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Rheumatoid arthritis (RA) synovitis is dominated by monocytes/macrophages with inflammatory patterns resembling microbial stimulation. In search of triggers, we reduced the intestinal microbiome in 20 RA patients (open label study DRKS00014097) by bowel cleansing and 7-day fasting (≤250 kcal/day) and performed immune monitoring and microbiome sequencing. Patients with metabolic syndrome (n = 10) served as a non-inflammatory control group. Scores of disease activity (DAS28/SDAI) declined within a few days and were improved in 19 of 20 RA patients after breaking the fast (median ∆DAS28 = −1.23; ∆SDAI = −43%) or even achieved remission (DAS28 < 2.6/n = 6; SDAI < 3.3/n = 3). Cytometric profiling with 46 different surface markers revealed the most pronounced phenomenon in RA to be an initially increased monocyte turnover, which improved within a few days after microbiota reduction and fasting. Serum levels of IL-6 and zonulin, an indicator of mucosal barrier disruption, decreased significantly. Endogenous cortisol levels increased during fasting but were insufficient to explain the marked improvement. Sequencing of the intestinal microbiota indicated that fasting reduced potentially arthritogenic bacteria and changed the microbial composition to species with broader metabolic capabilities. More eukaryotic, predominantly fungal colonizers were observed in RA, suggesting possible involvement. This study demonstrates a direct link between the intestinal microbiota and RA-specific inflammation that could be etiologically relevant and would support targeted nutritional interventions against gut dysbiosis as a causal therapeutic approach.

ObjectivesPorphyromonas gingivalis (P.g.) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g. CH2007 (RACH2007-PPAD) from a rheumatoid arthritis (RA) patient and a synthetic citrullinated PPAD peptide (CPP) containing the main autocitrullination site as potential targets for antibody reactivity in RA and to analyse the possibility of citrullinating native human proteins by PPAD in the context of RA.MethodsRecombinant RACH2007-PPAD was cloned and expressed in Escherichia coli. Purified RACH2007-PPAD and its enzymatic activity was analysed using two-dimensional electrophoresis, mass spectrometry, immunoblot and ELISA. Autoantibody response to different modified proteins and peptides was recorded and bioinformatically evaluated.ResultsRACH2007-PPAD was capable to citrullinate major RA autoantigens, such as fibrinogen, vimentin, hnRNP-A2/B1, histone H1 and multiple peptides, which identify a common RG/RGG consensus motif. 33% of RA patients (n=30) revealed increased reactivity for α-cit-RACH2007-PPAD before RA onset. 77% of RA patients (n=99) presented α-cit-specific signals to CPP amino acids 57–71 which were positively correlated to α-CCP2 antibody levels. Interestingly, 48% of the α-CPP-positives were rheumatoidfactor IgM/anti-citrullinated peptide/protein antibodies (ACPA)-negative. Anti-CPP and α-RACH2007-PPAD antibody levels increase with age. Protein macroarrays that were citrullinated by RACH2007-PPAD and screened with RA patient sera (n=6) and controls (n=4) uncovered 16 RACH2007-PPAD citrullinated RA autoantigens and 9 autoantigens associated with lung diseases. We showed that the α-CPP response could be an important determinant in parenchymal changes in the lung at the time of RA diagnosis (n=106; p=0.018).ConclusionsRACH2007-PPAD induced internal citrullination of major RA autoantigens. Anti-RACH2007-PPAD correlates with ACPA levels and interstitial lung disease autoantigen reactivity, supporting an infection-based concept for induction of ACPAs via enzymatic mimicry.

Objectives Pulmonary manifestations in RA are common comorbidities, but the underlying mechanisms are largely unknown. The added value of a multiplex of ACPA and genetic risk markers was evaluated for the development of pulmonary fibrosis (PF) in an inception cohort. Methods A total of 1184 patients with early RA were consecutively included and followed prospectively from the index date until death or 31 December 2016. The presence of 21 ACPA fine specificities was analysed using a custom-made microarray chip (Thermo Fisher Scientific, Uppsala, Sweden). Three SNPs, previously found related to PF were evaluated, rs2609255 (FAM13A), rs111521887 (TOLLIP) and rs35705950 (MUC5B). ACPA and genetic data were available for 841 RA patients, of whom 50 developed radiologically defined PF. Results In unadjusted analyses, 11 ACPA specificities were associated with PF development. In multiple variable analyses, six ACPA specificities were associated with increased risk of PF: vimentin (Vim)60–75, fibrinogen (Fib)β62–78 (72), Fibα621–635, Bla26, collagen (C)II359–369 and F4-CIT-R (P Conclusion The development of PF in an inception cohort of RA patients was associated with both presence of certain ACPA and the number of ACPA specificities and risk genes.

Various research groups at the German Rheumatism Research Center in Berlin, in close cooperation with the Department of Rheumatology and Clinical Immunology of the Medical Clinic at the Charité, have made important contributions to the significance of B cells and plasma cells in rheumatic diseases, which are relevant not only for rheumatology but for all clinical specialties in which antibody-mediated diseases play a role. In particular, the research addresses impaired B cell homeostasis, the importance of the IgM Fc receptor in the regulation of autoimmunity, the role of long-lived memory plasma cells in maintaining autoimmunity and ensuring its survival in specific niches organized by stromal cells in bone marrow and inflamed tissues. The research results have contributed to a better understanding of the immunological and molecular mechanisms in rheumatic diseases and their treatment. The identification of the long-lived memory plasma cell has led to promising treatment approaches with curative potential in autoimmune diseases.Verschiedene Arbeitsgruppen am Deutschen Rheuma-Forschungszentrum Berlin haben in enger Zusammenarbeit mit der Medizinischen Klinik mit Schwerpunkt Rheumatologie und Klinische Immunologie an der Charité wichtige Beiträge zur Bedeutung der B‑Zellen und Plasmazellen bei rheumatischen Erkrankungen geleistet, die nicht nur für die Rheumatologie, sondern für alle klinischen Fachgebiete, in denen antikörpervermittelte Erkrankungen eine Rolle spielen, relevant sind. Insbesondere wird auf die gestörte B‑Zell-Homöostase, die Bedeutung des Immunglobulin M(IgM)-Fc-Rezeptors für die Regulation der Autoimmunität, die Rolle der langlebigen Gedächtnis-Plasmazelle bei der Aufrechterhaltung der Autoimmunität sowie die Sicherung ihres Überlebens in speziellen, von Stromazellen organisierten Nischen im Knochenmark und in entzündeten Geweben eingegangen. Die Forschungsergebnisse haben zu einem besseren Verständnis der immunologischen und molekularen Mechanismen bei rheumatischen Erkrankungen und ihrer Therapie beigetragen. Die Identifizierung der langlebigen Gedächtnis-Plasmazelle hat zu vielversprechenden therapeutischen Ansätzen mit kurativem Potenzial bei Autoimmunerkrankungen geführt.

