Antibody responses to de novo identified citrullinated fibrinogen peptides in rheumatoid arthritis and visualization of the corresponding B cells

Abstract

BackgroundAntibodies against citrullinated proteins (ACPA) are common in patients with rheumatoid arthritis (RA). ACPA can appear before disease onset and target many self-antigens. Citrullinated fibrin/fibrinogen represents a classical ACPA target antigen, and mass spectrometry of RA synovial fluid reveals elevated citrullinated (cit) fibrinogen (Fib) peptides compared to non-RA controls. We investigated the extent to which these less-studied peptides represent autoantibody targets and sought to visualize the corresponding cit-Fib-reactive B cells in RA patients.MethodsAn in-house ELISA was established against four cit-Fib α-subunit peptides (cit-Fib α-35; cit-Fib α-216,218; cit-Fib α-263,271 and cit-Fib α-425,426) and serum from patients with established RA (n = 347) and disease controls with psoriatic arthritis (PsA) or ankylosing spondylitis (AS) (n = 236) were analyzed. RA patients were genotyped for HLA-DR alleles, PTPN22 R620W and screened for anti-CCP2 and cit-Fib protein antibodies. The cit-Fib peptides were also used to assemble antigen tetramers to identify cit-Fib-reactive B cells in peripheral blood by flow cytometry.ResultsThe frequencies of autoantibodies against different cit-Fib epitopes in RA patients compared to PsA/AS patients were: cit-Fib α-35 (RA 20%, vs PsA/AS 1%); cit-Fib α-216,218 (13% vs 0.5%); cit-Fib α-263,271 (21% vs 0.5%) and cit-Fib α-425,426 (17% vs 1%). The presence of autoantibodies against these peptides was associated with presence of anti-CCP2 and anti-cit-Fib protein antibodies. No association was found between HLA-DR shared epitope and antibodies to the different cit-Fib peptides. However, association was observed between the PTPN22 risk allele and positivity to cit-Fib α-35 and cit-Fib α-263,271. B cells carrying surface Ig reactive to these cit-Fib peptides were found in RA peripheral blood and these tend to be more common in PTPN22 risk allele carriers.ConclusionsOur data show that several cit-Fib peptides are targeted by autoantibodies in RA, but not in PsA/AS, implicating that these are not due to arthritis but more specific for RA etiology. The RA-associated anti-cit protein response is broad with many parallel immune responses. The association between cit-Fib autoantibodies and the PTPN22 R620W risk allele supports the hypothesis of altered B cell regulation, such as autoreactive B cells evading tolerance checkpoints.

Memory T cells specific to citrullinated α-enolase are enriched in the rheumatic joint

“…They are found in different isotypes, including IgM, IgA and several subtypes of IgG, such as IgG1, IgG2, IgG3 and IgG4 [274]. ACPAs can be studied after purification from plasma or synovial fluid while autoreactive ACPA-producing B cells can be identified and visualized by the use of B cell tetramers [275][276][277]. These reagents allow ex vivo analysis of the cells and, by combining this method with sequencing technologies, even make it possible to examine their BCRs and transcriptional profiles.…”

The Central Role of Acute Phase Proteins in Rheumatoid Arthritis: Involvement in Disease Autoimmunity, Inflammatory Responses, and the Heightened Risk of Cardiovascular Disease

“…[97][98][99] Fibrinogen is the soluble precursor of insoluble fibrin, the terminal component of the common coagulation pathway with thrombin responsible for the proteolysis of fibrinogen. 98,100 Thrombin, a trypsin-like serine protease, cleaves fibrinogen between Arg-16 and Gly-17 of the Aα chain and Arg-14 and Gly-14 of the Bβ chain to expose polymerization sites, resulting in release of fibrinopeptide A and fibrinopeptide from their respective N-terminal regions. 97,98 Fibrin monomers polymerize spontaneously, mediated by activated factor XIII (FXIIIa) that cross-link individual fibrin molecules to form a complex, stable, and insoluble branching network of fibrin fibers.…”

“…Several studies observed no significant T-cell response or arthritogenic induction to citrullinated-fibrinogen immunization of HLA-DRβ1 transgenic mice. 100,102,103 However, the presence of antibodies to citrullinated fibrinogen (ACF) is associated with human HLA-DRβ1-0404 Ã allele along with early disease onset and radiographic progression. 104 The arthritogenic potential of citrullinated-fibrinogen in HLA-DR4 transgenic mice has also been reported by Raijmakers et al 55 Possible reasons for these discrepancies include increased ACPA heterogeneity in human RA, increased number of sites available for citrullination on human fibrinogen, and differences between the T-cell repertoire of humans and murine models.…”

“…110 The presence of immune complexes containing citrullinated fibrin(ogen) and autoantibodies directed toward the citrullinated forms of fibrin(ogen) has been confirmed in human RA sera and synovial fluid in various studies. 55,57,100,101,108 The presence of circulating immune complexes containing citrullinated fibrinogen has been reported in half of anti-CCP þ RA patients in a study by Zhao et al 111 ACF antibodies have predominantly nonoverlapping reactivity, and target citrullinated fibrinogen peptides more favorably than native fibrinogen. 100 ACF-ELIZA detection tests display similar performance to commercial anti-CCP2 assays and are highly predictive of rapid radiographic progression in terms of RA specificity.…”

The Infectious Basis of ACPA-Positive Rheumatoid Arthritis

“…Although there may be few disagreements (Konig et al, 2016b ), the research data outlined above imply that citrullination, in conjunction with genetic factors, such as HLA-DRB1SE, protein tyrosine phosphatase nonreceptor type 22 (PTPN22) risk allele (Joshua et al, 2016 ) encoding an R620W amino acid change that allows survival of autoreactive B cells (Menard et al, 2011 ), is the key element in breaking tolerance. Thus citrullinated peptides may offer new therapeutic strategies for RA.…”