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Febrile temperature-regulated TRPV1 in CD4+ T cells mediates neuroinflammation in complex febrile seizures

Journal of Neuroinflammation volume 22, Article number: 103 (2025) Cite this article

Abstract

Background

Febrile seizures (FS) are the most prevalent convulsive disorder in children characterized by a high recurrence rate. However, the interaction between adaptive and innate immunity in the recurrence of FS remains poorly understood, and the molecular pathways involved are unclear. The objective of this study is to elucidate the role of Th17 cells in seizure susceptibility following complex febrile seizures (CFS), and to explore the regulatory mechanisms underlying Th17 cell differentiation and function under hyperthermic conditions through transient receptor potential vanilloid 1 (TRPV1).

Methods

RNA sequencing was employed to validate the seizure susceptibility following CFS and to explore the potential mechanisms by which high temperature contributes to Th17 cell differentiation. Neuronal excitability and damage were examined using Multi-electrode array (MEA) analysis and Nissl staining. Flow cytometry, chromatin immunoprecipitation (ChIP) analysis, and immunofluorescence (IF) were applied to examine how TRPV1 facilitates Th17 cell differentiation.

Results

Our study demonstrates that proinflammatory Th17 cells exhibit enhanced differentiation in a CFS mouse model and exacerbate blood–brain barrier (BBB) disruption. After infiltrating the central nervous system (CNS), Th17 cells promote neuroinflammation by activating microglia via IL-17A. Mechanistically, TRPV1 is critical for Th17 cell differentiation and function. Activated by febrile temperature both in vivo and in vitro, TRPV1 facilitates calcium ion influx, leading to the nuclear localization of nuclear factor of activated T cell 2 and 4 (NFAT2/4) and the phosphorylation of signal transducer and activator of transcription 3 (STAT3). Knockdown of TRPV1 attenuates Th17 cell differentiation and CNS infiltration, thereby protecting the BBB and reducing seizure susceptibility following CFS.

Conclusion

These results highlight the critical interplay between adaptive and innate immunity in CFS. The TRPV1/NFATs/STAT3 signaling pathway regulates Th17 cell differentiation and function under febrile conditions, revealing a promising therapeutic target for intervention.

Introduction

Febrile seizures (FSs) represent the most common convulsive disorder among infants and children aged 6 months to 5 years, with a global prevalence of approximately 2–5%[1–3]. FSs are classified as simple febrile seizures (SFS) and complex febrile seizures (CFS), with CFS characterized by prolonged seizures lasting at least 15 min or longer and/or recurrent within 24 h [4, 5]. Infants experiencing CFS face a heightened risk of developing temporal lobe epilepsy and hippocampal sclerosis later in life [6, 7]. Emerging evidence indicates that CFS is associated with distinct brain alterations that significantly differ from those in SFS patients [8]. These changes in CFS encompass synaptic remodeling, hippocampal structural damage, and memory impairment, suggesting a prolonged state of seizure susceptibility, often described as a “smoldering” phase following CFS [9–12]. Clinically, 33% of children experiencing their first febrile seizure are at risk of recurrent febrile seizures within one year [4]. Even after recovery from febrile seizures or during fever episodes without convulsions, children may remain in a seizure-prone state. However, the underlying immune response and mechanisms driving this heightened susceptibility remain poorly understood.

The period from birth to 5 years of age represents a critical stage of neurological development, during which their immune system is particularly vulnerable. This developmental stage is marked by a heightened susceptibility to FS, peaking at 2 years of age. Immune challenges and the resulting immune response, particularly neuroinflammation, are increasingly recognized as key contributors to the pathophysiology of seizure generation, seizure-related neuropathology, and epileptogenesis[13–15]. However, the interplay between adaptive and innate immunity during the recurrence of FSs, as well as the underlying molecular pathways remains unclear. Th17 cells, a subset of CD4+ T cells, are defined by their secretion of interleukin-17 (IL-17). In the healthy brain, Th17 cells are virtually absent [16–18]. They have been implicated in neurological disorders, including epilepsy[19, 20], multiple sclerosis (MS)[21–23], Alzheimer's disease (AD)[24, 25] and major depression disorder[26, 27]. Studies utilizing experimental autoimmune encephalomyelitis (EAE) animal model have shown that under pathological conditions where the blood–brain barrier (BBB) is compromised, peripherally differentiated Th17 cells can infiltrate the brain [28]. Importantly, febrile temperature (39.5 ℃) have been shown to selectively regulate the differentiation and pathogenicity of Th17 cells[29], suggesting the involvement ofTh17 cells in fever-induced febrile seizures.

A clinical study on 32 CFS patients has founded significantly higher levels of IL-17A in the cerebrospinal fluid, suggesting that Th17 cells may be present in the brain of CFS patients [30], thus may play a critical role in the onset and progression of CFS. Foreign immunogens and proinflammatory immune cells from the circulation can accumulate in the brain, potentially compromising the BBB and further activating central immune responses. Studies in EAE have suggested that IL-17A, IL-6 and other proinflammatory cytokines released from peripheral immune cells contribute to the BBB impairment and infiltrate the brain, where they activate the innate immune response in the CFS [31–33]. Once activated, microglia exhibit an enlarged soma and shorter branches, acting both as a target and a source of proinflammatory cytokines. Upon stimulation by pathological factors, microglia undergo rapid morphological and functional changes, thereby exacerbating neuroinflammation [34, 35]. Whether and how Th17 cells are involved in CFS, as well as the relationship between Th17 cell infiltration and microglia activation, warrants further investigation.

Transient receptor potential (TRP) channels, particularly TRPV1-4, have been identified as cellular thermosensors that can be activated by febrile temperatures [36–38]. As a critical regulator of body temperature, TRPV1 is expressed in many kinds of cells, including immune cells such as CD4+ T cells [39–41], CD8+ T cells [42] and macrophages [43–45]. Recent studies suggest that inhibition of TRPV1 and TRPV4 can block the febrile transition of Th2 cells [46]. However, the critical role of febrile temperature and TRPV1 in modulating Th17 cell differentiation and function especially in CFS remains unclear and further extensive inquiry is needed.

In the present study, the crosstalk between adaptive immunity and innate immunity was investigated in CFS mice model. Febrile temperatures modulate the directional differentiation of CD4+ T cells into Th17 cells, which subsequently contribute to blood–brain barrier disruption (BBBD) and promote infiltration into the brain, exacerbating neuroinflammation. This process depends on TRPV1 activity, as well as enhanced NFAT localization and STAT3 phosphorylation.

Materials and methods

Animals

C57BL/6 wild-type mice were approved by the Hubei Province Center for Animal Experiments and the 6.129X1-TRPV1 KO mice used in this study were provided by the Jackson Laboratory. IL-17A KO mice, whole-genome knockout for IL-17A in C57BL/6 mice, were purchased from Charles River Laboratories. All mice were housed in the SPF animal facility-Animal Biosafety Level III Laboratory (ABSL-III) at Wuhan University. All animal care and experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee of Wuhan University Medical School. Our study examined both male and female animals, and similar findings are reported for both sexes.

