Lactobacillus Reuteri Prevents Progression of Ankylosing Spondylitis in Mice by Restoring Gut Microbiota-Metabolism Homeostasis

Lactobacillus Reuteri Prevents Progression of Ankylosing Spondylitis in Mice by Restoring Gut Microbiota-Metabolism Homeostasis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Lactobacillus Reuteri Prevents Progression of Ankylosing Spondylitis in Mice by Restoring Gut Microbiota-Metabolism Homeostasis Lianjun Yang, Ke You, Kun Wang, Bin Liu, Tao Chen, Zhifei Cui, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6109718/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Journal of Translational Medicine → Version 1 posted 4 You are reading this latest preprint version Abstract Background: Ankylosing spondylitis (AS) is a chronic inflammatory disease characterized by progressive spinal fusion and systemic inflammation. Recent studies suggest that gut microbiota plays a crucial role in the pathogenesis of AS. Methods: This study investigated the therapeutic effects of Lactobacillus reuteri (L. reuteri) on AS progression and its underlying mechanisms using a proteoglycan (PG)-induced mouse model. Female BALB/c mice (n=10/group) were randomized into control group, PG group and PG + L. reuteri group. Disease severity was assessed via arthritis scores, Micro-CT images, and histopathology. Serum cytokines (IL-1β, IL-18, IL-17A, IL-23) were measured by ELISA. Intestinal barrier integrity was evaluated using FITC-dextran permeability, immunofluorescence (ZO-1, occludin), and colon histology. Gut microbiota (16S rRNA sequencing) and fecal metabolites (untargeted metabolomics) were analyzed. AhR/NLRP3 pathway activity was assessed via qRT-PCR (AhR, CYP1A1, CYP1B1) and Western blot (NLRP3). Results: Our findings demonstrated that L. reuteri significantly alleviated AS progression, as evidenced by reduced joint swelling and erythema, alongside a decreased arthritis index and paw thickness. Furthermore, treatment with L. reuteri resulted in a marked reduction in serum levels of pro-inflammatory cytokines, including IL-1β, IL-18, IL-17A, and IL-23, indicating its potential to modulate systemic inflammation. Additionally, L. reuteri enhanced intestinal mucosal barrier function, as demonstrated by improved histopathological integrity, reduced intestinal permeability, and restored expression of tight junction proteins ZO-1 and occludin. Moreover, L. reuteri treatment restored gut microbiota composition and metabolite profiles, aligning them more closely with control groups. Notably, L. reuteri also regulated the Aryl hydrocarbon receptor (AhR)/NLRP3 pathway, increasing mRNA levels of AhR, CYP1A1, and CYP1B1 while decreasing NLRP3 expression. Conclusion: In conclusion, L. reuteri effectively prevents the progression of AS in mice by restoring gut microbiota-metabolism homeostasis and modulating inflammatory pathways, highlighting its potential as a therapeutic agent for AS. Ankylosing spondylitis Lactobacillus reuteri gut microbiota metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Ankylosing spondylitis (AS) is a chronic inflammatory disease primarily affecting the axial skeleton, particularly the spine and sacroiliac joints[ 1 ]. It is characterized by inflammation that can lead to pain, stiffness, and eventual fusion of the vertebrae, resulting in a condition often referred to as "bamboo spine" due to its rigidity. The disease typically presents in young adults, with a higher prevalence in males than females, and is strongly associated with the HLA-B27 antigen, although not all individuals with this antigen develop AS. The prevalence of AS varies, with estimates ranging from 0.1–1.4% in the general population, and it can significantly impact quality of life due to chronic pain and functional limitations[2 , 3]. The pathogenesis of AS is complex and not fully understood, involving both genetic predisposition and environmental factors, and is increasingly linked to alterations in the gut microbiota. The relationship between AS and intestinal flora involves several interconnected mechanisms, including dysbiosis, immune response modulation, and genetic predisposition. Dysbiosis, or an imbalance in the gut microbiota, has been observed in AS patients. Studies have shown that AS is associated with a higher abundance of certain bacterial taxa, such as Proteobacteria , and a decrease in beneficial bacteria such as Bacteroidetes and Firmicutes [4 , 5]. This microbial imbalance can lead to increased intestinal permeability, often referred to as a "leaky gut," which allows microbial products to enter the bloodstream and potentially trigger systemic inflammation [6 , 7]. The presence of specific bacteria, such as Klebsiella pneumoniae , has been implicated in the pathogenesis of AS due to its structural similarities to human antigens, which may lead to molecular mimicry and autoimmune responses[ 8 ].In AS, the dysbiotic gut microbiome can influence the activation of T helper 17 (Th17) cells, which are known to produce pro-inflammatory cytokines such as IL-17. This cytokine is central to the inflammatory processes observed in AS[ 9 ]. The interaction between gut bacteria and the immune system can lead to an exaggerated immune response, contributing to the chronic inflammatory characteristic of AS. Furthermore, certain microbial metabolites, such as short-chain fatty acids (SCFAs), can modulate immune responses and have been shown to have anti-inflammatory effects, suggesting that healthy microbiota could potentially mitigate AS symptoms [ 6 ]. Current clinical treatments for AS primarily include non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), and biologics targeting specific inflammatory pathways. However, these therapies often have limited efficacy and can be associated with significant side effects, including gastrointestinal complications and increased risk of infections. Furthermore, many patients do not achieve adequate disease control, highlighting the need for alternative therapeutic strategies. Therapies aimed at restoring the gut microbiota balance, such as probiotics or fecal microbiota transplantation (FMT), are being explored as potential adjunctive treatments for AS. Lactobacillus reuteri ( L. reuteri ), a well-studied probiotic, has been shown to exert various beneficial effects on the immune system and gut health, which are crucial for preventing and managing immune-related disorders. In models of collagen-induced arthritis, pre-administration of heat-killed L. reuteri significantly reduced the severity and incidence of arthritis. This effect was associated with a decrease in pro-inflammatory cytokines (IL-6) and an increase in anti-inflammatory cytokines (IL-10)[ 10 ]. The ability of L. reuteri to regulate gut microbiota composition is another area of significant research. In colitis models, administration of L. reuteri has been associated with reduced intestinal inflammation and restored the balance of intestinal microbiota[ 11 ]. Moreover, L. reuteri plays a crucial role in maintaining intestinal barrier integrity, which is essential for preventing systemic inflammation and autoimmune responses[ 12 ]. Given the interplay between the gut microbiota and immune responses in AS, exploring the therapeutic potential of L. reuteri in preventing disease progression is a compelling avenue of research. However, while promising, current research on the effects of L. reuteri in AS remains limited, with a need for further investigation into the underlying mechanisms and the specific metabolic pathways involved. This study aims to investigate the effects of L. reuteri on the progression of AS in a murine model, focusing on its role in restoring gut microbiota composition and metabolic homeostasis. Understanding these mechanisms may provide insight into novel therapeutic strategies for managing AS. 2. Results 2.1. L. reuteri attenuated the disease progression and AS-associated symptoms in PG-induced AS Mice The assessment of the peripheral arthritis index and paw thickness provides both intuitive and objective measures to reflect the severity of AS. From the 7th week after induction, AS mice in the PG group gradually developed paw swelling and redness (Figure. 1B), with an increasing arthritis index (Figure. 1C) and paw thickness (Figure. 1D), whereas no such symptoms were observed in the control group, confirming the successful establishment of the AS mouse model. However, oral administration of L. reuteri significantly alleviated paw swelling and redness in AS mice. Compared to the PG group, the arthritis index and paw thickness in the L. reuteri -treated group were significantly reduced (P < 0.05). Pathological examination of the ankle joint with HE staining showed that the ankle joint tissues of the control group mice had a normal structure, with no signs of synovial hyperplasia, inflammation, cartilage degradation, or bone erosion, and the joint space was maintained normally (Figure. 1E). In contrast, the PG group mice displayed significant arthritis, characterized by pronounced synovial hyperplasia, inflammatory cell infiltration, cartilage erosion, and bone erosion (Figure. 1E). Notably, treatment with L. reuteri significantly reduced the synovial inflammation score, cartilage degeneration score, and bone erosion score in the L. reuteri group compared to the PG group (P < 0.05, Figure. 1H), indicating a significant suppression of arthritis. Meanwhile, in the control group, the intervertebral tissue structure appeared normal, with intact annulus fibrosus and nucleus pulposus and no infiltration of inflammatory cells. In contrast, AS mice in the PG group exhibited disrupted intervertebral structures (Figure.1F), extensive infiltration of inflammatory cells around the intervertebral discs, excessive bone matrix formation, and destruction of the intervertebral spaces. Following L. reuteri treatment, the infiltration of inflammatory cells around the intervertebral discs was significantly reduced, and the extent of bone matrix formation and intervertebral disc destruction was markedly alleviated (Figure. 1H, P < 0.01). Micro-CT scanning revealed comparable findings, with the PG group displaying bone destruction and loss, whereas mice treated with L. reuteri exhibited milder bone deterioration( Figure. 1G). 2.2. L. reuteri attenuates systemic inflammatory response Compared with the control group, serum levels of pro-inflammatory cytokines IL-1β, IL-18, IL-17A, and IL-23 were significantly elevated in the PG group (P < 0.01). These findings indicate an enhanced inflammatory response in the AS model. In contrast, treatment with L. reuteri significantly reduced the serum levels of IL-1β, IL-18, IL-17A, and IL-23 in AS mice (P < 0.05), suggesting that L. reuteri ameliorates systemic inflammation by modulating the inflammatory cytokine profile. 2.3. L. reuteri improves ileum tissue pathology and enhances intestinal mucosal barrier function in AS mice. Studies have shown that approximately 60% of AS patients exhibit subclinical intestinal inflammation upon colonoscopic examination[ 13 ]. Therefore, we used HE staining to determine whether intestinal mucosal changes accompany AS progression in mice. Pathological observation under a light microscope revealed dense, intact, and well-aligned ileal villi in the control group (Figure.3A). In contrast, the PG group exhibited disorganized villi, shortened villus length, increased crypt depth, and a reduced villus height-to-crypt depth ratio compared to the control group (Figure. 3B-D, P < 0.05). Following L. reuteri treatment, the ileal morphological abnormalities in the L. reuteri group were significantly improved compared to the PG group, with statistically significant differences (P < 0.05). To assess intestinal permeability, we measured serum DX-4000-FITC levels, a fluorescent tracer indicative of gut barrier integrity. Compared to the Control group, PG-induced AS mice exhibited significantly elevated serum DX-4000-FITC levels (P < 0.05), indicating increased gut permeability. Treatment with L. reuteri significantly reduced serum DX-4000-FITC levels in AS mice (P < 0.01), demonstrating its ability to restore gut barrier function (Figure.3H). Immunofluorescence analyses further revealed decreased protein expression levels of tight junction proteins ZO-1 and occludin in the ileum of the PG group compared to the Control group (P < 0.05). After L. reuteri treatment, the expression levels of ZO-1 and occludin were partially restored (Figure.3E-G, < 0.05), highlighting the protective effect of L. reuteri on intestinal epithelial barrier integrity in AS mice. 2.4. Effect of L. reuteri on the composition of gut microbiota in AS mice We used 16s rRNA amplicon sequencing to investigate if L. reuteri affected the intestinal bacterial composition in AS mice. Using Venn diagrams, we observed overlapping OTU data for the three groups, which helped us identify microbes across the groups. It was found that 364 of the 450 OTUs were shared by all groups (Fig. 4 A). Notably, the L. reuteri treatment group exhibited 11 unique microbes, while the groups designated as PG and control displayed 5 and 16 distinct microbes, respectively. Alpha diversity was measured using the Chao1 and observed species. The Chao1 and observed species indices showed a significant decrease in the PG group compared to the control groups (p < 0.05, Figure. 