Many cytokines such as interleukin 6 (IL-6) and interferon gamma (IFN-γ) are implicated in ictogenesis and epileptogenesis (Muller et al., 1993; Samland et al., 2003). IL-17A promotes the inflammatory response by activating neutrophils and stimulating production of proinflammatory cytokines such as IL-6 and IL-1β (Miossec et al., 2009). It is reportedly involved in the pathogenesis of a variety of inflammatory diseases such as multiple sclerosis (Jadidi-Niaragh & Mirshafiey, 2011). In addition, IL-17 receptor is highly expressed in multiple sclerosis lesions (Kebir et al., 2007) and cortical lesions in focal cortical dysplasia (He et al., 2013). However, a role of IL-17A in ictogenesis and epileptogenesis has not been demonstrated or suspected. In the current study, we determined the contents of IL-17A and other cytokines in the peripheral blood and cerebrospinal fluid (CSF) of patients with epilepsy and investigated whether they correlated with seizure severity.
Interleukin 17A (IL-17A) is implicated in the pathogenesis of several neuroimmunologic diseases. We aimed to evaluate the relationship between IL-17A and seizure severity in patients with epilepsy. Seventy patients with probable symptomatic epilepsy and 68 healthy controls were included. Interictal serum IL-17A and related cytokine (IL-23, IL-6, IL-1β, interferon gamma (IFN-γ), and IL-10) levels were measured. The relationship between seizure severity and cytokine concentrations was assessed by Spearman correlation and multivariate linear regression test. IL-17A levels in the cerebrospinal fluid (CSF) were tested in 30 additional patients with epilepsy, either in the postictal or interictal period and 15 patients with idiopathic inflammatory demyelinating diseases (IIDDs). Interictal serum IL-17A levels were significantly elevated in patients with epilepsy compared to controls. IL-6, IFN-γ, and IL-1β levels were also markedly elevated. Spearman correlation analysis revealed positive correlation between IL-17A, IL-6 levels and Veterans Administration Seizures Frequency and Severity Rating Scale score(VA score); IFN-γ, IL-10 levels, and National Hospital Seizure Severity Scale (NHS3) score. In addition, IL-17A levels correlated significantly with seizure frequency. Multivariate linear regression test showed that only IL-17A levels were independently positively correlated with VA scores (B = 0.288, p = 0.027). Postictal IL-17A levels in the CSF were significantly elevated compared to interictal patients and patients with IIDDs. Our results suggest that interictal IL-17A levels correlated highly with seizure severity.
We prospectively recruited 70 interictal patients with probable symptomatic epilepsy who sought treatment from June 2009 to April 2012 at our institution. No etiology was found after detailed history, physical, laboratory, and imaging studies. Major exclusion criteria were a history of autoimmune diseases, allergic response, immune deficiency disorder, diabetes, psychiatric illness, malignancy, severe cognitive impairment, or a systemic or central nervous system (CNS) infection 2 weeks before sample collection. Epilepsy was diagnosed by two experienced neurologists according to the 2006 International League Against Epilepsy (ILAE) Classification (Engel, 2006). All patients were evaluated for seizure frequency using seizure diaries and seizure severity using the National Hospital Seizure Severity Scale (NHS3) and the Veterans Administration Seizures Frequency and Severity Rating Scale score (VA score) (Cramer & French, 2001). Sixty-eight age- and sex-matched healthy volunteers were included as normal controls.
Thirty additional patients with probable symptomatic epilepsy and 15 age- and sex-matched patients with idiopathic inflammatory demyelinating diseases (IIDDs) (inflammatory demyelinating polyneuropathy, n = 10; CNS demyelinating diseases, n = 5) were recruited for CSF study. For patients with IIDDs, those with a family history of epilepsy or psychiatric illness; a history of seizure, febrile convulsions, or psychiatric illness; and a history of other autoimmune diseases, infectious diseases, allergic response, immune deficiency disorder, diabetes, malignancy, or severe cognitive impairment were excluded.