Mouse model of complex febrile seizures (CFS)

Female and male mice at 12 days of age (P12) were used in this study. Mice were randomized divided into Ctrl or CFS group, and group allocation during experiments was done blindly. The CFS model has been described previously[47, 48]. Mice were intraperitoneally injected with 0.9% saline (10 ml/kg) to prevent dehydration. Subsequently, pups were placed in a hyperthermia chamber set at 43.0 ± 0.5 ℃, where seizures were induced. Once hindlimb clonus (HLC) or even serious seizures appeared (Racine stage III or higher), animals were removed from the hyperthermic chamber for 2 minutes and then returned. This cycle was repeated every 2 hours, with mice re-exposed to the chamber for 30 minutes to induce FS. This procedure was repeated six times (FS×6) with 2-hour intervals to establish the CFS model. The behavioral characteristics of seizures were evaluated according to the Racine scale:Stage I, facial twitch; Stage II, head nodding; Stage III, unilateral forelimb clonus; Stage IV, bilateral forelimb clonus; Stage V, tonic–clonic seizure with running, jumping, and falling. Seizure latency was defined as the first time from the placement of pups in the hyperthermic chamber until the first onset of hindlimb clonus or generalized convulsions (seizure grade III, or IV or V) [49, 50]. The seizure grade corresponds to the highest level of seizure observed in the pups during the 30-min febrile seizure induction. Rectal temperatures of WT and TRPV1−/− mice were measured before and during hyperthermic exposure using a lubricated rectal thermistor probe (Huicheng Technology, China, #TH212). Basal body temperature was recorded before CFS induction. Following the onset of behavioral seizures (grade III or higher), rectal temperature was measured again. Body weight of WT and TRPV1−/− mice were assessed using a electronic balance (Sartrorius, Germany). No significant difference in basal body temperature or body weight were observed between the WT and TRPV1−/− mice at P12, consistent with previous report[51](data not shown).

Western blot analysis

Tissues or cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (Biosharp, China, #BL504A) supplemented with 1% phenylmethanesulfonyl fluoride (PMSF) (Beyotime Biotechnology, China, #P1005) and 1% phosphatase inhibitor cocktail (100 ×) (Beyotime Biotechnology, China, # P1049). Proteins were separated by SDS-PAGE sample loading buffer (Beyotime Biotechnology, China, # P0015N), transferred to nitrocellulose membrane. The membranes were blocked with 5% BSA, incubated in primary anti-body for 12 h and in secondary antibody for 2 h. The antibodies used in this study are listed in Table 1.. Original protein bands are shown in supplementary file 1.

Table 1. Antibodies applied in this study
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RNA expression analysis

Total RNA from cells or tissues was extracted using TRIzol reagent (Vazyme, China, #R401-01). Total RNA was then reversed-transcribed using HiScript II Reverse Transcriptase (Vazyme, China, #RL201-01). Quantitative real-time polymerase chain reaction (qPCR) analysis was performed using SYBR Green Real-Time PCR master mix kit (Vazyme, China, #Q141-02/03), following the manufacturer’s instructions. The primers used in this study are listed in Table 2. β-Actin was used as an internal control. Gene expression was analyzed with the 2−ΔΔCt relative quantification method.

Table 2 qPCR primer sequences
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Chromatin immunoprecipitation (ChIP) analysis

ChIP was performed as previously described[52]. Briefly, 37% formaldehyde was added to the sample to cross-link the target protein and the corresponding genomic DNA. Cross-linking was quenched with 125 mM glycine for 5 min. The sample was lysis with SDS buffer for 10 min and the chromatin was then sheared into DNA fragments of approximately 1 kb using sonication. The sample was incubated with antibody and protein A/G at 4 ℃ overnight. The antibodies used were as follow: IgG (1 μg per IP, SIGMA), NFAT2(1 μg per IP, SIGMA), NFAT4(1 μg per IP, SIGMA). Low Salt Immune Complex Wash Buffer, High Salt Immune Complex Wash Buffer, LiCl Immune Complex Wash Buffer and TE Buffer were used to wash the samples. The protein-DNA complex was eluted with Elution Buffer (1%SDS, 0.1 M NaHCO3) and incubated with 5 M NaCl at 65 ℃ for 4 h to remove the cross-linking between protein and genomic DNA. The sequences were verified by qPCR, and ChIP-qPCR primers were used in this study are shown in Table 3. The reagents used in ChIP analysis were obtained from ChIP Assay Kit (Biosharp, China, # P2078).

Table 3 ChIP qPCR primer sequences (STAT3)
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In vitro CD4+ T cells isolation and differentiation

CD4+ T cells were isolated using MojoSort™ Mouse CD4 Naïve T Cell Isolation Kit (BioLegend, China, # 480,039). Naïve CD4+ T cells were cultured in OptiVitro® T Cell Serum-Free medium (ExCell Bio) supplemented with 1 μg/ml αCD28 (BioLegend, China, #102,103) and differentiated in 6 or 12-well plates coated with 1 μg/ml αCD3 (BioLegend, China, #100,243). After 24 h, IL-6 (Absin, China, #abs04084), IL-1β (Absin, China, # abs04051) and IL-23 (Absin, China, # abs04583) were added to promote CD4+ T cell differentiation into Th17 cells, while IL-2 (BioLegend, China, #575,404) was included to support T cell survival.

Flow cytometry

For flow cytometry analysis, T cells were cultured in Cell Activation Cocktail (BioLegend, China, #423,303) for 6 h to inhibit the release of IL-17A. Flow cytometry was performed as previously described [29]. Briefly, T cells were re-suspended in PBS, then stained with fixable live/dead cell dye (BioLegend, China, #423,113) and surface markers: CD3 (BD Pharmingen, USA, #553,061) and CD4 (BD Pharmingen, USA, #553,051), followed by fixation using the Fixation Buffer (BioLegend, China, #420,801) for intracellular staining of IL-17A (BD Pharmingen, USA, #559,502). αCD16/CD32 (BioXCell, USA. #CP026-1MG) was used to block nonspecific binding of antibodies to T cells obtained from in vivo models before staining. Flow cytometry data were analyzed by CyExpert and FlowJo software.

Immunocytochemistry (ICC)

T cells were fixed with 4% PFA and then re-suspended in PBS of 1 million cells/mL. 200 μL of each cell suspension was added to a slide chamber coated with 100 μg/mL Poly-L-lysine (PLL). The slide chamber was placed in a vacuum oven at 37 ℃ until the PBS evaporated, then were permeabilized with 0.01% Triton X-100, blocked with 10% goat serum, and incubated with the primary antibody overnight. After washing the slide chambers with PBS, cells were incubated with fluorescent secondary antibody for 1 h at room temperature. Finally, the nucleus was stained with DAPI. Images were obtained using a confocal laser scanning microscope (Leica-LCS-SP8-STED, Leica, Wetzlar, Hessen, Germany) and analyzed with ImageJ.

