Differential ACPA Binding to Nuclear Antigens Reveals a PAD-Independent Pathway and a Distinct Subset of Acetylation Cross-Reactive Autoantibodies in Rheumatoid Arthritis

Abstract

Rheumatoid arthritis (RA) associated anti-citrullinated protein autoantibodies (ACPA) target a wide range of modified proteins. Citrullination occurs during physiological processes such as apoptosis, yet little is known about the interaction of ACPA with nuclear antigens or apoptotic cells. Since uncleared apoptotic cells and neutrophil extracellular trap (NET) products have been postulated to be central sources of autoantigen and immunostimulation in autoimmune disease, we sought to characterize the anti-nuclear and anti-neutrophil reactivities of ACPA. Serology showed that a subset of anti-CCP2 seropositive RA patients had high reactivity to full-length citrullinated histones. In contrast, seronegative RA patients displayed elevated IgG reactivity to native histone compared to controls, but no citrulline-specific reactivity. Screening of 10 single B-cell derived monoclonal ACPA from RA patients revealed that four ACPA exhibited strong binding to apoptotic cells and three of these had anti-nuclear (ANA) autoantibody reactivity. Modified histones were confirmed to be the primary targets of this anti-nuclear ACPA subset following immunoprecipitation from apoptotic cell lysates. Monoclonal ACPA were also screened for reactivities against stimulated murine and human neutrophils, and all the nuclear-reactive monoclonal ACPA bound to NETs. Intriguingly, one ACPA mAb displayed a contrasting cytoplasmic perinuclear neutrophil binding and may represent a different NET-reactive ACPA subset. Notably, studies of CRISPR-Cas9 PAD4 KO cells and cells from PAD KO mice showed that the cytoplasmic NET-binding was fully dependent on PAD4, whilst nuclear- and histone-mediated NET reactivity was largely PAD-independent. Our further analysis revealed that the nuclear binding could be explained by consensus-motif driven ACPA cross-reactivity to acetylated histones. Specific acetylated histone peptides targeted by the monoclonal antibodies were identified and the anti-modified protein autoantibody (AMPA) profile of the ACPA was found to correlate with the functional activity of the antibodies. In conclusion, when investigating monoclonal ACPA, we could group ACPA into distinct subsets based on their nuclear binding-patterns and acetylation-mediated binding to apoptotic cells, neutrophils, and NETs. Differential anti-modified protein reactivities of RA-autoantibody subsets could have an important functional impact and provide insights in RA pathogenesis.

The importance of differences; On environment and its interactions with genes and immunity in the causation of rheumatoid arthritis

“…Whereas the anti-CCP assay captures most ACPAs, there is a notable heterogeneity in reactivity to distinct citrullinated peptides and proteins with a large number of overlapping subsets being formed [17][18][19][20] (see also Figure 1). Subsequent to the recognition of reactivity against epitopes modified by citrullination, RA autoantibodies have been shown to also react with proteins/peptides modified by additional post-translational modifications, including homocitrullination (or carbamylation) [21][22][23][24] as well as acetylation [25,26]. Although most of these antibodies are seen in ACPA-positive patients, there are also smaller subsets of RA with antibodies against these alternative modifications, while being negative for ACPA or RF [18,20].…”

Pathogenic effector functions of ACPA: Where do we stand?

“…Adding to the complexity of the ACPA system is the extensive cross-reactivity of these autoantibodies towards different citrullinated antigens as well as other PTMs expressed by proteins 42 46 47. As these different modifications are recognised with varying avidities, attributing biological effects of monoclonal or polyclonal antibodies to the recognition of citrulline alone is difficult and it can be debated whether citrulline specificity and/or the ‘Peptidyl Arginine Deiminase-Citrulline’ pathway should be considered the predominant or indeed only auto-reactive phenomenon.…”

Neutrophil Extracellular Traps (NETs) Take the Central Stage in Driving Autoimmune Responses

“…NETs extrude novel autoantigens, such as citrullinated histones, which can promote the autoimmune response in RA [18,32]. Certainly ACPAs are reported to recognize autoantigens on NETs [7,30]; in particular citrullinated histones [31]. The NET-protein MPO was found elevated in RA synovial fluid (SF), skin and rheumatoid nodules, indicating that NETs are present in these inflamed areas (joint/synovium).…”