Juvenile Idiopathic Arthritis (JIA) is characterized by chronic arthritis, synovitis, and erosion of bone and cartilage. It is the most common rheumatic disease in young individuals, with an incidence ranging from 3.8 to 400 individuals per 100,000 (Costello et al., 2021). This condition not only damages the joints of affected children but often accompanies systemic involvement, such as macrophage activation syndrome (MAS), iridocyclitis, uveitis, and other multi-systemic manifestations (Arve-Butler et al., 2021). In untreated or severe cases, it can lead to deformities in hand joints or permanent eye damage, significantly impacting the physical and mental health of young individuals (Ravelli and Martini, 2007).

Over the course of evolutionary coexistence, the gut microbiota has formed a mutually dependent symbiotic relationship with the human body. The gut microbiota and its metabolic products play crucial roles in assisting digestion and absorption of food, synthesizing vitamins and energy, protecting the intestinal mucosal barrier, participating in essential metabolism, resisting invasion by pathogenic microbes, and regulating immune mechanisms (Brusca et al., 2014; Sathyabama et al., 2014). Alterations in the gut microbial community (dysbiosis) and decreased richness of the gut microbiota are emerging as relevant factors in the development of inflammatory and systemic autoimmune diseases (Lin et al., 2014; Collado et al., 2015; Rogers, 2015). Recent studies have indicated a potential connection between changes in the gut microbiota and JIA (Aggarwal et al., 2017; De Filippo et al., 2019; Öman et al., 2021; Chaudhary et al., 2023; Prinz et al., 2023; Shi et al., 2023).

In patients with enthesitis-related arthritis (ERA), an increase in the abundance of microbial genus such as Rikenellaceae and Dorea has been observed, while the abundance of Prevotellaceae is decreased (Aggarwal et al., 2017). Emmaline Prinz made a similar observation in mice, where an increase in Rikenellaceae altered the immune status of mice, making them more susceptible to arthritis (R = 0.43, p = 0.001) (Prinz et al., 2023). While animal models can provide valuable insights, they cannot substitute for clinical research. Currently, clinical studies on the relationship between gut microbiota and JIA are limited, and existing studies often suffer from small sample sizes. Observational studies are unable to eliminate confounding biases and reverse causation, and the pathophysiological mechanisms by which certain microbial genus operate can be partially compensated by dietary habits. Consequently, conflicting studies with opposing conclusions may arise. For instance, some studies suggest a decrease (Shi et al., 2023) or no difference (Öman et al., 2021) in Rikenellaceae and Dorea (De Filippo et al., 2019) in JIA, even though microbial differences may not impact the onset and progression of JIA (Chaudhary et al., 2023). Hence, a new approach is needed to clarify the causal relationship between gut microbiota and JIA.

The Mendelian Randomization (MR) study design is a robust tool in epidemiological research. Its core idea is to utilize genetic variations as instruments to assess the causal relationship between risk factors and specific diseases (Beeghly-Fadiel et al., 2020; Titova et al., 2020; Ahmed et al., 2021; Lu et al., 2021). Confounding factors are significant sources of interference in causal inference within epidemiological research. In MR studies, genetic variations follow the principles of mendelian inheritance, where alleles are randomly assigned to offspring, resembling the process of a randomized controlled trial (Davey Smith and Hemani, 2014; Davies et al., 2018). MR studies can effectively eliminate confounding factors and avoid reverse causation, which are challenges in observational studies (Hemani et al., 2018). Therefore, our aim is to employ MR to determine the causal relationship between gut microbiota and JIA, while also evaluating the extent to which plasma metabolites influence the association between gut microbiota and JIA.

In this large-scale MR analysis, we identified causal relationships between 5 genus and JIA, with Rikenellaceae and Dorea demonstrating unidirectional causality. Rikenellaceae showed a positive correlation with the risk of JIA. Mediation analysis indicated that Furaneol sulfate levels mediate the effect of Dorea on JIA by 19.94% (95% CI [8.86–31.03%]). This analysis underscores the connection between gut microbiota and JIA, emphasizing the mediating role of Furaneol sulfate levels (Figures 3, 4).