The study protocol was approved by the local institutional review boards at the authors' affiliated institutions. Informed consent was obtained from all the subjects or their legal surrogates.
Enzyme-Linked Immunosorbent Assay
Postictal samples were collected within 72 h after seizure attack, whereas interictal samples were collected at least 7 days from the last seizure attack. After an overnight fast, venous blood samples were taken in an advance vacuum tube (5 ml) and a lithium heparin vacuum tube (4 ml) separately. After lumbar puncture, 1 ml CSF was collected. Serum and CSF were centrifuged at 1000 g for 10 min at 4°C and the supernatant was saved and stored at −80°C for subsequent cytokine analysis. The concentrations of IL-17A, IFN-γ, IL-10, and IL-6 (all from PeproTech, Rocky Hill, NJ, U.S.A.), IL-1β (Bender MedSystems, Vienna, Austria), and IL-23 (Invitrogen, Carlsbad, CA, U.S.A.) were measured by enzyme-linked immunosorbent assay (ELISA) as instructed by the manufacturers. ELISAs were performed in duplicate and the data were expressed in pg/ml.
Numerical variables were expressed as mean ± standard deviation (SD) or median (interquartile range [IQR]). Differences were compared by chi-square test for categorized data, and unpaired Student's t-test for continuous roughly normally distributed data. Cytokine concentrations and CSF parameters were compared using nonparametric Mann-Whitney U-test. Correlation between cytokine concentrations and seizure severity was analyzed by Spearman correlation and multivariate linear regression analysis. In multivariate linear regression analysis, all of the continuous data had skewed distribution and were logarithmically transformed to fit normal distribution. We used stepwise methods to screen variables, including at the 0.05 level and excluding at the 0.1 level. We set outliers as data outside 3 SD. In this study, one outlier was found and excluded from regression models. Statistical analysis was performed using the SPSS 15.0 package (SPSS Inc., Chicago, IL, U.S.A.). All tests were two-sided with a significance level of p < 0.05 (two-tailed).
Interictal IL-17A levels are elevated in patients with epilepsy
The clinical features of epilepsy patients are shown in Table 1. The patients with epilepsy had significantly higher serum IL-17A levels (13.99 [66.62] pg/ml) than healthy controls (8.61 [5.00] pg/ml) (p = 0.017). They also had markedly higher contents of IFN-γ (11.61 [9.12] pg/ml vs. controls, 9.72 [8.32] pg/ml, p = 0.000), IL-1β, (10.50 [1.52] pg/ml vs. controls, 4.43 [6.96] pg/ml, p = 0.000), and IL-6 (6.12 [28.22] pg/ml vs. controls, 4.06 [5.08] pg/ml, p = 0.016). No significant difference was observed in the serum levels of IL-10 and IL-23 between the two groups (p > 0.05) (Fig. 1). Of note, no difference was found in the levels of IL-17A and other cytokines by seizure type, antiepileptic drug (AED) use, and disease duration (p > 0.05). Furthermore, CSF IL-17A content was significantly higher in patients with epilepsy postictally (14.53 [15.48] pg/ml) than that interictally (10.28 [3.69] pg/ml, p = 0.016). CSF IL-17A content in postictal epilesy patients was also significantly higher than that in IIDDs patients (8. 69 [4.10] pg/ml, p = 0.001). There were no differences between patients with epilepsy (in both the postictal and interictal period) and those with IIDDs in the number of white blood cells and glucose contents in the CSF.
