Epilepsy is a complex and chronic neurological disorder. Epileptic seizures occur when an area of the brain or the entire brain experiences abnormal synchronous discharge of neurons due to factors such as infection or metabolic disruption (Falco-Walter, 2020). It is pathologically characterized by sudden and abnormal brain discharges, which can lead to temporary brain dysfunction (Fisher et al., 2005). The estimated incidence rate of epilepsy is 50 per 100,000 people, affecting over 70 million people worldwide (Thijs et al., 2019). The Global Burden of Epilepsy Report estimated over 125,000 epilepsy-related deaths in 2016, 81% of which occurred in countries with a low or medium sociodemographic index (GBD 2016 Epilepsy Collaborators, 2019). Despite antiepileptic drugs being the main treatment, approximately 30% of epilepsy patients are resistant to drug therapy and rely on alternative interventions such as surgery (Laxer et al., 2014). Epilepsy not only significantly impacts the quality of life of patients but also increases their financial burden through treatment costs.

The development and progression of epilepsy involve complex biochemical processes. For a long time, researchers considered the brain as an immune-privileged area due to the presence of the blood–brain barrier (BBB). However, evidence indicates that both innate and acquired immunity can be rapidly induced within the central nervous system to respond to tissue damage caused by various reasons (Mukhtar, 2020). Moreover, increasingly more evidence shows the involvement of brain vascular dysfunction in the pathogenesis of epilepsy (Marchi et al., 2016; Vezzani et al., 2019). After the BBB is compromised, the infiltration of neural cells, brain microvascular endothelial cells, and peripheral immune cells into the brain parenchyma emerges as an important factor in the occurrence of epileptic seizures (Banks and Erickson, 2010). Immune cells, particularly microglial cells in the brain and T cells in the peripheral blood, are activated during epileptic seizures and release inflammatory factors such as cytokines and chemical mediators (Ravizza et al., 2008; Vezzani et al., 2011; Librizzi et al., 2012). These factors directly damage neurons, promoting the occurrence and persistence of epilepsy (Ravizza et al., 2008). Additionally, the inflammatory response alters the internal environment of the brain, making neurons more susceptible to abnormal discharge (Vezzani et al., 2011).

The association between plasma metabolites and epilepsy is increasingly becoming a focus of research, primarily because these metabolites reveal biochemical changes associated with epileptic seizures. Current studies indicate that more than 200 different metabolic diseases can trigger epileptic seizures, affecting the onset of epilepsy through various pathways (Almannai et al., 2021). For instance, compared to healthy individuals, patients with epilepsy show significant differences in the levels of amino acids, fatty acids, and energy metabolites (Auvin, 2012; Bejarano and Rodríguez-Navarro, 2015). These changes are related to not only alterations in neurotransmitter balance but may also involve inflammatory responses and changes in brain energy utilization and metabolic state (Almannai et al., 2021). Particularly, amino acids, such as the primary excitatory neurotransmitter glutamate and the key inhibitory neurotransmitter gamma-aminobutyric acid (GABA), play crucial roles in the neural transmission processes involved in epileptic seizures. The imbalance between these amino acids in epilepsy patients can cause excessive neuronal excitability, and thereby triggering seizures (Scharfman, 2007; Coulter and Steinhäuser, 2015). Additionally, certain plasma metabolites can interfere with neurotransmission. For example, glycine, acting as a co-agonist for the N-methyl-D-aspartate (NMDA) class of glutamate receptors, plays a crucial role in modulating the excitability of the nervous system. An abnormal increase in the concentration of glycine in the blood may lead to excessive stimulation of NMDA receptors, increasing the risk of epileptic seizures. This mechanism highlights the key role of metabolites in maintaining the balance of neurotransmission and their potential part in epilepsy pathogenesis (Kristiansen et al., 2016; (Kapur and Joshi, 2021).