Immunofluorescence histochemistry

Mouse brain tissue was paraffin-embedded and sectioned into 8 ~ 10 μm slices. The sections were dewaxed by xylene and dehydrated using a gradient of alcohol. After blocking with 10% goat serum for 2 h and incubated with primary antibody overnight. On the second day, washed with PBS, the sections were incubated with the secondary antibody for 2 h, followed by DAPI staining. Immunofluorescence images were captured using a confocal laser scanning microscope.

Calcium detection in living cells

T cells were washed 3 times with PBS and incubated with 2 μM Fluo4 AM (Beyotime Biotechnology, China, #S1060) in a cell incubator for 30 min followed by washing with PBS. Images were obtained under laser excitation at 488 nm using a confocal laser scanning microscope.

RNA-seq

The brain, after CFS, was collected for RNA-seq analysis. The RNA-seq library was constructed and sequenced with Illumina platform (insert size 300 bp, read length 125 bp) by Novogene Bioinformatics Technology Co., Ltd. CD4+ T cells cultured at 37 ℃ or 39.5 ℃ for 3 days were collected, and the total RNA was extracted with TRIzol. The RNA-seq library was then constructed and sequenced on the Illumina Nova Seq 6000 platform by DIATRE Biotech. Detailed data are provided in supplementary material 1 and 2.

Enzyme‑linked immunosorbent assay (ELISA)

The concentration of IL-17A in serum or T cell culture medium was quantified using an ELISA kit (4A biotech, China, #CME0041-096) according to the manufacturer’s instruction.

Scanning electron microscope (SEM)

Use a sharp blade to cut and harvest fresh brain cortex tissue blocks with the fixative (Solarbio, China, #P1127) quickly within 1–3 min. The size of mouse cortex blocks is about 1 mm3. The tissues are fixed at 4 ℃ for preservation and transportation. Using 0.1 M PB (pH = 7.4) to wash the tissues 3 times for 15 min each. SEM analysis was performed by Servicebio Co., Ltd.

Multi-electrode arrays (MEA)

The brain tissue was rapidly removed and placed in an ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM): 140 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose (pH 7.4 adjusted with NaOH). The brain tissue was then sliced into 300 μm thick and incubated in the same ACSF at room temperature until ready for the experiment. The baseline was recorded for 5–10 min, and then the brain slices were perfused by ACSF mixed with 5 mM 4-AP. The data were obtained and analyzed using MEA Master Pro (Axion BioSystems).

Statistical analysis

GraphPad Prism v9.00 (GraphPad, La Jolla, CA, USA) was used to analyze the data and construct the graphs and all data are presented as the mean ± standard error of mean. Student’s t-test and one-way analysis of variance (ANOVA) were used to determine significant differences between groups. Student’s t-test was used for statistical analysis of two groups, while one-way ANOVA was used for multiple groups. The differences in seizure susceptibility between the CFS group, in terms of seizure latency and severity, were assessed using the Kolmogorov–Smirnov test and repeated-measures one-way ANOVA. Analysis of group differences between WT + CFS and TRPV1−/− + CFS group in seizure latency and severity was performed using two-way analysis of variance (ANOVA) for repeated measures. For animal experiments, the n values represent the number of individual animals in each group. For in vitro studies, the n values refer to the number of independent replicates of the experiment in cultures derived from different mice. P < 0.05 indicated that the difference was statistically significant.

Results

Th17 cells are critical for seizure susceptibility following CFS

This study aims to investigate the mechanisms underlying increased seizure susceptibility following CFS. First, we examined alterations in neuronal excitability in murine models after CFS. CFS was induced following an established protocol [47], and 24 h later, the pups were subjected to hyperthermic conditions (43.0 ± 0.5 ℃) again to induce febrile seizures. Seizure susceptibility was then evaluated. Behavioral observations revealed an exacerbation of seizures post-CFS, as indicated by heightened seizure severity (p = 0.0005) (Fig. 1A). Subsequently, mouse brain tissues were isolated for RNA sequencing. Gene Ontology (GO) analysis revealed the enrichment of numerous pathways associated with neuronal excitability and molecular metabolism/transport, including glutamate/organic acid transport and secretion, suggesting potential hyperexcitability in the mouse brain post-CFS (Fig. 1B). To replicate the in vivo findings, mouse brain tissue was sectioned, and electrical activity was monitored using the multi-electrode array (MEA) recordings (Fig. 1C). For the assessment of epilepsy susceptibility, 4-aminopyridine (4-AP) was used to induce epileptic-like discharges in mouse brain slices following a stabilization period of 5–10 min. MEA data showed that 4-AP-induced aberrant discharges were profoundly elevated following CFS (p = 0.0067). These findings suggest that CFS murine models exhibited enhanced seizure susceptibility.

Fig. 1
figure 1

Mouse models exhibit increased susceptibility to epilepsy following CFS. A Schematic diagram of CFS model induction and the process for evaluating seizure susceptibility (top); seizure latency (middle) and seizure severity (bottom) (n = 5, Data are presented as mean ± SEM, Kolmogorov–Smirnov test). B Gene Ontology (GO) pathway analysis results comparing Ctrl group and CFS group (Ctrl n = 6, CFS n = 6). C Representative MEA voltage recordings from a mouse cortical slice exhibiting spontaneous action potentials firing, shown as spikes on the trace in artificial cerebrospinal fluid (ACSF)(left); the presence of 4-AP (5 mM) (right); the time calibration line in the Raster plot is 20 s. A representative trace recorded by MEA electrode, when the perfusion with 5 mM 4-AP, epileptiform activity was evoked. Top row shows voltage recording from Ctrl group and bottom row is from CFS group. The number of action potentials recorded by all electrodes within 0.1 s from Ctrl group (n = 7) or CFS group (n = 7). Data are presented as mean ± SEM. Student’s t test