Objectives To assess the association between venous thromboembolic (VTE) events and autoantibodies, following patients from RA diagnosis, measuring occurrence, levels and collective load of different autoantibodies against post-translational protein modifications, in particular recognizing citrullination (e.g. citrullinated fibrinogen) and RF by isotype. Methods A cohort of 2814 patients with newly diagnosed RA were followed for incident VTE through register linkages. Sera from RA diagnosis were centrally analysed for antibodies to second generation cyclic citrullinated peptides (anti-CCP2), 20 anti-citrullinated protein antibody (ACPA) fine-specificities, antibodies to additional protein modifications (carbamylation and acetylation) and RF by isotype. Association between baseline serology status and future VTE was analysed using Cox regression adjusted for age, sex and calendar period of RA diagnosis, overall and stratified by anti-CCP2 and RF positivity. Results During a median 16 years of follow-up, 213 first-ever VTE events were registered (5.0/1000 person-years). IgG anti-CCP2 (present in 65% of cohort) associated with VTE (hazard ratio [HR] = 1.33, 95% CI: 1.00, 1.78), in a dose-response manner. The risk of VTE increased with number of ACPA fine-specificities. IgM RF, but no other RF isotypes, associated with VTE (HR = 1.38, 95% CI: 1.04, 1.82). The associations were independent from smoking and HLA-DRB1 shared epitope alleles. None of the carbamylated or acetylated antibody reactivities associated with VTE. Conclusion Anti-CCP2, load of ACPA fine-specificities and IgM RF at RA diagnosis are associated with an increased risk of future VTE in RA. Antibodies to citrullinated fibrinogen did not differ substantially from other ACPA fine-specificities. Autoreactivity to other post-translational modifications was not associated with VTE risk.

BackgroundThere is a need for biomarker to identify patients ‘at risk’ for rheumatoid arthritis (risk-RA) and to better predict the therapeutic response and in this study we tested the hypothesis that novel native and citrullinated heterogeneous nuclear ribonucleoprotein (hnRNP)-DL autoantibodies could be possible biomarkers.MethodsUsing Protein macroarray and ELISA, epitope recognition against hnRNP-DL was analysed in sera from different developed RA disease and diagnosed SLE patients. Toll-like receptor (TLR) 7/9 and myeloid differentiation primary response gene 88 (MyD88)-dependency were studied in sera from murine disease models. HnRNP-DL expression in cultivated cells and synovial tissue was analysed by indirect immunofluorescence, immunoblot and immunohistochemistry. ResultshnRNP-DL was highly expressed in stress granules, citrullinated in the rheumatoid joint and targeted by autoantibodies either as native or citrullinated proteins in patient subsets with different developed RA disease. Structural citrullination dependent epitopes (SCEs) of hnRNP-DL were detected in 58% of the SLE patients although 98% of these sera were α-CCP2-negative. To obtain a specific citrullinated signal value, we subtracted the native antibody value from the citrullinated signal. This CNDL (Citrullinated-Native-hnRNP-DL)-index identified and the bioinformatic value was explored, as a new value for an “individual window of treatment success” in early RA and for the detection of RF-IgM/α-CCP2 seronegative RA patients (24-46%). Negative CNDL-index was found in SLE patients, risk-RA- and early RA-cohorts such as EIRA where the majority of these patients are DAS28-responders to methotrexate (MTX) treatment (87%). High positive CNDL-values were associated with more severe RA, shared epitope and parenchymal changes in the lung. Specifically, native α-hnRNP-DL is TLR7/9-dependent, associated with pain and ROC-analysis revealed an association to initial MTX or etanercept treatment response, especially in seronegative RA patients.ConclusionCNDL-index defines patients is a possible biomarker for develop RA and the “window of treatment success” thereby potentially closing the sensitivity gap.

Objective: Antibodies against posttranslationally modified proteins are a hallmark of rheumatoid arthritis (RA), but the emergence and pathogenicity of these autoantibodies are still incompletely understood. The aim of this study was to analyze the antigen specificities and mutation patterns of monoclonal antibodies (mAb) derived from RA synovial plasma cells and address the question of antigen cross-reactivity. Methods: IgG-secreting cells were isolated from RA synovial fluid, and the variable regions of the immunoglobulins were sequenced (n = 182) and expressed in full-length mAb (n = 93) and also as germline-reverted versions. The patterns of reactivity with 53,019 citrullinated peptides and 49,211 carbamylated peptides and the potential of the mAb to promote osteoclastogenesis were investigated. Results: Four unrelated anti-citrullinated protein autoantibodies (ACPAs), of which one was clonally expanded, were identified and found to be highly somatically mutated in the synovial fluid of a patient with RA. The ACPAs recognized >3,000 unique peptides modified by either citrullination or carbamylation. This highly multireactive autoantibody feature was replicated for Ig sequences derived from B cells from the peripheral blood of other RA patients. The plasma cell-derived mAb were found to target distinct amino acid motifs and partially overlapping protein targets. They also conveyed different effector functions as revealed in an osteoclast activation assay. Conclusion: These findings suggest that the high level of cross-reactivity among RA autoreactive B cells is the result of different antigen encounters, possibly at different sites and at different time points. This is consistent with the notion that RA is initiated in one context, such as in the mucosal organs, and thereafter targets other sites, such as the joints.

Publisher's version (útgefin grein), Objective: To investigate the relationship between anti–citrullinated protein antibodies (ACPAs), specific ACPA subspecificities, rheumatoid factor (RF) isotypes, and incident cardiovascular (CV) events in patients with rheumatoid arthritis (RA). Methods: Serum samples from Swedish patients with new-onset RA (diagnosed within 1 year of symptom onset between 1996 and 2009) were centrally typed for anti–cyclic citrullinated peptide 2 (anti-CCP2) antibodies, 20 ACPA subspecificities, and RF isotypes. Patients were followed up longitudinally in nationwide registers to monitor the occurrence of acute coronary syndrome (ACS), stroke, CV-related death, and major adverse CV events (MACE). The association between each serologic marker and CV outcome, and the impact of adjustment for the Disease Activity Score in 28 joints (DAS28), smoking status, and income at baseline, were assessed using Cox proportional hazards models. In addition, associations of serologic markers with all-cause mortality were explored. Results: In total, 2,814 patients with RA were included in the study. The median follow-up was 13 years, during which the CV end points of ACS, stroke, or CV-related death were reported to occur in 375 patients. Occurrence and/or levels of anti-CCP2 were associated with risk of incident ACS (hazard ratio [HR] 1.46, 95% confidence interval [95% CI] 1.03–2.06), stroke (HR 1.47, 95% CI 1.03–2.10), CV-related death (P = 0.024 for association with anti-CCP2 levels), and MACE (HR 1.34, 95% CI 1.06–1.70). Similarly, an association with the number of ACPA subspecificities was observed; however, this could not be attributed to any individual or group of ACPA subspecificities. Presence of IgM-RF was associated with all CV end points except ACS, and IgA-RF was exclusively associated with CV-related death. Adjustment for smoking status, income, and DAS28 scores decreased most of the HRs, whereas IgA-RF remained associated with CV-related death (HR 1.61, 95% CI 1.05–2.48). All of the assessed serologic makers were associated with all-cause mortality. Conclusion: RF isotypes and ACPAs are associated with future CV events in patients with RA. ACPA levels and number of subspecificities seem more important than the occurrence of particular subspecificities, and these associations were not explained by a history of ever smoking., Karolinska institutet (strategic research area epidemiology), The nordic research council (nordforsk), The rheumatology research foundation (foreum), Stiftelsen för Strategisk Forskning, Vetenskapsrådet, Stockholms Läns Landsting, The swedish heart lung foundation

The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from ten COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb CV07-209 neutralized authentic SARS-CoV-2 with IC50 of 3.1 ng/ml. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 Å revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2 neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy., HIGHLIGHTS • Characterization of potent human monoclonal SARS-CoV-2 neutralizing antibodies • Some SARS-CoV-2 antibodies reacted with mammalian self-antigens in different organs • Crystal structures of two antibodies in complex with SARS-CoV-2 RBD at 2.55/2.70 Å • Post-exposure antibody treatment protected from lung damage in infected hamsters, Kreye et al. report the isolation and characterization of monoclonal antibodies isolated from COVID-19 patients, some of which were found to display autoreactivity with mammalian self-antigens in different organs. Crystal structures of two antibodies in complex with SARS-CoV-2 Spike RBD reveal antibody engagement with the ACE2 binding site from different approach angles. One antibody is further evaluated for in vivo efficacy and was found to be both protective and efficacious post-challenge in a hamster infection model.

