Novel autoantibodies in patients with systemic sclerosis and gastrointestinal dysfunction provide insight into disease pathogenesis

ObjectiveGastrointestinal (GI) dysfunction in systemic sclerosis (SSc) is common and debilitating, yet its underlying mechanisms and related biomarkers are poorly understood. We sought to discover novel autoantibodies in patients with SSc-GI dysfunction and evaluate their clinical relevance. MethodsSera from 111 SSc patients enrolled in the Gastrointestinal Assessment Protocol (GAP) were screened for novel autoantibodies. Using immunoprecipitation of murine myenteric plexus lysates followed by mass spectrometry, autoantibodies targeting Argonaute RISC Catalytic Component 1/2 (AGO) and Dihydrolipoamide Branched Chain Transacylase E2 (DBT) were identified. Clinical associations were evaluated in two SSc cohorts. Expression of AGO and DBT in the murine enteric nervous system (ENS) was confirmed by immunohistochemistry. ENS-derived extracellular vesicles (EVs) from longitudinal muscle-myenteric plexus tissues were analyzed for AGO cargo using flow cytometry and western blotting. ResultsAnti-AGO antibodies occurred in 13.5% and anti-DBT in 4% of GAP patients. Anti-AGO antibodies were associated with severe constipation on the UCLA GIT 2.0 (26% vs. 9%, p=0.036), and anti-AGO2, specifically, associated with severe distention and bloating (16% vs. 4%, p=0.046). Higher AGO1/2 antibody levels associated with severe constipation (p=0.03). Anti-DBT patients exhibited less esophageal emptying at 10 seconds (30% vs. 81%, p=0.034) and less constipation [median 0 (IQR 0-0) vs. 0.75 (0.25-1), p=0.02]. Immunofluorescence studies revealed that anti-AGO antibodies target AGO2-containing gut-derived EVs, whereas anti-DBT antibodies recognize mesoderm- derived enteric neurons and smooth muscle. ConclusionSSc patient autoantibodies may reveal distinct clinical phenotypes and disease mechanisms that can define biomarkers, disease pathways, and targets for potential therapeutic strategies.