IL-6 as potential predictive biomarker of febrile seizures

IL-6 as potential predictive biomarker of febrile seizures | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article IL-6 as potential predictive biomarker of febrile seizures Jan Papež, Katarína Česká, Tomáš Loja, Jiří Šána, Marek Večeřa, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3964084/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Febrile seizures are the most common type of convulsions in children. Fever is induced by cytokines release during infection. Recent studies focusing on the identification of a possible role of cytokines in pathogenesis of febrile seizures have contributed conflicting results. Moreover, most of these studies investigated only a few cytokines, such as IL-1β, IL-6 and TNFα. The aim of this study was to investigate multiple cytokine-chemokine profiles that could be potentially associated with the development of febrile seizures. Methods Twenty-four febrile seizure cases (febrile seizure group) and two matched control groups were included in this study. Children with febrile illness without convulsion (febrile control group) and children without seizures and without fever (healthy control group) served as control groups. We investigated serum levels of IL-1β, IL-6, IL-8, IL-10, IL-18, CXCL10/IP-10, CCL2/MCP-1, CXCL13/BLC, TNFα, and fractalkine/CXC3CL1 in all children included in the study. Results The analysis of serum samples revealed a significant elevation of IL-6 (p = 0.0042) in the FS group compared to the febrile controls. Significantly higher levels of cytokines were also found in the FS group compared to healthy controls in IL-10 (p = 0.0039), TNFα (p = 0.0091) and MCP-1 (p = 0.0039). Conclusion Our study supports the hypothesis that IL-6 is involved in the pathogenesis of febrile seizures. We supposed that IL-6 could become a potential biomarker of the development of febrile seizures in children with febrile disease. This knowledge could be used in clinical practice to identify children at risk of developing of febrile convulsions. febrile seizures pathogenesis cytokines brain excitability Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Febrile seizures (FS) are the most common convulsions of childhood, and usually occur in children between the ages of six months and five years [ 1 ]. These children do not have any evidence of central nervous infection, electrolyte imbalance, metabolic disorders, or a history of febrile seizures[ 2 , 3 ]. FS are categorized into three types: simple, complex, and febrile status epilepticus. Simple FS are generalized, last for 15 minutes), focal, and recur within 24 hours. Febrile status epilepticus is the most severe type of FS, lasting for > 30 minutes [ 34 ]. While children with simple FS have an only slightly higher risk of subsequent epilepsy of around 1% compared to the general population (0.5%), the incidence of epilepsy observed in patients after complex FS is 4–6% [45]. The exact pathophysiology of FS remains unknown. FS are generally believed to result from a vulnerability of the developing brain to the fever, in combination with an underlying genetic predisposition and environmental factors [ 7 ]. Cytokines are a diverse family of intercellular signalling molecules that regulate inflammation and immune responses [ 8 ]. As a natural consequence of immune system activation during infection, both pro-inflammatory and anti-inflammatory cytokines are produced and released [ 9 ]. Subsequent fever leads to an increased neuronal excitability resulting in convulsions [ 10 ]. Cytokines include interleukins, chemokines, tumour necrosis factors, interferons, etc. Many studies investigating the role of cytokines in the pathogenesis of FS contributed inconsistent results. Moreover, most of these were focused on three major cytokines, e.g. IL-1β, IL-6 and TNFα [ 11 , 12 , 13 , 14 , 15 ]. Due to these conflicting results, we decided to explore the association of ten various pro-inflammatory and anti-inflammatory cytokines in the genesis of FS. Materials and methods Subjects This prospective case-control study was carried out at Children´s Hospital Brno affiliated with the Faculty of Medicine of Masaryk University in Brno, Czech Republic in the period from July 2021 to February 2022. All patients included in this prospective study with a case group of 40 were selected from children admitted to the hospital after an attack of simple FS. A total of 24 patients with febrile seizures were included in the study, with an average age of 22.54 ± 10.18 months. The group consisted of 13 male patients and 11 female patients. Inclusion criteria for the febrile seizure group were fever ≥ 38°C and the presence of either simple febrile seizures or complex febrile seizures. Patients with evidence of central nervous system infection, metabolic disorder, electrolyte imbalance, neurological deficits, and a history of febrile seizures were excluded from the study. Two additional groups of patients were defined in the project. The first group comprised patients with febrile illness but without febrile seizures. Diagnoses included in this group were nonspecific viral respiratory tract infections, viral bronchitis, laryngitis, and gastritis. There were eight subjects in this group, with an average age of 17 ± 13.47 months, consisting of three male and five female patients. The second group consisted of subjects without febrile illness. This group included a total of eight patients with an average age of 21.75 ± 13.04 months (five males and three females). Exclusion criteria were a history of neurological disease in the participant, febrile infection, or autoimmune disease. Detailed characteristics of the subjects are provided in Table. Table. Demographic characteristics of subjects Ethics All legal representatives of the patients have given their informed consent in this study, which has been approved by the Ethics Committee of the University Hospital Brno and the Faculty of Medicine, Masaryk University Brno. Methods Three millilitres of blood were taken from the peripheral vessels of participants in all the groups, and serum was obtained by centrifugation at 3,000 rpm for five minutes at 4 ̊C. The serum was then poured into acid-washed tubes and stored in a refrigerator at -20 ̊C. In the febrile seizure groups, blood samples were collected within two hours following the occurrence of a seizure episode. For quantitative detection of plasma levels of cytokines and chemokines, Luminex technology (multi-analyte profiling magnetic beads) was used to enable the detection and quantification of multiple protein targets simultaneously. We used a custom ProcartaPlex™ 9-Plex immunoassay panel (IL-6, IL-8, IL-10, IL-18, CXCL10/IP-10, CCL2/MCP-1, CXCL13/BLC, TNFα and fractalkine/CXC3CL1), the ProcartaPlex™ high sensitivity IL-1β simplex kit. All kits were used according