The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from ten COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb CV07-209 neutralized authentic SARS-CoV-2 with IC50of 3.1 ng/ml. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 Å revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2 neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy.

Objective Anti-citrullinated protein antibodies (ACPAs) have proven highly useful as biomarkers for rheumatoid arthritis (RA). However, composition and functionality of the associated autoreactive B cell repertoire have not been directly assessed. We aimed to selectively investigate citrullinated autoantigen-specific B cell receptors (BCRs) involved in RA and initiate studies on their pathogenicity. Methods Blood samples were obtained from patients in a University of Minnesota cohort with ACPA-positive RA (n = 89). Tetramer sets bearing citrullinated filaggrin peptide cfc1 or citrullinated α-enolase peptide were constructed to specifically capture autoreactive B cells from the unaltered, polyclonal repertoire in RA patients. Citrullinated peptide tetramer-bound B cells were subjected to flow cytometric cell sorting and single-cell IGH, IGK, and IGL gene sequencing for B cell lineage determinations. BCR gene sequences were also expressed as recombinant monoclonal antibodies (mAb) for direct evaluation of citrullinated autoantigen binding and effector functionality. Results Using citrullinated peptide tetramer enrichment to investigate single autoreactive blood B cells, we identified biased V-region gene usage and conserved junction arrangements in BCRs from RA patients. Parsimonious clustering of related immunoglobulin gene nucleotide sequences revealed clonal expansions of rare individual B cell clades, in parallel with divergent sequence mutations. Correspondingly, recombinant mAb generated from such BCR lineages demonstrated citrulline-dependent cross-reactivity extending beyond the citrullinated peptides used for B cell capture. A pair of citrullinated autoantigen-specific mAb with cross-reactive binding profiles also promoted arthritis in mice. Conclusion Our findings suggest that broad ACPA specificities in RA arise from a restricted repertoire of evolving citrulline-multispecific B cell clades with pathogenic potential.

Recombinant RARARA

Rheumatische Erkrankungen gehören zu den häufigsten chronisch entzündlichen Krankheiten. Neben ausgeprägter Schmerzhaftigkeit und progredienter Gelenkzerstörung reduzieren die rheumatoide Arthritis (RA), die Spondyloarthritiden (SpA) und die Psoriasisarthritis (PsA) die Arbeitsfähigkeit, die Lebensqualität und bei unzureichender Behandlung auch die Lebenserwartung. Seit der Einführung der Biologika zur Therapie dieser Erkrankungen hat die Suche nach geeigneten Biomarkern zur Frühdiagnostik und Vorhersage des Therapieerfolgs zunehmend an Bedeutung gewonnen. Das Hauptziel des Verbundes ArthroMark ist, neue Biomarker zu identifizieren und moderne Bildgebungsverfahren einzusetzen mit dem Ziel, die Diagnose, die Verlaufskontrolle und die Stratifizierung von Patienten mit RA, SpA und PsA zu verbessern. Mit der Entwicklung geeigneter Biomarker für diese Erkrankungen trägt dieses Vorhaben zur Gesundheitsforschung im Bereich chronischer Erkrankungen des Bewegungsapparates bei. Durch die Zusammenarbeit verschiedener nationaler Zentren sollen standortspezifische Ressourcen wie Probenbanken und klinische Studien gemeinsam nutzbar gemacht werden und individuelle Schwerpunkte in der Biomarkeranalyse mit einem entsprechenden Mehrwert vernetzt werden. Gemeinsames Datenmanagement und Vereinheitlichung der Datenerhebung sowie bestmögliche Charakterisierung der Patienten durch neue Bildgebungsmethoden sollen die Qualität der Markerprüfung optimieren.

OBJECTIVE Anti-citrullinated protein antibodies (ACPAs) are a hallmark of seropositive rheumatoid arthritis (RA). Yet, the precise disease-relevant autoantigens that are targeted by ACPAs remains a matter of debate. This study utilized patient-derived monoclonal ACPAs, rather than serum autoantibody analysis, to characterize the multireactivity to different protein modifications and to reveal autoantibody subsets in patients with RA. METHODS Twelve human monoclonal ACPAs (positive by the second-generation cyclic citrullinated peptide test) were generated from 6 RA patients, and a head-to-head comparison of their reactivities was performed. For profiling, we used a complementary DNA-based protein array (Engine GmbH) and 3 peptide-screening platforms with RA autoantigens (Thermo Fisher Scientific), citrullinated and carbamylated peptides (NimbleGen/Roche), or histone-derived peptides with different posttranslational modifications (JPT Histone Code), covering >207,000 peptides (>7,800 gene products). RESULTS The fine-specificity profiles of the investigated ACPAs varied, but all of the monoclonal ACPAs displayed multireactivity to a large number of citrullinated peptides/proteins, each characterized by specific binding properties. ACPA subsets could be defined by clone-distinct consensus binding motifs (e.g., Cit-Gly, Gly-Cit, or Arg-Cit-Asp), with the most common ACPA recognition being that of a Gly in the +1 flanking position, but with additional amino acid preferences. For ACPA protein recognition, we observed a preference for citrullinated RNA-binding proteins with high Arg/Gly content. Six of the 12 ACPA clones also bound acetylated lysine (KAc) or homocitrulline peptide motifs, displaying a similar affinity or higher apparent affinity than that for citrullinated peptides. CONCLUSION ACPAs and anti-modified protein autoantibodies represent overlapping facets of RA autoimmunity and bind to a wide variety of modified proteins, extending well beyond the historically recognized set of RA autoantigens. So far, KAc reactivity has been detected only in the context of anti-carbamylated and anti-citrullinated peptide autoantibody responses, postulating the existence of hierarchies of autoreactivity in RA. Future investigations of ACPA fine specificities and functionality should take into consideration the presence of consensus Cit/Carb/KAc motifs and the multireactivity of these autoantibodies in patients with RA.

