Novel autoantibody targets identified in patients with autoimmune hepatitis (AIH) by PhIP-Seq reveals pathogenic insights

Abstract

While the identification of autoantigens remains a critical challenge in understanding and treating autoimmune diseases, the role of autoantibodies in the pathophysiology of autoimmune hepatitis (AIH) is uncertain. To better characterize the antigenic landscape of AIH, we employed Phage Immunoprecipitation-Sequencing (PhIP-Seq) to identify novel autoantibodies specific to patients. Using these results, a logistic regression classifier was able to predict which patients had AIH, relative to healthy controls indicating the presence of a distinct humoral immune signature. To further investigate the autoantibodies most specific to AIH, significant peptides were identified with >98% specificity relative to a broad array of controls (298 patients with NAFLD, PBV, or healthy controls). Top ranked autoreactive targets included anti-SLA, a well-recognized autoantibody in AIH, and a second hit to disco interacting protein 2 homolog A (DIP2A). The autoreactive fragment of DIP2A shares a nearly identical 8 amino acid stretch with the U27 protein of HHV-6, a virus known to reside in the liver. Finally, peptides derived from the LRRNT domain of the relaxin family peptide receptor 1 (RXFP1) were highly enriched and specific to AIH. The enriched peptides map to a motif adjacent to the receptor binding domain, required for RXFP1 signaling. RXFP1 is a G-protein coupled receptor that binds relaxin-2, an anti-fibrogenic molecule shown to reduce the myofibroblastic phenotype of hepatic stellate cells. Eight of nine patients with antibodies to RXFP1 had evidence of advanced fibrosis (F3 or greater). Furthermore, serum from AIH patients positive for anti-RFXP1 antibody was able to significantly inhibit relaxin-2 signaling in THP1 cells relative to anti-RXFP1 negative control serum, while depletion of IgG abrogated this effect. These data demonstrate that PhIP-Seq is a powerful discovery tool, capable of identifying known and novel autoantibody targets in AIH, lending additional supporting evidence that HHV6 may play a role in the development of AIH, and pointing to a potential pathogenic role for anti-RXFP1 IgG in some patients. Identification of anti-RXFP1 in patient serum may enable risk stratification of AIH patients for fibrosis progression and lead to the development of novel strategies for disease intervention.