Juvenile idiopathic arthritis (JIA) is a heterogeneous condition characterised by arthritis of unknown origin manifesting before the age of 16 (1), often resulting in functional limitations and, in severe cases, disability (2). Globally, approximately three million children are affected by JIA (3, 4), making it the most prevalent chronic inflammatory rheumatic disease in childhood. Severe cases may exhibit persistent systemic symptoms, joint inflammation, severe drug side effects and macrophage activation syndrome (MAS) occurrence, which can pose life-threatening consequences, thereby placing a considerable burden on children’s health and socioeconomic systems (5). Juvenile idiopathic arthritis-associated uveitis (JIAU) is commonly acknowledged as a prevalent and severe extra-articular manifestation of JIA (6), characterised by chronic bilateral recurrent anterior uveitis, a leading cause of disability and visual impairment. The incidence of JIAU varies from 5.0% to 19.1% among JIA populations across different geographical regions (7). Although multiple factors, such as genetics, environment and immunity, are hypothesised to contribute to the pathogenesis and underlying mechanisms of JIA and JIAU (6, 8), their precise aetiology and pathogenesis remain unclear, necessitating further investigation to enhance diagnosis, treatment and reduce associated disease burden. The human intestinal microbiota, comprising approximately 100 trillion bacteria of 1000–1150 species, forms a symbiotic relationship with the host, playing critical roles in human metabolism, nutrient absorption, immune responses and other aspects (9). Additionally, the intestinal microbiota is intricately linked to the onset and progression of various human diseases. In the context of predictive, preventive and personalised medicine, systemic inflammation serves as an essential communication bridge between the human host and the gut microbiota (10). Recent evidence suggests a potential role of gut microbiota in immune-mediated diseases such as rheumatoid arthritis (RA), wherein dysbiosis of the gut microbiota disrupts intestinal barrier function, leading to increased permeability and immune imbalance. Consequently, this dysregulation allows immune cells to migrate to extraintestinal sites, including joints, thereby triggering localised inflammation (11, 12).

Recent case-control studies spanning three continents provide compelling evidence of the influence of gut microbiota dysbiosis on the onset and progression of JIA (13–16). A prospective study focussing on the gut microbiota in patients with JIA in Italy and the Netherlands reports the presence of gut microbial dysbiosis in this population. Moreover, significant differences in microbial diversity and composition exist between patients with JIA and healthy individuals (17). Another study conducted in China yielded similar findings, indicating that patients with JIA display a significantly lower abundance of Anaerostipes, Dorea, Lachnospira and Roseburia compared to the control group. The study also identified 12 genera that could potentially serve as biomarkers and predictive factors for JIA (18). Nevertheless, the causal relationship between JIA and the gut microbiota remains uncertain, necessitating further investigation. Furthermore, observational studies are prone to the impact of confounding variables and reverse causality, potentially biasing results (19).

To address these limitations, we employ Mendelian randomisation (MR) to investigate the genetic-level association between JIA or JIAU and gut microbiota. MR utilises single nucleotide polymorphisms (SNPs) as instrumental variables (IVs) to estimate potential causal links between exposure variables and health outcomes (20). Leveraging extensive genome-wide association studies (GWAS) data, we employ a bidirectional two-sample MR approach to explore the causal relationship between the gut microbiota and these diseases, offering novel perspectives into potential therapeutic strategies targeting the microbiota (21–23).

JIA is a common autoimmune rheumatic disease that poses a significant threat to the overall health and well-being of children, triggering joint deformities, functional impairments, stunted growth and osteoporosis, among other serious complications. Meanwhile, JIAU, a prevalent extra-articular complication of JIA, further complicates the clinical picture with chronic, non-granulomatous and non-infectious uveitis, often affecting the anterior segment of the eye. Approximately 10% to 14% of patients with JIA exhibit signs of uveitis before arthritis onset (41). Current treatment modalities for JIA and JIAU primarily focus on symptomatic relief, lacking definitive curative options (8). Previous studies have reported that siblings of patients with JIA have a higher risk of developing JIA than the general population, with the onset age, disease type and disease course of twin siblings exhibiting a similar trajectory (42). These findings indicate a genetic factor underlying JIA pathogenesis (43, 44). Moreover, subsequent studies have reported a genetic susceptibility locus for JIA, further confirming this speculation. However, as research advances, scientists have discovered that these genetic susceptibility factors can only explain 18% of the causes of JIA. Currently, the progression of JIA is speculated to be the outcome of the interaction between genetic susceptibility genes, environmental stimuli and immune dysregulation (45–47).

Emerging evidence underscores the microbiota’s involvement in immune-related disorders like RA and inflammatory bowel disease (IBD) (11). The intestinal microbiota predominately contributes to JIA pathogenesis by altering intestinal mucosal permeability and regulating the host immune system (48). Notably, altered microbial compositions, such as increased phyla Bacteroidetes and decreased Firmicutes, have been observed in paediatric patients with JIA compared to healthy individuals (15). This study suggests an increase in the abundance of class betaproteobacteria in patients with JIA, consistent with previous research findings (15).