Background Recent studies have shown divergent associations between rheumatoid arthritis (RA)-associated autoantibodies and RA onset phenotype. Whereas some studies report more inflammation in seronegative patients [1, 2], others report that rheumatoid factor (RF) associates with high baseline disease activity [3]. Objectives To define how individual RA-associated autoantibodies associate with individual disease activity score (DAS28, DAS28CRP) components at the time of RA diagnosis. Methods Sixteen individual ACPA reactivities, anti-CCP2, IgA-, IgG- and IgM-RF were analyzed centrally in baseline sera from 1600 RA patients classified according to the 1987 ACR criteria. All antibody cut-offs were determined as the 98th percentile among controls. The results were related to CRP, ESR, number of swollen and tender joints, DAS28, DAS28CRP, global disease activity (visual analogue scale), and health assessment questionnaire obtained at baseline. Results Individually, anti-CCP2, IgA RF and IgM RF associated with low counts of swollen and tender joints, with strongest association to anti-CCP2, and with high ESR. The association to low joint counts was stronger to number of individual ACPA reactivities than to anti-CCP2 levels (table). In multiple regression adjusted for age, sex and inclusion year, ACPA number associated strictly with low joint counts whereas IgM RF level associated with increased inflammatory markers, especially ESR (table). Also among IgM RF negative patients, occurrence of ACPA associated with low swollen and tender joint counts. Contrary, among ACPA negative patients, IgM showed no association to elevated ESR. The effect of RF on ESR increased with the number of individual ACPA reactivities present, most prominently for IgM RF (figure). Conclusion In early RA, ACPA associate with low counts of affected joints and IgM RF associates with elevated ESR in an ACPA-dependent manner. The latter finding relates to in vitro studies showing enhanced inflammatory effect of ACPA immune complexes with addition of RF[4, 5]. Future RA studies relating autoantibodies to disease activity may benefit from evaluating the impact of individual antibodies as well as distinct DAS components separately. References [1] Barra L, et al. J Rheumatol 2014;41:2361. [2] Nordberg LB, et al. Ann Rheum Dis 2017;76:341. [3] Aletaha D, et al. Arthritis Res Ther 2015;17:229. [4] Laurent L, et al. Ann Rheum Dis 2015;74:1425. [5] Sokolove J, et al. Arthritis Rheumatol 2014;66:813. Disclosure of Interests Eleftheria Pertsinidou: None declared, Vivek Anand Manivel: None declared, Lars Klareskog Grant/research support from: Yes, but not for the presented study., Lars Alfredsson: None declared, Linda Mathsson Employee of: employed by Thermo Fisher Scientific, Monika Hansson: None declared, Martin Cornillet: None declared, Guy Serre: None declared, Rikard Holmdahl: None declared, Karl Skriner: None declared, Per-Johan Jakobsson: None declared, Helga Westerlind: None declared, Johan Askling Grant/research support from: Karolinska Institutet (JA) has or has had research agreements with the following pharmaceutical companies, mainly in the context of the ATRIS national safety monitoring programme for rheumatology biologicals: Abbvie, BMS, MSD, Eli Lilly, Pfizer, Roche, Samsung Bioepis, and UCB., Consultant for: Karolinska Institutet has received remuneration for JA participating in ad boards arranged by Lilly, Novartis, and Pfizer., Saedis Saevarsdottir Employee of: Part-time employee at deCODE Genetics/Amgen Inc, working on genetic research unrelated to this project., Johan Ronnelid: None declared

Background The association and role of antibodies to citrullinated proteins (ACPA) on the risk of cardiovascular (CV) co-morbidity in patients with rheumatoid arthritis (RA) is incompletely understood. We have previously reported an association between high levels of CCP2 and risk of acute coronary syndrome (ACS) in RA1. Objectives To further investigate any association between specific ACPA or rheumatoid factor (RF) isotypes, and CV events (ACS, stroke, CV death, and major adverse CV event (MACE)). Methods 2533 patients with RA from the Swedish EIRA study were tested for ACPA specificities on a custom-made microarray chip. All antibodies (Ab) detected with a prevalence >= 10% were included in the analysis. “Ab load” was defined as the number of specificities expressed in an individual, and categorized into four groups. ACPA was also measured by a commercial anti-CCP2 assay and anti-CCP2 levels divided into 1500 arbitrary units/mL, where The data was linked to the National Patient Register and the Cause of Death register, and events of ACS (ICD10=I20.0, I21, or cause of death listed as “I21”), stroke (ICD10=I60-I64), CV death (cause of death listed as “I”), or MACE (any of the above) identified. We used Cox proportional hazard model, adjusted for sex, age and calendar period of RA diagnosis, to assess associations between ACPA/RF status and each CV outcome. Results Median age at diagnosis was 52 years, and median follow-up was 12 years. The incidences per 1000 person-years were 4.3 for ACS, 4.0 for stroke, 2.4 for CV deaths, and 9.4 for MACE. 66% were anti-CCP2 positive. This was associated with ACS, stroke and MACE. There was a trend with increasing CV risk with increasing anti-CCP2 levels (Table). For RF, the pattern was markedly different across isotypes; IgM RF associated with ACS, stroke and MACE whereas IgA RF associated to CV death (Table). 18 different ACPA specificities (Fil 307-324 (CPP-1), Vim 60-75, Vim 2-17, Fib β36-52, Fib alpha 580-600, Eno 5-21 (CEP-1), Fib α621-635, Fib α36-50, Fib β60-74, Ptm13, Ptm36, Pept Z1, Pept Z2, Pept 1, Pept 5, Bla26) had a frequency >10% and included in the analyses. Median Ab load was 6 (IQR:2). There was a significant p-value for trend between Ab load and CV risk for stroke and MACE. Fib β36-52 was the only Ab associated with all 4 outcomes, and, together with CPP3 and ptm13, the only ACPAs associated to ACS. No association to ACS remained when all Abs were jointly included in the model. For stroke, 13 of the 18 Abs associated with risk, but only Fib β60-74 remained (HR 1.91 (95% CI 1.07-3.42)) in the full model. For CV death and MACE, 14 Abs were associated, but no associations remained in the full model. Conclusion In patients with RA, RF and ACPA are linked to CV risk. For RF, CV risks differ with isotype. For ACPAs, very high levels, and the number of individual ACPAs, are linked to CV risk, suggesting that Ab load may be more important than individual ACPAs. Reference [1] Mantel A, et al, 2015. PMID 26138387 Disclosure of Interests Helga Westerlind: None declared, Johan Ronnelid: None declared, Monika Hansson: None declared, Lars Alfredsson: None declared, Linda Mathsson Employee of: employed by Thermo Fisher Scientific, Guy Serre: None declared, Martin Cornillet: None declared, Rikard Holmdahl: None declared, Per-Johan Jakobsson: None declared, Karl Skriner: None declared, Lars Klareskog Grant/research support from: Yes, but not for the presented study., Saedis Saevarsdottir Employee of: Part-time employee at deCODE Genetics/Amgen Inc, working on genetic research unrelated to this project., Johan Askling Grant/research support from: Karolinska Institutet (JA) has or has had research agreements with the following pharmaceutical companies, mainly in the context of the ATRIS national safety monitoring programme for rheumatology biologicals: Abbvie, BMS, MSD, Eli Lilly, Pfizer, Roche, Samsung Bioepis, and UCB., Consultant for: Karolinska Institutet has received remuneration for JA participating in ad boards arranged by Lilly, Novartis, and Pfizer.