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Inflammatory processes have been implicated in the pathophysiology and seizure susceptibility of epilepsy including FS [53, 54]. Recent research indicates that febrile temperatures can promote the differentiation of Th17 cells and enhance their pathogenicity [29]. Therefore, Th17 cells may play a critical role in the seizure susceptibility following CFS. To investigate this hypothesis, an immunofluorescence assay was performed to determine whether Th17 cells infiltrated the brain after CFS. The results revealed the presence of Th17 cells in the brains of the CFS group, whereas Th17 cells were nearly undetectable in the Ctrl group (p < 0.0001) (Fig. 2A). Considering the possibility of increased peripheral differentiation of Th17 cells infiltrating the brain, FCM was subsequently employed to quantify Th17 cells in the spleen and peripheral blood. The FCM data indicated a notable rise in the proportion of Th17 cells in both the spleen (p = 0.0109) and peripheral blood (p = 0.0014) of the CFS group (Fig. 2B). ELISA analysis also indicated a significant increase in IL-17A in the serum of the CFS group (p = 0.0031) (Fig. 2C). Besides Th17 cells, CD4+ T cells can differentiate into Th1/2 cells and Treg cells. The qPCR analysis was performed to detect the expression of specific transcription factors for Th1 cells (T-bet), Th2 cells (GATA3) and Treg cells (Foxp3) in the spleen of CFS mice. Surprisingly, the qPCR results revealed that the mRNA levels of T-bet, GATA3 and Foxp3 were similar in both control and CFS groups (Fig. 2D). In contrast, there was a significantly increase in key transcription factors for Th17 cells in the CFS group, namely RORγt (p = 0.0006) and STAT3 (p = 0.0006) accompanied by an upregulation of Th17 cell-specific cytokines: GM-CSF (p = 0.0167), IL-17A (p = 0.0203) and IL-17F (p = 0.0015), as well as their corresponding receptors: IL-1βR (p = 0.0013), IL-23R (p = 0.0005) and TGFβR (p = 0.0170). Additionally, one of the anti-inflammatory cytokines: IL-10 (p = 0.0108) was downregulated. The above findings substantiate a selective role of CFS-induced fever in vivo in promoting Th17 cell differentiation. More importantly, peripherally differentiated Th17 cells were observed to infiltrate the brain, suggesting their potential involvement in the pathogenesis of CFS.

Fig. 2
figure 2

Th17 cells involvement in seizure susceptibility following CFS. A Representative confocal images showing CD4+IL-17A+ cells (CD4: green IL-17A: red) in cortical from the mice of Ctrl or CFS group and the total number of CD4+IL-17A+ cells (Ctrl group: n = 6 CFS group: n = 7, Data are presented as mean ± SEM, Student’s t test). B Flow cytometry analysis was used to measure the counts of Th17 cells. left: Th17 cell from blood of Ctrl or CFS group. right: Th17 cell from spleen of Ctrl or CFS group (Ctrl group: n = 4 CFS group: n = 5, Data are presented as mean ± SEM, Student’s t test). C IL-17A in the serum of Ctrl group (n = 4) and CFS group (n = 5) measured by ELISA. Data are presented as mean ± SEM, Student’s t test. D The qPCR analysis showing mRNA levels of key transcription factor genes of Th1(T-bet), Th2(GATA3), Treg (FOXP3) and Th17(STAT3/RORγt); Th17 key cytokine genes, GM-CSF, IL-17A, IL-17F and IL-10; Th17 key receptor genes, IL-1βR and IL-23R (n = 4 for each group, Data are presented as mean ± SEM, Student’s t test)

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TRPV1 is upregulated on CD4+ T cells after hyperthermia

Transient receptor potential (TRP) channels, specifically TRPV1-4, have been reported to be activated by febrile temperatures [38, 55, 56]. Inhibition of TRPV1 or TRPV4 can suppress the Th1/2 switch under moderate heat (39 ℃)[46]. However, the role of TRPV1-4 in regulating the differentiation of CD4+ T cells into Th17 cells, or in the pathogenicity of Th17 cells under hyperthermia remains unclear. To investigate whether the TRPV1-4 are involved in CD4+ T cells differentiation under hyperthermia, CD4+ T cells were isolated from the spleen and cultured at either 37 ℃ or 39.5 ℃ for 3 days. The expression of TRPV1-4 is shown in the Fig. 3A. The quantitative PCR (qPCR) results indicated significant upregulation of TRPV1 (p = 0.0022), TRPV3 (p = 0.004), and TRPV4 (p = 0.0008) in the 39.5 ℃-treated group, but not TRPV2 (p = 0.004) (Fig. 3B). RNA-seq analysis revealed that TRPV1 and TRPV3 were upregulated in the 39.5 ℃-treated group compared to the 37 ℃-treated group (Fig. 3C). Moreover, protein analysis verified a significant upregulation of TRPV1 (p = 0.0028) and TRPV4 (p = 0.0007) in the 39.5 ℃-treated group (Fig. 3D). Immunofluorescence assay demonstrated enhanced intensity of TRPV1 (yellow) (p = 0.0001) and TRPV4 (green) (p = 0.0002) in CD4+ T cells under 39.5 ℃ (Fig. 3E). Furthermore, qPCR assay detected elevated mRNA levels of TRPV1-4 in the spleen of CFS mice model. Specifically, TRPV1, 3 and 4 (TRPV1, p = 0.0050; TRPV3, p = 0.0141; TRPV4, p = 0.0358) were upregulated in the CFS group compared with Ctrl group (Fig. S1). These findings collectively indicate an upregulated expression of TRPV channels in response to hyperthermia in CD4+ T cells, both in vitro and in vivo.

Fig. 3
figure 3

TRPV channels expression was upregulated in CD4+ T cells under hyperpyrexia. A Schematic of detecting the expression TRPV channels in naïve CD4+ T cells cultured at 37 ℃ or 39.5 ℃ for 3 days. B The qPCR analysis showing the mRNA level of TRPV1-4 after 3 day’s culture under 37 ℃ or 39.5 ℃ (n = 4 for each group, Data are presented as mean ± SEM, Student’s t test). C Heat map of genome-wide genes (top15, upregulated in 39.5 ℃) in CD4+ T cells cultured at 37 ℃ or 39.5 ℃ (n = 3 for each group). D Representative immunoblot bands and the bands analysis of TRPV1/4 in CD4+ T cells cultured at 37 ℃ or 39.5 ℃ (n = 4 for each group, Data are presented as mean ± SEM, Student’s t test). E Representative confocal immunofluorescent images of TRPV1/4 in CD4+ T cells cultured at 37 ℃ or 39.5 ℃ and the analysis of fluorescence intensity (n = 6 for each group, Data are presented as mean ± SEM, Student’s t test)

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TRPV1−/− prevents Th17 cell differentiation under hyperthermia

