Ischemic stroke (IS), a prevalent neurological disorder, results from inadequate cerebral blood flow, leading to neuronal tissue hypoxia and ischemia (Zhu et al., 2022). Globally, approximately 80% of all stroke cases are attributed to ischemic stroke, presenting a significant public health challenge (Herpich and Rincon, 2020; Rabinstein, 2020). The substantial incidence rate and profound implications on disability and mortality underscore its importance. Clinically, ischemic stroke manifests as an acute impairment of brain function, with progression and prognosis contingent upon the affected region and extent of cerebral damage (Feske, 2021). Given the unsatisfactory therapeutic results and substantial burden linked to IS, it is crucial to ascertain potential underlying factors that contribute to its onset.

In the exploration of risk factors for ischemic stroke, researchers have increasingly emphasized the significance of gut microbiota (Pluta et al., 2021; Peh et al., 2022) and immune cells (Qiu et al., 2021). Gut microbiota, a vast microbial community within the human body, is closely linked to host health, influencing the integrity and function of the blood-brain barrier (BBB). Metabolites like short-chain fatty acids (SCFAs) regulate BBB permeability and stability, and the activation of the autonomic nervous system, such as the vagus nerve, by gut microbiota also affects BBB function (Long et al., 2022; Zhang et al., 2023). Disruption of the BBB is a crucial pathological feature of ischemic stroke, leading to complications such as edema, hemorrhage, and inflammation, exacerbating brain damage (Battaglini et al., 2020).

Immune cells, vital for the body’s defense system, play a crucial role in ischemic stroke through interactions with brain endothelial cells and the release of inflammatory factors. Factors like LAMs, IL-1β, and TNF-α influence the permeability and integrity of cerebral blood vessels, while surface molecules or released inflammatory factors like CD40, CD40L, IL-6, and TNF-α regulate vascular tone in brain blood vessels by influencing the synthesis and release of nitric oxide (NO) (Iadecola et al., 2020; Xu et al., 2020; Endres et al., 2022). Both gut microbiota and immune cells have been identified as potential contributors to ischemic stroke development, influencing disease occurrence through interactions with the nervous system (Nam, 2019; Hu et al., 2022; Huang et al., 2022; Wang et al., 2022).

Mendelian randomization (MR), an effective approach to infer causality, employs genetic variants as instrumental variables (IVs) to investigate the causal impact of exposure on outcomes (Bowden and Holmes, 2019; Birney, 2022). In this study, gut microbiota and immune cells were selected as exposure variables, with IS as the outcome variable for MR analysis. The goal was to explore potential causal relationships, providing a theoretical foundation for further investigations into the intricate mechanisms and risk factors associated with IS.

In this MR analysis report, we present a pioneering study that establishes a potential causal link between gut microbiota/immune cells and IS. Through a meticulous two-sample MR investigation, we have uncovered compelling evidence of a causal correlation between four specific gut microbial taxa and 16 immune cells concerning IS (Figure 4). This groundbreaking discovery contributes valuable insights into the intricate relationship between gut microbiota, immune cells, and the development of IS. The robustness of our findings, supported by thorough analyses and sensitivity assessments, positions this study as a significant step forward in understanding the potential mechanisms underlying IS.