Background: Porphyromonas gingivalis (P g.) is involved in triggering self-reactive immune responses when cirtullinating bacterial or human proteins. However, first evidence to link anti-ribosomal T and B cells responses to rheumatoid arthritis (RA) has been published but the mechanism and its influence on therapy is not clear (1). Infection based autoimmunity induced by citrullination of human proteins with P g. peptidyl arginine deiminase from RA patient (RA-PPAD) and crossreactivity binding induced by P g. was investigated using patient sera, affinity purified RA patient antibodies and monoclonal antibodies to cit-RA-PPAD. Objectives: Antibodies to RA-PPAD isolated from an RA patients (RA-PPAD) was first time linked to target specific citrullinated ribosomal proteins and therapy. Methods: Screening of RA sera was conducted on 37.830 unique human proteins on protein marcoarrays (http://www.engine-gmbh.de) with 30 RA sera. The autoantibody response to 840 different proteins was recorded and bioinformatically evaluated. Protein arrays were citrullinated with human peptidyl arginine deiminase PAD 2, 4, rabbit PAD and RA-PPAD from P.g. Sera and affinity purified antibodies were used to detect reactivity to 840 autoantigens and 15aa CCP peptide form RA-PPAD. Sera from TBA treated sera anti-TNF (adalimumab, etanercept, certolizumab) treatment were tested with the cit-PPAD-peptide of 15aa (CPP). Results: A human protein macroarray consisting of 840 indentified autoantigens from RA patients was modified by human PAD2 and PAD4, rabbit PAD, and RA-PPAD form P g. Using cit specific monoclonal antibodies we identified the ribosomal proteins (RP), RPL18a, RPS27a, modified by PAD2, RPL18a and MRPS11 modifies by PAD4, and RPL7L1 modified by rabbit PAD specifically targeted. In addition 6 RA patient sera and 3 osteoarthritis (OA) control sera were used to identify the citrullinated RA-PPAD specific modified autoantigens not targeted when modified by human PAD2 or PAD4 or rabbit PAD. We identified the RA-PPAD citrullinated ribosomal Proteins RPL3, RPL21, RPS24, RPL9, RPL15, RPS24, RPS3a, MRPL28 specifically targeted by RA patients. This identifies ribosomal proteins as major specific RA-PPAD citrullination targets. Moreover, affinity purified antibodies bound to native and citrullinated RA-PPAD from 6 RA patient sera and 3 OA patient sera were tested for crossreactvity on citrullinated human proteinarray. Antibodies to citrullinated ribosomal proteins MRPS11, RPL21, RPS3a, RPL18a, RPS27a, MRPL28 were detected in the RA group but not in the OA control group. High antibody titre against the cit-PPAD-peptide of 15aa (CPP) derived from the autocitrullination site (R63) of RA-PPAD correlates with TNFα-inhibitor (TBA) non-response (n=61). DMARD patients refractory to different treatment regimes (n=61), receiving anti-TNF (adalimumab, etanercept, certolizumab), do not respond when maintaining high α-CPP IgG level. Conclusion: Failure of Porphyromonas gingivalis clearance in RA patients leads to infection induced enzymatic mimicry based autoreactivity targeting evolutionary conserved human ribosomal proteins. Autoimmunity to ubiquitous self-antigens may trigger localized tissue damage in RA.TBA non-response leads to the suggestion to clear Porphyromonas gingivalis infection before α-TNF treatment. Reference [1] :Ito, Y. et al. Detection of T cell responses to a ubiquitous cellular protein in autoimmune disease. Science346, 363–368 (2014) Disclosure of Interests: Madeleine Jenning: None declared, Bianka Marklein: None declared, Ute Nonhoff: None declared, Zoltan Konthur: None declared, Gerd Rudiger Burmester Consultant for: Roche, Sanofi-Genzyme, Speakers bureau: Roche, Sanofi-Genzyme, Karl Skriner: None declared

ANTI-MODIFIED PROTEIN AUTOANTIBODIES IN RA DISPLAY IMPORTANT PEPTIDE CROSS-REACTIVITY BUT YET PROTEIN RECOGNITION SELECTIVITY

Career situation of first and presenting author Young investigator. Introduction Porphyromonas gingivalis (P g.) is involved in triggering self-reactive immune responses when citrullinating bacterial or human proteins. However, first evidence to link anti-ribosomal T and B cells responses to rheumatoid arthritis (RA) has been published but the mechanism is still not clear.1 Infection based autoimmunity induced by citrullination of human proteins with P g. peptidyl arginine deiminase from RA patient (RA-PPAD) and crossreactivity binding induced by P g. was investigated using affinity purified RA patient antibodies and a monoclonal antibodies to cit-RA-PPAD. Objectives Antibodies to RA-PPAD isolated from an RA patients (RA-PPAD) was first time linked to target specific citrullinated ribosomal proteins. Methods Screening of RA sera was conducted on 37.830 unique human proteins on protein marcoarrays (http://www.engine-gmbh.de) with 30 RA sera. The autoantibody response to 840 different proteins was recorded and bioinformatically evaluated. Protein arrays were citrullinated with PAD2,4,rabbit PAD and RA-PPAD. Sera and affinity purified antibodies were used to detect reactivity to 840 autoantigens. Results A human protein macroarray consisting of 840 indentified autoantigens from RA patients was modified by human PAD2 and PAD4, rabbit PAD, and RA-PPAD form P g..Using cit specific monoclonal antibodies we identified the ribosomal proteins (RP), RPL18a, RPS27a, modified by PAD2, RPL18a and MRPS11 modifies by Pad4, and RPL7L1 modified by rabbit PAD specifically targeted. In addition 6 RA patient sera and 3 osteoarthritis (OA) control sera were used to identify the citrullinated RA-PPAD specific modified autoantigens not targeted when modified by human PAD2 or PAD4 or rabbit Pad. We identified the RA-PPAD citrullinated ribosomal Proteins RPL3, RPL21, RPS24, RPL9, RPL15, RPS24.RPS3a, MRPL28 specifically targeted by RA patients. This identifies ribosomal proteins as major specific RA-PPAD citrullination targets. Moreover, affinity purified antibodies bound to native and citrullinated RA-PPAD from 6 RA patient sera and 3 OA patient sera were tested for crossreactvity on citrullinated human proteinarray. Antibodies to citrullinated ribosomal proteins MRPS11, RPL21, RPS3a, RPL18a, RPS27a, MRPL28 were detected in the RA group but not in the OA control group. Conclusions Failure of Porphyromonas gingivalis clearance in RA patients leads to infection induced enzymatic mimicry based autoreactivity targeting evolutionary conserved human ribosomal proteins. Autoimmunity to ubiquitous self-antigens may trigger localized tissue damage in RA. Reference Ito Y, et al. Detection of T cell responses to a ubiquitous cellular protein in autoimmune disease. Science 2014;346:363–368. Disclosure of Interest None declared.

Career situation of first and presenting author Young investigator. Introduction Anti-citrullinated protein autoantibodies (ACPA) in RA patients target a wide range of modified proteins and recent findings reveal that monoclonal ACPA are cross-reactive due to recognition of shared consensus citrulline-peptide motifs. Among physiological targets of ACPA, citrullination in neutrophil extracellular trap (NET) products, have been postulated to be important. Objectives We sought to characterize the anti-nuclear and anti-neutrophil reactivities of different patient-derived monoclonal ACPA. Methods The study included ten recombinant single B-cell derived RA monoclonal ACPA-IgG with CCP2 reactivity from different cell subsets and compartments (1, 2). They were screened for HEp-2 ANA reactivity, and binding to apoptotic cells or stimulated neutrophils. Binding was compared to CRISPR PAD4 KO cells and neutrophils from PAD2 and PAD4 KO mice. Immunoprecipitation and mass spectrometry were used to identify modified nuclear ACPA targets, and ELISA and Western blot for mAb binding to acetylated epitopes. Results Four out of ten ACPA clones exhibited strong binding to apoptotic cells, nuclear binding to activated neutrophils, and reactivity to NETs. Three of these were ANA positive. Another NET-reactive ACPA instead displayed a cytoplasmic binding pattern. This cytoplasmic NET-binding was PAD4-dependent, whilst nuclear-mediated NET reactivity was PAD-independent. Using apoptotic cells, acetylated histones were confirmed to be the primary targets of the nuclear reactivity, which could be explained by consensus-motif driven ACPA cross-reactivity. Specifically targeted acetylated histone peptides were identified and the anti-modified protein autoantibody (AMPA) profiles of the ACPA were found to correlate with cell binding. Conclusions When investigating monoclonal ACPA, our novel data reveal a distinct subset of ACPA with nuclear binding patterns and AMPA activity with acetylated histones (and not citrullinated proteins) in NETs and apoptotic cells. References Titcombe PJ, Wigerblade G, Sippl N, Zhang N, Shmagel AK, et al. Pathogenic citrulline-multispecific B cell receptor clades in rheumatoid arthritis. Arthritis Rheumatol 2018. doi:10.1002/art.40590 Steen J, Forsstrom B, Sahlstrom P, Odowd V, Israelsson L, et al. Human plasma cell derived monoclonal antibodies to posttranslationally modified proteins recognize amino acid motifs rather than specific proteins. Arthritis Rheumatol 2018. doi:10.1002/art.40699 Disclosure of Interest None declared.