4B-C). Conversely, we found that Chao1 and observed species indices were significantly increased in the L. reuteri treatment group compared with the PG group, aligning them more closely with the control group’s levels. Principal-coordinate analysis (PCoA) of the unweighted UniFrac distances was performed to evaluate the β diversity among the three groups. It can be observed from the PCoA plot (Fig. 4 C-D) that the samples in the PG group and the control group show significant separation, indicating a substantial difference in microbial community structure between the two groups. In contrast, the distance between the L. reuteri group and the control group is reduced, suggesting a higher similarity in microbial community structure between these two groups. Linear discriminant analysis (LDA) combined with effect size analysis (LEfSe) was used to identify different bacterial taxa among different groups (p 2.0). The alterations in intestinal microbiota in AS and L. reuteri -treated mice were further analyzed at both the phylum and genus levels. At the phylum level, the gut microbiota of mice in each group was predominantly composed of Bacteroidetes , Firmicutes , Proteobacteria , and Verrucomicrobia , collectively accounting for approximately 99% of the intestinal microbiota in all groups ( Figure.5A). In the control group, Bacteroidetes had the highest proportion at 61.1%, followed by Firmicutes at 33.9%. Compared to the control group, the proportion of Firmicutes in the PG group increased significantly to 54.4% (p < 0.05), while Bacteroidetes decreased to 38.3% (p < 0.05). After treatment with L. reuteri , the proportion of Bacteroidetes increased to 65.2%, while the proportion of Firmicutes decreased to 31.5%. The Bacteroidetes/Firmicutes ratio was 1.8 in the control group, decreased to 0.7 in the PG group, and increased to 2.07 in the L. reuteri -treated group. At the genus level, we observed that the microbial composition in the PG group differed significantly from that in the control group (Figure.5B). We identified a total of 11 genera that exhibited significant differences among the groups (p < 0.05). These differences at the genus level were visualized using a heatmap (Figure.5C). Compared to the control group, the abundances of Alloprevotella , Barnesiella , and Eubacterium were significantly decreased in the PG group, while Brachyspira , Parabacteroides , and Rikenella were significantly increased (p < 0.05). Compared to the PG group, the L. reuteri group showed a significant increase in the abundances of Alloprevotella , Barnesiella , and Eubacterium , and a significant decrease in the abundances of Brachyspira and Parabacteroides (p < 0.05). Furthermore, compared to the other two groups, the L. reuteri group exhibited increased abundances of Veillonella and Paraprevotella . Finally, the PICRUSt analysis based on KEGG database used to analyze the functional differences between different groups (Figure.5D). In the control group, 3 KEGG pathways were enriched; in the PG group, 6 KEGG pathways were enriched; and in the L. reuteri group, 9 KEGG pathways were enriched.The enriched KEGG pathways in the control group mainly included Amino Acid Metabolism, Energy Metabolism, and Transport and Catabolism. In the PG group, the enriched KEGG pathways primarily involved Membrane Transport, Cell Motility, Transcription, Signal Transduction, Environmental Adaptation, and Xenobiotics Biodegradation and Metabolism.For the L. reuteri group, the enriched KEGG pathways were primarily associated with Replication and Repair, Nucleotide Metabolism, Metabolism of Other Amino Acids, Biosynthesis of Other Secondary Metabolites, Cell Growth and Death, Digestive System, Signaling Molecules and Interaction, Immune System Diseases, and Metabolic Diseases. 2.5. Effect of L. reuteri on serum metabolomics in AS mice Based on untargeted serum metabolomics for multivariate analyses, principal component analysis (PCA) showed that a clear separation could be observed between the Control and PG group, and the L. reuteri group located much closer to Control group both in positive and negative ion mode (Figure. 6A-B). The clustering pattern further evidenced that L. reuteri could protect against AS. Furthermore, the findings of the serum metabolomics analysis revealed significant differences in the expression of 11 metabolites (P 2, and VIP > 1) between the three groups. The heatmap in Figure. 6C illustrates the changes in the levels of these 11 metabolites across the different groups. Compared to the control group, the levels of maleic acid, D-malic acid, phenol sulfate, cis-aconitate, citric acid, 3-indoxyl sulfate, 4-methylumbelliferone, and 2,4-dihydroxyacetophenone 5-sulfate were significantly decreased in the PG group (p < 0.05). After treatment with L. reuteri , the levels of these metabolites increased significantly. Additionally, compared to the control group, the levels of seselin and LysoPS 18:0 were elevated in the PG group (p 1 and an impact > 0.05), Figure. 6D highlights the most affected metabolic path ways, including Citrate cycle (TCA cycle) and Glyoxylate and dicarboxylate metabolismmetabolism. 2.6. Correlation analysis To investigate the potential relationships between these differential serum metabolites, and the abundance of different species, we conducted Spearman correlation analysis.To assess the strength of the association between a specific metabolite and a microorganism, the corresponding correlation coefficient is used. From the Figure. 6E, it can be observed that Eubacterium shows a strong positive correlation with 2,4-dihydroxyacetophenone 5-sulfate, 4-methylumbelliferone, and maleic acid, while displaying a negative correlation with LysoPS 18:0 and seselin. Alloprevotella exhibits a strong positive correlation with D-malic acid, 4-methylumbelliferone, maleic acid, cis-aconitate, and 2,4-dihydroxyacetophenone 5-sulfate, and a moderate negative correlation with seselin. Barnesiella shows a positive correlation with 3-indoxyl sulfate and citric acid.These findings suggest complex interactions between the gut microbiota and serum metabolites. 2.7. The intervention effect of L. reuteri on AhR/NLRP3 Pathway The results of the metabolic profile suggest that treatment with L. reuteri can effectively increase the content of the 3-indoxyl sulfate which is an Aryl hydrocarbon receptor(AhR) agonist. Recent research studies have found that AhR can negatively regulate NLRP3 expression[ 24 ].Therefore,our plan is to investigate whether L. reuteri can impact the AhR/NLRP3 pathway.The RT-qPCR was performed to evaluate the effects of L. reuteri on the expression of related genes in the AhR/NLRP3 pathway. As shown in Figure.7, the relative mRNA expression levels of AhR, CYP1A1, and CYP1B1 were significantly decreased, whereas NLRP3 expression was increased in the PG group (p < 0.01), compared to the control group. Following treatment with L. reuteri , the mRNA expression levels of AhR, CYP1A1and CYP1B1 were elevated, while NLRP3 expression was markedly reduced. These findings indicate that L. reuteri modulates the AhR/NLRP3 pathway and plays a crucial role in inhibiting inflammation in AS mice. 3. Discussion AS is associated with significant morbidity and can lead to progressive spinal fusion, resulting in debilitating pain and impaired mobility. The etiology of AS remains largely elusive, although genetic predisposition, particularly the presence of the HLA-B27 antigen, is recognized as a key factor. Emerging evidence suggests that dysbiosis of gut microbiota may contribute to the onset and progression of AS by modulating immune responses and promoting systemic inflammation. Probiotic interventions have been shown to reshape gut microbiota and have a positive impact on inflammatory diseases. In light of these complexities, this study investigates the therapeutic potential of L. reuteri in modulating gut microbiota to address AS. L. reuteri is known to influence the gut microbiome and its metabolic outputs, which can have systemic effects on the host. For instance, a study demonstrated that L. reuteri alleviates the severity of autoimmune arthritis by promoting the peripheral migration of gut-derived Tregs[ 14 ].Utilizing a well-defined animal model of AS, our research aims to elucidate the mechanisms by which L. reuteri may inhibit disease progression. Key findings indicate that L. reuteri not only alleviates joint and spinal damage but also exerts anti-inflammatory effects by reducing levels of pro-inflammatory cytokines and restoring gut microbiota-metabolism homeostasis. These results provide a promising foundation for developing novel therapeutic strategies targeting gut microbiota in the management of AS. Studies have highlighted the potential of L. reuteri in reducing inflammation and improving gut barrier function, which are crucial in managing chronic inflammatory conditions like AS. Our results show a significant reduction in serum levels of IL-1β, IL-18, IL-17A and IL-23 after L. reuteri treatment, suggesting this probiotic can attenuate the inflammatory response in AS. Elevated levels of these cytokines are hallmarks of AS pathogenesis, contributing to inflammation, bone erosion, and pathological new bone formation. The observed reduction in IL-17A and IL-23 is particularly significant, given the central role of the IL-23/IL-17 axis in AS pathogenesis. By decreasing IL-23 and IL-17A levels, L. reuteri likely disrupts this axis, mitigating the inflammatory response. These findings are consistent with recent studies demonstrating the anti-inflammatory effects of L. reuteri . For example, Gao et al.reported that Lactobacillus strains can decrease IL-8, IL-17A, IL-6, and TNF-α levels levels in intestinal epithelial injury model, thereby further supporting the immunomodulatory effects of L. reuteri [ 15 ].Similarly, Jiang et al. indicated that L. reuteri alleviated the elevation of serum inflammatory cytokines (IL-1α, IL-2, and IL-18) and partially restored gut microbiota and metabolome dysbiosis in acute liver failure. This shift in cytokine levels lends further credence to the notion that L. reuteri not only halts disease progression but actively reconfigures the inflammatory landscape associated with AS. Up to 70% of AS patients have evidence of terminal ileitis resembling IBD, suggesting gut inflammation is important in disease pathogenesis[ 16 ]. ZO-1 and occludin are crucial components of the intestinal epithelial barrier, which regulates the permeability of the gut and prevents the translocation of harmful substances and pathogens. Disruption of these proteins can lead to increased intestinal permeability, often referred to as "leaky gut," which has been implicated in various inflammatory diseases, including AS[ 17 ].Our findings align with these observations, as PG-induced AS mice displayed significantly higher serum DX-4000-FITC levels, indicative of compromised gut barrier function. In contrast, L. reuteri treatment significantly reduced gut permeability and restored ZO-1 and occludin expression. Our study corroborates reports linking altered gut permeability to systemic inflammation in AS and adds evidence of L. reuteri 's reparative effect on tight junction proteins. This restoration may facilitate gut-homeostasis, ultimately leading to improved systemic health outcomes. What sets our study apart is its emphasis on the connection between gut microbiota restoration and immune regulation in AS. While previous research has primarily focused on immune cells in inflammation, our study suggests that L. reuteri may alleviate inflammation by restoring gut microbiota balance. Our findings indicate that L. reuteri treatment reverses gut microbiota dysbiosis in AS mice by restoring the Firmicutes/Bacteroidetes balance and promoting the growth of beneficial bacteria such as Alloprevotella and Barnesiella while reducing harmful bacteria like Brachyspira . Increased abundances of beneficial genera such as Alloprevotella and Barnesiella following L. reuteri treatment may enhance short-chain fatty acid production, which has been shown to reduce inflammation and support gut barrier integrity[ 18 ].Our KEGG pathway analysis further confirms that L. reuteri improves metabolic and immune functions, particularly pathways associated with amino acid metabolism and immune regulation. The observed changes in gut microbiota composition, with L. reuteri restoring the relative abundance of beneficial bacteria, further support the hypothesis that gut dysbiosis plays a crucial role in the development of AS [ 19 ]. In addition to its effects on gut microbiota, L. reuteri has been shown to impact the metabolomic profile of the host. Compared to prior studies that focused on inflammatory cytokines, our work integrates microbiota and metabolomic changes, providing a holistic view of AS pathophysiology. The metabolomic findings demonstrate that L. reuteri treatment effectively modulates key metabolic pathways disrupted in AS. The observed recovery of metabolites such as citric acid and cis-aconitate suggests that L. reuteri may modulate the TCA cycle, which plays a central role in cellular energy metabolism and is often dysregulated in inflammatory conditions[ 20 ]. Additionally, reductions in pro-inflammatory metabolites like LysoPS 18:0,a phospholipid associated with immune cell