In our study, we observed a positive correlation between genus.Paraprevotella.id.962 and genus.Streptococcus.id.1853 and the risk of ischemic stroke (IS), while genus.Barnesiella.id.944 and genus.LachnospiraceaeNK4A136group.id.11319 demonstrated a negative correlation. These findings underscore the potential role of gut microbiota in the development of IS. Dysbiosis in gut microbiota may impact IS through various pathways. Firstly, gut microbiota plays a crucial role in regulating the host immune system, influencing the activity and quantity of immune cells and potentially impacting the inflammatory processes underlying IS. Secondly, certain bacteria may produce metabolites affecting host metabolism, subsequently influencing vascular health and increasing the risk of IS. Additionally, gut microbiota may exert remote effects on the central nervous system by influencing neurotransmitter synthesis and release. In murine models of middle cerebral artery occlusion (MCAO), bacterial imbalance peaked 12–24 h after the ischemic event and gradually returned to normal levels by day 7 (Xu et al., 2021). Studies have shown that stroke-induced changes in mucosal microbiota composition lead to an increase in Clostridia and Akkermansia muciniphila (Stanley et al., 2018). However, clinical studies have produced varying results regarding taxonomic classification and function. One study found a decrease in Lactobacillus, a major type of host probiotic bacteria, following experimental stroke in monkeys (Chen et al., 2019). Recent cohort studies revealed notable enrichment of Enterobacteriaceae, Ruminococcaceae, and Akkermansia at the family level, while Bacteroidaceae, Prevotella, and Faecalibacterium were significantly reduced after IS (Yamashiro et al., 2017). Opportunistic pathogenic Proteobacteria, such as Enterobacter, Megasphaera, Oscillibacter, and Desulfovibrio, are more abundant in IS patients with transient ischemic attack (TIA). E. coli overgrowth may exacerbate brain infarction via lipopolysaccharide (LPS)-mediated systemic inflammation. Short-chain fatty acids (SCFAs) help maintain gut integrity by reducing inflammation (Tan et al., 2021); depletion of SCFA-producing bacteria like Roseburia, Bacteroides, Lachnospiraceae, Faecalibacterium, Blautia, and Anaerostipes can represent dysbiosis among IS patients due to low fecal SCFA levels (Koh et al., 2016). However, a different study reported that SCFA producers like Odoribacter and Akkermansia were enriched among IS patients (Li et al., 2019). These diverse findings emphasize the need for further research on the relationship between post-stroke dysbiosis and clinical outcomes.

The most effective method for modulating the gut microbiota is through fecal microbiota transplantation (FMT), a process that involves introducing a suspension of fecal matter from a donor into the recipient’s intestinal tract. FMT has been proven to be highly successful in treating recurrent Clostridioides difficile infections (Duquenoy et al., 2020). Researchers utilizing FMT observed that germ-free mice receiving post-middle cerebral artery occlusion (MCAO) donors’ microbiota had significantly larger brain infarct volumes and reduced behavioral performance compared to those receiving sham-surgery microbiota (Singh et al., 2016). A higher stroke dysbiosis index was associated with more severe brain damage and poorer outcomes (Xia et al., 2019). However, there is still limited understanding regarding the changes in microbiome composition among different enterotypes and their potential impact on the host. All human fecal samples were collected from inpatients after experiencing a stroke, leaving uncertainty about whether these alterations in the gut microbiome occurred before or were induced by stress shortly after the stroke event. Existing clinical studies are primarily cross-sectional, necessitating further longitudinal investigations to uncover the temporal dynamics and remodeling of the microbiome, with the goal of establishing an association between changes in gut microbiota composition and IS. Nonetheless, human microbiota research faces limitations due to significant heterogeneity among subjects. Factors such as genetic variations, dietary habits, lifestyles, age, and co-morbidities/co-medications can all influence the composition of the intestinal flora (Wen and Wong, 2017). These variables highlight the complexity of studying the gut microbiota and emphasize the need for comprehensive and longitudinal investigations to unravel the intricate relationship between microbiome changes and IS.

Immune cells also play a crucial role in the pathogenesis of ischemic stroke (IS). Our study identified 11 types of immune cells positively correlated with the risk of developing IS, while 5 types were negatively correlated. This suggests that the activity and balance of the immune system may have complex effects on the occurrence and progression of IS. Abnormal activation of immune cells can potentially contribute to IS through multiple pathways. Firstly, overactivated immune cells trigger local immune inflammation, leading to endothelial cell damage and ultimately facilitating the development of IS (Fernandez et al., 2019). Secondly, the activity of immune cells affects the coagulation system, increasing the risk of thrombus formation. Additionally, negatively correlated immune cells may reduce the risk of developing IS by regulating immunotolerance and decreasing abnormal immune responses (Li et al., 2022).