In animal experiments, an extraperitoneal injection of intestinal bacterial cell wall components into mice has been demonstrated to induce arthritis in a conventional environment, but not in a sterile environment (49), suggesting a close relationship between gut microbiota alterations and arthritis development. Early investigations by Malin et al. (50) highlighted elevated bacterial urease activity in the stool samples of children with JIA compared to those of healthy children, suggesting an anaerobic dysbiosis of the intestinal flora. Interestingly, oral Lactobacillus administration to patients with JIA reduced urease activity. Results from a multicentre study conducted by van Dijkhuizen et al. (78 Italian children and 21 Dutch children) (17) revealed that Italian children with JIA exhibited an elevated abundance of Faecalibacterium prausnitzii, Erysipelotrichaceae, Enterococcus, Parabactteroides and Ruminococccaceae, but reduced levels of Allobaculum, Gemellaceae, Propionibacterium acnes and Turicibacter compared to the healthy control group. Similarly, our study also suggests an increase in the abundance of genera Ruminococcaceae UCG013 and Ruminococcaceae UCG003 in patients with JIA. Additionally, a significant decrease in the level of intestinal flora has been reported in samples from active and inactive states compared to healthy children (17). Kindgren et al.’s study on population queues (51) revealed a higher content of Acidaminococcales, Prevotella 9 and Veillonella parvula in JIA cases, while Coprococcus, Subdoligranulum, Phascolarctobacterium, Dialister spp, Bifidobacterium breve, Fusicatenibacter saccharivorans, Roseburia intestinalis and Akkermansia muciniphila exhibited reduced abundance. Notably, studies have linked the presence of Parabacteroides distasonis to an increased risk of subsequent JIA occurrence, alongside shorter breastfeeding duration and increased antibiotic exposure, particularly in genetically susceptible populations. Therefore, it can be concluded that environmental stimuli have a stronger effect on genetically predisposed infants and that microbial dysbiosis during infancy may initiate or accelerate the development of JIA. However, t our MR study findings indicate an increase in the abundance of genus Phascolarctobacterium in JIAU, contrasting previous results. This inconsistency may stem from JIAU’s nature as a complication of JIA, potentially exerting different effects on the gut microbiota. However, these findings support the notion that environmental stimuli exert a significant influence. In another cross-sectional investigation, Qian et al. reported that the JIA group exhibited a significant reduction in the relative abundance of four genera (Anaerostipes, didiister, Lachnospira and Roseburia) compared to the control group (18). These four genera are known to produce short-chain fatty acids (SCFAs), whose decrease is associated with severe clinical complications. Additionally, the study identified the genus Lachnospiraceae UCG001 as potentially reducing the risk of JIA. However, contrary to previous findings (18), an increase in the level of genus Roseburia in JIA was observed in our study.

Previous research consistently highlights differences in the gut microbiota composition between children with JIA and their healthy counterparts. However, the causal relationship between gut microbiota and the disease remains unclear. It is uncertain whether microbiota imbalance precedes JIA or arises as a consequence of long-term inflammation, abnormal metabolism or behavioural changes associated with JIA symptoms. Given the limitations of previous studies, including susceptibility to confounding factors and reverse causation effects, further investigation is warranted to elucidate the link between JIA or JIAU and the gut microbiota. In our study, leveraging GWAS data and MR analysis, we investigated the causality between JIA or JIAU and gut microbiota. In addition to our primary findings, we observed an elevated risk of FamilyXI, while genera Rikenellaceae RC9 gut group, Intestinimonas and Clostridium innocuum group were linked to decreased JIA risk. Conversely, a reduced abundance of genus Prevotella7 was associated with a decreased likelihood of JIAU. Furthermore, the abundance of order Burkholderiales, family Alcaligenaceae and genus Anaerofilum increased, while the abundance of genera Olsenella, Coprococcus2, Romboutsia and Eisenbergiella decreased in JIA cases. JIAU decreased the abundance of phylum Lentisphaerae, class Lentisphaeria, order Victivallales, family Peptostreptococcaceae, family Victivallaceae and genus Slackia. These novel findings contribute to existing knowledge by uncovering previously unexplored associations.

