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Introduction
Status epilepticus is one of the most common neurological emergencies in children and adults. Febrile status epilepticus cases are often associated with inflammatory neurological diseases caused by specific pathogens or antineuronal autoimmunity. In addition, there is a subgroup of super-refractory status epilepticus triggered by fever and having no known cause.1 This condition is designated as either acute encephalitis with refractory, repetitive partial seizures (AERRPS)2 or febrile infection-related epilepsy syndrome (FIRES).3 The pathogenesis of AERRPS/FIRES is currently unknown. A close relationship between febrile illness and status epilepticus suggests deleterious effects of inflammation and autoimmunity on the onset and progression of seizure. However, immune mechanisms in human status epilepticus associated with isolated fever have not been fully elucidated.
We report a comprehensive study of the inflammatory mediators in paediatric cases of AERRPS. We show a marked upregulation of proinflammatory cytokines and chemokines in the cerebrospinal fluid (CSF) of patients with this condition.
Methods
We defined AERRPS using the criteria shown in online supplementary table S1. Between April 2010 and July 2013, 14 patients with AERRPS and 14 patients with other inflammatory neurological diseases (OIND) were enrolled in the study. Serum and CSF specimens from patients with AERRPS and OIND were collected between 0 and 39 days from the onset of neurological symptoms. Eighteen patients with non-inflammatory neurological diseases (NIND) served as a control group. Additionally, the Shizuoka Institute of Epilepsy and Neurological Disorders provided us with 13 conserved CSF specimens from patients with AERRPS who fulfilled the same criteria, which we also included in our analysis. There was no significant difference in age or gender among AERRPS, OIND and NIND groups (see online supplementary table S2). Serum and CSF levels of cytokines and chemokines were measured by Bio-Plex Suspension Array System (Bio-Rad, Hercules, California, USA) according to the manufacturer's protocols. Statistical significance among the three groups was analysed by non-parametric Kruskal-Wallis tests followed by post hoc Steel-Dwass non-parametric multiple comparison procedures. Relationships between two variables were determined by Spearman's partial rank correlation coefficient. Compensation for multiple comparisons was not performed. The results are shown as means±SD and p<0.05 was considered significant (see online supplementary methods for details).
Results
Serum analysis showed significant differences in the concentrations of 16 analytes among the three groups: 6 analytes were upregulated and 10 were downregulated in the AERRPS group compared with either NIND and/or OIND groups (see online supplementary table S3). Analysis of CSF demonstrated significant differences in the concentrations of 22 analytes and all except CXCL12 and vascular endothelial growth factor were upregulated in the AERRPS group compared with either NIND and/or OIND groups (see online supplementary table S4). Among them, interleukin (IL)-6, IL-8 and CXCL10 were increased most strikingly (figure 1A). Although serum and CSF concentrations of IL-6 (figure 1B, E), IL-8 (figure 1C, F) and CXCL10 (figure 1D, G) in the AERRPS group were significantly higher than that of the NIND group, the changes were more drastic in CSF than in serum.
Serum and CSF concentrations of cytokines and chemokines. (A) The most highly upregulated cytokines and chemokines in the CSF of patients with acute encephalitis with refractory, repetitive partial seizures (AERRPS). The fold change was determined by comparing the mean (left) and median (right) concentrations of each cytokine/chemokine in the AERRPS and non-inflammatory neurological diseases (NIND) groups (calculated as AERRPS group per NIND group). The 20 most upregulated analytes in the CSF of the AERRPS group are listed. Asterisk indicates analytes that were immeasurable in more than one-half of the serum samples. (B–D) The serum levels of IL-6 (B) and IL-8 (C) in the AERRPS group were significantly higher than the levels in the NIND group, and CXCL10 (D) in the AERRPS group was significantly higher than the levels in NIND and other inflammatory neurological diseases (OIND) groups. (E–G) The CSF levels of IL-6 (E), IL-8 (F) and CXCL10 (D) in the AERRPS group were significantly higher than the levels in the NIND group. The y-axis is presented in log scale (B–G). *p<0.05 and **p<0.01, Steel-Dwass tests.