to the manufacturer’s instructions (Affymetrix eBioscience, Hatfield, UK). Flow cytometry analysis was performed using BD FACS Verse (BD Biosciences, San Jose, CA, USA). Each plasma sample was tested in triplicate and the results were calculated according to the manufacturer’s instructions. Statistical analysis Between-group statistics (i.e. samples of patients with febrile seizures vs. febrile controls & healthy controls) were calculated using the Mann-Whitney nonparametric test, significance level set at alpha = 0.05. P-values were corrected for multiple comparison with the Bonferroni correction and p-values of < 0.05 were considered statistically significant. Between-grade comparison was performed using the Kruskal-Wallis nonparametric test. To strengthen the results, Mann-Whitney non-parametric tests were performed between all grades for all the analysed molecules. Results Luminex technology (multi-analyte profiling magnetic beads) was used to enable the detection and quantification of multiple protein targets simultaneously (plasma levels of cytokine and chemokine) in samples of patients with FS versus the group with febrile infection without seizures and the control afebrile group. We compared a total of ten cytokines and chemokines in samples from patients. The analysis of serum samples revealed a significant elevation of IL-6 (p = 0.0042) in the FS group compared to FC (see Fig. 1 here). Significantly higher levels of cytokines were also found in the FS group compared to HC in IL-10 (p = 0.0039), TNFα (p = 0.0091) and MCP-1 (p = 0.0039) (Fig. 2–4 here). Figure 1. IL-6 serum level Figure 2. IL-10 serum level Figure 3. TNFα serum level Figure 4. MCP-1 serum level Discussion In the present study we examined a total of ten cytokines, including pro-inflammatory and anti-inflammatory cytokines. The multiplex cytokine analysis revealed significantly elevated serum levels of IL-6 in the FS group compared to the FC group. No statistical difference was found between the FS group and the HC group, nor between the FC group and the HC group. On the other hand, we found significantly higher levels of cytokines IL-10, TNFα and MCP-1 in the FS group compared to the HC group (see results and Fig. 1–4 here). IL-6 is a pleiotropic, pro-inflammatory cytokine that is secreted by activated T-lymphocytes, macrophages, endothelial cells, and epithelial cells. It has a broad range of biological activities in immune regulation, haematopoiesis, inflammation and has a strong correlation with fever, and it can promote other cells [ 16 ]. The function of IL-6 in the body is variable, but according to many studies, its proactive role in exacerbating seizures by stimulating the preoptic area in the hypothalamus has been confirmed [ 17 , 18 ]. The concentration of IL-6 is mainly regulated at the level of expression, because IL-6 has a short half-life (20–60 minutes) and is rapidly cleared from the plasma [ 18 ]. Several studies have been performed to explore the role of IL-6 in FS. Similarly to our study, Kim et al. reported that serum IL-6 was significantly higher in children with FS than in FC group [ 19 ]. Chen et al. also reported that the level of IL-6 was higher in the FS group compared to the FC group and the difference was statistically significant [ 19 ]. Azab et al. also found that children with FS had significantly higher serum IL-6 levels than the FC group. Moreover, they found significant elevation of serum IL-6 in the FS group compared to the HC group [ 20 ]. A study by Kumar et al. also found higher levels of IL-6 in FS patients than in the FC group [ 21 ]. However, the difference did not reach statistical significance. Similarly to a previous study, two other studies reported that serum levels of IL-6 in FS were elevated compared to FC but the difference was not statistically significant [ 22 , 23 ]. Meta-analysis by Kwon et al. focused on the role of serum IL-1β, IL-6, TNFα in FS concluded that only IL-6 levels in blood serum were significantly associated with FS [ 24 ]. According to the work of Indian authors, a correlation between IL-6 concentration and the severity of febrile seizures was even demonstrated. Specifically, in patients with complex febrile seizures, the IL-6 concentration was significantly higher than in subjects with simple febrile seizures and controls. The findings of this study are supported by the results of Azab et al. [ 20 , 25 ]. Contrary to our results, Gupta et al. reported that serum IL-6 levels were significantly lower in children with FS as compared to FC [ 26 ]. The researchers hypothesized that one of the reasons for low serum IL-6 levels in children with febrile seizures could be the possibility of consumption of cytokines in the genesis of febrile seizures. Some inconsistency in the results between certain studies may be attributed to the age of patients, gender, timing of serum sample collection, aetiology of fever, associated illnesses that can modify cytokine profiles, and chronic medication of patients [ 26 ]. Hu et al. investigated the serum cytokine profiles of children with FS and severe acute encephalitis. IL-6, -10, -12p70, -17A, -2, -4, -5, -9, -13, -22, and − 1β, IF-γ, and TNF-α. IL-6 were measured. The study revealed a significant increase of IL-6 in the serum of the FS patients compared to those with severe acute encephalitis or encephalopathy, suggesting that interleukin-6 is activated predominantly during the acute stage of FS [ 27 ]. Some studies also analysed the IL-6 levels in cerebrospinal fluid (CSF). Virta et al. reported that CSF IL-6 levels were detectable in all 16 FS children [ 11 ]. They hypothesized that detectable levels of cytokines in CSF in FS patients could be a cause for seizure, or they could be produced because of seizure activity. However, we did not perform lumbar puncture to analyse the cytokine levels in CSF for ethical reasons. There are also studies that investigated the association between IL-6 gene polymorphisms and the risk of FS development [ 20 , 28 , 29 , 30 ]. Chen et al. performed a meta-analysis and found that IL-6 (− 572, − 174, −597) polymorphisms were significantly associated with susceptibility to FS [ 31 ]. The rules for analysing cytokine profiles and interpreting results of cytokine concentrations are still not entirely clarified. Cytokines are unstable molecules with a short biological half-life and different peak maxima concentrations. In the presented studies, a significant elevation of IL-6 has been repeatedly demonstrated in patients with FS. The second cytokine that is consistently demonstrated as a significant marker of febrile seizures in studies is IL-1RA. According to the work of Hautala et al., the cytokine IL1RA is strongly associated with FS, even when comparing its levels to those with febrile illness without seizures. [ 32 ]. Based on our results and earlier studies clinical attention should be paid to monitoring changes of IL-6 levels. In our study, we observed an increased level of TNF-alpha in patients with FS compared to the HC group. TNF-alpha, a pro-inflammatory cytokine primarily produced by activated macrophages, is particularly prominent in the acute phase, especially during the febrile stage of the disease [ 33 ]. The TNF-alpha level is elevated in various neuropathological processes, including ischemia, trauma, epilepsy, and degenerative brain diseases [ 34 ]. According to numerous original studies, it has been demonstrated that TNF-alpha is involved in the cytokine cascade in patients with FS, and genetic polymorphism also plays a role in its function [ 35 ]. In our study, we did not observe a significant difference in the concentration of this cytokine between the FS and FC groups of patients. According to the study by Tutuncuoglu et al.