Given the upregulation of TRPV1 both in vitro (~ two fold change) and in vivo (~ five fold change), we hypothesized that TRPV1 is required for CD4+ T cells differentiation into Th17 cells. Firstly, FCM was used to test whether Th17 cell differentiation could be inhibited by TRPV1 deficiency. As expected, TRPV1−/− prevents Th17 cell differentiation in both peripheral blood (WT + CFS vs WT, p = 0.001; WT + CFS vs TRPV1−/− + CFS, p = 0.0032) and spleen (WT + CFS vs WT, p < 0.0001; WT + CFS vs TRPV1−/− + CFS, p = 0.0007) in the CFS group compared with that in Ctrl group (Fig. 4A). At the mRNA level, the expression of key Th17 cell cytokine genes, including IL-17A (WT + 39.5 ℃ vs TRPV1−/− + 39.5 ℃, p = 0.0006) and IL-17F (WT + 39.5 ℃ vs TRPV1−/− + 39.5 ℃, p < 0.0001), key transcription factor genes Rorγt (WT + 39.5 ℃ vs TRPV1−/− + 39.5 ℃, p = 0.0239) and STAT3 (WT + 39.5 ℃ vs TRPV1−/− + 39.5 ℃, p < 0.0001), as well as cytokine receptor genes IL-1βR (WT + 39.5 ℃ vs TRPV1−/− + 39.5 ℃, p = 0.0084) and TGF-βR (WT + 39.5 ℃ vs TRPV1−/− + 39.5 ℃, p < 0.0001) were upregulated in WT + 39.5 ℃ group compared with WT + 37.5 ℃ group, which were alleviated by TRPV1 deficiency (Fig. 4B). To investigate the in vitro effect of TRPV1 in Th17 cell differentiation, naïve CD4+ T cells were cultured under Th17 cell-polarizing conditions at a high temperature (39.5 ℃) for 3 days, followed by FCM analysis. It was observed that febrile temperature (39.5 ℃) caused a significant enhancement of Th17 cell differentiation (WT + 39.5 ℃ vs WT + 37 ℃, p = 0.0023) which was reduced by TRPV1 deletion (WT + 39.5 ℃ vs TRPV1−/− + 39.5 ℃, p = 0.0111) (Fig. 4C).An ELISA assay was also performed to detect the content of IL-17A in the medium of Th17 cells. Treatment with 39.5 ℃ upregulated IL-17A (WT + 39.5 ℃ vs WT + 37 ℃, p < 0.0001), which was inhibited by TRPV1 deletion (WT + 39.5 ℃ vs TRPV1−/− + 39.5 ℃, p = 0.0205) (Fig. 4D). These data strongly suggest an intrinsic role of TRPV1 in regulating Th17 cell differentiation both in vivo and in vitro under hyperthermia.

Fig. 4
figure 4

TRPV1 deficiency inhibited Th17 cell differentiation under hyperpyrexia. A Representative pseudocolor plots and statistical date of Th17 cells proportions of blood (left) and splenic (right) Th17 cells in WT and TRPV1−/− group with or without CFS (n = 4 for each group, Data are presented as mean ± SEM, One-way ANOVA). B The qPCR analysis showing Th17 cell cytokine genes, IL-17A, IL-17F, GM-CSF and IL-10; Th17 cell key transcription factors, RORγt and STAT3; Th17 cell receptor, IL-1βR and IL-23R; (n = 4 for each group, Data are presented as mean ± SEM, One-way ANOVA). C FCM analysis and statistical data of naive CD4+ T cell, isolated from the spleen of WT or TRPV1−/− mice, cultured at Th17 cell-polarized condition at 37 ℃ and 39.5 ℃ (middle and left panels) (n = 4 for each group, Data are presented as mean ± SEM, One-way ANOVA) D ELISA analysis for IL-17A from the medium of WT or TRPV1−/− Th17 cells cultured at 37 ℃ or 39.5 ℃ (WT + 37 ℃: n = 4; TRPV1−/− + 37 ℃ n = 4; WT + 39.5 ℃: n = 6; TRPV1−/− + 39.5 ℃ n = 6; Data are presented as mean ± SEM, One-way ANOVA)

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To understand the mechanism on the involvement of TRPV1 in febrile Th17 cell differentiation, we focused on its function as a cation channel that mediates Ca2+ influx. Several genes encoded Ca2+-related proteins were significantly upregulated in CD4+ T cells following hyperthermic treatment (39.5 ℃), as shown by RNA-seq analysis (Fig. S2A). Notably, NFIAc3, which is activated by Ca2+ and translocated to the nucleus to promote the expression of target genes[57], was among the upregulated genes. Additionally, the expression of NFAT2/4 and their nuclear localization were also enhanced after hyperthermic treatment (39.5 ℃). However, TRPV1 deletion reduced Ca2+ influx and the nuclear localization of NFAT2/4 in CD4+ T cells under hyperthermia (Fig. S2 B-D). Prediction analysis using the JASPAR database (http://jaspar.genereg.net/) revealed that NFAT2/4 has a high binding affinity for STAT3, a critical transcription factor for Th17 cell activation and differentiation[22, 58] (Fig. S3). ChIP analysis showed a direct binding of NFAT2/4 to the promoter of STAT3 (Fig. S4 A-B). Under specific cytokine stimulation, such as IL-6, STAT3 is recruited to the plasma membrane, phosphorylated by JAK2, and subsequently migrates to the nucleus to promote the expression of downstream target genes (RORγt and IL-17A), thereby facilitating Th17 cell differentiation. The phosphorylation and nuclear localization of STAT3 in CD4+ T cells were enhanced under hyperthermic conditions (39.5 ℃), but these effects were reversed by TRPV1 deficiency (Fig. S4 C-D). Together, these findings suggest that TRPV1 regulates Th17 cell differentiation through the downstream signaling pathways involving NFATs and STAT3.

Th17 cells cause blood–brain barrier disruption (BBBD) through IL-17A

The BBB is crucial for maintaining CNS homeostasis[59]. Our results have confirmed an increase of Th17 cells in both peripheral and cortex after CFS. Other studies have proved that a compromised BBB contributes to the aggravation of disease progression, such as in epilepsy [60, 61]. The qPCR assay was performed to detect mRNA expression levels of cortical tight junctions (TJs) (Fig. 5A). The expression of ZO-1/Ocldn/Cldn5 was downregulated after CFS, with partial recovery observed in TRPV1−/− mice (WT + CFS vs WT, ZO-1, p = 0.0005; Ocldn, p = 0.0373; Cldn5, p = 0.0257) (Fig. 5B). Subsequently, electron microscopy revealed pronounced swelling and compromised TJs in endothelial cells from the WT + CFS group, whereas damage was less severe in the TRPV1−/− + CFS group (Fig. 5C). Finally, western blot analysis conformed the significant downregulated of TJs in the WT + CFS group, whereas the TRPV1−/− + CFS group exhibited partial reversal of this downregulation (WT + CFS vs WT, ZO-1, p = 0.0035; Ocldn, p = 0.0024; Cldn5, p = 0.0052) (Fig. 5D).