activation, indicates that L. reuteri may modulate immune responses[ 21 ].Notably, the identification of specific metabolites like indole-3-lactic acid which is a potent endogenous agonist for AhR and have immunomodulatory effects that may mitigate AS-related inflammation. Recent study has shown that bacterial metabolites of tryptophan, such as indoxyl-3-sulfate, exhibit significant anti-inflammatory effects. In experimental autoimmune encephalomyelitis (EAE) mice fed a tryptophan-deficient diet, the disease course was prolonged, and symptoms worsened. However, upon re-administration of indoxyl-3-sulfate, the condition of the EAE mice improved. This study also revealed that indoxyl-3-sulfate act on microglia through the AhR, thereby regulating astrocytes to exert anti-inflammatory effects[ 22 ].Moreover,indoxyl-3-sulfate can promote an anti-inflammatory and tolerogenic environment by modulating dendritic cell function and indirectly reducing T cell-mediated inflammation[ 23 ]. The upregulation of AhR and its downstream targets, CYP1A1 and CYP1B1, suggests that L. reuteri enhances the activation of pathways involved in anti-inflammatory responses. L. reuteri and its metabolites, including indole-3-lactic acid, have been shown to interact with AhR pathway which plays a crucial role in regulating immune responses and maintaining intestinal homeostasis. The activation of AhR can lead to the suppression of NLRP3 inflammasome activation, thereby reducing the production of pro-inflammatory cytokines and alleviating inflammation[ 24 ].The involvement of the NLRP3 inflammasome in the pathogenesis of AS has been highlighted in various studies, where it is known to contribute to the production of pro-inflammatory cytokines such as IL-1β and IL-18, which exacerbate the inflammatory response[ 25 ].Our results show that by promoting a healthy balance of gut bacteria, L. reuteri can influence the production of metabolites that activate AhR, thereby indirectly inhibiting the NLRP3 expression and reducing inflammation. This probiotic's ability to restore gut microbiota balance and its interaction with the AhR/NLRP3 pathway suggest a promising therapeutic strategy for managing ankylosing spondylitis. In conclusion, this research provides compelling evidence that L. reuteri can inhibit the progression of ankylosing spondylitis in a mouse model, primarily through the modulation of gut microbiota, improvement of metabolic profiles, and regulation of AhR/NLRP3 inflammatory pathway. These findings not only highlight the therapeutic potential of L. reuteri in managing AS but also underscore the critical role of gut health in maintaining systemic immune homeostasis. However, this study has limitation. while the murine AS model provides valuable insights, the extrapolation of these findings to human AS patients requires further investigation. 4. Materials and Methods 4.1. Experimental design L. reuteri (strain designation 100 − 23, DSMZ 17509) was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany)[ 26 ]. L. reuteri strains were inoculated into MRS medium (Guangdong Huankai Microbial Technology Co., Ltd, China) and incubated anaerobically at 37°C for 18 hours. Then, L. reuteri strains were collected by centrifugation and resuspended in saline to achieve a concentration of 5×10 9 CFU/mL. Female BALB/c mice at 8 months were purchased from Beijing Huafukang Biotechnology Co., Ltd. These mice were housed in a temperature-controlled condition with a suitable indoor temperature and humidity, ad libitum access to water and a 12-hour light-dark cycle. All animal experiments were carried out with the approval of the Institutional Animal Care and Ethics Committee of Southern Medical University. We chose the proteoglycan (PG) induced inflammation mouse model as the AS animal which were induced using previously described standard methods[27 , 28]. Briefly, on days 0, 21, and 42, mice were intraperitoneally injected with emulsified 100 µg PG (Sigma-Aldrich, USA) and 2 mg of dimethyl dioctadecylammonium bromide (Sigma-Aldrich, USA). After modeling, the mice were randomly assigned to three groups (n = 10 per group): Control group: healthy BALB/c mice receiving equal volumes of saline during the schedule, AS model group (PG group): AS mice receiving equal volumes of saline during the schedule, L. reuteri group: AS mice were administered 0.2 mL of the L. reuteri suspension(1x10 9 CFU/day/mouse) for 4 weeks continuously. The dosage of L. reuteri used was based on a previous publication [ 26 ]and our preliminary experiments. Treatment was started in the 10th week after PG induction and lasted for 4 weeks. The total experiment time last 14 weeks. 4.2. Clinical and histological assessments of arthritis and spondylitis The assessment of peripheral arthritis severity was conducted daily by observing changes in the swelling of the mice's paws. The criteria for scoring arthritis were established as follows (0–4 points)[ 29 ]: 0 = normal, indicating no arthritis; 1 = slight swelling and/or redness in a single joint; 2 = moderate swelling and redness found in one or more joints of the paw; 3 = substantial erythema and swelling across all joints and ankles; 4 = extensive swelling affecting the entire paw. To evaluate the severity and progression of the disease, the cumulative score from all paws of each mouse was utilized, with a maximum potential score of 16 points. The greatest thickness recorded in either of the two hind paws of each mouse was concurrently measured using a Vernier caliper. A micro-CT was performed for each mouse with ScancoMicroCT u40 (Scanco, Bassersdorf, Switzerland) at week 14. At the 14-week mark, following euthanasia, tissues from the paw and spine were collected, fixed in 4% paraformaldehyde, decalcified using 10% EDTA for one month, embedded in paraffin, sectioned into slices of 5µm thickness, and subsequently stained with hematoxylin and eosin (H&E).Histological scoring of the ankle joint: After the ankle joint of the mice was sliced, H&E staining was conducted to assess pathological changes in the tissues and to assign scores. The scoring focused on synovial inflammation, cartilage degeneration, and bone erosion. Histological scoring of the spine: The grading of spondylitis histology was categorized from 1 to 4:1 = minor accumulation of inflammatory cells and/or involvement of the annulus fibrosus surrounding the intervertebral disc (IVD); 2 = mild inflammation with IVD absorption or erosion (less than 50% of IVD); 3 = severe inflammation affecting the IVD with significant loss of intervertebral space (greater than 50%); 4 = total cartilaginous or bony ankylosis. Each mouse's total score was determined by averaging the scores of 10 intervertebral joints, as previously described. 4.3. Measurement of cytokines in the serum At the end of week 14, the mice were anesthetized using isoflurane, and blood samples were obtained through cardiac puncture. Once the blood coagulated, serum samples were extracted by centrifugation at 1500×g for 10 min and preserved at -80°C. The concentrations of inflammatory cytokines IL-1β, IL-18, IL-17A, and IL-23 were measured in accordance with the ELISA kit's(ABclonal Technology TM, Wuhan, China)guidelines. 4.4. In vivo intestinal permeability assessment During the 14th week of the experiment, the mice underwent a fasting period lasting six hours, during which they were not provided with any water. Following this fasting period, the mice received an administration of 4 kDa fluorescently labeled dextran (DX-4000-FITC, Sigma-Aldrich) at a dose of 600 mg/kg body weight (125 mg/ml). After one hour had elapsed from the administration of the fluorescent dextran, blood samples of 300 µl were obtained through the eye socket for the purpose of preparing serum necessary for fluorescence measurement. To ensure accurate analysis, the collected serum sample was diluted with an equal volume of phosphate-buffered saline (PBS). The concentration of FITC in the diluted serum was then determined using a fluorescence spectrophotometer, set to an excitation wavelength of 485 nm and an emission wavelength of 535 nm. To facilitate this quantitative analysis, a standard curve was created for the concentration of DX-4000-FITC by systematically diluting the DX-4000-FITC solution with PBS. 4.5. Histopathologic evaluation and immunofluorescence staining Following the euthanasia of the mice, the tissue from the small intestine was promptly extracted and preserved in a 4% paraformaldehyde solution. Subsequently, standard procedures for paraffin embedding were employed to process the tissue. The resulting intestinal tissue blocks were sliced into standard 5µm sections and then stained with H&E for histological examination. The measurement of villus height was taken from the apex of the villus down to the villus-crypt junction, whereas crypt depth was characterized as the measurement from the villus base to the muscularis layer. In each section, the heights of 9 intact villi and the depths of 9 crypts were recorded. Immunofluorescence staining: The slides were blocked with 5% fetal bovine serum (FBS) for 30 min at 37°C and incubated with primary antibodies (Occludin and ZO-1) overnight at 4°C as fellow: Occludin (1: 500, Thermo Fisher Scientific, USA), ZO-1 (1: 500, Thermo Fisher Scientific, USA). The slides were washed three times with PBS-Triton and incubated with FITC-conjugated goat anti-rabbit secondary antibody (Bioss, China) or Cy3-conjugated goat anti-rabbit secondary antibody (Bioss, China) at 37°C for 30 min. Slides were washed three times with PBS and the nuclei were stained with DAPI (Bioss, China) at room temperature for 10 min. Slides were observed under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany), and the pictures were analyzed by Image-Pro plus 6.0. 4.6.DNA extraction and 16S rRNA gene sequencing Fecal samples were meticulously collected in sterile containers twelve hours following the last gavage and were promptly frozen at a temperature of -80°C to preserve their integrity. To isolate microbial DNA from the fecal material of each sample, the researchers employed the QIAamp DNA Stool Minikit(Qiagen Ltd, Strasse, Germany). Subsequently, next-generation sequencing of the 16S rDNA was conducted using the Illumina HiSeq PE250 platform, with the sequencing process carried out by the Realbio Genomics Institute located in Shanghai, China. The amplification of the V3–V4 region of the 16S rDNA gene sequence was executed from the genomic DNA utilizing the specific primer pair F341 (5′-ACTCCTACGGGRSGCAGCAG-3′) and R806 (5′-GGACTACVVGGGTATCTAATC-3′). Following the amplification, the raw sequencing data underwent a stringent quality control procedure utilizing the UPARSE software. In the subsequent step, the clustered reads were sorted into operational taxonomic units (OTUs) exhibiting at least 97% similarity using Usearch, a widely recognized tool for microbial analysis. To analyze the data thoroughly, principal coordinate analysis (PCoA) was performed, alongside heatmap analysis and species abundance assessments, utilizing the statistical programming language R. Furthermore, to investigate the differences in the relative abundance of gut microbiota among the control, PG, and L. reuteri groups, linear discriminant analysis effect size (LEfSe) was employed in conjunction with the Kruskal-Wallis rank sum test. The analysis established a threshold for significance in the LEfSe results based on a log linear discriminant analysis (LDA) score greater than 2. PICRUS was utilized to predict metagenome functional profiles derived from the 16S rRNA data obtained from the sequencing to further enhance understanding of the microbial community and its functions[ 27 ]. 4.7. Metabolomics analysis Untargeted LC-MS metabolite profiling analysis on serum samples was conducted by Hangzhou Lian-Chuan Biotechnology Co., Ltd. The details of experimental protocols were performed as described previously[ 30 – 32 ]. An LC-MS system (TripleTOF 5600, AB SCIEX, USA) was utilized to analyze the metabolomics in positive and negative ion modes. The identified metabolites were annotated using the KEGG database ( https://www.genome.jp/kegg/pathway.html ) and the HMDB database ( https://hmdb.ca/metabolites).T o obtain the correlation of the samples, principal component analysis (PCA) was conducted on the standardized data to provide an overview of group clustering and to identify potential outliers. Supervised PLS-DA was executed utilizing metaX to differentiate the diverse variables across the groups. The default criteria for differential metabolite screening were VIP > 1, P value < 0.05, and FC ≥ 2 or FC ≤ 0.5. A heatmap was generated to visualize the hubs of significantly changed metabolites as a function of quercetin treatment duration. The pathway analysis was performed using the website MetaboAnalyst ( http://www.metaboanalyst.ca/ ). This research examined the correlation between metabolites and intestinal microbiota through the application of the Pearson correlation coefficient to evaluate linear associations. 