Gut microbiota regulates immune responses through two main mechanisms: direct recognition by immune cells and metabolite-mediated immune response modulation. Short-chain fatty acids (SCFAs) not only regulate immune responses in the gut but also impact systemic multi-system functions. SCFAs act as inhibitors of histone deacetylases (HDACs) and ligands for G protein-coupled receptors (GPCRs), serving as signaling molecules that influence the immune system. SCFAs (butyrate, propionate, and acetate) as HDAC inhibitors affect peripheral blood mononuclear cells, leading to the inactivation of NF-κB and reduced production of the pro-inflammatory cytokine TNF-α (Usami et al., 2008). Another study further demonstrated that butyrate can regulate the function of intestinal macrophages by inhibiting HDAC, downregulating the production of pro-inflammatory factors, including NO, IL-6, and IL-12 (Chang et al., 2014). Bacteria such as Faecalibacterium, Ruminococcaceae, Parabacteroides, Clostridiales vadin BB60 group, and Roseburia are known producers of butyrate (Louis and Flint, 2009; Louis and Flint, 2017). Parabacteroides and Clostridiales vadin BB60 group also produce other SCFAs such as acetate, propionate, and hexanoate (Che et al., 2019; Lei et al., 2021). Our results suggest that the protective effects of Eubacteriumrectale, Catenibacterium, and Dorea against JIA may be attributed to their role in SCFA production. Studies have indicated a close association between Dorea and autoimmune diseases (Wang et al., 2023), with involvement in regulating the body’s immune checkpoint inhibition response (Liu et al., 2023), suggesting a potential link between Dorea and JIA.

Observational studies generally suggest a close association between gut microbiota and autoimmune diseases. In a comparison of baseline samples from Italian patients and healthy controls, patients exhibited an increase in the abundance of Erysipelotrichaceae, Faecalibacterium prausnitzii, Fusobacterium, Enterococcus, and Ruminococcaceae, while Allobaculum, Gemellaceae, Propionibacterium acnes, and Turicibacter were less abundant compared to healthy controls (van Dijkhuizen et al., 2019). Although increasing evidence indicates the role of gut microbiota dysbiosis in JIA, this field is still in its early stages. To date, studies on the composition and changes in the gut microbiota of JIA children, compared to healthy subjects, have been descriptive, and the potential functions of the microbiota remain speculative, making it challenging to establish causal relationships between microbial changes and JIA (De Filippo et al., 2019). Recent research suggests that metabolic processes and metabolites can influence disease risk and provide therapeutic targets. Understanding the causal role of metabolites in disease etiology can offer actionable intervention points for treatment. One approach to assessing the role of metabolites in disease outcomes is through human genetics. Many metabolite levels have a high heritability, providing an opportunity for MR, a causal inference method that uses genetic variation as IVs to test the role of exposures in disease outcomes. As alleles are randomly assigned at conception, this randomization process often breaks the confounding with most risk factors, thereby reducing the tendency to confound results (Chen et al., 2023). Interestingly, research has found that dysbiosis of the gut microbiota can influence autoimmunity (Bellés et al., 2022) and the progression (Chung and Kasper, 2010; Stoll et al., 2014) of inflammatory diseases by altering metabolite levels and ratios (Chen et al., 2023). To our knowledge, there is currently no in-depth study on the relationship between plasma metabolites and JIA. Our study results suggest that Furaneol sulfate levels may bridge the causal relationship between gut microbiota and JIA, laying the groundwork for exploring the intersection of gut microbiota and JIA and potentially inspiring new strategies for JIA treatment.