Fig. 5
figure 5

TRPV1−/− or IL-17A−/− alleviates the reduction of tight junction-associated proteins after CFS. A Scheme of in vivo experiments of TJs analysis in WT and TRPV1−/− mice after CFS or not. B mRNA expression levels of TJs (ZO-1/Ocldn/Cldn5) (n = 4 for each group, Data are presented as mean ± SEM, One-way ANOVA). C SEM data showing TJs between endothelial cells; arrows: tight junctions between two endothelial cells. D Representative protein bands of TJs (ZO-1/Ocldn/Cldn5) in cortical brain homogenates and corresponding statistical analysis (n = 4 for each group, Data are presented as mean ± SEM, One-way ANOVA). E Scheme of in vivo experiments of TJs analysis in the cortical region in WT and IL-17A−/− mice with or without CFS. F mRNA expression levels of TJs (ZO-1/Ocldn/Cldn5) (n = 6 for each group, Data are presented as mean ± SEM, One-way ANOVA). G SEM data showing disrupted tight junctions between endothelial cells; arrows indicate tight junctions between two endothelial cells. H Representative protein bands of TJs (ZO-1/Ocldn/Cldn5) in WT and IL-17A−/− mice after CFS or not, and corresponding statistical analysis (n = 3 for each group, Data are presented as mean ± SEM, One-way ANOVA)

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It has been demonstrated that inflammatory cytokines released by peripheral immune cells can disrupt the BBB, participating in multiple CNS diseases [62]. We have shown an increase in Il-17A both in vivo and in vitro after CFS or under febrile temperature. To study the integrity of the BBB, the endothelial cell line Bend.3 was treated with IL-17A (100 ng/μl) for 24 h to examine the expression of TJs. Western blot results showed significant downregulation of ZO-1 (p = 0.0036)/Cldn5 (p < 0.0001), suggesting that IL-17A can decrease endothelial TJs (Fig. S5). Accordingly, we detect BBBD in IL-17A−/− mice (Fig. 5E); qPCR results showed that IL-17A deficiency alleviated the downregulation of TJs after CFS (WT + CFS vs WT, ZO-1, p < 0.0001; Ocldn, p = 0.0085; Cldn5, p = 0.0005) (Fig. 5F). Electron microscopy further confirmed that IL-17A deletion could attenuate the TJs damage between two endothelial cells following CFS (Fig. 5G). Western blot analysis showed that IL-17A deficiency alleviated TJs downregulation after CFS (WT + CFS vs WT, ZO-1, p = 0.0005; Ocldn, p = 0.0085; Cldn5, p < 0.0001) (Fig. 5H). These results collectively demonstrate that the increased peripheral Th17 cells after CFS led to BBBD through the release of IL-17A.

Th17 cells exacerbate the inflammation and activate microglia after CFS

Microglia, as the first responders under pathological conditions, play a critical role in immune surveillance. Therefore, we explored the effect of infiltrating Th17 cells on local immune microenvironment and their subsequent impact on microglia. Immunofluorescence assay was employed to detect Th17 cell infiltration. The result showed that the number of Th17 cells was significantly decreased in the TRPV1−/− + CFS group (WT + CFS vs TRPV1−/− + CFS, p = 0.0059) (Fig. 6A). Consequently, the activation of cortical microglia was alleviated after CFS in the TRPV1−/− mice, as evidenced by a decreased expression of activated microglial markers, cytokine genes and chemokine genes at mRNA level (WT + CFS vs TRPV1−/− + CFS, IL-1β, p = 0.0015; IL-6, p = 0.0027; MHC II, p < 0.0001; CD80, p = 0.0001; CD86, p = 0.0026; CCL3, p = 0.0083; CCL4, p < 0.0001; CCL9, p = 0.0007; CXCL10, p < 0.0001; CXCL12, p = 0.0244; CCL20, p = 0.0063)(Fig. S6A). The protein levels of activated microglial markers also decreased in TRPV1−/−+CFS group compared with WT+CFS group (WT + CFS vs TRPV1−/− + CFS, P-P38, p = 0.0008; IBA1, p = 0.0008; CD68, p = 0.0158) (Fig. S6B). The immunofluorescence assay further demonstrated that the number of IBA1+CD68+ microglia was decreased in TRPV1−/− + CFS group (WT + CFS vs TRPV1−/− + CFS, p = 0.0465), with a longer microglial branching length (WT + CFS vs TRPV1−/− + CFS, p = 0.0063) (Fig. S6C). These results indicate that knockout of TRPV1 alleviates Th17 cell infiltration after CFS, thereby helping restore central immune microenvironment and reducing microglial activation.

Fig. 6
figure 6

Th17 cells activate microglia and exacerbate the inflammation via IL-17A following CFS. A Representative fluorescence images of cortical Th17 cells infiltration in WT and TRPV1−/− mice after CFS and statistical analysis of Th17 cells numbers (n = 6 for each group, Data are presented as mean ± SEM, One-way ANOVA). B Relative protein analysis of microglial activation marker CD68/P-P38 and inflammatory cytokines IL-6/TNFα in cortical homogenates from WT and IL-17A−/− mice with or without CFS; corresponding quantitative data (n = 3 for each group, Data are presented as mean ± SEM, One-way ANOVA). C Representative fluorescent images of activated microglia (IBA1+CD68+ cells) in cortical regions from WT and IL-17A−/− mice with or without CFS; the number of IBA1+CD68+ cells/0.1mm2 (n = 6 for each group, Data are presented as mean ± SEM, One-way ANOVA); quantification of microglial branch length (n = 12 for each group, Data are presented as mean ± SEM, One-way ANOVA). D The mRNA expression levels of microglial activation marker CD80/CD86/MHC II and inflammatory factors iNOS/ IL-6/IL-1β in cortical homogenates from WT and IL-17A−/− mice with or without CFS (n = 4 for each group, Data are presented as mean ± SEM, One-way ANOVA)

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To further determine the role of Th17 cells in the activation of microglia, primary microglia were treated with IL-17A. After 24 h, the microglia exhibited a significant upregulation of CD68 expression (p = 0.0017), increased cell body size, and shortened branches (p = 0.0011) (Fig. S7). Subsequent protein immunoblotting experiments revealed that microglia activation markers CD68/P-P38 and inflammatory factors IL-6/TNF-α were significantly lower in the IL-17A−/− + CFS group compared to the WT + CFS group (WT + CFS vs IL-17A−/− + CFS, P-P38, p = 0.0036; TNF-α, p = 0.0192; IL-6, p = 0.0463) (Fig. 6B). Furthermore, immunofluorescence analysis showed a significantly decreased number of IBA1+CD68+ microglia (WT + CFS vs IL-17A−/− + CFS, p = 0.0017) with longer branches (WT + CFS vs IL-17A−/− + CFS, p = 0.0443) in the IL-17A−/− + CFS group, indicating markedly reduced microglia activation (Fig. 6C). The qPCR results showed that the microglia activation genes: CD80 (p = 0.0005), CD86 and MHC II (p = 0.0118) and inflammatory cytokines genes: iNOS, IL-6 (p = 0.0081) and IL-1β (p < 0.0001) were significantly decreased in the IL-17A−/− + CFS group compared to the WT + CFS group (Fig. 6D). These findings suggest that reducing the infiltration of Th17 cells or IL-17A secretion can alleviate microglial activation and the inflammatory response in CNS.