4.8. Quantitative reverse transcription PCR(RT-qPCR) Total RNA was isolated from the spine tissues of mice using an RNA extraction kit (Solarbio, Beijing, China) following the manufacturer’s protocol. The total RNA was then reversely transcribed into cDNA by an iScript™ cDNA Synthesis kit (Bio-Rad, USA), and quantitative PCR reactions were performed with SsoAdvanced Universal SYBR Green Supermix(Bio-Rad, USA). The sequence of the primers used is presented in supplemental table S1 . RT-qPCR was performed using a CFX96 Real-Time PCR System (Bio-Rad, USA). The changes in gene expression were calculated using the 2–ΔΔCt method, and the house-keeping gene β-actin was used as the endogenous control for normalization. 4.9. Statistical analysis Data were analyzed using a t-test or one-way ANOVA with SPSS statistical software. All data in this research are expressed as mean ± standard error of at least three independent experiments. The difference was considered significant at p < 0.05. Abbreviations The following abbreviations are used in this manuscript: L.Reuteri/Lreu Lactobacillus reuteri AS Ankylosing spondylitis PG Proteoglycan AhR aryl hydrocarbon receptor Declarations Author Contributions: Data curation, Validation, Methodology, writing - review & editing, Funding acquisition,L.J.Y; Methodology, Formal analysis,K.Y; Review & editing,K.W; Resources,T.C.; Resources,B.L.;Methodology ,Z.F.C.; Resources, D.W.Z.; Methodology,Z.H.S.; Resources,discussed the results,.X.L.; Conceptualization, Supervision, Resources,H.L.; All authors have read and agreed to the published version of the manuscript. Authorship must be limited to those who have contributed substantially to the work reported. Funding: This study was supported by the Natural Science Foundation of China 82302035 (L. Yang); Project of Administration of Traditional Chinese Medicine of Guangdong Province of China（NO.20241046 L. Yang）; The Basic and Applied Basic Research Foundation of Guangdong Province, Grant Number 2023A1515111101 (K. Wang). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data and materials used to support the findings of this study are available from the corresponding author upon request. Conflicts of Interest: The authors declare no conflict of interest. References Wang R, Dasgupta A, Ward MM. Predicting Probability of Response to Tumor Necrosis Factor Inhibitors for Individual Patients With Ankylosing Spondylitis. JAMA Netw open. 2022;5:e222312. 10.1001/jamanetworkopen.2022.2312 . Zambrano-Zaragoza JF, Agraz-Cibrian JM, González-Reyes C, Durán-Avelar MDJ. Vibanco-Pérez, N. Ankylosing Spondylitis: From Cells to Genes. International Journal of Inflammation 2013, 2013, 1–16. 10.1155/2013/501653 Nossent JC, Sagen-Johnsen S, Bakland G. 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Xing J, Niu T, Zou B, Yang G, Shi C, Yan Q, Sun M, Yu T, Zhang S, Feng X, et al. Gut microbiota-derived LCA mediates the protective effect of PEDV infection in piglets. Microbiome. 2024;12. 10.1186/s40168-023-01734-4 . Supplementary Table 1 Supplemental table 1 is not available with this version. Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Journal of Translational Medicine → Version 1 posted Reviewers agreed at journal 23 Mar, 2025 Reviewers invited by journal 23 Mar, 2025 Editor assigned by journal 19 Mar, 2025 First submitted to journal 18 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6109718","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":432640550,"identity":"78b665cf-9059-44ac-b5d4-de7d86a6b220","order_by":0,"name":"Lianjun Yang","email":"","orcid":"","institution":"The Fifth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Lianjun","middleName":"","lastName":"Yang","suffix":""},{"id":432640551,"identity":"c2b35d08-26fd-41aa-ba01-2f4d8927b960","order_by":1,"name":"Ke You","email":"","orcid":"","institution":"University of Macau Faculty of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"You","suffix":""},{"id":432640552,"identity":"a4c1e96f-199f-4206-b0be-9f8929c3813d","order_by":2,"name":"Kun Wang","email":"","orcid":"","institution":"The Fifth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Kun","middleName":"","lastName":"Wang","suffix":""},{"id":432640553,"identity":"024f401b-6ac4-42cc-b601-45d6bf3b0d72","order_by":3,"name":"Bin Liu","email":"","orcid":"","institution":"Jinan University First Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Liu","suffix":""},{"id":432640554,"identity":"90224181-d1e4-43a0-8f1f-e0c5f423a7f0","order_by":4,"name":"Tao Chen","email":"","orcid":"","institution":"The Fifth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Chen","suffix":""},{"id":432640555,"identity":"dfed62e6-8264-49f3-bb2f-ded37ed73e7d","order_by":5,"name":"Zhifei Cui","email":"","orcid":"","institution":"The Fifth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Zhifei","middleName":"","lastName":"Cui","suffix":""},{"id":432640556,"identity":"6927e5e1-1b56-4e9c-b1ed-3a2abe4aa1d6","order_by":6,"name":"Dawei Zhang","email":"","orcid":"","institution":"The Fifth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Dawei","middleName":"","lastName":"Zhang","suffix":""},{"id":432640557,"identity":"4dbbd570-b7c6-4479-abc9-b2aee15ca330","order_by":7,"name":"Zhihai Su","email":"","orcid":"","institution":"The Fifth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Zhihai","middleName":"","lastName":"Su","suffix":""},{"id":432640558,"identity":"a0168873-e3ee-4ef1-b4f8-f1d060a0f7c4","order_by":8,"name":"Xiang Liu","email":"","orcid":"","institution":"The Fifth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Liu","suffix":""},{"id":432640559,"identity":"3904762b-6cc2-47ed-8048-6beccc639523","order_by":9,"name":"Hai Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYBACPgYeBoaECgYGNna4WAJ+LWxgLWeADGaStDC2AVnEa5HIPfjh4bxt8nzMDMyfef4cZuBnzzFg+LkDn5a8ZInEbbcN25gZ2KR52w4zSPa8MWDsPYNHC88ZA5AWRpAWZt6GwwwGN3IMmMFOxa3F+EfinNv2bTCH2RPUwt5jJpHYcDsRqIVBmocNaIsEEVosEo7dTm4DKpOc25bOI3HmWcHBXjxa+Jl5jG/+qLltO7+9+fCHN3+s5fjbkzc++IlHCxJgbGACxhEPiHmAKA1gTT+IVjoKRsEoGAUjCQAABFBGsEc89k8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-4212-3282","institution":"Fifth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":true,"prefix":"","firstName":"Hai","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2025-02-26 04:40:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6109718/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6109718/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12967-025-06681-2","type":"published","date":"2025-07-01T15:58:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79823236,"identity":"fc171248-02f6-4d43-b5d6-a822351f2e93","added_by":"auto","created_at":"2025-04-03 09:09:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12827513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eL. reuteri\u003c/em\u003e alleviates AS progression and diminishes disease severity in mice.\u003c/p\u003e\n\u003cp\u003e(A) Diagram depicting the experimental design. (B) Representative images of hind paws from each group (Scale bar: 2 mm). (C) Arthritis scores and (D) hind paw thickness were assessed in the three groups (n = 10 mice per group). (E) Histological sections of ankle joints displaying synovial inflammation (black arrowhead), joint space narrowing, cartilage erosion (red arrowhead), and bone destruction (yellow arrowhead) in the PG group (H\u0026amp;E staining; Scale bar: 500 µm). (F) Vertebral joint sections showing severe inflammatory cell infiltration around the disc (black arrowhead), excessive bone matrix deposition (yellow arrowhead), and partial intervertebral disc destruction (red arrowhead) in the PG group (Scale bar: 50 µm). (G) Micro-CT images from the three groups indicating bone destruction (green arrow) and bone loss (yellow arrow) in the PG group (Scale bar: 1 cm). (H) Histological analysis of paw joint sections (n = 6 mice per group). (I) Histological evaluation of vertebral joint sections (n = 6 mice per group). Data are presented as means ± SD (\u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.01 vs. control; *P \u0026lt; 0.05, **P \u0026lt; 0.01 vs. PG).\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6109718/v1/340d25ad1b307be83b3d36d2.png"},{"id":79823156,"identity":"35535d07-4f22-40b2-844c-fe97176c3dfb","added_by":"auto","created_at":"2025-04-03 09:09:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2264873,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eL. reuteri\u003c/em\u003e decreases the expression of inflammatory cytokines in AS mice. The serum levels of IL-1β, IL-18, IL-17A, and IL-23 were determined using ELISA. Data are presented as means ± standard deviation from 10 mice per group. (A) IL-1β; (B) IL-18; (C) IL-17A; (D) IL-23 (\u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.01 vs. control; *P \u0026lt; 0.05, and **P \u0026lt; 0.01 vs. PG alone).\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6109718/v1/5815c5a90d6429c48b7eaf0a.png"},{"id":79823145,"identity":"d1f74844-2803-424a-a369-f1f71437b9dd","added_by":"auto","created_at":"2025-04-03 09:09:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":25449563,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eL. reuteri\u003c/em\u003e improves ileum tissue pathology and enhances intestinal mucosal barrier function in AS Mice(A)Ileum sections were stained with H\u0026amp;E (Scale bar\u0026nbsp;is 50μm). Statistic data of villus height(B), crypt depth(C), and villus/crypt ratio(D). (E)Representative photomicrographs of immunofluorescence staining of occludin(red) and ZO-1 (green) with DAPI (blue) in ileum sections. (F-G) Quantification of the immunofluorescence intensity of occludin and ZO-1.(H) Gut permeability was measured by the DX-4000-FITC levels in the serum(n =10 mice per group). (\u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.01 vs. control; *P \u0026lt; 0.05, and **P \u0026lt; 0.01 vs. PG alone).\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6109718/v1/31530b0aa851466125ea54b1.png"},{"id":79823164,"identity":"afe6b23a-5a51-4da3-ac9d-265546904fc4","added_by":"auto","created_at":"2025-04-03 09:09:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4563755,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eL. reuteri\u003c/em\u003e influences the composition of the gut microbiota in AS mice. (A) Venn diagram of shared and unique OTUs among different groups. (B)Chao1 index and (C) observed species index for the three groups. (D)PCoA score plot. (E) Cladogram generated from LEfSe analysis. (F) LEfSe analysis identified the taxa with the greatest differences in abundance between the three groups (p \u0026lt; 0.05, linear discriminant analysis \u0026gt;2 logs).\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6109718/v1/b7d8382e453a14e5f42b2292.png"},{"id":79824165,"identity":"d1158991-297b-4cf3-b855-ebb02b26a002","added_by":"auto","created_at":"2025-04-03 09:17:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3139654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eL. reuteri\u003c/em\u003e effects on alterations in intestinal microbial abundance and function.Analysis of the composition of bacteria present at phylum (A) and genus levels (B).(C) Heatmap displaying differentially abundant bacterial species.(D) KEGG functional prediction (levels 2)of bacterial communities based on PICRUSt.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6109718/v1/07bd02914dccb0ea6cef46dd.png"},{"id":79823219,"identity":"617458ba-9d9c-4224-9334-7e1046276574","added_by":"auto","created_at":"2025-04-03 09:09:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3994968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eL. reuteri\u003c/em\u003e alters gut microbiota-related metabolites. PCA plot between the three groups in positive(A) and negative modes(B). (C) Heatmap of differentially expressed metabolites. (D) Analysis of the metabolic pathway of differentially expressed metabolites. (E) Heatmap visualizing the correlations between differentially serum metabolites and the gut microbiota. The color-coding scale in the heatmap represents Spearman’s correlation coefficient (red signifies a positive correlation, and blue signifies a negative correlation,where white indicate very weak or no correlation).\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6109718/v1/227beefbda3cb49bd0c6eead.png"},{"id":79823154,"identity":"9f68a169-0558-42f3-a4be-2026ab3984dc","added_by":"auto","created_at":"2025-04-03 09:09:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2802032,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eL. reuteri\u003c/em\u003e on AhR/NLRP3 pathway in AS mice. RT-qPCR analysis of AhR, CYP1A1, CYP1B1 and NLRP3 mRNA levels in sipne tissues. (\u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.01 vs. control; *P \u0026lt; 0.05, and **P \u0026lt; 0.01 vs. PG alone; n = 5 per group).\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6109718/v1/768dfb64a65591a108122800.png"},{"id":86179155,"identity":"3176703b-ea8c-4fb4-b3e9-cceaa1423d40","added_by":"auto","created_at":"2025-07-07 16:16:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":52173909,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6109718/v1/16723c7a-5867-4e6c-8bd2-677c02a4e427.pdf"}],"financialInterests":"","formattedTitle":"Lactobacillus Reuteri Prevents Progression of Ankylosing Spondylitis in Mice by Restoring Gut Microbiota-Metabolism Homeostasis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAnkylosing spondylitis (AS) is a chronic inflammatory disease primarily affecting the axial skeleton, particularly the spine and sacroiliac joints[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is characterized by inflammation that can lead to pain, stiffness, and eventual fusion of the vertebrae, resulting in a condition often referred to as \"bamboo spine\" due to its rigidity. The disease typically presents in young adults, with a higher prevalence in males than females, and is strongly associated with the HLA-B27 antigen, although not all individuals with this antigen develop AS. The prevalence of AS varies, with estimates ranging from 0.1\u0026ndash;1.4% in the general population, and it can significantly impact quality of life due to chronic pain and functional limitations[2\u003csup\u003e,\u003c/sup\u003e3].