Objective Anti-citrullinated protein antibodies (ACPAs) are characteristic of rheumatoid arthritis. However, their presence years before onset of clinical RA is perplexing. Although multiple putative citrullinated antigens have been identified, no studies have demonstrated the specific capacity of these antigens to initiate inflammatory arthritis. We sought to recapitulate the transition from preclinical to clinical RA and to demonstrate the capacity of local citrullination to facilitate this transition. Methods We performed proteomic analysis of activated human neutrophils to identify citrullinated proteins including those targeted as part of the RA immune response. Using ELISA we compared RA and OA synovial fluid for levels of citrullinated histone 2B (cH2B) and its immune complex. Using macrophage activation assays we assessed the effect of histone citrullination on immunostimulatory capacity and evaluated the stimulatory capacity of native and citrullinated H2B immune complexes. Finally, we assessed the potential for anti-cH2B antibodies to mediate arthritis in vivo. Results We identified robust targeting of neutrophil-derived citrullinated histones by the ACPA immune response. Over 90% of RA patients have anti-cH2B antibodies. Histone citrullination increases innate immunostimulatory capacity and immune complexes containing citrullinated histones activate macrophage cytokine production and propagate neutrophil activation. Finally, we demonstrate that immunization with H2B is arthritogenic, but only in the setting of underlying articular inflammation. Conclusion We identify citrullinated histones, specifically cH2B as an antigenic target of the ACPA immune response. Furthermore, local generation of citrullinated antigen during low grade articular inflammation provides a mechanistic model for the conversion from preclinical autoimmunity to inflammatory arthritis.

Conflict of interest:ILW is a founder of Forty Seven Inc., in which he and SSP own equity.

BackgroundThe pathophysiology of osteoarthritis (OA) involves wear and tear, and a state of low-grade inflammation. Tissue repair responses include transforming growth factor beta (TGFβ)-induced myofibroblast production of extracellular matrix. Fibronectins are an essential part of the extracellular matrix, and injection of fibronectin fragments into rabbit joints is a previously established animal model of OA. Fibronectin containing the ED-A domain is currently being used as drug delivery target in the development of anti-inflammatory drugs (e.g. Dekavil).MethodsIn this study, samples of synovial membrane were obtained from patients with knee OA undergoing joint replacement surgery. Immunostaining for ED-A fibronectin and the myofibroblast marker alpha smooth muscle actin (αSMA) was performed on fibroblast-like synovial cells (FLS) and synovial membranes. RAW 264.7 macrophages were incubated with recombinant ED-A fibronectin.ResultsThe staining of ED-A fibronectin in OA FLS was increased by TGFβ but not by TNFα, lipopolysaccharide, or IL-6 (n = 3). ED-A fibronectin co-stained with the myofibroblast marker αSMA in both the OA FLS (n = 3) and in the OA synovial membranes (n = 8). ED-A fibronectin staining was associated with both number of lining layer cells (rho = 0.85 and p = 0.011) and sublining cells (rho = 0.88 and p = 0.007) in the OA synovium (n = 8), and co-distributed with TNFα (n = 5). Recombinant ED-A fibronectin increased the production of TNFα by RAW 264.7 macrophages (n = 3).ConclusionsThe disease process in OA shares features with the chronic wound healing response. Our findings support utilizing ED-A fibronectin for drug delivery or therapeutic targeting to reduce pro-inflammatory responses in OA.