IntroductionAnticitrullinated peptide antibody (ACPA) responses for 22 citrullinated peptides in patients with early rheumatoid arthritis (RA) were analysed and related to radiological and clinical outcome during the first 2 years in a prospective inception cohort.MethodsThe ACPA reactivities were assessed in 1022 patients with early RA (symptoms ResultsFrequency of ACPA reactivities varied between 13.3% and 63.1%. Of the anticyclic citrullinated peptide-2 (anti-CCP2) antibody-negative patients, ACPA reactivities were positive in 32.6%. Smoking, human leucocyte antigen-shared epitope (HLA-SE), anti-CCP2/rheumatoid factor, protein tyrosine phosphatase non-receptor type 22 (1858C/T) and DAS28 were significantly associated with number of ACPA reactivities. The ACPA reactivities modified differently the development of DAS28 over 24 months (identified using trajectories). Anti-Filaggrin307-324, anti-hnRNP (Peptide)-Z1 and anti-F4-CIT-R antibodies anticipated lower DAS28 values (pConclusionsSeveral ACPA reactivities modified significantly the DAS28 development during the first 24 months and were significantly associated with Larsen score at baseline, 24 months and radiological progression.

Introduction Differences in enzymatic activity and pathogenic impact of Porphyromonas gingivalis (P.g.) peptidylarginine deiminase (PPAD) for development of RA have been published, confounded by different PPAD variations and methods used. Objectives Enzymatic active PPAD isolated from an RA patient (RA-PPAD) was first time linked to citrullination of RA autoantigens, diagnosis, therapy response and RA-onset. Methods Recombinant RA-PPAD cloned, verified by DNA sequencing and expressed in Escherichia coli and purified. RA-PPAD and its enzymatic activity was analysed using 2D-Elektrophoresis, mass spectrometry (MS), immunoblot and ELISA. Results RA-PPAD autocitrullinates amino acid position 63 (aa63) and exhibits so far two new amino acid mutations aa73 F to L and aa447 E to V. Anti-citrullinated RA-PPAD antibodies were detected in 38% (n=36) of patients with RA, but were absent in Systemic lupus erythematosus (n=30), Osteoarthritis (n=36) and control sera (n=23). Twenty percent of RA patients (n=30) showed an increase in antibody-titre against citrullinated RA-PPAD after RA onset. High antibody titre against the cit-PPAD-peptide of 15aa (CPP) derived from the autocitrullination site (R63) correlates with TNFα-inhibitor (TBA) non-response (n=61). Anti-CPP levels correlate with DAS28,rheumatoid factor, α-CCP 2 levels and increase with age. RA-PPAD is able to citrullinate internal arginines in fibrinogen, vimentin, hnRNP-A2/B1 and histone H1. This internal citrullination-sites are recognised by RA sera and able to bind HLA401. Conclusions Failure of P.g. clearance in RA patients may lead to excessive exposure of citrullinated self-antigens and bacterial antigens inducing immune-mimicry. P.g. infection can be linked to RA and its correlation to TBA non-response leads to the suggestion to clear P.g infection before α-TNF treatment. Disclosure of interest None declared

IntroductionThe second generation anticycliccitrullinated peptide (anti-CCP2) assay detects the majority but not all anticitrullinated protein/peptide antibodies (ACPA). Anti-CCP2-positive rheumatoid arthritis (RA) is associated with HLA-DRB1* shared epitope (SE) alleles and smoking. Using a multiplex assay to detect multiple specific ACPA, we have investigated the fine specificity of individual ACPA responses and the biological impact of additional ACPA reactivity among anti-CCP2-negative patients.MethodsWe investigated 2825 patients with RA and 551 healthy controls with full data on anti-CCP2, HLA-DRB1* alleles and smoking history concerning reactivity against 16 citrullinated peptides and arginine control peptides with a multiplex array.ResultsThe prevalence of the 16 ACPA specificities ranged from 9% to 58%. When reactivity to arginine peptides was subtracted, the mean diagnostic sensitivity increased by 3.2% with maintained 98% specificity. Of the anti-CCP2-negative patients, 16% were found to be ACPA positive. All ACPA specificities associated with SE, and all but one with smoking. Correction for arginine reactivity also conveyed a stronger association with SE for 13/16 peptides. Importantly, when all ACPA specificities were analysed together, SE and smoking associated with RA in synergy among ACPA positive, but not among ACPA-negative subjects also in the anti-CCP2-negative subset.ConclusionsMultiplexing detects an enlarged group of ACPA-positive but anti-CCP2-negative patients with genetic and environmental attributes previously assigned to anti-CCP2-positive patients. The individual correction for arginine peptide reactivity confers both higher diagnostic sensitivity and stronger association to SE than gross ACPA measurement.

Background We have described a planar microarray for the determination of antibodies against individual citrullinated peptides (ACPA; Hansson M et al. Arthritis Res Ther 2012;14:R201). We have also developed a method for the quantification of autoantibodies in immune complexes (IC; Sohrabian et al. Ann Rheum Dis 2015;74(Suppl 1):A74). Here we have combined these techniques to determine ACPA profiles in RA IC. Objectives To investigate if measurement of specific ACPA in synovial fluids (SF) and in IC from sera and SF can provide more prognostic information than conventional measurement of total ACPA and rheumatoid factor (RF) in serum. Methods Seventy-seven RA patients with knee synovitis were treated with intra-articular triamcinolone hexacetonide, and followed until relapse. DAS28 and radiographic joint damage according to Larson-Dale were recorded. Anti-CCP2, IgM and IgA RF and circulating C1q-binding immune complexes (CIC) were determined in paired sera and SF. IC were purified from sera and SF by binding to C1q-coated beads, and thereafter eluted with a procedure developed in our laboratory. Antibodies against 19 citrullinated peptides were investigated with a custom-made microarray assay based on the ImmunoCAP ISAC system (Phadia AB, Sweden) in sera and SF as well as in IC from sera and SF. The target peptides were filaggrin 307–324 (CCP1), vimentin peptides 60–75 and 2–17, fibrinogen peptides α36–50,, α563–583, α580–600, α621–635, β36–52, β60–74, β62–81 (with citrullination in positions 72 and 74, respectively), α-enolase 5–21 (CEP-1), peptides 1, 5, Z1, Z2 and Bla26 from hnRNP, and histone 4 peptides 14–34 and 31–50. Cutoffs were established in relation to healthy controls. Backward stepwise regression was used to investigate what factors determined Larsen Dale index, DAS28, and duration of remission after steroid treatment. Independent factors were anti-CCP2, IgM RF, IgA RF, CIC, number of ACPA peptide reactivities, and number of ACPA reactivities in IC, all measured both in serum and paired SF. Results A considerable proportion of anti-CCP2 negative patients had multiple ACPA in SF, and in IC fractions. High DAS28 associated with reactivity against 7/19 peptides in serum and 9/19 in SF. High Larsen score associated with number of specific ACPA in SF IC and with CIC in SF. DAS28 levels associated with IgM RF in SF and with CIC in SF, and steroid response duration with number of specific ACPA in serum and in SF IC. Conclusions We found ACPA in SF, and especially in the IC fraction of SF, in a sizeable fraction of anti-CCP2 negative patients. Number of peptide-specific ACPA (but not anti-CCP2 levels) associated with radiological destruction and length of remission after intra-articular steroid therapy. Our data do not support a role for any unique ACPA specificity in RA pathogenesis. Instead, the number of individual ACPA specificities may be important. Disclosure of Interest None declared