[ 14 ] an elevation of TNF-alpha in cerebrospinal fluid was demonstrated in patients with FS compared to the control group. However, according to our study, there is no significant difference in serum between the two aforementioned groups, as confirmed by several other studies [ 36 ]. These findings contrast with certain other studies that reported a statistical difference in TNF-alpha concentration between patients with FS and FC groups [ 37 ]. Given that our original study did not reveal a significant difference in TNF-alpha levels between the FS and FC groups, we hypothesize that the elevation of TNF-alpha plays a significant role in the pathophysiology of seizures. This assumption is also supported by the fact that TNF-alpha freely enters the central nervous system along nerve roots and secondarily increases levels of pro-inflammatory cytokines in the central nervous system [ 38 , 41 ]. Another significant finding in our study is the statistically significant elevation of CCL2/MCP-1 in patients with febrile seizures compared to HC. The level of the cytokine CCL2/MCP-1 peaks in the serum approximately 12 hours after febrile seizures and decreases within 48 hours. IL-10 and IL-1RA, as anti-inflammatory cytokines, exhibit a different dynamic in their value reduction. It is known that CCL2/MCP-1 is a key mediator in the molecular pathway that links peripheral inflammatory processes to neuronal hyperexcitability [ 39 ]. Several studies have demonstrated an association between the elevation of the CCL2/MCP-1 cytokine and pharmacoresistant epilepsy [ 12 , 13 ]. Elevated levels of CCL2/MCP-1 may indicate pharmacoresistance in patients with epilepsy [ 40 ]. A clear association between the elevation of CCL2/MCP-1 in the serum of patients with febrile seizures has not been published, making this result significant and important. According to our findings, we demonstrated a significant up-regulation of IL-10 in serum samples from patients with febrile seizures compared to afebrile controls. IL-10 belongs to anti-inflammatory cytokines, produced by macrophages, lymphocytes, and microglia [ 42 ]. The exact mechanism of IL-10 involvement in the pathogenesis of febrile seizures is still not entirely clear. Animal models have demonstrated that after the injection of recombinant human IL-10, a higher temperature threshold for the development of febrile seizures was observed [ 43 ]. Our results align with the findings of Korean authors, who demonstrated a significant elevation of IL-10 in febrile seizures as a compensatory factor with anti-inflammatory and anticonvulsive effects. According to the cited study [ 19 ] IL-10, along with another anti-inflammatory factor IL1Ra, could potentially represent a therapeutic target in the early stages of epileptogenesis. Conclusion In our study we identified a clear difference in IL-6 concentrations in patients with FS compared to the group with febrile illness without convulsions. We supposed that IL-6 could become a potential predictive biomarker of the development of FS in children with febrile disease. The ability to predict the occurrence of FS in children with fever would find its great application in clinical practice. Simultaneously, we demonstrate the significant elevation of IL-10, TNFα and CCL2/MCP-1 in patients with FS compared to HC. Most importantly, elevation of CCL2/MCP-1 has been described only in patients with epilepsy as potential biomarkers of epileptogenesis and also pharmacoresistance in epilepsy [ 12 , 13 , 40 ]. It is known that release of pro-inflammatory molecules and the activation of with inflammatory cascades has the potential to modulate brain excitability in terms of hyperexcitability. The neuromodulatory abilities of some pro-inflammatory molecules (cytokines, chemokines) binding to their specific receptors on the surface of neurons directly affect their excitability. We believe that the elevation of these pro-inflammatory cytokines in FS lead to hyperexcitability of the immature child's brain in response to fever. Abbreviations FS: febrile seizures FC: febrile controls HC: healthy controls IL-6: interleukin 6 TNFα: tumour necrosis factor IL-10: interleukin 10 CCL2/MCP-1: chemokine ligand 2/monocyte chemoattractant protein 1 IL-1β: ineterleukin 1βeta IL-8: interleukin 10 IL-18: interleukin 18 CXCL10/IP-10: C-X-C motif chemokine ligand 10/Interferon gamma-induced protein 1 CXCL13/BLC: Chemokine (C-X-C motif) ligand 13/B lymphocyte chemoattractant CXC3CL1: C-X3-C ligand 1 protein Declarations Acknowledgements The authors would like to thank all children who participated in this study. Authors’ contributions JP and KČ collected the data and prepared 1st draft of the manuscript. TJ, JŠ a MV performed the measurements, analyzed and interpreted the data. PJ reviewed and revisited the manuscript, ŠA supervised the project and its conceptualization and critically reviewed the manuscript. All authors read and approved the final manuscript. The datasets used and analysed during the current study available from the corresponding author on reasonable request. Funding This work was supported by the Ministry of Health, Czech Republic, Conceptual Development of Research Organization (FNBr, 65269705) and by the Ministry of Health of the Czech Republic by the Czech Health Research Council (Project No. NU21-04-00305) Ethics approval and consent to participate All legal representatives of the patients have given their informed consent in this study, which has been approved by the Ethics Committee of the University Hospital Brno and the Faculty of Medicine, Masaryk University Brno. 