We estimated the correlation among the CSF concentrations of eight cytokines and chemokines with p values<0.001 in online supplementary table S4 (CXCL9, CXCL10, IL-6, IL-8, CXCL1, macrophage colony-stimulating factor, CCL13 and CCL26, online supplementary table S5). CXCL9 and CXCL10 (R=0.905, p=9.09×10−11, online supplementary figure A), and IL-8 and CXCL1 (R=0.724, p=1.96×10−5, online supplementary figure B) were correlated the strongest.
Discussion
There is growing evidence to support the involvement of neuroinflammation in ictogenic and epileptogenic processes. Proinflammatory cytokines play a crucial role in this process. Studies using experimental animal models have demonstrated IL-1β expression in microglia and astrocytes after seizures and that IL-1β itself can enhance neuronal excitability.4 Based on these findings, a hypothesis has been proposed that the vicious cycle consisting of seizure activity and inflammation contribute to the further progression of inflammation-mediated status epilepticus.1 However, this paradigm has not been definitively demonstrated in human status epilepticus.
The pathogenesis of AERRPS/FIRES remains controversial. Based on occasional CSF pleocytosis, an immune-mediated pathogenesis has been implicated in FIRES and AERRPS.2 The biphasic clinical course and the absence of infectious agents suggest a possible infection-triggered process rather than an infectious disease.5 Central nervous system (CNS) pathology of FIRES is devoid of inflammatory infiltrations,3 suggesting little contribution of systemic adaptive immunity to this condition.
This study demonstrates for the first time the upregulation of certain cytokines and chemokines in AERRPS. It is noteworthy that proinflammatory cytokines (IL-6, macrophage migration inhibitory factor (MIF) etc) and chemokines (CXCL10, IL-8, etc) were selectively upregulated. In sharp contrast, most T-cell-associated cytokines (IL-2, IL-17A, etc) and homoeostatic chemokines (CCL21, CXCL12, etc) remained unchanged or were downregulated. These findings provide strong evidence for the involvement of innate inflammation in the pathogenesis of AERRPS. Changes in cytokine and chemokine levels were more prominent in CSF than in serum, suggesting that inflammation primarily takes place in CNS. It is currently unknown whether intrathecal inflammation is a cause or effect, and it is possible that an exaggerated immune response is a secondary phenomenon due to refractory status epilepticus. It is difficult to use patients with non-inflammatory status epilepticus as a control group because patients with this condition are usually not recommended for CSF analysis.
In conclusion, the present study unveiled a previously unrecognised relationship between a group of proinflammatory cytokines/chemokines and refractory status epilepticus in a human disease. These findings may provide important clues to understand the pathomechanism of and to develop effective treatment strategies for inflammation-related seizure disorders.
Acknowledgments
The authors thank Dr H Fujita, Dr M Amamoto, Dr A Nakayama, Dr Y Nonoda, Dr N Matsumoto, Dr H Tsuji, Dr K Doi, Dr S Watanabe, Dr S Fukumura, Dr K Maruta, Dr T Fukuyama and Dr H Akaike for providing clinical information and patients’ samples.
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Files in this Data Supplement:
- Data supplement 1 - Online supplement
- Data supplement 2 - Online supplement
Footnotes
Contributors HS, YT, MS and MH were involved in conception, design and interpretation of the data. HS, NT and IK were involved in analysis of the data. HS, NT, IK, MS and MH were involved in drafting of the paper. All authors were involved in approval of the final version.
Funding This work was supported by Health Labour Sciences Research Grant for Research on Measures for Intractable Diseases from the Japanese Ministry of Health, Labour and Welfare (H22-Nanchi-Ippan-029 and H23-Nanchi-Ippan-107, HS, NT, IK), Research Grant from the Japan Epilepsy Research Foundation (H22–5, HS) and Research Support from Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics (20-Young-3, HS).
Competing interests None.
Ethics approval Institutional Review Board of the National Center of Neurology and Psychiatry (No. 21-9-2) and the Tokyo Metropolitan Institute of Medical Science (No. 12–34).
Provenance and peer review Not commissioned; externally peer reviewed.