Background The present study was undertaken to investigate novel anti-hnRNPs autoantibodies in rheumatic diseases. Objectives Novel mutated hnRNP A3 was cloned out of RA synovial tissue linking it directly to Warburg effect and lactate production in RA. Increased lactate production in RA synovia and tumors is klinked to alternative splicing process from PKM1 to PKM2 hnRNPs dependent. Methods After immunobloting and 2D-gel-eletrophoresis of a semipurified hnRNP fraction two protein spots were sequenced and identified to be highly similar to hnRNPA3. The hnRNP A3 variants were cloned from RA synovial tissue, which identified the isoforms found on protein level. 3700 RA sera were screened for the presence of mutated anti-hnRNP A3 autoantibodies using recombinant proteins and peptides thereof. Binding of RNA to hnRNP A3 (MA3) and mutated citrullinated A3 peptides (MCA3) the epitope recognition was investigated. Expression of hnRNP A3 in synovial tissue was analysed by Results Autoantibodies to MA3 protein were detected in 13% of RA (n=215) patients, in 9% SLE (n=154), in 27% of MCTD patients (n=44/10) and in less than 5% of 129 patients with other rheumatic disorders but not at all in healthy controls on immunoblot. When using renaturated MA3 on ELISA 22% of RA patients were detected and 87% of these patients had erosive arthritis. Same modification as in cancer cells were identified in synovial tissue and verified by MS and DNA sequencing. Using 2–3 citrullinated MCA3 peptides up to 81% of patients (n=150) with established and 67% (n=2926) of patients with an early RA with a specificity of 97% were detected. In early RA 27% and 25% in established RA of CCP2 negative and 93% of CCP2 positive patients were identified. By combining with the already established CCP2 and the new MCA3, 72% of early patients are positive. MCA3 autoantibodies predominantly occur (p Conclusions Mutated hnRNP-A3 is as a novel Toll7/9 dependent autoantigen in systemic rheumatic diseases. These mutated proteins are major components of RNA and DNA containing alternative splicing complexes leading to the Warburg effect and and autoantibodies predominantly occurre in an erosive, and severe courses of RA. Disclosure of Interest None declared

Background Rheumatoid arthritis triggers the formation of prion-like stress granules. To investigate which members of the heterogeneous nuclear ribonucleoprotein (hnRNP)-family, components of functionally important subcellular particles are targeted by autoantibodies from RA and other systemic rheumatic diseases. Methods Using a protein macroarray we identified JKTBP in humans and animal models of inflammatory rheumatic diseases. Bacterially expressed recombinant JKDBP proteins were used to confirm the obtained data. Epitope, TLR7/9 and MyD88 dependency was determined by ELISA. JKTBP expression in cultivated cells and synovial tissue was analysed by indirect immunofluorescence, immunoblot and immunohistochemistry. Results Anti-JKTBP autoantibodies were detected in 46% of the patients with systemic lupus erythematosus (n=103), in 20–30% of the patients with rheumatoid arthritis (n=286), in 10% of the patients with mixed connective tissue disease (n=20) or spondyloathropathy (n=20), and in In localization, experiments anti-JKTBP autoantibodies specifically stained stress granules (SG) in the cytoplasm. Immunohistochemical studies revealed JKTBP to be highly expressed in SG in the cytoplasm of RA synovial tissue different from OA and normal control tissue Conclusions These data identify SG as targeted particle in RA and JKTBP as a novel autoantigen in RA, SLE patients and mouse models of inflammatory rheumatic diseases. In combination with the hnRNPs AUF1 and hnRNP-B1, JKTBP autoantibodies close the sensitivity gap in RA left by rheumatoid factor and anti-CCP2 antibodies. Disclosure of Interest None declared

Objectives Rituximab (RTX) has shown clinical efficacy but up to 40% of RTX treated rheumatoid arthritis (RA) patients are poor responders (Ann-Rheum-Dis. 2005 Feb;64(2):246–52) and the commonly used RA biomarkers ( RF/ACPA) are poor predictors for therapy response. In this study the autoantibody repertoire analysed on protein macorarrays from RA patients under RTX treatment was correlated to clinical DAS28 response. Methods Screening of RA sera was conducted on 37.830 unique human proteins on protein marcoarrays (http://www.engine-gmbh.de)with sera taken before and 24 weeks after treatment. The autoantibody response of different immunoglobulin classes IgD, IgA, and IgG was recorded and bioinformatically evaluated. Response was determined according to DAS28 criteria. DAS 28 scores in the responder group before treatment was from 5.4 – 7.8 and in the non-responder group 5,6 – 6,8. We analyzed 26 RA patient sera (9 responder, 7 non-responder and 10 patients with blinded response classification) investigated the data of found autoantigens in-silico and by hierarchical clustering Results In the cohort of 26 patients 1292 different autoantigens (IgD,IgA,IgG) were detected. Using protein array we investigated clusters of autoantigen responses that disappeared or developed during RTX treatment of RA patients. RA autoantigenic patterns before and 6 month after RTX treatment were patient-specific and no relevant autoantigenic cluster was found that was shared between patients or associated with response. However, RTX reduced the repertoire of autoantibodies after 24 weeks of treatment in the tested RA patient cohort on average by 60%. RA patients which do not respond are generating on average 63% new autoantibodies. In good responders to RTX only 5,5% (+/-3%) new autoantibodies can be detected. The IgA and IgG autoantibody repertoire in the serum after 24 weeks of RTX treatment is reduced (IgA: 41%, IgG: 31%) in good responders whereas it is increased (IgA: 1,3%, IgG: 24%) in non responders to RTX. Conclusions After 6 month of RTX treatment the autoantibody repertoire in all good responding RA patients is reduced and non responders to RTX change their autoantibody repertoire directed against new but patient specific antigens. The fast rebuilding of functional B cells is only detected in non-responders to rituximab. Disclosure of Interest None declared

Acpa against different citrullinated peptides identify specific phenotypes of rheumatoid arthritis

Objectives Previous reports showed that peptidylarginine deiminase (PPAD) form Porphyromonas gingivalis (P.g.) is not able to citrullinate proteins internaly. New mutated PPAD (mPPAD) from P.g. involved in periodontal disease (PD) cloned out of P.g. strain was characterized and analyzed for its reactivity in sera from patients with systemic autoimmune diseases Methods We cloned a new enzymatically active recombinant mutated PPAD from P.g. mPPAD mutations and citrullination sites were analyzed by DNA sequencing and/or protein mass spectrometry. Autocitrullination activity it9s enzymatic-activity and human autoantigen protein citrullination was investigated by 2D-Elektrophoresis, MS, immunoblot analysis and ELISA. Furthermore we tested anti-mPPAD/cit-mPPAD with human sera (n=93) from early RA before and after onset of RA (n=30), established RA (n=32), SLE (n=16) and healthy blood donors (n=15) in ELISA assays. To study a potential impact on the RA mouse model (CAIA), mPPAD-containing vesicles from P.g. were injected by intraperitoneal injection (IP). Results Recombinant mPPAD lacks 43 amino acids at the N-terminus and exhibits so far two new amino acid mutations (amino acid position 73 (F>L) and 447 (E>V). We were able to demonstrate, mPPAD is enzymatically active over a huge pH-range (3–10) and autocitrullinates at amino acid position 63 the arginine to citrulline. Moreover mPPAd citrullinates major autoantigens in RA (Fibrinogen, Vimentin and hnRNP-A2/B1) which are detectable by RA patient sera and specific anti-citrulline monoclonal antibodies. mPPAD citrullinates HeLa-protein extracts and these specific citrullinated proteins are recognized by RA patient sera. Anti-citrullinated mPPAD antibodies were detected in 41% (n=32) of patients with RA but not in SLE (n=16) and control sera (n=15). In a RA follow-up study (n=30), we detected nearly similar antibody-sensitivities for citrullinated mPPAD before and after onset of RA (13/20%). Only a minority (7%) of RA patients show higher mPPAD antibody levels after RA diagnosis. In the Collagen antibody-induced arthritis (CAIA) RA mouse model mPPAD containing P.g. vesicles when injected IP showed a TLR2-dependent protective anti- inflammatory effect like P.g. LPS and Lipomannan. Conclusions Pg. infection and RA disease diagnosis occurs on different timepoints and Pg. infection induces a TLR2-dependent protective anti-inflammatory effect.We show the first time that mPPAD can citrullinate major human autoantigens internally and their immunologically and diagnostic relevance. Disclosure of Interest None declared