Mendelian Randomization (MR) is an emerging statistical method that draws on Mendel’s laws of inheritance, specifically the law of segregation and the law of independent assortment, to explore potential causal relationships between exposures and diseases (Sanderson et al., 2022). The rise and development of MR are closely linked to advancements in large-scale Genome-Wide Association Studies (GWASs) technology. GWAS has enabled the widespread identification of genetic variants associated with diseases, providing a wealth of genetic instrumental variables for MR analysis. MR uses genetic variations, particularly single-nucleotide polymorphisms (SNPs), as instrumental variables (IVs) to simulate the random allocation mechanism of randomized controlled trials (RCTs) (Smith and Ebrahim, 2003). The core advantage of MR is its ability to construct potential causal relationships between variables while avoiding the limitations of traditional observational studies where correlation does not imply causation. As genetic variations are determined at birth and do not change due to the onset of disease, the MR method can effectively avoid the problems of confounding factors and reverse causality (Davies et al., 2018). This makes MR an ideal alternative research strategy for exploring the causal relationships between exposure factors and outcomes in situations where RCTs cannot be conducted or where conducting RCTs poses ethical challenges. A recent study investigated the causal relationship between metabolism and immune cells and female reproductive diseases with excellent results through MR (Shen et al., 2024). Mendelian randomization analysis has many advantages, it also has certain limitations due to the difficulty in satisfying the horizontal pleiotropy assumption, the challenges in effectively correcting for the influence of a few genetic regulatory factors through sensitivity analysis, and the small sample sizes in most studies on metabolic quantitative trait loci.

The current understanding of the causal effect of immune cells and plasma metabolites on epilepsy is limited. This study used GWAS and a two-sample MR approach to assess the potential causal association of 731 immune cells and 1,400 plasma metabolites with the occurrence of epilepsy. Further, we investigated whether the identified candidate immune cells influence the development of epilepsy through their effects on plasma metabolites. The findings of this study not only deepen our understanding of the pathophysiological basis of epilepsy but also provide the groundwork for the development of effective epilepsy screening and prevention measures, which are significant for improving clinical outcomes.

An increasing number of studies are exploring the genetic, immunologic, and metabolic bases of epilepsy, given by fast-growing prevalence (Ravizza et al., 2008; Vezzani et al., 2011; Auvin, 2012; Bejarano and Rodríguez-Navarro, 2015; Almannai et al., 2021). Advances in immunology and metabolomics have provided valuable insights into understanding the pathogenesis of epilepsy. In contrast to these observational studies, we employed MR using genetic variation as instrumental variables and summary statistics from GWAS to reveal causal relationships between epilepsy risk and levels of immune cells and metabolites.

Our findings revealed that 28 immune phenotypes were causally associated with epilepsy. Of these immune phenotypes, 18 were correlated with a reduced risk of epilepsy, whereas 10 were associated with an increased risk of epilepsy. Additionally, we found that among the 1,400 metabolites investigated, 14 were causally linked to the occurrence of epilepsy—seven were positively correlated and seven were negatively correlated with epilepsy occurrence. Furthermore, eight metabolites mediated the effects of six immune cells on epilepsy. In particular, IgD+ CD38dim %B cell on 1-oleoyl-GPC (18:1) levels, memory B cell %lymphocyte on AMP levels, CD39+ resting Treg AC on DMTPA levels, CD45RA+ CD8br AC on DMTPA levels, CD45RA+ CD8br AC on X-12680 levels, DN (CD4−CD8−) AC on 3-hydroxyoctanoylcarnitine (2) levels, CD3 on Naive CD4+ on 1-oleoyl-GPC (18,1) levels, and FSC-A on myeloid DC on X-12544 levels were associated with reduced epilepsy risk. Whereas, IgD+ CD24+ %B cell on threonine levels, CD39+ CD8br %T cell on branched-chain, straight-chain, or cyclopropyl 10:1 fatty acids, FSC-A and myeloid DC on 1-(1-enyl-stearoyl)-2-linoleoyl-GPE (p-18:0/18:2) levels, and CD11c on monocyte on X-21441 levels were associated with increased epilepsy risk.

Our results indicate that CD3 and CD39 may play important roles in various types of T cells. Inflammation mediated by T cells is considered a key factor in the occurrence of epilepsy (Vezzani et al., 2011). The activation of CD3 promotes the differentiation and proliferation of T cells, releasing various inflammatory factors, such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). These inflammatory factors can enhance the activity of immune cells in the brain and promote the production of inflammatory mediators, further affecting the excitability of neurons, leading to the occurrence and progression of epilepsy (Smith-Garvin et al., 2009). In addition, the regulatory role of CD3 may also affect the subset distribution and function of T cells, such as the dysfunction of regulatory T cells (Treg), thereby affecting the immune system’s response to the brain (Riazi et al., 2010). CD39 is a cell surface enzyme highly expressed in astrocytes and endothelial cells; its main function is to catalyze the hydrolysis of extracellular ATP and ADP, generating AMP (Braun et al., 2000). Extracellular ATP, as an inflammatory mediator, can activate immune cells and promote the development of inflammatory reactions. Moreover, a previous study has shown that targeting purinergic P2 receptors as pharmacological targets can effectively regulate the occurrence of epilepsy, the inflammatory process, and brain damage caused by epileptic seizures (Engel et al., 2016).