\u003c/p\u003e \u003cp\u003eThe pathogenesis of AS is complex and not fully understood, involving both genetic predisposition and environmental factors, and is increasingly linked to alterations in the gut microbiota. The relationship between AS and intestinal flora involves several interconnected mechanisms, including dysbiosis, immune response modulation, and genetic predisposition. Dysbiosis, or an imbalance in the gut microbiota, has been observed in AS patients. Studies have shown that AS is associated with a higher abundance of certain bacterial taxa, such as \u003cem\u003eProteobacteria\u003c/em\u003e, and a decrease in beneficial bacteria such as \u003cem\u003eBacteroidetes\u003c/em\u003e and \u003cem\u003eFirmicutes\u003c/em\u003e [4\u003csup\u003e,\u003c/sup\u003e5]. This microbial imbalance can lead to increased intestinal permeability, often referred to as a \"leaky gut,\" which allows microbial products to enter the bloodstream and potentially trigger systemic inflammation [6\u003csup\u003e,\u003c/sup\u003e7]. The presence of specific bacteria, such as \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, has been implicated in the pathogenesis of AS due to its structural similarities to human antigens, which may lead to molecular mimicry and autoimmune responses[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].In AS, the dysbiotic gut microbiome can influence the activation of T helper 17 (Th17) cells, which are known to produce pro-inflammatory cytokines such as IL-17. This cytokine is central to the inflammatory processes observed in AS[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The interaction between gut bacteria and the immune system can lead to an exaggerated immune response, contributing to the chronic inflammatory characteristic of AS. Furthermore, certain microbial metabolites, such as short-chain fatty acids (SCFAs), can modulate immune responses and have been shown to have anti-inflammatory effects, suggesting that healthy microbiota could potentially mitigate AS symptoms [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrent clinical treatments for AS primarily include non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), and biologics targeting specific inflammatory pathways. However, these therapies often have limited efficacy and can be associated with significant side effects, including gastrointestinal complications and increased risk of infections. Furthermore, many patients do not achieve adequate disease control, highlighting the need for alternative therapeutic strategies. Therapies aimed at restoring the gut microbiota balance, such as probiotics or fecal microbiota transplantation (FMT), are being explored as potential adjunctive treatments for AS. \u003cem\u003eLactobacillus reuteri\u003c/em\u003e(\u003cem\u003eL. reuteri\u003c/em\u003e ), a well-studied probiotic, has been shown to exert various beneficial effects on the immune system and gut health, which are crucial for preventing and managing immune-related disorders. In models of collagen-induced arthritis, pre-administration of heat-killed \u003cem\u003eL. reuteri\u003c/em\u003e significantly reduced the severity and incidence of arthritis. This effect was associated with a decrease in pro-inflammatory cytokines (IL-6) and an increase in anti-inflammatory cytokines (IL-10)[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The ability of \u003cem\u003eL. reuteri\u003c/em\u003e to regulate gut microbiota composition is another area of significant research. In colitis models, administration of \u003cem\u003eL. reuteri\u003c/em\u003e has been associated with reduced intestinal inflammation and restored the balance of intestinal microbiota[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, \u003cem\u003eL. reuteri\u003c/em\u003e plays a crucial role in maintaining intestinal barrier integrity, which is essential for preventing systemic inflammation and autoimmune responses[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the interplay between the gut microbiota and immune responses in AS, exploring the therapeutic potential of \u003cem\u003eL. reuteri\u003c/em\u003e in preventing disease progression is a compelling avenue of research. However, while promising, current research on the effects of \u003cem\u003eL. reuteri\u003c/em\u003e in AS remains limited, with a need for further investigation into the underlying mechanisms and the specific metabolic pathways involved. This study aims to investigate the effects of \u003cem\u003eL. reuteri\u003c/em\u003e on the progression of AS in a murine model, focusing on its role in restoring gut microbiota composition and metabolic homeostasis. Understanding these mechanisms may provide insight into novel therapeutic strategies for managing AS.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. \u003cem\u003eL. reuteri\u003c/em\u003e attenuated the disease progression and AS-associated symptoms in PG-induced AS Mice\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe assessment of the peripheral arthritis index and paw thickness provides both intuitive and objective measures to reflect the severity of AS. From the 7th week after induction, AS mice in the PG group gradually developed paw swelling and redness (Figure. 1B), with an increasing arthritis index (Figure. 1C) and paw thickness (Figure. 1D), whereas no such symptoms were observed in the control group, confirming the successful establishment of the AS mouse model. However, oral administration of \u003cem\u003eL. reuteri\u003c/em\u003e significantly alleviated paw swelling and redness in AS mice. Compared to the PG group, the arthritis index and paw thickness in the \u003cem\u003eL. reuteri\u003c/em\u003e -treated group were significantly reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003ePathological examination of the ankle joint with HE staining showed that the ankle joint tissues of the control group mice had a normal structure, with no signs of synovial hyperplasia, inflammation, cartilage degradation, or bone erosion, and the joint space was maintained normally (Figure. 1E). In contrast, the PG group mice displayed significant arthritis, characterized by pronounced synovial hyperplasia, inflammatory cell infiltration, cartilage erosion, and bone erosion (Figure. 1E). Notably, treatment with \u003cem\u003eL. reuteri\u003c/em\u003e significantly reduced the synovial inflammation score, cartilage degeneration score, and bone erosion score in the \u003cem\u003eL. reuteri\u003c/em\u003e group compared to the PG group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Figure. 1H), indicating a significant suppression of arthritis.\u003c/p\u003e\n \u003cp\u003eMeanwhile, in the control group, the intervertebral tissue structure appeared normal, with intact annulus fibrosus and nucleus pulposus and no infiltration of inflammatory cells. In contrast, AS mice in the PG group exhibited disrupted intervertebral structures (Figure.1F), extensive infiltration of inflammatory cells around the intervertebral discs, excessive bone matrix formation, and destruction of the intervertebral spaces. Following \u003cem\u003eL. reuteri\u003c/em\u003e treatment, the infiltration of inflammatory cells around the intervertebral discs was significantly reduced, and the extent of bone matrix formation and intervertebral disc destruction was markedly alleviated (Figure. 1H, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Micro-CT scanning revealed comparable findings, with the PG group displaying bone destruction and loss, whereas mice treated with \u003cem\u003eL. reuteri\u003c/em\u003e exhibited milder bone deterioration( Figure. 1G).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2.\u003cem\u003eL. reuteri\u003c/em\u003e attenuates systemic inflammatory response\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eCompared with the control group, serum levels of pro-inflammatory cytokines IL-1\u0026beta;, IL-18, IL-17A, and IL-23 were significantly elevated in the PG group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These findings indicate an enhanced inflammatory response in the AS model. In contrast, treatment with \u003cem\u003eL. reuteri\u003c/em\u003e significantly reduced the serum levels of IL-1\u0026beta;, IL-18, IL-17A, and IL-23 in AS mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suggesting that \u003cem\u003eL. reuteri\u003c/em\u003e ameliorates systemic inflammation by modulating the inflammatory cytokine profile.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3.\u003cem\u003eL. reuteri\u003c/em\u003e improves ileum tissue pathology and enhances intestinal mucosal barrier function in AS mice.\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eStudies have shown that approximately 60% of AS patients exhibit subclinical intestinal inflammation upon colonoscopic examination[\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, we used HE staining to determine whether intestinal mucosal changes accompany AS progression in mice. Pathological observation under a light microscope revealed dense, intact, and well-aligned ileal villi in the control group (Figure.3A). In contrast, the PG group exhibited disorganized villi, shortened villus length, increased crypt depth, and a reduced villus height-to-crypt depth ratio compared to the control group (Figure. 3B-D, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Following \u003cem\u003eL. reuteri\u003c/em\u003e treatment, the ileal morphological abnormalities in the \u003cem\u003eL. reuteri\u003c/em\u003e group were significantly improved compared to the PG group, with statistically significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eTo assess intestinal permeability, we measured serum DX-4000-FITC levels, a fluorescent tracer indicative of gut barrier integrity. Compared to the Control group, PG-induced AS mice exhibited significantly elevated serum DX-4000-FITC levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating increased gut permeability. Treatment with \u003cem\u003eL. reuteri\u003c/em\u003e significantly reduced serum DX-4000-FITC levels in AS mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), demonstrating its ability to restore gut barrier function (Figure.3H). Immunofluorescence analyses further revealed decreased protein expression levels of tight junction proteins ZO-1 and occludin in the ileum of the PG group compared to the Control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). After \u003cem\u003eL. reuteri\u003c/em\u003e treatment, the expression levels of ZO-1 and occludin were partially restored (Figure.3E-G, \u0026lt; 0.05), highlighting the protective effect of \u003cem\u003eL. reuteri\u003c/em\u003e on intestinal epithelial barrier integrity in AS mice.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cem\u003e2.4.\u003c/em\u003e Effect of \u003cem\u003eL. reuteri\u003c/em\u003e on the composition of gut microbiota in AS mice\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eWe used 16s rRNA amplicon sequencing to investigate if \u003cem\u003eL. reuteri\u003c/em\u003e affected the intestinal bacterial composition in AS mice. Using Venn diagrams, we observed overlapping OTU data for the three groups, which helped us identify microbes across the groups. It was found that 364 of the 450 OTUs were shared by all groups (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Notably, the \u003cem\u003eL. reuteri\u003c/em\u003e treatment group exhibited 11 unique microbes, while the groups designated as PG and control displayed 5 and 16 distinct microbes, respectively. Alpha diversity was measured using the Chao1 and observed species. The Chao1 and observed species indices showed a significant decrease in the PG group compared to the control groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Figure. 4B-C). Conversely, we found that Chao1 and observed species indices were significantly increased in the \u003cem\u003eL. reuteri\u003c/em\u003e treatment group compared with the PG group, aligning them more closely with the control group\u0026rsquo;s levels.\u003c/p\u003e\n \u003cp\u003ePrincipal-coordinate analysis (PCoA) of the unweighted UniFrac distances was performed to evaluate the \u0026beta; diversity among the three groups. It can be observed from the PCoA plot (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC-D) that the samples in the PG group and the control group show significant separation, indicating a substantial difference in microbial community structure between the two groups. In contrast, the distance between the \u003cem\u003eL. reuteri\u003c/em\u003e group and the control group is reduced, suggesting a higher similarity in microbial community structure between these two groups. Linear discriminant analysis (LDA) combined with effect size analysis (LEfSe) was used to identify different bacterial taxa among different groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, LDA score\u0026thinsp;\u0026gt;\u0026thinsp;2.0).