B cells have a complex role in the immune response to stroke, exhibiting both harmful and beneficial effects. Following an ischemic stroke, the compromised blood-brain barrier may allow B cells and other peripheral immune cells to enter the affected brain tissue (Malone et al., 2023). These cells play a critical role in regulating the progression of brain injury caused by ischemia and influencing the subsequent inflammatory response after a stroke (Schuhmann et al., 2017; Maheshwari et al., 2023). However, our findings align with this complexity, demonstrating that certain phenotypes associated with B cell activity, such as CD19 on CD24+ CD27+, CD20 on IgD- CD27-, CD25 on naive-mature B cell, CD27 on IgD- CD38br, IgD+CD38dim %B cell are linked to an increased risk of stroke. This highlights the necessity for further investigation into understanding the nuanced role of these specific B cell-related characteristics in strokes.

Similarly, T cells are primarily recognized for their pro-inflammatory effects in ischemic stroke as they infiltrate the blood-brain barrier, choroid plexus, and meninges (Huang et al., 2021). They contribute to the exacerbation of post-stroke inflammation by producing inflammatory cytokines like interferon (IFN)-gamma, interleukin (IL)-21, tumor necrosis factor (TNF), and IL-17 (Meng et al., 2019). The interaction between T lymphocytes and platelets can also worsen microvascular dysfunction and inflammation during ischemic stroke progression (Stoll and Nieswandt, 2019). However, our analysis indicates that T cells have a dual role in stroke pathogenesis due to their regulatory function in inflammation. This regulation is crucial since it impacts both brain injury and repair processes. T cells play a pivotal role in maintaining this balance. Our findings further support the inverse association between CD45RA on naive CD4+ cells and CD8 on CM CD8b with stroke occurrence. Notably, we highlight the significance of CD39 on activated CD4 Tregs as an essential immune element for neuroprotection during ischemic stroke. TD CD4+ %T cell and CM CD8br %CD8br are linked to an increased risk of stroke.

Previous studies suggest that initially, Tregs mitigate acute brain injury by modulating the immune response through their immunosuppressive capabilities and interactions with other immune cells such as neutrophils (Wang et al., 2015). Our findings demonstrate the intricate nature of Treg phenotypes in relation to stroke. While certain Treg phenotypes, such as CD28+ CD45RA+ CD8br %CD8br and Activated & resting Treg %CD4+, exhibit neuroprotective effects, others like CD127 on granulocyte, CD3 on CD39+ secreting Treg, CD4 on activated & secreting Treg, DN (CD4-CD8-) AC are associated with an elevated risk of stroke. This highlights the necessity for further investigation into the nuanced role played by different Treg phenotypes in the context of stroke.

The benefits of this study are notable. MR utilizes genetic variations as substitutes for environmental exposure, establishing a causal link between exposure and disease occurrence. Since genetic variants are assumed to be randomly determined before birth, they are independent of environmental factors, firmly established long before illness onset. This characteristic helps overcome issues associated with residual confounding and reverse causation common in traditional observational studies. Openly accessible datasets were employed in this research, providing more precise estimates and increased statistical power due to extensive sample sizes in GWAS. The findings remained unaffected by horizontal pleiotropy or other variables, ensuring the study’s statistical power to detect a significant association between gut microbiota/immune cells and IS. However, it’s important to acknowledge certain limitations. Firstly, to mitigate population stratification bias, participants of European descent were primarily included, potentially introducing bias into the findings. Secondly, the absence of demographic information such as gender and ethnicity in the original dataset prevented subgroup analyses. Thirdly, due to the insufficient number of SNPs meeting the genome-wide significance threshold (p < 5 × 10^–8), the study focused solely on SNPs reaching the locus-wide significance level (p < 5 × 10^–5). These constraints might limit the generalizability of results and potentially influence the study’s accuracy.

In summary, our extensive investigations reveal a correlation between specific gut microbiota features and immune cells with the occurrence of IS. Four bacterial characteristics and eleven immune cells exhibit a strong positive relationship with IS, suggesting their potential role as significant contributors. Conversely, two other bacterial features and five immune cells demonstrate an inverse relationship with IS, indicating a potential protective effect. These specific microbial strains and immune cell phenotypes emerge as promising biomarkers, offering valuable insights for the development of innovative strategies in the prevention and treatment of IS.

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

HS: Writing – original draft, Writing – review & editing. ZW: Writing – original draft. YX: Resources, Writing – original draft. YG: Validation, Writing – original draft. HM: Supervision, Writing – original draft. JG: Funding acquisition, Writing – review & editing.