The impact of AMP on the occurrence of epilepsy is multifaceted. First, AMP can be metabolized into adenosine, which acts through G protein-coupled adenosine receptors to participate in the onset of epilepsy (Boison and Jarvis, 2021). Additionally, the accumulation of AMP within cells can lead to the activation of AMP-activated protein kinase (AMPK), which enhances the cell’s energy utilization efficiency (Liu et al., 2020), however, excessive activation may lead to cellular dysfunction, thereby triggering epileptic seizures. AMP may also be involved in the pathogenesis of epilepsy through other mechanisms, such as affecting the function of ion channels or regulating the release of neuroexcitatory agents (Benarroch, 2013). DMTPA is an amino acid metabolite derived from S-adenosylmethionine, significantly associated with acute kidney injury and hypertension (Bajaj et al., 2021; Wang et al., 2021). It may promote the occurrence of epilepsy by affecting cerebral hemodynamic changes such as ischemia, microangiopathy, and cerebral arteriosclerosis (Gasparini et al., 2019). Yang et al. indicated that X-12680 is associated with Alzheimer’s disease (AD) risk and has a role in reducing this risk (Yang et al., 2024). In our study, we found similar results. Both AD and epilepsy are related to abnormal nervous system function. We hypothesize that X-12680 levels may be beneficial for neuronal repair, thereby protecting nervous system function.

Metabolite research has been conducted for neurological diseases. Darst et al. revealed that 7 out of 1,097 metabolites are related to the executive function trajectory of AD (Darst et al., 2021). A recent study suggests a significant bidirectional causal relationship between generalized convulsive epilepsy and inflammatory factors (Wang et al., 2023), suggesting the potential mechanisms by which inflammatory metabolites influence the occurrence of epilepsy.

In this study, we used new metabolite datasets to explore their impact on epilepsy risk and found a causal relationship between 14 metabolites and the occurrence of epilepsy. These findings highlight the significant role that metabolites play in the development of epilepsy. Of these, threonine metabolism plays a crucial role in the epileptic mechanism in humans, rats, and mice (Lai et al., 2022). Threonine participates in the synthesis of neurotransmitters such as glycine and serine, triggers excitatory neurotransmission, and thereby influences the excitatory and inhibitory balance of neurons, promoting the onset and development of epilepsy (Hernandes and Troncone, 2009).

Additionally, in our study, we found that six types of immune cells mediate the regulation of eight metabolites, influencing the occurrence of epilepsy. The mechanism by which immune cells regulate metabolites to influence the occurrence of epilepsy is not yet clear. We hypothesize that this mediating effect may be related to energy metabolism, neurotransmitter transmission, and inflammatory responses (Vezzani and Friedman, 2011; Marchi et al., 2014).

Our study’s novelty is first reflected in our use of diverse immune cell characteristics and metabolites as research subjects, revealing their potential causal relationship with the occurrence of epilepsy. Second, by employing mediation analysis, we provide a theoretical basis for understanding the mechanism by which immune cells influence the development of epilepsy through the regulation of metabolites (refer to Supplementary Figure S6). This offers valuable references and insights for future research.

Our study still has some limitations. First, the GWAS data we used were all from European cohorts. Given the genetic diversity between different races, the validity of our findings may not apply to other ethnic populations. Second, the sample size of the immune cell and metabolite data used in this study is relatively small, which may affect the reliability and generalizability of the results. Therefore, in the future, the results must be validated in a larger sample to improve the statistical power of the study and ensure the robustness and credibility of the conclusions. Finally, our study focused on the general population of patients with epilepsy without distinguishing between different subtypes of epilepsy, limiting the exploration of personalized treatment strategies for individuals with epilepsy.

Our study revealed the critical roles of immune cells and metabolites in the development of epilepsy. It has identified potential targets for future interventions, such as modulating levels of CD3, CD39, or threonine, which could become new strategies for preventing and treating epilepsy. Future research should include functional studies to further explore the roles and levels of immune cells and metabolites in cellular and animal models. These studies will deepen our understanding of the specific pathways and interactions involving immune and metabolic features in the pathogenesis of epilepsy, thereby validating our findings and enhancing their clinical translational potential.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

KW: Writing – original draft, Software, Investigation, Formal analysis. JY: Writing – original draft, Methodology, Data curation. WX: Writing – review & editing, Supervision, Investigation. LW: Writing – review & editing, Visualization, Methodology. YW: Writing – review & editing, Supervision, Methodology.