|Serum (n = 70)||Cerebrospinal fluid|
|Postictal patients (n = 15)||Interictal patients (n = 15)|
|Mean duration of disease, years||7.00 (15.00)||0.67 (9.98)||2.00 (10.83)|
|Mean age of onset, years||20.51 ± 9.76||28.67 ± 13.96||31.46 ± 16.83|
|Frequency per month||1.00 (2.68)||1.00 (0.83)||0.33 (0.17)|
|NHS3 score||15.13 ± 6.56||13.73 ± 4.67||13.60 ± 3.91|
|VA score||28.50 (53.35)||18.00 (37.00)||7.00 (15.20)|
Interictal IL-17A levels correlate with and are an independent risk factor for seizure severity
Spearman correlation analysis revealed that IL-17A content significantly correlated with VA (r = 0.372, p = 0.002) and seizure frequency (r = 0.253, p = 0.045). IFN-γ and IL-10 concentrations positively correlated with NHS3 scores (r = 0.361, p = 0.002; r = 0.269, p = 0.026, respectively). IL-6 content correlated significantly with VA (r = 0.300, p = 0.017). IL-23 and IL-1β contents did not correlate with patient clinical features. Furthermore, epilepsy duration and age of onset did not correlate with IL-17A and other cytokines (data not shown). Furthermore, multivariate linear regression analysis with seizure frequency, NHS3, and VA scores as dependents, and age, duration, age of onset, levels of IL-17A, IL-23, IFN-γ, IL-10, IL-6, and IL-1β as covariates revealed that only serum IL-17A concentration displayed positive correlation with VA scores (B = 0.288, p = 0.027), whereas other cytokines did not enter the regression model.
Inflammatory processes within the brain may constitute a common and crucial mechanism in the pathophysiology of seizures and epilepsy (Vezzani et al., 2011). Patients with epilepsy had elevated proinflammatory cytokine levels (IL-6 and IFN-γ) in the peripheral blood (Lehtimaki et al., 2004), which were further confirmed by our results. Here, we present the first clinical evidence for elevated IL-17A levels in the peripheral blood and CNS of patients with epilepsy. We also show that IL-17A level is an independent factor of seizure severity. Our findings further implicate immune factors such as IL-17A in the disease process of epilepsy.
IL-17A is implicated in the development of inflammatory diseases and correlates with disease severity (Montes et al., 2009). Our Spearman's rank correlation test revealed that both IL-17A and IL-6 positively correlated with VA scores. In addition, IL-17A positively correlated with seizure frequency. Regression analysis further displayed that only IL-17A was an independent risk factor of VA scores. IFN-γ and IL-10 also positively correlated with NHS3 scores, but we did not find any independent risk factor factors that correlated with NHS3 scores. Because the VA scale focuses on seizure type and frequency while NHS3 emphasizes seizure manifestation (Cramer & French, 2001), we assumed that IL-17A levels were more associated with seizure frequency than seizure manifestation. Notably, our multiple linear regression revealed that IL-17A level correlated independently with seizure severity, but not IFN-γ and IL-6, which were previously considered proepileptic. This may be due to perpetuation of inflammation by IL-17A via stimulating the production of several proinflammatory cytokines such as IL-6, IL-1β, and tumor necrosis factor (Miossec et al., 2009).
We also found that CSF IL-17A content was dramatically increased after acute seizure attack. We were unable to obtain CSF samples from healthy subjects because of lack of consent. However, interictal IL-17A content was also elevated, as it was comparable to that of patients with IIDDs, a group that has been previously demonstrated to have elevated IL-17 levels and activated Th17 cells in the CSF (Li et al., 2012). We inferred that CSF IL-17A level was significantly elevated in patients with epilepsy, which further increased after acute seizure attack. The precise mechanism underlying elevated postictal CSF IL-17A levels still remains unclear. We speculate that IL-17A disrupts the integrity of the tight junctions, which promote the transmigration of Th17 or other active T cells across the blood–brain barrier (Kebir et al., 2007), which in turn aggravates seizure attack.
In conclusion, our study offers evidence of increased levels of IL-17A, both in the CSF and peripheral blood in epilepsy patients, which correlated with seizure severity. We speculate an important role of IL-17A in the aggravation of epilepsy. Targeting IL-17A may provide an intriguing option to improve the prognosis of epilepsy.
This work was supported by project grants from Shanghai Municipal Committee of Science and Technology (Code 10JC1402800, Code 13DJ1400301), the National Natural Science Foundation of China (Code 81200999, Code 31271188), and research fund for the Doctoral Program of Higher Education of China (Code 20100071110059).
None of the authors has any conflict of interest related to this manuscript. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.