These responses significantly expand in chronic CIA (day 55) to target a broad spectrum of native and citrullinated molecules (Determine2d). Non-citrullinated native variants of all citrullinated peptides were included on the microarrays and for the vast majority of the reactive citrullinated peptides the native variants were non-reactive. of diseases, the autoantibody responses expanded to target multiple citrullinated epitopes in both CIA and EAE. Z-VAD(OH)-FMK Using immunoblotting and mass spectrometry analysis, we identified citrullination of multiple polypeptides in CIA joint and EAE brain tissue Z-VAD(OH)-FMK that have not previously been described as citrullinated. == Conclusions == Our results suggest that anti-citrulline antibody responses develop in the early stages of CIA and EAE, and that autoimmune inflammation results in citrullination of joint proteins in CIA and brain proteins in EAE, thereby creating neoantigens that become additional Z-VAD(OH)-FMK targets in epitope spreading of autoimmune responses. == Introduction == Post-translational modifications can produce neo-antigens that become targets of autoimmune responses [1,2]. A post-translational modification of importance in rheumatoid arthritis (RA) is the conversion of peptidylarginine to peptidylcitrulline by peptidyl arginine deiminase (PAD) [3,4]. This enzymatic modification is usually termed citrullination, and detection of antibodies targeting citrullinated epitopes has become a key diagnostic marker for the diagnosis of RA, with a sensitivity of about 60% and a specificity of 95% [5-10]. Nevertheless, the mechanisms by which anti-citrullinated protein antibody (ACPA) responses develop remain poorly understood. In this study, we used protein microarrays to characterise ACPA responses in rodent models of RA and multiple sclerosis (MS). In RA, ACPA responses are currently assayed in clinical laboratories by detection of antibodies targeting cyclic citrullinated peptides (CCPs) derived from filaggrin [7]. These CCPs probably represent molecular mimics of the true citrullinated autoantigens in RA synovium. In RA and other inflammatory arthritides, multiple joint proteins become citrullinated [10-12]. Recent evidence suggests that cigarette smoking induces citrullination of lung proteins, and in patients possessing the shared epitope of human leucocyte antigen (HLA) DR, smoking is usually associated with about a 20-fold increased risk of developing anti-CCP antibody-positive RA [13]. Further, Kuhn and colleagues showed that a monoclonal antibody specific for citrullinated fibrinogen exacerbated tissue injury in rodent models of arthritis [14], although it remains possible that this non-affinity matured IgM antibody might cross-react with a native cartilage component as described for anti-citrulline IgG responses in RA [15]. Thus, it is possible that cigarette smoking represents an environmental trigger that induces anti-citrulline antibody responses in genetically susceptible individuals and thereby contributes to the development of RA [13]. Alterations in the citrullination of myelin proteins and autoimmune targeting of citrullinated myelin proteins have been observed in MS. Myelin basic protein (MBP) is usually partially citrullinated (C8 isoform) in normal brain tissue, although there is a significant increase in the relative amount of this partially citrullinated form in MS brain tissue [15]. An extensively Z-VAD(OH)-FMK citrullinated form of MBP is usually associated with Marburg encephalitis, a fulminant autoimmune demyelinating disease [16]. There have also been reports of anti-citrullinated-MBP T cell reactivity in MS [17], as well as evidence that citrullinated MBP can serve as an autoantigen in experimental autoimmune encephalomyelitis (EAE) [18]. In this study we applied synovial and myelin protein arrays to investigate the development and evolution of ACPA responses in rodent models of RA and MS, with the objective of gaining further insights into the aetiology, evolution and potential pathogenic role of such responses in human RA and MS. We demonstrate targeting of a limited number of citrullinated polypeptides in pre-disease samples derived from mice with collagen-induced arthritis (CIA) and early disease samples derived from mice with EAE, and growth of responses to target multiple Rabbit polyclonal to CCNB1 citrullinated molecules in established and long-standing disease. Mass spectroscopy analysis identified citrulline-modifications in multiple proteins in inflamed CIA joint and EAE brain tissue. Our results suggest that citrullination of synovial proteins in CIA and brain proteins in EAE generate neoantigens capable of provoking anti-citrulline antibody responses. == Materials and methods == == CIA and EAE == All animal experiments were conducted under approval from the Stanford University Institutional Animal Care and Use Committee. CIA was induced in DBA1/J mice by an intradermal injection of collagen type II (CII) (100 g/mouse) emulsified in complete Freund’s adjuvant (CFA) made up of 5 mg/mL heat-killedMycobacterium tuberculosisH37Ra (Difco Laboratories, Detroit, MI, USA), followed by boosting with CII (100 g/mouse) emulsified in incomplete Freund’s adjuvant.