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe alterations in intestinal microbiota in AS and \u003cem\u003eL. reuteri\u003c/em\u003e -treated mice were further analyzed at both the phylum and genus levels. At the phylum level, the gut microbiota of mice in each group was predominantly composed of \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eProteobacteria\u003c/em\u003e, and \u003cem\u003eVerrucomicrobia\u003c/em\u003e, collectively accounting for approximately 99% of the intestinal microbiota in all groups ( Figure.5A). In the control group, \u003cem\u003eBacteroidetes\u003c/em\u003e had the highest proportion at 61.1%, followed by \u003cem\u003eFirmicutes\u003c/em\u003e at 33.9%. Compared to the control group, the proportion of Firmicutes in the PG group increased significantly to 54.4% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while \u003cem\u003eBacteroidetes\u003c/em\u003e decreased to 38.3% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). After treatment with \u003cem\u003eL. reuteri\u003c/em\u003e, the proportion of \u003cem\u003eBacteroidetes\u003c/em\u003e increased to 65.2%, while the proportion of \u003cem\u003eFirmicutes\u003c/em\u003e decreased to 31.5%. The \u003cem\u003eBacteroidetes/Firmicutes\u003c/em\u003e ratio was 1.8 in the control group, decreased to 0.7 in the PG group, and increased to 2.07 in the \u003cem\u003eL. reuteri\u003c/em\u003e-treated group.\u003c/p\u003e\n \u003cp\u003eAt the genus level, we observed that the microbial composition in the PG group differed significantly from that in the control group (Figure.5B). We identified a total of 11 genera that exhibited significant differences among the groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These differences at the genus level were visualized using a heatmap (Figure.5C). Compared to the control group, the abundances of \u003cem\u003eAlloprevotella\u003c/em\u003e, \u003cem\u003eBarnesiella\u003c/em\u003e, and \u003cem\u003eEubacterium\u003c/em\u003e were significantly decreased in the PG group, while \u003cem\u003eBrachyspira\u003c/em\u003e, \u003cem\u003eParabacteroides\u003c/em\u003e, and \u003cem\u003eRikenella\u003c/em\u003e were significantly increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared to the PG group, the \u003cem\u003eL. reuteri\u003c/em\u003e group showed a significant increase in the abundances of \u003cem\u003eAlloprevotella\u003c/em\u003e, \u003cem\u003eBarnesiella\u003c/em\u003e, and \u003cem\u003eEubacterium\u003c/em\u003e, and a significant decrease in the abundances of \u003cem\u003eBrachyspira\u003c/em\u003e and \u003cem\u003eParabacteroides\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, compared to the other two groups, the \u003cem\u003eL. reuteri\u003c/em\u003e group exhibited increased abundances of \u003cem\u003eVeillonella\u003c/em\u003e and \u003cem\u003eParaprevotella\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eFinally, the PICRUSt analysis based on KEGG database used to analyze the functional differences between different groups (Figure.5D). In the control group, 3 KEGG pathways were enriched; in the PG group, 6 KEGG pathways were enriched; and in the \u003cem\u003eL. reuteri\u003c/em\u003e group, 9 KEGG pathways were enriched.The enriched KEGG pathways in the control group mainly included Amino Acid Metabolism, Energy Metabolism, and Transport and Catabolism. In the PG group, the enriched KEGG pathways primarily involved Membrane Transport, Cell Motility, Transcription, Signal Transduction, Environmental Adaptation, and Xenobiotics Biodegradation and Metabolism.For the \u003cem\u003eL. reuteri\u003c/em\u003e group, the enriched KEGG pathways were primarily associated with Replication and Repair, Nucleotide Metabolism, Metabolism of Other Amino Acids, Biosynthesis of Other Secondary Metabolites, Cell Growth and Death, Digestive System, Signaling Molecules and Interaction, Immune System Diseases, and Metabolic Diseases.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cem\u003e2.5.\u003c/em\u003e Effect of \u003cem\u003eL. reuteri\u003c/em\u003e on serum metabolomics in AS mice\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eBased on untargeted serum metabolomics for multivariate analyses, principal component analysis (PCA) showed that a clear separation could be observed between the Control and PG group, and the \u003cem\u003eL. reuteri\u003c/em\u003e group located much closer to Control group both in positive and negative ion mode (Figure. 6A-B). The clustering pattern further evidenced that \u003cem\u003eL. reuteri\u003c/em\u003e could protect against AS. Furthermore, the findings of the serum metabolomics analysis revealed significant differences in the expression of 11 metabolites (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, FC\u0026thinsp;\u0026gt;\u0026thinsp;2, and VIP\u0026thinsp;\u0026gt;\u0026thinsp;1) between the three groups. The heatmap in Figure. 6C illustrates the changes in the levels of these 11 metabolites across the different groups.\u003c/p\u003e\n \u003cp\u003eCompared to the control group, the levels of maleic acid, D-malic acid, phenol sulfate, cis-aconitate, citric acid, 3-indoxyl sulfate, 4-methylumbelliferone, and 2,4-dihydroxyacetophenone 5-sulfate were significantly decreased in the PG group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). After treatment with \u003cem\u003eL. reuteri\u003c/em\u003e, the levels of these metabolites increased significantly. Additionally, compared to the control group, the levels of seselin and LysoPS 18:0 were elevated in the PG group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Following \u003cem\u003eL. reuteri\u003c/em\u003e treatment, the levels of these metabolites were significantly reduced.Based on metabolic pathway analysis (\u0026minus;\u0026thinsp;Log (P)\u0026thinsp;\u0026gt;\u0026thinsp;1 and an impact\u0026thinsp;\u0026gt;\u0026thinsp;0.05), Figure. 6D highlights the most affected metabolic path ways, including Citrate cycle (TCA cycle) and Glyoxylate and dicarboxylate metabolismmetabolism.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cem\u003e2.6.\u003c/em\u003e Correlation analysis\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTo investigate the potential relationships between these differential serum metabolites, and the abundance of different species, we conducted Spearman correlation analysis.To assess the strength of the association between a specific metabolite and a microorganism, the corresponding correlation coefficient is used. From the Figure. 6E, it can be observed that \u003cem\u003eEubacterium\u003c/em\u003e shows a strong positive correlation with 2,4-dihydroxyacetophenone 5-sulfate, 4-methylumbelliferone, and maleic acid, while displaying a negative correlation with LysoPS 18:0 and seselin. \u003cem\u003eAlloprevotella\u003c/em\u003e exhibits a strong positive correlation with D-malic acid, 4-methylumbelliferone, maleic acid, cis-aconitate, and 2,4-dihydroxyacetophenone 5-sulfate, and a moderate negative correlation with seselin. \u003cem\u003eBarnesiella\u003c/em\u003e shows a positive correlation with 3-indoxyl sulfate and citric acid.These findings suggest complex interactions between the gut microbiota and serum metabolites.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cem\u003e2.7.\u003c/em\u003e The intervention effect of \u003cem\u003eL. reuteri\u003c/em\u003e on AhR/NLRP3 Pathway\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe results of the metabolic profile suggest that treatment with \u003cem\u003eL. reuteri\u003c/em\u003e can effectively increase the content of the 3-indoxyl sulfate which is an Aryl hydrocarbon receptor(AhR) agonist. Recent research studies have found that AhR can negatively regulate NLRP3 expression[\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e].Therefore,our plan is to investigate whether \u003cem\u003eL. reuteri\u003c/em\u003e can impact the AhR/NLRP3 pathway.The RT-qPCR was performed to evaluate the effects of \u003cem\u003eL. reuteri\u003c/em\u003e on the expression of related genes in the AhR/NLRP3 pathway.\u003c/p\u003e\n \u003cp\u003eAs shown in Figure.7, the relative mRNA expression levels of AhR, CYP1A1, and CYP1B1 were significantly decreased, whereas NLRP3 expression was increased in the PG group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), compared to the control group. Following treatment with \u003cem\u003eL. reuteri\u003c/em\u003e, the mRNA expression levels of AhR, CYP1A1and CYP1B1 were elevated, while NLRP3 expression was markedly reduced. These findings indicate that \u003cem\u003eL. reuteri\u003c/em\u003e modulates the AhR/NLRP3 pathway and plays a crucial role in inhibiting inflammation in AS mice.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAS is associated with significant morbidity and can lead to progressive spinal fusion, resulting in debilitating pain and impaired mobility. The etiology of AS remains largely elusive, although genetic predisposition, particularly the presence of the HLA-B27 antigen, is recognized as a key factor. Emerging evidence suggests that dysbiosis of gut microbiota may contribute to the onset and progression of AS by modulating immune responses and promoting systemic inflammation. Probiotic interventions have been shown to reshape gut microbiota and have a positive impact on inflammatory diseases. In light of these complexities, this study investigates the therapeutic potential of \u003cem\u003eL. reuteri\u003c/em\u003e in modulating gut microbiota to address AS. \u003cem\u003eL. reuteri\u003c/em\u003e is known to influence the gut microbiome and its metabolic outputs, which can have systemic effects on the host. For instance, a study demonstrated that \u003cem\u003eL. reuteri\u003c/em\u003e alleviates the severity of autoimmune arthritis by promoting the peripheral migration of gut-derived Tregs[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].Utilizing a well-defined animal model of AS, our research aims to elucidate the mechanisms by which \u003cem\u003eL. reuteri\u003c/em\u003e may inhibit disease progression. Key findings indicate that \u003cem\u003eL. reuteri\u003c/em\u003e not only alleviates joint and spinal damage but also exerts anti-inflammatory effects by reducing levels of pro-inflammatory cytokines and restoring gut microbiota-metabolism homeostasis. These results provide a promising foundation for developing novel therapeutic strategies targeting gut microbiota in the management of AS.\u003c/p\u003e \u003cp\u003eStudies have highlighted the potential of \u003cem\u003eL. reuteri\u003c/em\u003e in reducing inflammation and improving gut barrier function, which are crucial in managing chronic inflammatory conditions like AS. Our results show a significant reduction in serum levels of IL-1β, IL-18, IL-17A and IL-23 after \u003cem\u003eL. reuteri\u003c/em\u003e treatment, suggesting this probiotic can attenuate the inflammatory response in AS. Elevated levels of these cytokines are hallmarks of AS pathogenesis, contributing to inflammation, bone erosion, and pathological new bone formation. The observed reduction in IL-17A and IL-23 is particularly significant, given the central role of the IL-23/IL-17 axis in AS pathogenesis. By decreasing IL-23 and IL-17A levels, \u003cem\u003eL. reuteri\u003c/em\u003e likely disrupts this axis, mitigating the inflammatory response. These findings are consistent with recent studies demonstrating the anti-inflammatory effects of \u003cem\u003eL. reuteri\u003c/em\u003e. For example, Gao et al.reported that Lactobacillus strains can decrease IL-8, IL-17A, IL-6, and TNF-α levels levels in intestinal epithelial injury model, thereby further supporting the immunomodulatory effects of \u003cem\u003eL. reuteri\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].Similarly, Jiang et al. indicated that \u003cem\u003eL. reuteri\u003c/em\u003e alleviated the elevation of serum inflammatory cytokines (IL-1α, IL-2, and IL-18) and partially restored gut microbiota and metabolome dysbiosis in acute liver failure. This shift in cytokine levels lends further credence to the notion that \u003cem\u003eL. reuteri\u003c/em\u003e not only halts disease progression but actively reconfigures the inflammatory landscape associated with AS.\u003c/p\u003e \u003cp\u003eUp to 70% of AS patients have evidence of terminal ileitis resembling IBD, suggesting gut inflammation is important in disease pathogenesis[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. ZO-1 and occludin are crucial components of the intestinal epithelial barrier, which regulates the permeability of the gut and prevents the translocation of harmful substances and pathogens. Disruption of these proteins can lead to increased intestinal permeability, often referred to as \"leaky gut,\" which has been implicated in various inflammatory diseases, including AS[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].Our findings align with these observations, as PG-induced AS mice displayed significantly higher serum DX-4000-FITC levels, indicative of compromised gut barrier function. In contrast, \u003cem\u003eL. reuteri\u003c/em\u003e treatment significantly reduced gut permeability and restored ZO-1 and occludin expression. Our study corroborates reports linking altered gut permeability to systemic inflammation in AS and adds evidence of \u003cem\u003eL. reuteri\u003c/em\u003e 's reparative effect on tight junction proteins. This restoration may facilitate gut-homeostasis, ultimately leading to improved systemic health outcomes.