Characterization of Autoantigens Targeted by Anti-Citrullinated Protein Antibodies In Vivo: Prominent Role for Epitopes Derived from Histone 4 Proteins

Abstract

Anti-citrullinated protein antibodies (ACPA) have become an integral part of the clinical definition of rheumatoid arthritis, and are hypothesized to be important in the immunopathogenesis of this autoimmune disease. Several citrullinated proteins have been demonstrated to serve as candidate autoantigens for the ACPA, based on in vitro immune reactions between citrullinated peptides/proteins and RA sera. Yet it remains unclear whether the autoantigens identified in vitro are indeed directly and specifically targeted by the ACPA in vivo. Moreover, it is unclear whether ACPA present in RA sera are directed towards the same spectrum of autoantigens as the ACPA present within the synovial compartment. In this study, we isolated ACPA immune complexes from RA synovial fluids (SF) and sera by using immobilized cyclic citrullinated peptides (CCP3) based immune affinity, and characterized the proteins that are directly and specifically associated with them by mass spectrometry. The results demonstrate that four histone proteins are prominent ACPA autoantigens, with the frequency of detection being histone H4 (89%), H2B (63%), H3 (63%), and H2A (58%) in ACPA positive RA SF. We further demonstrate that a histone 4 peptide containing citrulline at position Cit39 was recognized by 100% of ACPA positive RA SF. An adjacent citrulline residue at Cit40 was recognized by 34% of ACPA positive RA SF. An H4 peptide containing Cit39-40 was recognized in the serum of 94% ACPA positive RA, 77% ACPA positive first-degree relatives (FDR) of RA patients, and 2.5% of healthy controls. The Cit39-40 peptide substantially blocked the ACPA reactivity in both SF and serum. Although the spectrum of ACPA we identified was limited to those isolated using immobilized CCP3 peptides, the findings indicate that H4 is a widely recognized RA autoantigen in both the synovial and serum compartments. The identification of this immunodominant ACPA epitope may be valuable in designing approaches to immune tolerance induction in RA.

Proteomic analysis of extracellular vesicles reveals an immunogenic cargo in rheumatoid arthritis synovial fluid

“…Nine citrullinated peptides were identified, all of which were only detected in EVs from RA joints with high‐level inflammation (Table 2). These included citrullinated peptides derived from fibrinogen alpha (FGA), actin [β‐actin (ACTB)/ACTG] and histone H3, which are known to be recognised by anti‐citrullinated peptide antibodies (ACPA) 14–16 . Consistent with this, the majority of patients, in whom citrullinated EV peptides were observed, were ACPA‐positive.…”

A Prospective Study of the Development of Inflammatory Arthritis in the Family Members of Indigenous North American People With Rheumatoid Arthritis

“…In the available samples tested, the calculated ACPA score correlated with anti‐CCP titers (r = 0.55, P = 0.01; n = 214). ACPA levels directed toward an immunodominant citrullinated epitope derived from the histone 4 protein showed a high degree of correlation between the levels of anti–Cit‐H4 antibodies and the ACPA score in sequential samples from the progressors (r = 0.66, P = 0.0001; n = 42).…”

“…Levels of ACPA targeting the specific peptide Cit‐H4 were closely correlated with increasing ACPA scores, indicating a relationship between these 2 events. We have previously shown that this peptide is an immunodominant autoantigen in RA synovial fluid, where ongoing NETosis may fuel the autoimmune response . These findings suggest that the autoimmune response to Cit‐H4 likely develops outside the joint as part of a broader ACPA response, and then becomes amplified in the inflammatory synovial microenvironment.…”

“…To generate an ACPA score, each ACPA value was divided by the mean value of the cohort, then all ACPA values were added together, calculated as [Σ(ACPA value/mean ACPA value)] as previously described . An ELISA assay was used to test for serum anti–citrullinated histone 4 antibodies (anti–Cit‐H4), as we have previously reported .…”

Relative efficiencies of peptidylarginine deiminase 2 and 4 in generating target sites for anti-citrullinated protein antibodies in fibrinogen, alpha-enolase and histone H3

“…PAD2 and PAD4 are expressed in RA synovium and their presence correlates with inflammatory markers [ 11 – 13 ]. Among the best described PAD substrates is fibrinogen, which is present in the synovium of RA patients [ 14 – 18 ] together with numerous other citrullinated self-antigens including vimentin, alpha-enolase (ENO1), collagen type II and histones, all of which have proposed pathogenic relevance [ 19 – 24 ]. PAD2 seems less restricted by the amino acid composition surrounding the acceptor arginine residue than PAD4 [ 25 ].…”