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Additional Declarations No competing interests reported. Supplementary Files Table1.xlsx Table. Demographic characteristics of subjects Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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University","correspondingAuthor":false,"prefix":"","firstName":"Katarína","middleName":"","lastName":"Česká","suffix":""},{"id":276188394,"identity":"0460b85d-2ca0-4f88-89e9-6c268ca6404c","order_by":2,"name":"Tomáš Loja","email":"","orcid":"","institution":"Central European Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Tomáš","middleName":"","lastName":"Loja","suffix":""},{"id":276188395,"identity":"78e70e05-7cf5-4a7d-9a5e-a8fd1d3c80cf","order_by":3,"name":"Jiří Šána","email":"","orcid":"","institution":"Central European Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Jiří","middleName":"","lastName":"Šána","suffix":""},{"id":276188396,"identity":"4b6f88d4-b78c-42c0-9890-8eec0af52507","order_by":4,"name":"Marek Večeřa","email":"","orcid":"","institution":"Central European Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Marek","middleName":"","lastName":"Večeřa","suffix":""},{"id":276188397,"identity":"9db6ecc1-115a-4b9a-9843-6706f4fa92d9","order_by":5,"name":"Petr Jabandžiev","email":"","orcid":"","institution":"University Hospital, Masaryk University","correspondingAuthor":false,"prefix":"","firstName":"Petr","middleName":"","lastName":"Jabandžiev","suffix":""},{"id":276188398,"identity":"b0556895-21c4-4af4-8752-81a3577c4586","order_by":6,"name":"Stefania Aulicka","email":"","orcid":"","institution":"University Hospital, Masaryk University","correspondingAuthor":false,"prefix":"","firstName":"Stefania","middleName":"","lastName":"Aulicka","suffix":""},{"id":276188399,"identity":"06508386-c0be-4dbb-bbf6-3b9256d05a44","order_by":7,"name":"Štefania Aulická","email":"data:image/png;base64,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","orcid":"","institution":"University Hospital, Masaryk University","correspondingAuthor":true,"prefix":"","firstName":"Štefania","middleName":"","lastName":"Aulická","suffix":""}],"badges":[],"createdAt":"2024-02-17 12:45:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3964084/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3964084/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52034589,"identity":"d109a74d-5c96-4d6a-a7d5-51be701657f3","added_by":"auto","created_at":"2024-03-05 16:50:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":257588,"visible":true,"origin":"","legend":"\u003cp\u003eIL-6 serum level\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3964084/v1/f1185122066aa723fd6b91b0.png"},{"id":52033766,"identity":"d89c8b0d-f394-46cc-9031-493c3b037b3e","added_by":"auto","created_at":"2024-03-05 16:42:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":226311,"visible":true,"origin":"","legend":"\u003cp\u003eIL-10 serum level\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3964084/v1/16577ee796a1c1785f9cba08.png"},{"id":52033769,"identity":"05d48ef3-ab88-4672-b1a4-02b25db3575f","added_by":"auto","created_at":"2024-03-05 16:42:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":274778,"visible":true,"origin":"","legend":"\u003cp\u003eTNFα serum level\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3964084/v1/311163e7d7b4f16f0b9c8a40.png"},{"id":52034590,"identity":"596d88fd-e29d-4d6a-b78d-04c15986f22b","added_by":"auto","created_at":"2024-03-05 16:50:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":264185,"visible":true,"origin":"","legend":"\u003cp\u003eMCP-1 serum level\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3964084/v1/a81f9f4c5e06eb57f00b6120.png"},{"id":59934300,"identity":"68d6d888-2365-4777-a542-eb2bf48a1766","added_by":"auto","created_at":"2024-07-09 13:29:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1515891,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3964084/v1/df4f95a0-5084-46ab-857c-c97eb77c5d36.pdf"},{"id":52033767,"identity":"575ae9f4-cc2a-41cf-a321-562152b6fe19","added_by":"auto","created_at":"2024-03-05 16:42:41","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10351,"visible":true,"origin":"","legend":"\u003cp\u003eTable. Demographic characteristics of subjects\u003c/p\u003e","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3964084/v1/83e79bc935f42f2a10a85824.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"IL-6 as potential predictive biomarker of febrile seizures","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFebrile seizures (FS) are the most common convulsions of childhood, and usually occur in children between the ages of six months and five years [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These children do not have any evidence of central nervous infection, electrolyte imbalance, metabolic disorders, or a history of febrile seizures[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. FS are categorized into three types: simple, complex, and febrile status epilepticus. Simple FS are generalized, last for \u0026lt;\u0026thinsp;15 minutes, and do not recur within 24 hours. Complex FS are more prolonged (\u0026gt;\u0026thinsp;15 minutes), focal, and recur within 24 hours. Febrile status epilepticus is the most severe type of FS, lasting for \u0026gt;\u0026thinsp;30 minutes [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. While children with simple FS have an only slightly higher risk of subsequent epilepsy of around 1% compared to the general population (0.5%), the incidence of epilepsy observed in patients after complex FS is 4\u0026ndash;6% [45].\u003c/p\u003e \u003cp\u003eThe exact pathophysiology of FS remains unknown. FS are generally believed to result from a vulnerability of the developing brain to the fever, in combination with an underlying genetic predisposition and environmental factors [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCytokines are a diverse family of intercellular signalling molecules that regulate inflammation and immune responses [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. As a natural consequence of immune system activation during infection, both pro-inflammatory and anti-inflammatory cytokines are produced and released [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Subsequent fever leads to an increased neuronal excitability resulting in convulsions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Cytokines include interleukins, chemokines, tumour necrosis factors, interferons, etc. Many studies investigating the role of cytokines in the pathogenesis of FS contributed inconsistent results. Moreover, most of these were focused on three major cytokines, e.g. IL-1β, IL-6 and TNFα [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Due to these conflicting results, we decided to explore the association of ten various pro-inflammatory and anti-inflammatory cytokines in the genesis of FS.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSubjects\u003c/h2\u003e \u003cp\u003eThis prospective case-control study was carried out at Children\u0026acute;s Hospital Brno affiliated with the Faculty of Medicine of Masaryk University in Brno, Czech Republic in the period from July 2021 to February 2022. All patients included in this prospective study with a case group of 40 were selected from children admitted to the hospital after an attack of simple FS.