Objective Gastrointestinal (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.

Methods Sera 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.

Results Anti-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.

Conclusion SSc patient autoantibodies may reveal distinct clinical phenotypes and disease mechanisms that can define biomarkers, disease pathways, and targets for potential therapeutic strategies.

The authors have declared no competing interest.

This work was supported by the Rheumatology Research Foundation, the Scleroderma Research Foundation, the Chresanthe Stauraluakis Memorial Discovery Fund to the Johns Hopkins Scleroderma Center, a pilot and feasibility award from Harvard Digestive Disease Core to SK, the Sara and Alex Othon Fund for Scleroderma Research, the Donald B and Dorothy L Stabler Foundation, and NIH grants R01 AR081382 to ZM, K24 AR080217 to AS, and R01AG066768 to SK. The Rheumatic Diseases Research Core Center, where the autoantibodies were assayed, is supported by NIH P30-AR070254. The mass spectrometry was performed at the JHU School of Medicine Mass Spectrometry and Proteomics Core Facility.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Johns Hopkins University and the University of Texas Health IRBs approved these studies, and all individuals provided informed consent.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

Funding Statement: This work was supported by the Rheumatology Research Foundation, the Scleroderma Research Foundation, the Chresanthe Stauraluakis Memorial Discovery Fund to the Johns Hopkins Scleroderma Center, a pilot and feasibility award from Harvard Digestive Disease Core to SK, the Sara and Alex Othon Fund for Scleroderma Research, the Donald B and Dorothy L Stabler Foundation, and NIH grants R01 AR081382 to ZM, K24 AR080217 to AS, and R01AG066768 to SK. The Rheumatic Diseases Research Core Center, where the autoantibodies were assayed, is supported by NIH P30-AR070254. The mass spectrometry was performed at the JHU School of Medicine Mass Spectrometry and Proteomics Core Facility.

Conflict of interest statement: None of the authors received any financial support or other benefits from commercial sources for the work reported in this manuscript, nor do any of the authors have any financial interests, which could create a potential conflict of interest or appearance thereof.

All data produced in the present work are contained in the manuscript