Given the pivotal role of gut microbiota in arthritis, targeted probiotics are emerging as a novel therapeutic avenue for rheumatic diseases. Since the mature and stable state of the microbiota once formed is difficult to change, and childhood is a critical period to acquire basic functions (such as immune tolerance to commensal microbiota), these findings present a unique opportunity for early intervention and potentially modifying the disease progression by targeting the microbiota (12). Clinical trials investigating prebiotics and probiotics are on the rise, with an expanding body of evidence reporting favourable tolerance and potential benefits for restoring infant microbiota to health (52, 53). However, a randomised controlled trial of probiotics by Shukl et al. demonstrated good tolerance in patients with Enthesitis-related arthritis (ERA) but did not show any favourable clinical or immunological effects compared to non-steroidal anti-inflammatory medication therapy (54). Therefore, to assess the efficacy and safety of probiotics for JIA, further clinical data are warranted to ensure better recommendations for clinical practice.

Our research offers several advantages. To the best of our knowledge, this is the first investigation to explore the causal relationship between JIA/JIAU and gut microbiota abundance using MR analyses. Relative to other research methods, MR analyses have several advantages in the study of the causal relationship between gut microbiota and JIA or JIAU. First, in terms of evidence-based medicine levels of evidence, MR has the third highest evidence-based rating after systematic reviews of randomised controlled trials (RCTs) and RCTs when RCTs are feasible, and the highest evidence-based rating when RCTs are not feasible, ranking first (55). Whereas, due to practical and ethical reasons, random assignment of specific gut flora cannot be done, RCT studies are not feasible when conducting causal studies of gut flora with JIA and JIAU. Observational study data are relatively more readily available and closer to real-world situations, but problems such as smaller sample sizes, confounding factors, and causal inversions often limit inferences about cause and effect (56), so appropriate causal modelling is needed to infer causal associations between exposure factors and disease outcomes. Animal studies have an even lower evidence-based rating. Therefore, for the study of the causal relationship of gut microbiota with JIA or JIAU, MR studies provide an effective way to solve the above problems. By utilising MR, which relies on the random allocation of allelic genes at conception, we established a temporal relationship (‘cause before effect’) between genetic variations and disease development, which is free from the influence of postnatal environmental factors and social behaviours. This approach minimised the risk of reverse causal linkages and allowed for more effective control of confounding variables. Moreover, to ensure the robustness of the auxiliary variables used in the MR analysis, we sourced GWAS data for gut microbiota, JIA and JIAU from the largest available database. Furthermore, to minimise the possible effects of weak IV bias, we adopted appropriate thresholds for the genomic instruments based on a threshold of p< 1.0×10^-5 in this bidirectional study. This criterion was selected based on the availability of an adequate number of SNPs with suitable statistical power for the majority of gut microbiota to effectively prevent confounding.

While our study boasts several strengths, it also has certain limitations. Firstly, although our analysis yields statistically significant p-values, they may not be as robust as those corrected using the Bonferroni method for significance. Nevertheless, our research is hypothesis-driven and supported by substantial biological data and past studies that establish the epidemiological link between gut microbiota and JIA or JIAU. However, future research may need to include samples from a larger population of patients with JIA and JIAU to further strengthen these results. Secondly, adjusting p-values for multiple comparisons may increase the risk of false positives and potentially weaken the number and multi-level structure of microbial communities (abundance and correlation between microbial strains) as well as the correlation between JIA or JIAU. Therefore, cautious interpretation is warranted when considering unfavourable outcomes or potentially significant p-values. Furthermore, the two-sample MR method is a theoretical causal analysis method, and the conclusions of our study may contradict epidemiological studies suggesting the impact of environmental risk factors on JIA or JIAU. Additionally, as the specific mechanisms of the gut microbiome in the onset of JIA or JIAU remain unclear, further research, including clinical studies and other ex vivo and in vivo studies such as animal experiments, is needed to verify the results of this study. New research techniques and methods such as metagenomics and metabolomics technologies, where available, are needed to further validate the results of the present study and to understand the relationship between the observed intestinal flora and JIA, as well as the mechanisms behind the relationship of JIAU. Lastly, as our study primarily includes participants of European descent in the JIA group, generalising our results to other ethnic populations should be done with caution. When GWAS summary data on JIA, JIAU and gut microbiota from other races become available in the future, we also hope to analyse and study them to improve the applicability and generalisability of these findings.

This study presents compelling evidence supporting a causal association between genetically predicted gut microbiota and JIA or JIAU development. This study underscores the significant interactive influence of intestinal flora on these conditions, suggesting their potential as novel biomarkers for diagnosis and prevention. These findings offer valuable insights that can aid in addressing and mitigating the impact of JIA or JIAU.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

J-BH: Conceptualization, Writing – original draft, Writing – review & editing, Formal analysis, Methodology, Validation, Visualization. Y-XC: Formal analysis, Writing – original draft, Writing – review & editing, Methodology, Validation, Visualization. Z-YS: Formal analysis, Validation, Writing – original draft, Writing – review & editing. X-YC: Methodology, Validation, Writing – review & editing. Y-NL: Conceptualization, Validation, Writing – review & editing. J-HY: Conceptualization, Funding acquisition, Writing – review & editing, Validation.