ABSTRACT: Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic joint inflammation resulting in progressive cartilage and bone destruction. In two thirds of early RA patients, rheumatoid factor (RF) and/or anti-citrullinated protein antibodies (ACPAs) are present. While these antibodies are well-established markers of RA, antinuclear antibodies (ANAs) are found in 25% - 77% of RA patients, a prevalence that is significantly higher than the prevalence of ANA in the general population1,2. However, in most ANA positive samples from RA patients no specific autoantigen can be identified. In this study, we set out to expand our knowledge on the ANA landscape in RA by applying a recently described 3–5 unbiased immunoprecipitation-mass spectrometry (IP-MS) approach, which allows identification of known and unknown autoantigens. IP-MS was performed on 55 ANA-positive (titer ≤1/160, nuclear or cytoplasmatic staining pattern) samples from newly diagnosed, DMARD-naïve RA patients, selected out of 191 newly diagnosed RA patients included in three differentcohorts. Autoantigens were identified in 10 of the 55 samples. IP-MS identified antibodies to SSA and SSB (#1), Ku (#2), fibrillarin (#3) and the mitochondrial dihydrolipoyllysine-residue acetyltransferase and pyruvate dehydrogenase complex (#4, 7, 10), protein argonaute (AGO) 1, 2 and 3 (#5,6), dihydrolipoamide branched chain transacylase E2 (DBT) (#7), T-cell intracytoplasmic antigen 1(TIA1)/Nucleolysin TIAR (TIAL1) (#8), the N-terminal acetyltransferase C (NatC) complex (#9) and the mitochondrial chaperone protein BCS1 (#10). Anti-SSA and anti-SSB antibodies are most commonly detected in primary Sjögren’s syndrome (pSjS) and systemic lupus erythematosus (SLE), but they can also occur, less frequently, in other connective tissue diseases, including rheumatoid arthritis (RA) and systemic sclerosis (SSc), and only rarely in idiopathic inflammatory myopathies (IIM) or antisynthetase syndromes (ASyS). Anti-Ku antibodies are classically associated with overlap syndromes and may be present in patients with systemic sclerosis, SLE, or IIM. Anti-fibrillarin antibodies exhibit high specificity for systemic sclerosis (SSc), particularly the diffuse cutaneous subtype, and are only infrequently observed in overlap syndromes. Considering the high specificity for anti-fibrillarin antibody positivity with SSc, a careful monitoring during routine follow-up was provided. At present, however, the patient does not manifest any clinical features indicative of SSc. Antibodies to AGO2 (anti-Su) are found in various rheumatic and neurological diseases and in apparently healthy individuals at low prevalence 6,7. Similarly, antibodies to TIA1/TIAL1 have been described in several rheumatic diseases (SLE, SSc and RA) and in healthy individuals8. DBT has previously been identified as an antigenic component of anti-mitochondrial M2 antibodies and is predominantly associated with SSc. The N-terminal acetyltransferase C (NatC) complex was identified in sample (#9) with a cytoplasmic reticular staining. The NatC complex is engaged in N-terminal protein acetylation (a post-translational modification) and regulates protein stability and function. BCS1L was found in a sample together with DLAT, PDHA1 and PDHB, subunits known as representing antimitochondrial antibodies (#10). BCS1 is a mitochondrial chaperone protein and a requisite for respiratory chain complex III assembly. Autoantibodies against mitochondrial proteins have been described in RA9, but BCS1L has not yet been identified as mitochondrial autoantigen in the literature. Antibodies to the NatC complex and to BCS1L were not found in samples from 43 healthy controls (analyzed in 82 runs with cytoplasmatic extract and 127 runs with nuclear extract as protein source for IP). These were not present in samples from 102 inflammatory controls, including individuals with anti-synthetase syndrome (n=14 with 18 separate MS runs), IIM (n=12), pSjS (n=12), SLE (n=36) and SSc (n=28). In conclusion, IP-MS identified autoantigens in 10/55 ANA positive samples. A multitude of autoantigenic targets was revealed, including novel autoantigens. Further studies are needed to explore the potential clinical relevance and pathogenicity of autoantibodies against the NatC complex and BCS1.
Birthe Michiels 