\u003c/p\u003e \u003cp\u003eWhat sets our study apart is its emphasis on the connection between gut microbiota restoration and immune regulation in AS. While previous research has primarily focused on immune cells in inflammation, our study suggests that \u003cem\u003eL. reuteri\u003c/em\u003e may alleviate inflammation by restoring gut microbiota balance. Our findings indicate that \u003cem\u003eL. reuteri\u003c/em\u003e treatment reverses gut microbiota dysbiosis in AS mice by restoring the \u003cem\u003eFirmicutes/Bacteroidetes\u003c/em\u003e balance and promoting the growth of beneficial bacteria such as \u003cem\u003eAlloprevotella\u003c/em\u003e and \u003cem\u003eBarnesiella\u003c/em\u003e while reducing harmful bacteria like \u003cem\u003eBrachyspira\u003c/em\u003e. Increased abundances of beneficial genera such as \u003cem\u003eAlloprevotella\u003c/em\u003e and \u003cem\u003eBarnesiella\u003c/em\u003e following \u003cem\u003eL. reuteri\u003c/em\u003e treatment may enhance short-chain fatty acid production, which has been shown to reduce inflammation and support gut barrier integrity[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].Our KEGG pathway analysis further confirms that \u003cem\u003eL. reuteri\u003c/em\u003e improves metabolic and immune functions, particularly pathways associated with amino acid metabolism and immune regulation. The observed changes in gut microbiota composition, with \u003cem\u003eL. reuteri\u003c/em\u003e restoring the relative abundance of beneficial bacteria, further support the hypothesis that gut dysbiosis plays a crucial role in the development of AS [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to its effects on gut microbiota, \u003cem\u003eL. reuteri\u003c/em\u003e has been shown to impact the metabolomic profile of the host. Compared to prior studies that focused on inflammatory cytokines, our work integrates microbiota and metabolomic changes, providing a holistic view of AS pathophysiology. The metabolomic findings demonstrate that \u003cem\u003eL. reuteri\u003c/em\u003e treatment effectively modulates key metabolic pathways disrupted in AS. The observed recovery of metabolites such as citric acid and cis-aconitate suggests that \u003cem\u003eL. reuteri\u003c/em\u003e may modulate the TCA cycle, which plays a central role in cellular energy metabolism and is often dysregulated in inflammatory conditions[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Additionally, reductions in pro-inflammatory metabolites like LysoPS 18:0,a phospholipid associated with immune cell activation, indicates that \u003cem\u003eL. reuteri\u003c/em\u003e may modulate immune responses[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].Notably, the identification of specific metabolites like indole-3-lactic acid which is a potent endogenous agonist for AhR and have immunomodulatory effects that may mitigate AS-related inflammation. Recent study has shown that bacterial metabolites of tryptophan, such as indoxyl-3-sulfate, exhibit significant anti-inflammatory effects. In experimental autoimmune encephalomyelitis (EAE) mice fed a tryptophan-deficient diet, the disease course was prolonged, and symptoms worsened. However, upon re-administration of indoxyl-3-sulfate, the condition of the EAE mice improved. This study also revealed that indoxyl-3-sulfate act on microglia through the AhR, thereby regulating astrocytes to exert anti-inflammatory effects[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].Moreover,indoxyl-3-sulfate can promote an anti-inflammatory and tolerogenic environment by modulating dendritic cell function and indirectly reducing T cell-mediated inflammation[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe upregulation of AhR and its downstream targets, CYP1A1 and CYP1B1, suggests that \u003cem\u003eL. reuteri\u003c/em\u003e enhances the activation of pathways involved in anti-inflammatory responses. \u003cem\u003eL. reuteri\u003c/em\u003e and its metabolites, including indole-3-lactic acid, have been shown to interact with AhR pathway which plays a crucial role in regulating immune responses and maintaining intestinal homeostasis. The activation of AhR can lead to the suppression of NLRP3 inflammasome activation, thereby reducing the production of pro-inflammatory cytokines and alleviating inflammation[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].The involvement of the NLRP3 inflammasome in the pathogenesis of AS has been highlighted in various studies, where it is known to contribute to the production of pro-inflammatory cytokines such as IL-1β and IL-18, which exacerbate the inflammatory response[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].Our results show that by promoting a healthy balance of gut bacteria, \u003cem\u003eL. reuteri\u003c/em\u003e can influence the production of metabolites that activate AhR, thereby indirectly inhibiting the NLRP3 expression and reducing inflammation. This probiotic's ability to restore gut microbiota balance and its interaction with the AhR/NLRP3 pathway suggest a promising therapeutic strategy for managing ankylosing spondylitis.\u003c/p\u003e \u003cp\u003eIn conclusion, this research provides compelling evidence that \u003cem\u003eL. reuteri\u003c/em\u003e can inhibit the progression of ankylosing spondylitis in a mouse model, primarily through the modulation of gut microbiota, improvement of metabolic profiles, and regulation of AhR/NLRP3 inflammatory pathway. These findings not only highlight the therapeutic potential of \u003cem\u003eL. reuteri\u003c/em\u003e in managing AS but also underscore the critical role of gut health in maintaining systemic immune homeostasis. However, this study has limitation. while the murine AS model provides valuable insights, the extrapolation of these findings to human AS patients requires further investigation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Experimental design\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cem\u003eL. reuteri\u003c/em\u003e (strain designation 100\u0026thinsp;\u0026minus;\u0026thinsp;23, DSMZ 17509) was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany)[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. \u003cem\u003eL. reuteri\u003c/em\u003e strains were inoculated into MRS medium (Guangdong Huankai Microbial Technology Co., Ltd, China) and incubated anaerobically at 37\u0026deg;C for 18 hours. Then, \u003cem\u003eL. reuteri\u003c/em\u003e strains were collected by centrifugation and resuspended in saline to achieve a concentration of 5\u0026times;10\u003csup\u003e9\u003c/sup\u003eCFU/mL.\u003c/p\u003e\u003cp\u003eFemale BALB/c mice at 8 months were purchased from Beijing Huafukang Biotechnology Co., Ltd. These mice were housed in a temperature-controlled condition with a suitable indoor temperature and humidity, ad libitum access to water and a 12-hour light-dark cycle. All animal experiments were carried out with the approval of the Institutional Animal Care and Ethics Committee of Southern Medical University. We chose the proteoglycan (PG) induced inflammation mouse model as the AS animal which were induced using previously described standard methods[27\u003csup\u003e,\u003c/sup\u003e28]. Briefly, on days 0, 21, and 42, mice were intraperitoneally injected with emulsified 100 \u0026micro;g PG (Sigma-Aldrich, USA) and 2 mg of dimethyl dioctadecylammonium bromide (Sigma-Aldrich, USA).\u003c/p\u003e\u003cp\u003eAfter modeling, the mice were randomly assigned to three groups (n\u0026thinsp;=\u0026thinsp;10 per group): Control group: healthy BALB/c mice receiving equal volumes of saline during the schedule, AS model group (PG group): AS mice receiving equal volumes of saline during the schedule, \u003cem\u003eL. reuteri\u003c/em\u003e group: AS mice were administered 0.2 mL of the \u003cem\u003eL. reuteri\u003c/em\u003e suspension(1x10\u003csup\u003e9\u003c/sup\u003eCFU/day/mouse) for 4 weeks continuously. The dosage of \u003cem\u003eL. reuteri\u003c/em\u003e used was based on a previous publication [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]and our preliminary experiments. Treatment was started in the 10th week after PG induction and lasted for 4 weeks. The total experiment time last 14 weeks.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Clinical and histological assessments of arthritis and spondylitis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe assessment of peripheral arthritis severity was conducted daily by observing changes in the swelling of the mice's paws. The criteria for scoring arthritis were established as follows (0\u0026ndash;4 points)[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]: 0\u0026thinsp;=\u0026thinsp;normal, indicating no arthritis; 1\u0026thinsp;=\u0026thinsp;slight swelling and/or redness in a single joint; 2\u0026thinsp;=\u0026thinsp;moderate swelling and redness found in one or more joints of the paw; 3\u0026thinsp;=\u0026thinsp;substantial erythema and swelling across all joints and ankles; 4\u0026thinsp;=\u0026thinsp;extensive swelling affecting the entire paw. To evaluate the severity and progression of the disease, the cumulative score from all paws of each mouse was utilized, with a maximum potential score of 16 points. The greatest thickness recorded in either of the two hind paws of each mouse was concurrently measured using a Vernier caliper. A micro-CT was performed for each mouse with ScancoMicroCT u40 (Scanco, Bassersdorf, Switzerland) at week 14.\u003c/p\u003e \u003cp\u003eAt the 14-week mark, following euthanasia, tissues from the paw and spine were collected, fixed in 4% paraformaldehyde, decalcified using 10% EDTA for one month, embedded in paraffin, sectioned into slices of 5\u0026micro;m thickness, and subsequently stained with hematoxylin and eosin (H\u0026amp;E).Histological scoring of the ankle joint: After the ankle joint of the mice was sliced, H\u0026amp;E staining was conducted to assess pathological changes in the tissues and to assign scores. The scoring focused on synovial inflammation, cartilage degeneration, and bone erosion. Histological scoring of the spine: The grading of spondylitis histology was categorized from 1 to 4:1\u0026thinsp;=\u0026thinsp;minor accumulation of inflammatory cells and/or involvement of the annulus fibrosus surrounding the intervertebral disc (IVD); 2\u0026thinsp;=\u0026thinsp;mild inflammation with IVD absorption or erosion (less than 50% of IVD); 3\u0026thinsp;=\u0026thinsp;severe inflammation affecting the IVD with significant loss of intervertebral space (greater than 50%); 4\u0026thinsp;=\u0026thinsp;total cartilaginous or bony ankylosis. Each mouse's total score was determined by averaging the scores of 10 intervertebral joints, as previously described.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Measurement of cytokines in the serum\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAt the end of week 14, the mice were anesthetized using isoflurane, and blood samples were obtained through cardiac puncture. Once the blood coagulated, serum samples were extracted by centrifugation at 1500\u0026times;g for 10 min and preserved at -80\u0026deg;C. The concentrations of inflammatory cytokines IL-1β, IL-18, IL-17A, and IL-23 were measured in accordance with the ELISA kit's(ABclonal Technology TM, Wuhan, China)guidelines.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.4. In vivo intestinal permeability assessment\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDuring the 14th week of the experiment, the mice underwent a fasting period lasting six hours, during which they were not provided with any water. Following this fasting period, the mice received an administration of 4 kDa fluorescently labeled dextran (DX-4000-FITC, Sigma-Aldrich) at a dose of 600 mg/kg body weight (125 mg/ml). After one hour had elapsed from the administration of the fluorescent dextran, blood samples of 300 \u0026micro;l were obtained through the eye socket for the purpose of preparing serum necessary for fluorescence measurement. To ensure accurate analysis, the collected serum sample was diluted with an equal volume of phosphate-buffered saline (PBS). The concentration of FITC in the diluted serum was then determined using a fluorescence spectrophotometer, set to an excitation wavelength of 485 nm and an emission wavelength of 535 nm. To facilitate this quantitative analysis, a standard curve was created for the concentration of DX-4000-FITC by systematically diluting the DX-4000-FITC solution with PBS.