\u003c/p\u003e \u003cp\u003eA total of 24 patients with febrile seizures were included in the study, with an average age of 22.54\u0026thinsp;\u0026plusmn;\u0026thinsp;10.18 months. The group consisted of 13 male patients and 11 female patients.\u003c/p\u003e \u003cp\u003eInclusion criteria for the febrile seizure group were fever\u0026thinsp;\u0026ge;\u0026thinsp;38\u0026deg;C and the presence of either simple febrile seizures or complex febrile seizures. Patients with evidence of central nervous system infection, metabolic disorder, electrolyte imbalance, neurological deficits, and a history of febrile seizures were excluded from the study.\u003c/p\u003e \u003cp\u003eTwo additional groups of patients were defined in the project. The first group comprised patients with febrile illness but without febrile seizures. Diagnoses included in this group were nonspecific viral respiratory tract infections, viral bronchitis, laryngitis, and gastritis. There were eight subjects in this group, with an average age of 17\u0026thinsp;\u0026plusmn;\u0026thinsp;13.47 months, consisting of three male and five female patients.\u003c/p\u003e \u003cp\u003eThe second group consisted of subjects without febrile illness. This group included a total of eight patients with an average age of 21.75\u0026thinsp;\u0026plusmn;\u0026thinsp;13.04 months (five males and three females). Exclusion criteria were a history of neurological disease in the participant, febrile infection, or autoimmune disease.\u003c/p\u003e \u003cp\u003eDetailed characteristics of the subjects are provided in Table.\u003c/p\u003e \u003cp\u003eTable. Demographic characteristics of subjects\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEthics\u003c/h2\u003e \u003cp\u003e All legal representatives of the patients have given their informed consent in this study, which has been approved by the Ethics Committee of the University Hospital Brno and the Faculty of Medicine, Masaryk University Brno.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThree millilitres of blood were taken from the peripheral vessels of participants in all the groups, and serum was obtained by centrifugation at 3,000 rpm for five minutes at 4 ̊C. The serum was then poured into acid-washed tubes and stored in a refrigerator at -20 ̊C. In the febrile seizure groups, blood samples were collected within two hours following the occurrence of a seizure episode. For quantitative detection of plasma levels of cytokines and chemokines, Luminex technology (multi-analyte profiling magnetic beads) was used to enable the detection and quantification of multiple protein targets simultaneously. We used a custom ProcartaPlex\u0026trade; 9-Plex immunoassay panel (IL-6, IL-8, IL-10, IL-18, CXCL10/IP-10, CCL2/MCP-1, CXCL13/BLC, TNFα and fractalkine/CXC3CL1), the ProcartaPlex\u0026trade; high sensitivity IL-1β simplex kit. All kits were used according to the manufacturer\u0026rsquo;s instructions (Affymetrix eBioscience, Hatfield, UK). Flow cytometry analysis was performed using BD FACS Verse (BD Biosciences, San Jose, CA, USA). Each plasma sample was tested in triplicate and the results were calculated according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eBetween-group statistics (i.e. samples of patients with febrile seizures vs. febrile controls \u0026amp; healthy controls) were calculated using the Mann-Whitney nonparametric test, significance level set at alpha\u0026thinsp;=\u0026thinsp;0.05. P-values were corrected for multiple comparison with the Bonferroni correction and p-values of \u0026lt;\u0026thinsp;0.05 were considered statistically significant. Between-grade comparison was performed using the Kruskal-Wallis nonparametric test. To strengthen the results, Mann-Whitney non-parametric tests were performed between all grades for all the analysed molecules.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eLuminex technology (multi-analyte profiling magnetic beads) was used to enable the detection and quantification of multiple protein targets simultaneously (plasma levels of cytokine and chemokine) in samples of patients with FS versus the group with febrile infection without seizures and the control afebrile group. We compared a total of ten cytokines and chemokines in samples from patients. The analysis of serum samples revealed a significant elevation of \u003cb\u003eIL-6\u003c/b\u003e (p\u0026thinsp;=\u0026thinsp;0.0042) in the FS group compared to FC (see Fig.\u0026nbsp;1 here). Significantly higher levels of cytokines were also found in the FS group compared to HC in \u003cb\u003eIL-10\u003c/b\u003e (p\u0026thinsp;=\u0026thinsp;0.0039), \u003cb\u003eTNFα\u003c/b\u003e (p\u0026thinsp;=\u0026thinsp;0.0091) and \u003cb\u003eMCP-1\u003c/b\u003e (p\u0026thinsp;=\u0026thinsp;0.0039) (Fig.\u0026nbsp;2\u0026ndash;4 here).\u003c/p\u003e \u003cp\u003eFigure 1. IL-6 serum level\u003c/p\u003e \u003cp\u003eFigure 2. IL-10 serum level\u003c/p\u003e \u003cp\u003eFigure 3. TNFα serum level\u003c/p\u003e \u003cp\u003eFigure 4. MCP-1 serum level\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study we examined a total of ten cytokines, including pro-inflammatory and anti-inflammatory cytokines. The multiplex cytokine analysis revealed significantly elevated serum levels of IL-6 in the FS group compared to the FC group. No statistical difference was found between the FS group and the HC group, nor between the FC group and the HC group. On the other hand, we found significantly higher levels of cytokines IL-10, TNFα and MCP-1 in the FS group compared to the HC group (see results and Fig.\u0026nbsp;1\u0026ndash;4 here).\u003c/p\u003e \u003cp\u003eIL-6 is a pleiotropic, pro-inflammatory cytokine that is secreted by activated T-lymphocytes, macrophages, endothelial cells, and epithelial cells. It has a broad range of biological activities in immune regulation, haematopoiesis, inflammation and has a strong correlation with fever, and it can promote other cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The function of IL-6 in the body is variable, but according to many studies, its proactive role in exacerbating seizures by stimulating the preoptic area in the hypothalamus has been confirmed [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The concentration of IL-6 is mainly regulated at the level of expression, because IL-6 has a short half-life (20\u0026ndash;60 minutes) and is rapidly cleared from the plasma [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Several studies have been performed to explore the role of IL-6 in FS.