Immune Responses Targeting Homocitrullinated Peptides Drive Rheumatoid Arthritis-like Pathology in HLA-DR4 Transgenic Mice

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by persistent synovial inflammation and progressive joint destruction. A hallmark feature of RA is the presence of anti-citrullinated protein autoantibodies (ACPAs), which have been extensively studied in disease pathogenesis. The development of these autoantibodies is strongly associated with the expression of HLA-DR4, containing the shared epitope. More recently, anti-homocitrullinated protein autoantibodies (AHCPAs) have also been implicated in RA and demonstrate a similar genetic association. Therefore, this study aimed to evaluate the role of homocitrulline immune responses in RA pathogenesis. HLA-DR4 transgenic (DR4tg) and B6 mice were immunized with a synthetic homocitrullinated peptide (HomoCitJED) or PBS followed by a booster 21 days later. Beginning on day 75 post-primary immunization mice received knee intra-articular (i.a.) injections of HomoCitJED once a week for three consecutive weeks. Swelling post i.a. injection was measured using digital calipers. IgG antibodies against homocitrullinated peptides/proteins were measured using enzyme-linked immunosorbent assay (ELISA). Knees were sectioned and analyzed for histopathological damage and citrullinated/homocitrullinated protein levels. HomoCitJED immunized DR4tg mice exhibited significantly greater swelling and incidence of anti-HomoCitJED and -homocitrullinated fibrinogen IgG autoantibodies. Additionally, 30% had responses against CitJED, a synthetic citrullinated peptide. HomoCitJED immunized DR4tg mice also experienced greater total histopathological damage; however, the immunizations/injections did not affect citrullinated/homocitrullinated protein levels. In conclusion, homocitrulline-specific immune responses can induce RA-like disease in DR4tg mice, supporting the pathogenic role of AHCPAs in RA.