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.5. Histopathologic evaluation and immunofluorescence staining\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFollowing the euthanasia of the mice, the tissue from the small intestine was promptly extracted and preserved in a 4% paraformaldehyde solution. Subsequently, standard procedures for paraffin embedding were employed to process the tissue. The resulting intestinal tissue blocks were sliced into standard 5\u0026micro;m sections and then stained with H\u0026amp;E for histological examination. The measurement of villus height was taken from the apex of the villus down to the villus-crypt junction, whereas crypt depth was characterized as the measurement from the villus base to the muscularis layer. In each section, the heights of 9 intact villi and the depths of 9 crypts were recorded.\u003c/p\u003e \u003cp\u003eImmunofluorescence staining: The slides were blocked with 5% fetal bovine serum (FBS) for 30 min at 37\u0026deg;C and incubated with primary antibodies (Occludin and ZO-1) overnight at 4\u0026deg;C as fellow: Occludin (1: 500, Thermo Fisher Scientific, USA), ZO-1 (1: 500, Thermo Fisher Scientific, USA). The slides were washed three times with PBS-Triton and incubated with FITC-conjugated goat anti-rabbit secondary antibody (Bioss, China) or Cy3-conjugated goat anti-rabbit secondary antibody (Bioss, China) at 37\u0026deg;C for 30 min. Slides were washed three times with PBS and the nuclei were stained with DAPI (Bioss, China) at room temperature for 10 min. Slides were observed under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany), and the pictures were analyzed by Image-Pro plus 6.0.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.6.DNA extraction and 16S rRNA gene sequencing\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFecal samples were meticulously collected in sterile containers twelve hours following the last gavage and were promptly frozen at a temperature of -80\u0026deg;C to preserve their integrity. To isolate microbial DNA from the fecal material of each sample, the researchers employed the QIAamp DNA Stool Minikit(Qiagen Ltd, Strasse, Germany). Subsequently, next-generation sequencing of the 16S rDNA was conducted using the Illumina HiSeq PE250 platform, with the sequencing process carried out by the Realbio Genomics Institute located in Shanghai, China. The amplification of the V3\u0026ndash;V4 region of the 16S rDNA gene sequence was executed from the genomic DNA utilizing the specific primer pair F341 (5\u0026prime;-ACTCCTACGGGRSGCAGCAG-3\u0026prime;) and R806 (5\u0026prime;-GGACTACVVGGGTATCTAATC-3\u0026prime;). Following the amplification, the raw sequencing data underwent a stringent quality control procedure utilizing the UPARSE software. In the subsequent step, the clustered reads were sorted into operational taxonomic units (OTUs) exhibiting at least 97% similarity using Usearch, a widely recognized tool for microbial analysis. To analyze the data thoroughly, principal coordinate analysis (PCoA) was performed, alongside heatmap analysis and species abundance assessments, utilizing the statistical programming language R. Furthermore, to investigate the differences in the relative abundance of gut microbiota among the control, PG, and \u003cem\u003eL. reuteri\u003c/em\u003e groups, linear discriminant analysis effect size (LEfSe) was employed in conjunction with the Kruskal-Wallis rank sum test. The analysis established a threshold for significance in the LEfSe results based on a log linear discriminant analysis (LDA) score greater than 2. PICRUS was utilized to predict metagenome functional profiles derived from the 16S rRNA data obtained from the sequencing to further enhance understanding of the microbial community and its functions[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.7. Metabolomics analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eUntargeted LC-MS metabolite profiling analysis on serum samples was conducted by Hangzhou Lian-Chuan Biotechnology Co., Ltd. The details of experimental protocols were performed as described previously[\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. An LC-MS system (TripleTOF 5600, AB SCIEX, USA) was utilized to analyze the metabolomics in positive and negative ion modes. The identified metabolites were annotated using the KEGG database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp/kegg/pathway.html\u003c/span\u003e\u003cspan address=\"https://www.genome.jp/kegg/pathway.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the HMDB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hmdb.ca/metabolites).T\u003c/span\u003e\u003cspan address=\"https://hmdb.ca/metabolites).T\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003eo obtain the correlation of the samples, principal component analysis (PCA) was conducted on the standardized data to provide an overview of group clustering and to identify potential outliers. Supervised PLS-DA was executed utilizing metaX to differentiate the diverse variables across the groups. The default criteria for differential metabolite screening were VIP\u0026thinsp;\u0026gt;\u0026thinsp;1, P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and FC\u0026thinsp;\u0026ge;\u0026thinsp;2 or FC\u0026thinsp;\u0026le;\u0026thinsp;0.5. A heatmap was generated to visualize the hubs of significantly changed metabolites as a function of quercetin treatment duration. The pathway analysis was performed using the website MetaboAnalyst (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.metaboanalyst.ca/\u003c/span\u003e\u003cspan address=\"http://www.metaboanalyst.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This research examined the correlation between metabolites and intestinal microbiota through the application of the Pearson correlation coefficient to evaluate linear associations.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.8. Quantitative reverse transcription PCR(RT-qPCR)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTotal RNA was isolated from the spine tissues of mice using an RNA extraction kit (Solarbio, Beijing, China) following the manufacturer\u0026rsquo;s protocol. The total RNA was then reversely transcribed into cDNA by an iScript\u0026trade; cDNA Synthesis kit (Bio-Rad, USA), and quantitative PCR reactions were performed with SsoAdvanced Universal SYBR Green Supermix(Bio-Rad, USA). The sequence of the primers used is presented in supplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. RT-qPCR was performed using a CFX96 Real-Time PCR System (Bio-Rad, USA). The changes in gene expression were calculated using the 2\u0026ndash;ΔΔCt method, and the house-keeping gene β-actin was used as the endogenous control for normalization.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.9. Statistical analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eData were analyzed using a t-test or one-way ANOVA with SPSS statistical software. All data in this research are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of at least three independent experiments. The difference was considered significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eThe following abbreviations are used in this manuscript:\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"944\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.6239%;\"\u003e\n \u003cp\u003e\u003cem\u003eL.Reuteri/Lreu\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.3761%;\"\u003e\n \u003cp\u003e\u003cem\u003eLactobacillus reuteri\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.6239%;\"\u003e\n \u003cp\u003eAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.3761%;\"\u003e\n \u003cp\u003eAnkylosing spondylitis\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.6239%;\"\u003e\n \u003cp\u003ePG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.3761%;\"\u003e\n \u003cp\u003eProteoglycan\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.6239%;\"\u003e\n \u003cp\u003eAhR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.3761%;\"\u003e\n \u003cp\u003earyl hydrocarbon receptor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Data curation, Validation, Methodology, writing - review \u0026amp; editing, Funding acquisition,L.J.Y;\u0026nbsp;Methodology, Formal analysis,K.Y; Review \u0026amp; editing,K.W;\u0026nbsp;Resources,T.C.;\u0026nbsp;Resources,B.L.;Methodology\u0026nbsp;,Z.F.C.;\u0026nbsp;Resources, D.W.Z.;\u0026nbsp;Methodology,Z.H.S.;\u0026nbsp;Resources,discussed the results,.X.L.;\u0026nbsp;Conceptualization, Supervision, Resources,H.L.;\u0026nbsp;All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eAuthorship must be limited to those who have contributed substantially to the work reported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This study was supported by the Natural Science Foundation of China 82302035 (L. Yang); Project of Administration of Traditional Chinese Medicine of Guangdong Province of China（NO.20241046 L. Yang）; The Basic and Applied Basic Research Foundation of Guangdong Province, Grant Number 2023A1515111101 (K. Wang).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e Data and materials used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang R, Dasgupta A, Ward MM. 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Microbiome. 2024;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s40168-023-01734-4\u003c/span\u003e\u003cspan address=\"10.1186/s40168-023-01734-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Supplementary Table 1","content":"\u003cp\u003eSupplemental table 1 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ankylosing spondylitis, Lactobacillus reuteri, gut microbiota, metabolism","lastPublishedDoi":"10.21203/rs.3.rs-6109718/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6109718/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Ankylosing spondylitis (AS) is a chronic inflammatory disease characterized by progressive spinal fusion and systemic inflammation. Recent studies suggest that gut microbiota plays a crucial role in the pathogenesis of AS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e This study investigated the therapeutic effects of \u003cem\u003eLactobacillus reuteri (L. reuteri)\u003c/em\u003e on AS progression and its underlying mechanisms using a proteoglycan (PG)-induced mouse model. Female BALB/c mice (n=10/group) were randomized into control group, PG group and PG + \u003cem\u003eL. reuteri\u003c/em\u003e group. Disease severity was assessed via arthritis scores, Micro-CT images, and histopathology. Serum cytokines (IL-1β, IL-18, IL-17A, IL-23) were measured by ELISA. Intestinal barrier integrity was evaluated using FITC-dextran permeability, immunofluorescence (ZO-1, occludin), and colon histology. Gut microbiota (16S rRNA sequencing) and fecal metabolites (untargeted metabolomics) were analyzed. AhR/NLRP3 pathway activity was assessed via qRT-PCR (AhR, CYP1A1, CYP1B1) and Western blot (NLRP3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eOur findings demonstrated that \u003cem\u003eL. reuteri\u003c/em\u003e significantly alleviated AS progression, as evidenced by reduced joint swelling and erythema, alongside a decreased arthritis index and paw thickness. Furthermore, treatment with \u003cem\u003eL. reuteri\u003c/em\u003e resulted in a marked reduction in serum levels of pro-inflammatory cytokines, including IL-1β, IL-18, IL-17A, and IL-23, indicating its potential to modulate systemic inflammation. Additionally, \u003cem\u003eL. reuteri\u003c/em\u003eenhanced intestinal mucosal barrier function, as demonstrated by improved histopathological integrity, reduced intestinal permeability, and restored expression of tight junction proteins ZO-1 and occludin. Moreover, \u003cem\u003eL. reuteri\u003c/em\u003e treatment restored gut microbiota composition and metabolite profiles, aligning them more closely with control groups. Notably, \u003cem\u003eL. reuteri\u003c/em\u003ealso regulated the Aryl hydrocarbon receptor (AhR)/NLRP3 pathway, increasing mRNA levels of AhR, CYP1A1, and CYP1B1 while decreasing NLRP3 expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eIn conclusion, \u003cem\u003eL. reuteri\u003c/em\u003e effectively prevents the progression of AS in mice by restoring gut microbiota-metabolism homeostasis and modulating inflammatory pathways, highlighting its potential as a therapeutic agent for AS.\u003c/p\u003e","manuscriptTitle":"Lactobacillus Reuteri Prevents Progression of Ankylosing Spondylitis in Mice by Restoring Gut Microbiota-Metabolism Homeostasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-03 09:08:30","doi":"10.21203/rs.3.rs-6109718/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-03-23T10:02:22+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-23T08:18:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-19T14:11:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2025-03-18T04:12:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d6a97433-3c1b-4679-a33b-522cdff05198","owner":[],"postedDate":"April 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:05:42+00:00","versionOfRecord":{"articleIdentity":"rs-6109718","link":"https://doi.org/10.1186/s12967-025-06681-2","journal":{"identity":"journal-of-translational-medicine","isVorOnly":false,"title":"Journal of Translational Medicine"},"publishedOn":"2025-07-01 15:58:10","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-04-03 09:08:30","video":"","vorDoi":"10.1186/s12967-025-06681-2","vorDoiUrl":"https://doi.org/10.1186/s12967-025-06681-2","workflowStages":[]},"version":"v1","identity":"rs-6109718","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6109718","identity":"rs-6109718","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}