\u003c/p\u003e \u003cp\u003eSimilarly to our study, Kim et al. reported that serum IL-6 was significantly higher in children with FS than in FC group [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Chen et al. also reported that the level of IL-6 was higher in the FS group compared to the FC group and the difference was statistically significant [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Azab et al. also found that children with FS had significantly higher serum IL-6 levels than the FC group. Moreover, they found significant elevation of serum IL-6 in the FS group compared to the HC group [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. A study by Kumar et al. also found higher levels of IL-6 in FS patients than in the FC group [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, the difference did not reach statistical significance. Similarly to a previous study, two other studies reported that serum levels of IL-6 in FS were elevated compared to FC but the difference was not statistically significant [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Meta-analysis by Kwon et al. focused on the role of serum IL-1β, IL-6, TNFα in FS concluded that only IL-6 levels in blood serum were significantly associated with FS [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. According to the work of Indian authors, a correlation between IL-6 concentration and the severity of febrile seizures was even demonstrated. Specifically, in patients with complex febrile seizures, the IL-6 concentration was significantly higher than in subjects with simple febrile seizures and controls. The findings of this study are supported by the results of Azab et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eContrary to our results, Gupta et al. reported that serum IL-6 levels were significantly lower in children with FS as compared to FC [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The researchers hypothesized that one of the reasons for low serum IL-6 levels in children with febrile seizures could be the possibility of consumption of cytokines in the genesis of febrile seizures.\u003c/p\u003e \u003cp\u003eSome inconsistency in the results between certain studies may be attributed to the age of patients, gender, timing of serum sample collection, aetiology of fever, associated illnesses that can modify cytokine profiles, and chronic medication of patients [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHu et al. investigated the serum cytokine profiles of children with FS and severe acute encephalitis. IL-6, -10, -12p70, -17A, -2, -4, -5, -9, -13, -22, and \u0026minus;\u0026thinsp;1β, IF-γ, and TNF-α. IL-6 were measured. The study revealed a significant increase of IL-6 in the serum of the FS patients compared to those with severe acute encephalitis or encephalopathy, suggesting that interleukin-6 is activated predominantly during the acute stage of FS [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSome studies also analysed the IL-6 levels in cerebrospinal fluid (CSF). Virta et al. reported that CSF IL-6 levels were detectable in all 16 FS children [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. They hypothesized that detectable levels of cytokines in CSF in FS patients could be a cause for seizure, or they could be produced because of seizure activity. However, we did not perform lumbar puncture to analyse the cytokine levels in CSF for ethical reasons.\u003c/p\u003e \u003cp\u003eThere are also studies that investigated the association between IL-6 gene polymorphisms and the risk of FS development [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Chen et al. performed a meta-analysis and found that IL-6 (\u0026minus;\u0026thinsp;572, \u0026minus;\u0026thinsp;174, \u0026minus;597) polymorphisms were significantly associated with susceptibility to FS [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe rules for analysing cytokine profiles and interpreting results of cytokine concentrations are still not entirely clarified. Cytokines are unstable molecules with a short biological half-life and different peak maxima concentrations. In the presented studies, a significant elevation of IL-6 has been repeatedly demonstrated in patients with FS. The second cytokine that is consistently demonstrated as a significant marker of febrile seizures in studies is IL-1RA. According to the work of Hautala et al., the cytokine IL1RA is strongly associated with FS, even when comparing its levels to those with febrile illness without seizures. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Based on our results and earlier studies clinical attention should be paid to monitoring changes of IL-6 levels.\u003c/p\u003e \u003cp\u003eIn our study, we observed an increased level of TNF-alpha in patients with FS compared to the HC group. TNF-alpha, a pro-inflammatory cytokine primarily produced by activated macrophages, is particularly prominent in the acute phase, especially during the febrile stage of the disease [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The TNF-alpha level is elevated in various neuropathological processes, including ischemia, trauma, epilepsy, and degenerative brain diseases [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. According to numerous original studies, it has been demonstrated that TNF-alpha is involved in the cytokine cascade in patients with FS, and genetic polymorphism also plays a role in its function [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our study, we did not observe a significant difference in the concentration of this cytokine between the FS and FC groups of patients. According to the study by Tutuncuoglu et al.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] an elevation of TNF-alpha in cerebrospinal fluid was demonstrated in patients with FS compared to the control group. However, according to our study, there is no significant difference in serum between the two aforementioned groups, as confirmed by several other studies [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese findings contrast with certain other studies that reported a statistical difference in TNF-alpha concentration between patients with FS and FC groups [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven that our original study did not reveal a significant difference in TNF-alpha levels between the FS and FC groups, we hypothesize that the elevation of TNF-alpha plays a significant role in the pathophysiology of seizures. This assumption is also supported by the fact that TNF-alpha freely enters the central nervous system along nerve roots and secondarily increases levels of pro-inflammatory cytokines in the central nervous system [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother significant finding in our study is the statistically significant elevation of CCL2/MCP-1 in patients with febrile seizures compared to HC. The level of the cytokine CCL2/MCP-1 peaks in the serum approximately 12 hours after febrile seizures and decreases within 48 hours. IL-10 and IL-1RA, as anti-inflammatory cytokines, exhibit a different dynamic in their value reduction.