It is noteworthy that research results on the differences in gut flora in JIA patients are inconsistent. In a study of Italian children, the gut microbial diversity in JIA patients was significantly reduced compared to healthy subjects, with an increased abundance of the Dorea genus in JIA patients (De Filippo et al., 2019). The microbial composition of JIA children did not show significant differences from their healthy siblings (Öman et al., 2021). Reasons for result heterogeneity include, firstly, studies may not have considered potential confounding factors such as gender, race, diet, delivery mode, and medication use (Xin et al., 2021). Overall, JIA is more common in girls than boys (ratio of 2: 1). Systemic juvenile idiopathic arthritis is believed to occur in both sexes, while enthesitis-related arthritis is more common in boys than girls (Thierry et al., 2014). Different regions and races have different dietary habits; a high-fiber diet increases the ratio of Bacillota/Bacteroidota in the gut, promoting SCFA production, while a low-fiber, high-protein diet increases pro-inflammatory cytokine levels like IL-2 and IL-6 (De Filippo et al., 2010, 2019). Secondly, bacterial classification may differ between studies, contributing to result heterogeneity.

In systemic juvenile idiopathic arthritis (sJIA) patients, elevated levels of IL-6 have been found in blood and synovial fluid, and they are associated with disease activity (De Benedetti et al., 1997). IL-6 antagonists have been shown to be a potential therapy for refractory inflammatory diseases, similar to traditional corticosteroids (Ataie-Kachoie et al., 2013). In mice, copper disrupts the ecological balance and diversity of the gut microbiota, increasing Enterobacteriaceae while reducing the abundance of Bacteroidaceae, Ruminococcaceae, and Lachnospiraceae. However, Bacillus subtilis reverses copper toxicity by increasing taurine and L-glutamate levels while decreasing phosphatidylcholine and phosphatidylethanolamine, moving toward alleviating metabolic disruption (Gao et al., 2023). Olive oil supplementation prevents type 1 diabetes in NOD mice by modulating the gut microbiota and serum metabolites (Wang et al., 2023). Therefore, we hypothesize that finding suitable drugs or targets to reduce Furaneol sulfate levels may slow down the progression of JIA.

We were the first to employ MR to investigate the causal relationships among gut microbiota, plasma metabolites, and JIA. We not only utilized various common sensitivity analyses but also mitigated the impacts of confounding factors and reverse causation. Our preliminary findings suggest a causal relationship between gut microbiota and JIA, as well as the intermediary factors. This provides further theoretical support for the treatment and prevention of JIA and introduces new approaches to its management. For instance, JIA could initially be controlled through the regulation of specific gut microbiota via diet, medications, or other means. Additionally, co-regulating plasma metabolite levels may lead to significant breakthroughs in JIA prevention and treatment.

However, our study has several limitations. Firstly, the analyzed population is predominantly of European descent, somewhat limiting the generalizability of the findings. Secondly, due to limited GWAS data, we did not explore each subtype of JIA individually. Thirdly, our results remain theoretical and have not been validated through clinical or animal experiments, leaving the specific mechanisms unclear. Further cellular and animal experiments are needed to elucidate these mechanisms. Subsequently, we will seek to confirm the reliability of the results at the population level through randomized clinical trials. Lastly, we observed that only 19.94% of the effect was mediated by plasma metabolite Furaneol sulfate levels, which is relatively low, necessitating more research to quantify other mediators.

Our mediation analysis using MR indicates a causal relationship among gut microbiota, plasma metabolites, and JIA. Specifically, the metabolic pathway involving Furaneol sulfate mediates the regulatory effect of Dorea on JIA. The genetic evidence provided by our study underscores the connections between gut microbiota, plasma metabolites, and JIA. This suggests that future interventions could focus on improving gut microbiota and co-regulating Furaneol sulfate levels through medications, thereby enhancing prevention and treatment strategies for JIA.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

Ethical approval was not required for the studies involving humans because this two-sample MR study is based on publicly available summary data from genome-wide association studies (GWAS). All of these studies have obtained approval from the relevant institutional review boards, and participants have provided informed consent. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.

BG: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. ZW: Writing – original draft, Writing – review & editing, Conceptualization, Data curation, Investigation, Methodology, Software, Supervision. KW: Conceptualization, Data curation, Investigation, Methodology, Software, Supervision, Writing – original draft, Writing – review & editing. YL: Formal analysis, Funding acquisition, Project administration, Resources, Validation, Visualization, Writing – original draft, Writing – review & editing. YZ: Conceptualization, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. ZZ: Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. JC: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.