Background: The human gut microbiota plays a fundamental role in sustaining mucosal integrity and immune homeostasis. Gut dysbiosis in ankylosing spondylitis (AS) is characterised by depletion of health-associated commensal bacteria that produce metabolites essential for epithelial maintenance and immune regulation. Despite their clinical relevance, the strain-level metabolic and immunomodulatory capacities of many commensals remain poorly defined. This imbalance contributes to impaired mucosal tolerance and persistent inflammation, which is increasingly recognised as a contributing factor to AS pathogenesis.

Objectives: To investigate the genomic, metabolic, and functional properties of five health-associated gut bacterial species— Bacteroides uniformis , B. xylanisolvens , Bittarella massiliensis , Clostridium scindens , and C. symbiosium —to elucidate their contributions to gut barrier protection and host immune modulation. These species belong to bacterial families underrepresented in individuals with AS.

Methods: Comparative genomic analysis was performed on three novel strains versus reference strains. Carbohydrate utilisation, short-chain fatty acid (SCFA) production, and metabolite profiles were assessed under axenic conditions using an integrated genotype-to-phenotype framework. Untargeted metabolomics confirmed metabolite production. Bacterial supernatants were tested individually and in combination on differentiated Caco-2 monolayers under homeostatic and inflammatory conditions, assessing transepithelial electrical resistance, permeability, and tight junction protein expression. Signalling pathway analysis investigated IL-23-induced STAT3 activation and TNF-α-mediated NF-κB activation. Immunomodulatory effects were profiled on THP-1 monocytes and peripheral blood mononuclear cells (PBMCs) from healthy donors and AS patients using flow cytometry and cytokine analysis.

Results: Untargeted metabolomics confirmed production of immunomodulatory metabolites including SCFA, indole derivatives, vitamins, antimicrobial compounds, and tricarboxylic acid across all strains. B. xylanisolvens , C. symbiosium , and C. scindens demonstrated significant protective effects against cytokine-induced barrier disruption. B. xylanisolvens , C. symbiosium , and B. uniformis inhibited IL-23-induced STAT3 activation, while NF-κB activation was not suppressed and, in some cases, was modestly enhanced. PBMCs from AS patients exhibited heightened reactivity to bacterial metabolites compared to healthy controls, demonstrating disease-associated dysregulation of host-microbe signalling. Under inflammatory conditions, bacterial supernatants showed context-dependent immunoregulatory effects, with differential modulation of monocyte activation markers and cytokine production.

Conclusions: Health-associated gut commensals exert complementary metabolic and immunomodulatory functions that reinforce epithelial barrier resilience and regulate inflammatory signalling. The disease-dependent responses of AS PBMCs suggest dysregulation of host-microbe interactions in disease. These mechanistic insights support the rational development of microbial or postbiotic interventions for AS and other chronic inflammatory diseases.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: None declared.