\u003c/p\u003e \u003cp\u003eIt is known that CCL2/MCP-1 is a key mediator in the molecular pathway that links peripheral inflammatory processes to neuronal hyperexcitability [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Several studies have demonstrated an association between the elevation of the CCL2/MCP-1 cytokine and pharmacoresistant epilepsy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Elevated levels of CCL2/MCP-1 may indicate pharmacoresistance in patients with epilepsy [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. A clear association between the elevation of CCL2/MCP-1 in the serum of patients with febrile seizures has not been published, making this result significant and important.\u003c/p\u003e \u003cp\u003eAccording to our findings, we demonstrated a significant up-regulation of IL-10 in serum samples from patients with febrile seizures compared to afebrile controls. IL-10 belongs to anti-inflammatory cytokines, produced by macrophages, lymphocytes, and microglia [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe exact mechanism of IL-10 involvement in the pathogenesis of febrile seizures is still not entirely clear. Animal models have demonstrated that after the injection of recombinant human IL-10, a higher temperature threshold for the development of febrile seizures was observed\u003c/p\u003e \u003cp\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Our results align with the findings of Korean authors, who demonstrated a significant elevation of IL-10 in febrile seizures as a compensatory factor with anti-inflammatory and anticonvulsive effects. According to the cited study [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] IL-10, along with another anti-inflammatory factor IL1Ra, could potentially represent a therapeutic target in the early stages of epileptogenesis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn our study we identified a clear difference in IL-6 concentrations in patients with FS compared to the group with febrile illness without convulsions. We supposed that IL-6 could become a potential predictive biomarker of the development of FS in children with febrile disease. The ability to predict the occurrence of FS in children with fever would find its great application in clinical practice.\u003c/p\u003e \u003cp\u003eSimultaneously, we demonstrate the significant elevation of IL-10, TNFα and CCL2/MCP-1 in patients with FS compared to HC. Most importantly, elevation of CCL2/MCP-1 has been described only in patients with epilepsy as potential biomarkers of epileptogenesis and also pharmacoresistance in epilepsy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is known that release of pro-inflammatory molecules and the activation of\u003c/p\u003e \u003cp\u003ewith inflammatory cascades has the potential to modulate brain excitability in terms of hyperexcitability. The neuromodulatory abilities of some pro-inflammatory molecules (cytokines, chemokines) binding to their specific receptors on the surface of neurons directly affect their excitability.\u003c/p\u003e \u003cp\u003eWe believe that the elevation of these pro-inflammatory cytokines in FS lead to hyperexcitability of the immature child's brain in response to fever.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eFS: febrile seizures\u003c/p\u003e\n\u003cp\u003eFC: febrile controls\u003c/p\u003e\n\u003cp\u003eHC: healthy controls\u003c/p\u003e\n\u003cp\u003eIL-6: interleukin 6\u003c/p\u003e\n\u003cp\u003eTNF\u0026alpha;: tumour necrosis factor\u003c/p\u003e\n\u003cp\u003eIL-10: interleukin 10\u003c/p\u003e\n\u003cp\u003eCCL2/MCP-1:\u0026nbsp;chemokine ligand 2/monocyte chemoattractant protein 1\u003c/p\u003e\n\u003cp\u003eIL-1\u0026beta;: ineterleukin 1\u0026beta;eta\u003c/p\u003e\n\u003cp\u003eIL-8: interleukin 10\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIL-18: interleukin 18\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCXCL10/IP-10: C-X-C motif chemokine ligand 10/Interferon gamma-induced\u0026nbsp;protein 1\u003c/p\u003e\n\u003cp\u003eCXCL13/BLC: Chemokine (C-X-C motif) ligand 13/B lymphocyte chemoattractant\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCXC3CL1: C-X3-C ligand 1 protein\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank all children who participated in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJP and KČ collected the data and prepared 1st draft of the manuscript. TJ, J\u0026Scaron; a MV performed the measurements, analyzed and interpreted the data. PJ reviewed and revisited the manuscript, \u0026Scaron;A supervised the project and its conceptualization and critically reviewed the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the\u0026nbsp;Ministry of Health, Czech Republic, Conceptual Development of Research Organization\u0026nbsp;(FNBr,\u0026nbsp;65269705) and by the\u0026nbsp;Ministry of Health of the Czech Republic\u0026nbsp;by the\u0026nbsp;Czech Health Research Council\u0026nbsp;(Project No.\u0026nbsp;NU21-04-00305)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll legal representatives of the patients have given their informed consent in this study, which has been approved by the Ethics Committee of the University Hospital Brno and the Faculty of Medicine, Masaryk University Brno.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors affirm that there are no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLeung AK, Hon KL, Leung TN. 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PMID: 29017522; PMCID: PMC5635534.\u003c/li\u003e\n\u003cli\u003eAzab SF, Abdalhady MA, Almalky MA, Amin EK, Sarhan DT, Elhindawy EM, Allah MA, Elhewala AA, Salam MM, Hashem MI, Soliman AA, Akeel NE, Abdellatif SH, Elsamad NA, Rass AA, Arafat MS. Serum and CSF adiponectin, leptin, and interleukin 6 levels as adipocytokines in Egyptian children with febrile seizures: a cross-sectional study. Ital J Pediatr. 2016 Apr 12;42:38. doi: 10.1186/s13052-016-0250-y. PMID: 27068222; PMCID: PMC4828849.\u003c/li\u003e\n\u003cli\u003eKumar KJ, Kurvari G, Kumar HCK, Tejashree A, Manjunath VG. A Comparative Analysis of Serum Interleukin-6 Levels in Children with Febrile Seizures and Febrile Controls. J Neurosci Rural Pract. 2022;13(2):336-8.\u003c/li\u003e\n\u003cli\u003eChoi J, Min HJ, Shin JS. Increased levels of HMGB1 and pro-inflammatory cytokines in children with febrile seizures. 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Serum Interleukin-6 Levels in Children with Febrile Seizures. Indian Pediatr. 2018 May 15;55(5):411-413. Epub 2018 Feb 9. PMID: 29428914.\u003c/li\u003e\n\u003cli\u003eHu MH, Huang GS, Wu CT, Lin JJ, Hsia SH, Wang HS, et al. Analysis of plasma multiplex cytokines for children with febrile seizures and severe acute encephalitis. J Child Neurol. 2014;29(2):182-6.