Background/Purpose: Antiphospholipid syndrome (APS) is an autoimmune clotting disorder of unknown etiology targeting a major autoantigen, β₂-glycoprotein I (β₂GPI). Infectious triggers have been implicated in transient autoantibody production, but the persistent stimuli for anti-β₂GPI antibodies remain unknown. Given the vast antigenic potential of the gut microbiota, we hypothesize that human gut commensal bacteria induce and sustain autoreactivity via cross-reactivity. To this end, we characterized APS PBMC reactivity to in silico candidates and determined fecal autoantibody production.

Methods: Protein BLAST and Clustal Omega were used to identify commensal protein sequences with high homology to β₂GPI-dominant epitopes. Using anaerobic cultures, we grew isolated candidate and control strains. Blood and stool samples were obtained from anti-β₂GPI-positive patients, non-autoimmune thrombophilia patients, and healthy controls. Stool DNA was isolated using the MoBio extraction kit. A novel species-specific real-time PCR strategy was developed and validated using isolated strains and defined fecal microbiomes. In vitro proliferation of PBMC to bacterial protein extracts was assessed by [³H]-thymidine incorporation. An in-house ELISA was established with high-binding plates to analyze anti-β₂GPI levels in plasma and fecal supernatants.

Results: Systematic in silico searches revealed Roseburia intestinalis as a major candidate for cross-reactivity. R. intestinalis is a common colonic gram-positive, flagellated, mucus adhering commensal containing high homology to the main B and T cell epitopes of β₂GPI. R. intestinalis colonization load was semi-quantified in patients and controls using real-time PCR. APS PBMC proliferated significantly more to protein extracts from R. intestinalis versus control subjects (n=5-6; p=0.0002), and also compared to the closely phylogenetically related, but mimic-deficient gut commensal Eubacterium rectale (n=6, p=0.020). Importantly, we were also able to detect anti-β₂GPI IgA antibodies in APS fecal supernatants, which differed significantly compared to controls (n=14-15; p=0.0019).

Conclusion: We have identified a major cross-reactive commensal candidate in silico with high homology to dominant β₂GPI epitopes and developed a highly specific real-time PCR-based screening strategy. APS PBMCs proliferated significantly more to candidate protein extracts compared to controls. Furthermore, we report, to our knowledge, for the first time fecal autoantibody production in a non-gut autoimmune disease. Production of fecal anti-β₂GPI IgA in patients with peripheral blood anti-β₂GPI IgG supports our hypothesis of a gut mucosal, cross-reactive trigger in APS, which we are actively pursuing.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/lymphocyte-proliferation-to-a-cross-reactive-gut-commensal-candidate-in-antiphospholipid-syndrome/