Reduction of Th17 cells infiltration alleviates neuronal loss and seizure susceptibility after CFS

We have shown that TRPV1 knockout reduces infiltration of Th17 cells and activation of microglia. Next, we investigated whether reduction of Th17 cells may affect the seizure susceptibility and neuronal damage in CFS mouse model. Behavioral observation showed a significant decrease in seizure severity (p = 0.0097) in TRPV1−/− + CFS group, with no apparent change in seizure latency (Fig. 7A). Nissl staining showed a severe neuronal damage and loss after CFS (WT + CFS vs TRPV1−/− + CFS, p = 0.0105), which was partially rescued in TRPV1−/− mice (Fig. 7B). To further determine the changes of seizure susceptibility after CFS, brain slices from CFS were obtained to assess the 4-AP-induced epileptiform discharges. A significant increase in epileptiform discharges was identified in the WT + CFS group, whereas abnormal firing was mitigated in the TRPV1−/− + CFS group (WT + CFS vs TRPV1−/− + CFS, p = 0.0441) (Fig. 7C). These results collectively demonstrate that TRPV1−/− reduces the infiltration of Th17 cells, leading to significant attenuation of neuronal damage and loss, thereby reducing seizure susceptibility after CFS.

Fig. 7
figure 7

TRPV1−/− alleviates seizure susceptibility flowing CFS. A Evaluation of seizure latency and seizure severity in WT and TRPV1−/− mice following CFS (WT + CFS: n = 7; TRPV1−/− + CFS: n = 5, Data are presented as mean ± SEM, two-way ANOVA). B Representative Nissl-stained images in WT and TRPV1−/− mice with or without CFS; arrows indicate loosely arranged neurons with condensed and deeply stained nuclei; quantification of damaged neurons (n = 6 for each group, Data are presented as mean ± SEM, One-way ANOVA). C Representative MEA voltage recordings from WT or TRPV1−/− mice cortical slice showing spontaneous action potentials firing, shown as spikes on the trace in ACSF and then perfused with 4-AP (5 mM); the time calibration line in the Raster plot is 20 s. A representative trace recorded by MEA electrode, when the perfusion with 5 mM 4-AP, epileptiform activity was evoked. The number of action potentials recorded by all electrodes within 0.1 s (n = 8 for each group, Data are presented as mean ± SEM, One-way ANOVA)

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Discussion

In this study, we validated that TRPV1 regulates the directional differentiation of CD4+ T cells into Th17 cells, thereby promoting seizure susceptibility in a CFS mouse model. Following a CFS-induced attack, the mice exhibit a seizure-prone state. We further demonstrated that peripheral differentiation and infiltration of Th17 cells into the CNS were enhanced after CFS. Additionally, temperature-sensitive TRPV channels were upregulated in CD4+ T cells both in vivo and in vitro. Consequently, CD4+ T cells with TRPV1 deficiency showed a significant reduction in Th17 cell differentiation and IL-17A secretion. Febrile-temperature-induced Th17 cell differentiation may depend on TRPV1 and Ca2+-related NFATs and STAT3 pathway, specifically via STAT3 phosphorylation, which can be reversed by TRPV1 deficiency at 39.5 ℃. These results support the idea that repeated exposures to hyperthermia can lead to hyperexcitability of neuronal activity.

Besides the direct role of hyperthermia in ion channel dysfunction, such as Nav1.1[63], and chloride channel[64], studies in the immature rat hippocampus have found that hyperthermia can reduce GABA release from presynaptic terminals, in part by blocking the adenylyl cyclase-protein kinase A signaling pathway and activating presynaptic 4-aminopyridine-sensitive K+ channels[65]. Furthermore, genetic factors [66, 67], brain pH [68], and inflammation [69, 70] have also been shown to contribute to the initiation and development of CFS. Additionally, heat-related proteins, such as heat shock protein-27 (Hsp27), are upregulated in the brain following febrile seizures [71]. Another study demonstrated that the local partial pressure of oxygen (pO2) rapidly increased, then decreased, ultimately returning to near baseline during behavioral febrile seizures [72]. Following FS, a rapid reduction in synaptic calcium-permeable (CP)-AMPA receptors and a decrease in calcium permeability in the membranes of principal neurons in both the hippocampus and the entorhinal cortex have been observed. These alterations may contribute to excessive excitotoxicity and neuronal death [73]. Following CFS, the BBBD was aggravated by Th17 cells infiltration, while IL-17A knockdown attenuated this effect. Conversely, IL-17A deficiency alleviates the microglia activation and inflammation in the CNS (Fig. 8). Along with the reduction of infiltrating Th17 cells and attenuation of CNS inflammation, neuronal damage and seizure susceptibility were also alleviated. Our study demonstrates that febrile-temperature-induced Th17 cells play a key role in enhancing seizure susceptibility after CFS through the TRPV1/NFATs/STAT3 signaling pathway.

Fig. 8
figure 8

Schematic diagram of TRPV1 mediating Th17 cells differentiation to promote CFS in mice. In complex febrile seizures (CFS) model, febrile temperature induces Th17 cells differentiation through the TRPV1/NFATs/STAT3 pathway. The proinflammatory Th17 cells enhance the disruption of blood brain barrier (BBB) and consequently infiltration into CNS to activate microglia through IL-17A, therefore exacerbating neuroinflammation. Collectively, the infiltrated Th17 cells and activated microglia cause neuron damage through releasing inflammatory cytokines, finally increasing the seizure susceptibility following CFS

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Over the past 30 years, CNS inflammation has been validated as a contributor to the initiation and maintenance of febrile seizures (FS) [74]. In our previous study, we demonstrated that TRPV1, a protein once thought to function solely as ion channels, promotes microglia activation and CNS inflammation, thereby enhancing seizure susceptibility in a recurrent febrile seizure model [75]. Furthermore, high temperature not only induces CNS inflammation but also stimulates the peripheral immune system, leading to systemic inflammation [76]. Research on the thermal regulation of immunity strongly supports the idea that fever-range temperatures activate peripheral immune cells, including neutrophils [77], macrophages [78], dendritic cells [79] (DCs), antigen-presenting cells (APCs) [80] and T cells [81]. A recent finding also shows that febrile temperature could promote Th17 cell differentiation and enhance their pathogenicity [29]. In this study, we explored how febrile temperatures enhance Th17 cell differentiation, which play a pivotal role in CFS development.