\u003c/li\u003e\n\u003cli\u003eChou IC, Lin WD, Wang CH, Tsai CH, Li TC, Tsai FJ. Interleukin (IL)-1beta, IL-1 receptor antagonist, IL-6, IL-8, IL-10, and tumor necrosis factor alpha gene polymorphisms in patients with febrile seizures. J Clin Lab Anal. 2010;24(3):154-9.\u003c/li\u003e\n\u003cli\u003eNur BG, Kahramaner Z, Duman O, Dundar NO, Sallakcı N, Yavuzer U, et al. Interleukin-6 gene polymorphism in febrile seizures. Pediatr Neurol. 2012;46(1):36-8.\u003c/li\u003e\n\u003cli\u003eShahrokhi A, Zare-Shahabadi A, Soltani S, Ashrafi MR, Zoghi S, Hosseini SA, et al. Association of IL6 single nucleotide polymorphisms with febrile seizures. J Neurol Sci. 2014;342(1-2):25-8.\u003c/li\u003e\n\u003cli\u003eChen Q, Li M, Zhang X, Zhong R, Lin W. Association between interleukin-6 gene polymorphisms and febrile seizure risk: A meta-analysis. Medicine (Baltimore). 2019;98(39):e17167.\u003c/li\u003e\n\u003cli\u003eHautala, M.K., Helander, H.M., Pokka, T.ML. et al. Recurrent febrile seizures and serum cytokines: a controlled follow-up study. Pediatr Res 93, 1574\u0026ndash;1581 (2023). https://doi.org/10.1038/s41390-022-02282-7.\u003c/li\u003e\n\u003cli\u003eWajant H, Siegmund D. TNFR1 and TNFR2 in the Control of the Life and Death Balance of Macrophages. Front Cell Dev Biol. 2019 May 29;7:91. doi: 10.3389/fcell.2019.00091.\u003c/li\u003e\n\u003cli\u003eBalosso S, Ravizza T, Perego C, Peschon J, Campbell IL, De Simoni MG, Vezzani A. Tumor necrosis factor-alpha inhibits seizures in mice via p75 receptors. Ann Neurol. 2005 Jun;57(6):804-12. doi: 10.1002/ana.20480. PMID: 15852477.\u003c/li\u003e\n\u003cli\u003eManiu I, Costea R, Maniu G, Neamtu BM. Inflammatory Biomarkers in Febrile Seizure: A Comprehensive Bibliometric, Review and Visualization Analysis. Brain Sci. 2021 Aug 17;11(8):1077. doi: 10.3390/brainsci11081077.\u003c/li\u003e\n\u003cli\u003eIchiyama T, Suenaga N, Kajimoto M, Tohyama J, Isumi H, Kubota M, Mori M, Furukawa S. Serum and CSF levels of cytokines in acute encephalopathy following prolonged febrile seizures. Brain Dev. 2008 Jan;30(1):47-52. doi: 10.1016/j.braindev.2007.05.008. Epub 2007 Jun 26. PMID: 17597322.\u003c/li\u003e\n\u003cli\u003eHa J, Choi J, Kwon A, Kim K, Kim SJ, Bae SH, Son JS, Kim SN, Kwak BO, Lee R. Interleukin-4 and tumor necrosis factor-alpha levels in children with febrile seizures. Seizure. 2018 May;58:156-162. doi: 10.1016/j.seizure.2018.04.004. Epub 2018 Apr 6. PMID: 29729582.\u003c/li\u003e\n\u003cli\u003eAsano T, Ichiki K, Koizumi S, et al. IL-8 in cerebrospinal fluid from children with acute encephalopathy is higher than in that from children with febrile seizure. Scand J Immunol. 2010 Jun;71(6):447-51.\u003c/li\u003e\n\u003cli\u003eCerri C, et al. The Chemokine CCL2 Mediates the Seizure-enhancing Effects of Systemic Inflammation. J Neurosci 2016 Mar 30; 36(13): 3777-88.\u003c/li\u003e\n\u003cli\u003eČesk\u0026aacute; K, Papež J, O\u0026scaron;lej\u0026scaron;kov\u0026aacute; H, Slab\u0026yacute; O, Radov\u0026aacute; L, Loja T, Lib\u0026aacute; Z, Svěr\u0026aacute;kov\u0026aacute; A, Br\u0026aacute;zdil M, Aulick\u0026aacute; \u0026Scaron;. CCL2/MCP-1, interleukin-8, and fractalkine/CXC3CL1: Potential biomarkers of epileptogenesis and pharmacoresistance in childhood epilepsy, European Journal of Paediatric Neurology, Volume 46, 2023, Pages 48-54, ISSN 1090-3798, https://doi.org/10.1016/j.ejpn.2023.06.001.\u003c/li\u003e\n\u003cli\u003eLabh R, Gupta R, Narang M, et al. Effect of valproate and add-on levetiracetam on inflammatory biomarkers in children with epilepsy. Epilepsy Behav 2021 Dec;125:108358.\u003c/li\u003e\n\u003cli\u003eWilliams K, Dooley N, Ulvestad E, Becher B, Antel JP. IL-10 production by adult human derived microglial cells. Neurochem Int. 1996 Jul;29(1):55-64. doi: 10.1016/0197-0186(95)00138-7. PMID: 8808789.\u003c/li\u003e\n\u003cli\u003eIshizaki Y, Kira R, Fukuda M, Torisu H, Sakai Y, Sanefuji M, Yukaya N, Hara T. Interleukin-10 is associated with resistance to febrile seizures: Genetic association and experimental animal studies. Epilepsia, 2009 Apr;50(4):761-7; doi: 10.1111/j.1528-1167.2008.01861.x. Epub 2008 Dec 4.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"febrile seizures, pathogenesis, cytokines, brain excitability","lastPublishedDoi":"10.21203/rs.3.rs-3964084/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3964084/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eFebrile seizures are the most common type of convulsions in children. Fever is induced by cytokines release during infection. Recent studies focusing on the identification of a possible role of cytokines in pathogenesis of febrile seizures have contributed conflicting results. Moreover, most of these studies investigated only a few cytokines, such as IL-1β, IL-6 and TNFα. The aim of this study was to investigate multiple cytokine-chemokine profiles that could be potentially associated with the development of febrile seizures.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTwenty-four febrile seizure cases (febrile seizure group) and two matched control groups were included in this study. Children with febrile illness without convulsion (febrile control group) and children without seizures and without fever (healthy control group) served as control groups. We investigated serum levels of IL-1β, IL-6, IL-8, IL-10, IL-18, CXCL10/IP-10, CCL2/MCP-1, CXCL13/BLC, TNFα, and fractalkine/CXC3CL1 in all children included in the study.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe analysis of serum samples revealed a significant elevation of IL-6 (p\u0026thinsp;=\u0026thinsp;0.0042) in the FS group compared to the febrile controls. Significantly higher levels of cytokines were also found in the FS group compared to healthy controls in IL-10 (p\u0026thinsp;=\u0026thinsp;0.0039), TNFα (p\u0026thinsp;=\u0026thinsp;0.0091) and MCP-1 (p\u0026thinsp;=\u0026thinsp;0.0039).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur study supports the hypothesis that IL-6 is involved in the pathogenesis of febrile seizures. We supposed that IL-6 could become a potential biomarker of the development of febrile seizures in children with febrile disease. This knowledge could be used in clinical practice to identify children at risk of developing of febrile convulsions.\u003c/p\u003e","manuscriptTitle":"IL-6 as potential predictive biomarker of febrile seizures","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-05 16:42:36","doi":"10.21203/rs.3.rs-3964084/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"187c8bb3-7789-4e3a-9d0f-1437290970b7","owner":[],"postedDate":"March 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-09T13:21:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-05 16:42:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3964084","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3964084","identity":"rs-3964084","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}