IDENTIFICATION OF NOVEL ACETYLATED ANTIGENS IN IMMUNE COMPLEXES OF SYNOVIAL FLUID FROM RA PATIENTS

Background Rheumatoid arthritis (RA) is an autoimmune disease characterized by genetic predisposition and associated immunological features including the production of autoantibodies against various epitopes. Several pathways have been implicated in the induction of autoantibodies against novel epitopes through post-translational modifications such as citrullination, carbamylation and acetylation (1). However, the contributions of these anti-modified protein antibodies (AMPAs) in the pathogenesis and formation of immune complexes (IC) in the synovial fluid has not been fully uncovered. Objectives To analyze reactivity of monomeric and IC antibodies of RA patients to citrullinated, carbamylated and acetylated modified antigens in comparison to disease control group and healthy donors. Methods Synovial fluid (SF) was collected from 17 RA patients and 40 individuals from a disease control group (CG) including reactive arthritis and psoriasis arthritis. Paired serum and SF samples were used from 16 RA and 19 CG compared to serum from 70 healthy donors (HD). Immune complexes (IC) were purified from sera and SF by applying a technique using purified human C1q, which was immobilised on magnetic tosylactivated microparticles (Dynabeads M-280) according to the manufacturer’s recommendations for activation of amine groups. Additionally, sucrose gradient centrifugation was used to fractionate SF and sera into 21 fractions and according to protein markers, protein G was used to isolate monomeric IgG from fractions 3-9 and IC IgG from fractions 11-22. ELISA kits for anti CCP-IgG, anti-mutated citrullinated vimentin (MCV)-IgG, anti-acetylated Lysine Antibody (anti-HC55)-IgG and anti-carbamylated vimentin (carbVim)-IgG were used to test reactivity against modified proteins. Extracted antigens from the IC were investigated with Western blot for specific modifications or individual antigens with the corresponding antibodies. Citrullinated and acetylated vimentin was identified as part of IC via mass spectroscopy. Purified AMPAs from serum or SF (isolated from IC or as soluble antibodies) were investigated on a custom-made LineBlot array containing 24 different antigens either citrullinated, carbamylated, acetylated or unmodified/native counterparts. Results Titers of anti-CCP, anti-MCV and anti-acetylated (anti-HC55) reactivity were higher in SF of RA patients compared to CG. AMPAs were detected in SF in IC as well as free antibodies. Some isolated IgG (monomeric and IC) showed reactivities against citrullinated and acetylated peptides that could not be detected in native samples. Surprisingly, in addition to citrullinated vimentin, the acetelylated form was also detected in the IC eluted fractions. Haptoglobin and fibrinogen fragments were identified as carbamylated modified proteins in small concentrations. The spectrum of identified acetylated proteins included human proteins such as histones as well as fragments of bacterial filament proteins. SF of RA patients showed reactivity against MCV and citrullinated desmin in LineBlot array. Conclusion AMPAs recognize different modified antigens and form immune complexes in the SF of RA patients. The elevated titer of anti-acetylated protein antibodies and the inclusion of its epitope in immune complexes in the synovium points to its contribution in the pathogenesis of RA. Figure 1. The identification of acetylated antigens in the synovium of RA patients is especially intriguing, as infections (bacteria) are known to not only acetylate their own proteins, but also modify host proteins. References [1]Grönwall C, Liljefors L, Bang H, et al. A Comprehensive Evaluation of the Relationship Between Different IgG and IgA Anti-Modified Protein Autoantibodies in Rheumatoid Arthritis. Front Immunol. 2021;12:627986-. Acknowledgements This work was supported by a grant from DFG. Disclosure of Interests Khetam Ghannam: None declared, Holger Bang Employee of: Employee of ORGENTEC Diagnostika GmbH, Thomas Häupl: None declared, Sarah Ohrndorf: None declared, Jan Zernicke: None declared, Ulrike Kuckelkorn: None declared, Eugen Feist: None declared, Gerd Rüdiger Burmester: None declared