Cytokines secreted by immune cells play a key role in the CFS pathogenesis, where the balance between pro- and anti-inflammatory cytokines is disrupted[82]. Mounting evidence indicates an increase in inflammatory cells or cytokines peripherally in patients with febrile seizures [83, 84]. Earlier basic studies and clinical trials have confirmed the increased secretion of IL-1ß, and it has even been used as a basis for the diagnosis of febrile seizures. Other noteworthy inflammatory cytokines include IL-6, prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNF-α) in the periphery [85]. PGE2 enters the hypothalamic region and, in turn, induces a fever. Abnormally increased IL-1β levels also progressively increase excitatory (glutamatergic) neurotransmission, and decreases inhibitory (GABAergic) neurotransmission, thus mediating the pathogenesis of convulsions [86]. Meanwhile, multiplex immunoassay studying CSF from pediatric patients showed that cytokines/chemokines (IL-1β, CXCL9, CXCL10, CXCL11, and CCL19) were elevated in febrile status epilepticus (FSE) compared to afebrile status epilepticus (ASE), although the median seizure duration and timing of CSF testing were similar between the two groups [87]. A recent study demonstrated that inflammatory cytokines reduced the threshold for hyperthermic seizures by exacerbating thermal hyperpnea-induced respiratory alkalosis, primarily through the sensitization of peripheral TRPV1 receptors in the vagus nerve [88]. And vagal TRPV1 has been shown to exert pro-convulsant effects by increasing the rate of expired CO2, resulting from an exaggerated ventilatory response to hyperthermia [89]. It is worth noting that although numerous studies have observed changes in peripheral and central cytokines, the association between cytokine levels and FSs remains inconclusive. Further studies of large-scale, case-controlled trials are needed to evaluate the precise concentrations of certain cytokines, especially IL-17 in FS patients. These clinical studies are crucial for developing novel therapies target specific cytokines in FS.

Our study identifies TRPV1 as a positive regulator in febrile-temperature-induced Th17 cell differentiation and peripheral inflammation, aligning with the previous findings that TRPV1 is critical for CNS inflammation [75, 90]. Recent studies also suggest that TRPV1 activation plays a role in the immunomodulatory effect of dendritic cells [91] and in the polarization of macrophages [44]. Another study validates that febrile temperature can directly promote differentiation of CD4+ T cells through a TRPV1-regulated, Notch-dependent pathway [46]. These results confirm that TRPV1 is essential for the function and differentiation of immune cells under hyperthermia conditions.

In summary, we have identified the critical role of Th17 cell differentiation in CFS through a novel pathway involving the ion channel TRPV1. In CFS models, elevated temperature can directly and selectively promote Th17 cell differentiation via the TRPV1/NFATs/STAT3 axis. The recruitment of Th17 cells and their associated toxicity can aggravate the BBBD and microglia activation. This work is the first to explore the ‘TRPV1-CD4-Th17-Microglia’axis from the perspective of systemic inflammation to clarify the recurrent nature of CFS. It also suggests that peripheral CD4+ T cells could serve as a promising therapeutic target for CFS.

Conclusion

This study demonstrated that the Th17 cell differentiation is enhanced in CFS, leading to aggravated BBBD via IL-17A and infiltration into the CNS. Th17 cells in the CNS activate microglia and exacerbate neuroinflammation. Mechanistically, the febrile temperatures activated TRPV1 to facilitate calcium ion influx, leading to the nuclear localization of NFAT2 and 4 and the phosphorylation of STAT3. TRPV1 deletion attenuates Th17 cell differentiation and CNS infiltration, protecting the BBB and reducing the seizure susceptibility following CFS. These findings not only provide insight into the mechanisms underlying Th17 cell involvement in febrile seizures but also suggest a potential target for intervention.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

TRPV1:

Transient receptor potential vanilloid 1

CFS:

Complex febrile seizures

BBB:

Blood brain barrier

CNS:

Central nervous system

NFAT2/4:

Nuclear factor of activated T cell 2 and 4

STAT3:

Signal transducer and activator of transcription 3

MS:

Multiple sclerosis

AD:

Alzheimer’s disease

ChIP:

Chromatin immunoprecipitation

MEA:

Multi-electrode arrays

IF:

Immunofluorescence

BBBD:

Blood brain barrier disruption

ICC:

Immunocytochemistry

SEM:

Scanning electron microscope

qPCR:

Quantitative real-time polymerase chain reaction

IL-17A:

Interleukin-17 A

IL-17F:

Interleukin-17 F

T-bet:

T-box transcription factor

GATA3:

GATA binding protein 3

FOXP3:

Forkhead box P3

RORγt:

RAR-related orphan receptor gamma t

GMCSF:

Granulocyte–macrophage colony-stimulating factor

TGF-βR:

Transforming growth factor beta receptor

IL-10:

Interleukin-10

TNF-α:

Tumor necrosis factor-α

IL-6:

Interleukin-6

IL-1β:

Interleukin-1 beta

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Funding

This research is funded by the National Natural Science Foundation of China (Grant No. 82171452), Basic and clinical research of neuro-immune-related diseases (Grant No. WHWF001), 2023–2024 Key Project of Hubei Provincial Administration of Traditional Chinese Medicine (Grant No. ZY2023Z018).

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Authors and Affiliations

  1. Department of Physiology, Wuhan University School of Basic Medical Sciences, 185 Donghu Road, Wuhan, 430071, Hubei, China

    Shuo Kong, Xianglei Jia, Xin Liang, Jingyi Liang, Yan Zhang, Ningyang Wu, Taoxiang Chen, Xiaohua He, Jun Yin, Song Han, Wanhong Liu, Yuanteng Fan, Jian Xu & Biwen Peng

  2. Department of Genetics, Shandong Second Medical University, Weifang, 261053, China

    Yu Chen

  3. Epilepsy Center, Jinan Children’s Hospital, 23976 Jingshi Road, Jinan, 250022, Shandong, China

    Song Su

  4. Clinical Laboratory, Weifang Maternal and Child Health Hospital, 407 Qingnian Road, Weifang, 261011, Shandong, China

    Jian Xu

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  1. Shuo Kong
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  2. Xianglei Jia
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  3. Xin Liang
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  8. Song Su
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  16. Biwen Peng
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Contributions

Shuo Kong, Biwen Peng, Jian Xu conceived and designed the experiments. Shuo Kong, Xianglei Jia, Xin Liang, Yu Chen, Jingyi Liang performed the experiments. Shuo Kong, Yan Zhang, Ning-Yang Wu analyzed the data. Song Su, Xiaohua He, Jun Yin, Song Han, Wanhong Liu, Yuanteng Fan, Taoxiang Chen contributed to the reagents, materials, and analysis tools. Shuo Kong and Biwen Peng wrote the manuscript. All authors reviewed and approved the final manuscript.

Corresponding authors

Correspondence to Jian Xu or Biwen Peng.

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This study has been conducted with the highest regard for animal welfare and in strict compliance with ethical guidelines. We affirm that all procedures followed in this research adhere to the established principles of ethical animal experimentation. All animal experiments were approved by Institutional Animal Care and Use Committee Wuhan University.

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Kong, S., Jia, X., Liang, X. et al. Febrile temperature-regulated TRPV1 in CD4+ T cells mediates neuroinflammation in complex febrile seizures. J Neuroinflammation 22, 103 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12974-025-03421-7

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Keywords

Journal of Neuroinflammation

ISSN: 1742-2094