role between innate and adaptive immunity in vivo and IL-17and IL-17R areupregulated in, and play a critical role in determining the pathological outcomes of, severalneurological disorders, including autoimmune diseases, neurodegeneration, and ischemicbrain injury. Studies have demonstrated that IL-17can disrupts the BBB, induces the releaseof cytokines (e.g., IL-1β, IL-6and TNF-α) and glutamate, and enhanced excitability of neuron.Thus, IL-17may play an important role in the generation and recurrence of seizures. Recenteviendence showed proinflammatory cytokines (e.g., IL-1β, TNF-α) can activate TRPV1which could regulate neurotransmitter release, synaptic plasticity, and neuronal death. Inaddition, IL-17can induce the release of IL-1β and TNF-α in CNS. Therefore, we speculatethat IL-17exerts its epileptogenetic role through altering the protein expression and activityof TRPV1. Accordingly, in this study, we used surgically resected MCDs and MCD model of miceto investigate the expression and specific cellular distribution of IL-17system and TRPV1inMCDs，to analyze the correlation between IL-17system protein levels and different clinicalvariables of all the surgical specimens of MCDs, to elucidate the role of activation of TRPV1in MCDs.Methods:The experiments were conducted in two parts. In the first part, the CTX from autopsyand surgical specimens (FCDs and TSC) from patients was adopted to measure the proteinlevel of IL-17, IL-17R and downstream factors of IL-17system signaling (ACT1andNFκB-p65) by western blot. Meanwhile, the expression and the specific cellular distributionof IL-17, IL-17R and downstream factors of IL-17system signaling were identified byimmunohistochemistry and double immunofluorescence labeling. In order to confirm the roleof IL-17system in the refractory epilepsy with MCDs, we investigated the characteristic ofmolecular phenotype of IL-17, IL-17R and downstream factors of IL-17system signaling inthe epileptic foci from patients with MCDs and analyzed the correlation between IL-17system protein levels and different clinical variables of all the surgical specimens of MCDs.In the second part, IL-17-treated astrocytes and neurons were adopted to measure the proteinlevels of TRPV1via western blot. We have established F-MCD model of mice with focalfreeze lesions. The CTX from sham mice and microgyral from F-MCD model of mice wasalso adopted to measure the protein level of TRPV1by western blot. Meanwhile, theexpression and the specific cellular distribution of TRPV1were identified byimmunohistochemistry and double immunofluorescence labeling. In addition,Electroencephalograms (EEGs) and behavior of WT and TRPV1-/-F-MCD model of miceinduced epilepsy by penicillin was monitored via video-EEG monitor in vivo. In order toconfirm the role of activation of TRPV1in the refractory epilepsy with MCDs, weinvestigated the characteristic of molecular phenotype of TRPV1in the microgyral fromF-MCD model of mice and the effect of activation of TRPV1on epileptic seizure.Results:I. Expression and cellular distribution of IL-17system in FCDs1．In the control specimens, the expression of IL-17and its receptor, IL-17R, weredetected at the protein level by Western blot, Immunohistochemistry data demonstrated that IL-17and IL-17R were mainly distributed in neurons, glial cells and endothelial cells.2．The protein levels of IL-17and IL-17R were markedly elevated in FCDIa, FCDIIa andFCDIIb compared with CTX tissues. Moreover, the protein levels of IL-17and IL-17R inFCDs were positively correlated with the frequency of seizures. Immunohistochemistry anddouble immunofluorescence labeling indicated that IL-17and IL-17R were highly expressedin neuronal microcolumns, dysmorphic neurons (DNs), balloon cells (BCs), astrocytes and theendothelial cells of blood vessels. Additionally, we detected a few IL-17-positiveCD4-positive T-lymphocytes in FCDIIa and FCDIIb but not in FCDIa. These resultssuggested that IL-17and IL-17R may be involved in the epileptogenicity of cortical lesions inFCDs.3．A pronounced increase in ACT1and NFκB-p65protein levels was detected in FCDs(FCDIa, FCDIIa and FCDIIb) compared with CTX tissues. Moreover, ACT1and NFκB-p65were widely distributed in the FCDs cortical lesions. Our data indicate that, in FCDs, IL-17may be involved in the epileptogenicity of cortical lesions in FCDs through the activation ofACT1/NFκB-p65signaling pathways.II. Expression and cellular distribution of IL-17system in TSC1．The protein levels of IL-17and IL-17R were clearly upregulated in cortical tubercompared with CTX tissues. This result suggested that an association between theoverexpression of IL-17and IL-17R and the epileptogenicity of cortical tubers in TSC.2．Immunostaining displayed that IL-17system was specifically distributed in the innateimmunity cells (DNs, GCs, astrocytes, and microglia) and adaptive immunity cells(T-lymphocytes) as well as the endothelial cells of blood vessels, suggesting that these cellsare the major sources of IL-17and it may be involved in the epileptogenicity of cortical tubersin TSC by accelerating the severity of inflammatory response and BBB disruptionIII. The role of activation of TRPV1by IL-17in F-MCD mice model1．In this study,48hours100ng/ml IL-17-treated astrocytes and neurons were adopted tomeasure the protein levels of TRPV1via western blot. The protein levels of TRPV1weremarkedly elevated after48h100ng/ml IL-17-treated as compared with the normal control.This result suggested that IL-17may be involved in the epileptogenicity of cortical lesions inFCDs through altering the protein expression and activity of TRPV1.2．We have established a mice model of F-MCD. The changes of pathology in F-MCD mice model were similar to those in F-MCD rat model, including microgyral malformation,disorganized lamination and the presence of misshapen cells.3．The protein levels of TRPV1were markedly elevated in microgyrus of F-MCD micemodel group compared with sham group. Immunohistochemistry and doubleimmunofluorescence labeling indicated that TRPV1was mainly distributed in neurons andglial cells.4．Electroencephalograms (EEGs) and behavior of WT and TRPV1-/-F-MCD model ofmice induced epilepsy by penicillin was monitored via video-EEG monitor in vivo.The spikefrequencies and duration of seizure in TRPV1-/-F-MCD model of mice group weresignificantly reduced compared with WT F-MCD model of mice group, and there wassignificant difference in behavioral changes between the two groups. These results suggestedthat the activation of TRPV1may play an important role in the epileptogenesis of MCD inrefractory epilepsy, thus possibly representing a novel target for antiepileptic therapy.Conclusion:1．IL-17may play an important role in the epileptogenesis of MCD in refractoryepilepsy，and maybe exerts its epileptogenetic role through the ACT1/NFκB-p65pathwaysand altering the protein expression and activity of TRPV1.2．we have established a mice model of F-MCD, the changes of pathology in F-MCDmice model were similar to those in F-MCD rat model, which is benefit to study in the future.