Seit Einfuhrung der Biologika sind Biomarker zur Fruhdiagnostik und Therapieentscheidung von zunehmender Bedeutung. Sie sollen im BMBF-geforderten Forschungsverbund ArthroMark fur die rheumatoide Arthritis und die Spondylarthritis etabliert werden. Biobanken und Vorarbeiten zu Genotypen, Genexpressions- und Autoantigenprofilen dienen als Grundlage. Bioinformatische Vernetzung soll die Untersuchungen aufeinander abstimmen und eine Studie mit modernen Bildgebungsverfahren die funktionelle Bedeutung der neuen Biomarker bestmoglich charakterisieren. Zur Validierung der diagnostischen Marker wird eine schrittweise Erweiterung des Netzwerkes angestrebt.

Objective In addition to releasing proteins and mediators, cells also release membrane vesicles (exosomes and apoptotic blebs) into the extracellular environment. Apoptotic blebs contain multiple autoantigens, but few data are available concerning the protein content of exosomes. Exosomes are formed during an immune response and can directly stimulate T cells or bind to dendritic cells. The aim of this study was to identify the nature of synovial exosomes from patients with different rheumatic diseases and to examine their potential autoantigenic content, which may be involved in the induction of an autoimmune response. Methods Synovial exosomes from patients with rheumatoid arthritis (RA), patients with reactive arthritis, and patients with osteoarthritis were purified, analyzed by electron microscopy, and labeled with immunogold to detect IgG and IgM molecules. Autoantigen content was identified by 2-dimensional electrophoresis–immunoblotting and subsequent mass spectrometry. In order to investigate the presence of citrullinated proteins, immunoblotting with anticitrulline antibodies was performed. Results Citrullinated proteins were observed in all exosome preparations, in contrast to other autoantigenic proteins (e.g., BiP and heterogeneous nuclear RNP A2) that were previously observed in RA and other autoimmune diseases. These citrullinated proteins included the fibrin α-chain fragment, fibrin β-chain, fibrinogen β-chain precursor, fibrinogen D fragment, and the Spα (CD5 antigen-like protein) receptor. Purification of synovial exosomes led to the detection of citrullinated fibrinogen and citrullinated Spα associated with IgM and IgG. Conclusion Synovial exosomes contain citrullinated proteins, which are known to be autoantigens in RA. Although immune mechanisms in which exosomes carry citrullinated peptides could play an important role in the induction and distribution of citrullinated proteins, there must be a specific recognition of these proteins that is unique to the RA immune system.

Human TNF-α transgenic (hTNFtg) mice develop erosive arthritis closely resembling rheumatoid arthritis (RA). To investigate mechanisms leading to pathological autoimmune reactions in RA, we examined hTNFtg animals for the presence of RA-associated autoantibodies including Abs to citrullinated epitopes (anti-cyclic citrullinated peptide), heterogeneous nuclear ribonucleoprotein (hnRNP)-A2 (anti-RA33), and heat shock proteins (hsp) (anti-hsp). Although IgM anti-hsp Abs were detected in 40% of hTNFtg and control mice, IgG anti-hsp Abs were rarely seen, and anti-cyclic citrullinated peptide Abs were not seen at all. In contrast, >50% of hTNFtg mice showed IgG anti-RA33 autoantibodies, which became detectable shortly after the onset of arthritis. These Abs were predominantly directed to a short epitope, which was identical with an epitope previously described in MRL/lpr mice. Incidence of anti-RA33 was significantly decreased in mice treated with the osteoclast inhibitor osteoprotegerin and also in c-fos-deficient mice lacking osteoclasts. Pronounced expression of hnRNP-A2 and a smaller splice variant was seen in joints of hTNFtg mice, whereas expression was low in control animals. Although the closely related hnRNP-A1 was also overexpressed, autoantibodies to this protein were infrequently detected. Because expression of hnRNP-A2 in thymus, spleen, brain, and lung was similar in hTNFtg and control mice, aberrant expression appeared to be restricted to the inflamed joint. Finally, immunization of hTNFtg mice with recombinant hnRNP-A2 or a peptide harboring the major B cell epitope aggravated arthritis. These findings suggest that overproduction of TNF-α leads to aberrant expression of hnRNP-A2 in the rheumatoid joint and subsequently to autoimmune reactions, which may enhance the inflammatory and destructive process.

Chronic inflammation is a major trigger of local and systemic bone loss. Disintegration of cell–matrix interaction is a prerequisite for the invasion of inflammatory tissue into bone. CD44 is a type I transmembrane glycoprotein that connects a variety of extracellular matrix proteins to the cell surface. Tumor necrosis factor (TNF) is a major inducer of chronic inflammation and its overexpression leads to chronic inflammatory arthritis. By generating CD44−/− human TNF-transgenic (hTNFtg) mice, we show that destruction of joints and progressive crippling is far more severe in hTNFtg mice lacking CD44, which also develop severe generalized osteopenia. Mutant mice exhibit an increased bone resorption due to enhanced number, size, and resorptive capacity of osteoclasts, whereas bone formation and osteoblast differentiation are not affected. Responsiveness of CD44-deficient osteoclasts toward TNF is enhanced and associated with increased activation of the p38 mitogen-activated protein kinase. These data identify CD44 as a critical inhibitor of TNF-driven joint destruction and inflammatory bone loss.

The role of autoimmune reactions in the pathogenesis of rheumatoid arthritis (RA) is poorly understood. To address this issue we have investigated the spontaneous T cell response to two well-characterized humoral autoantigens in RA patients and controls: 1) the heterogeneous nuclear ribonucleoprotein A2, i.e., the RA33 Ag (A2/RA33), and 2) filaggrin in unmodified and citrullinated forms. In stimulation assays A2/RA33 induced proliferative responses in PBMC of almost 60% of the RA patients but in only 20% of the controls (patients with osteoarthritis or psoriatic arthritis and healthy individuals), with substantially stronger responses in RA patients (p < 0.00002). Furthermore, synovial T cells of seven RA patients investigated were also clearly responsive. In contrast, responses to filaggrin were rarely observed and did not differ between RA patients and controls. Analysis of A2/RA33-induced cytokine secretion revealed high IFN-γ and low IL-4 production in both RA and control PBMC, whereas IL-2 production was mainly observed in RA PBMC (p < 0.03). Moreover, A2/RA33-specific T cell clones from RA patients showed a strong Th1 phenotype and secreted higher amounts of IFN-γ than Th1 clones from controls (p < 0.04). Inhibition experiments performed with mAbs against MHC class II molecules showed A2/RA33-induced T cell responses to be largely HLA-DR restricted. Finally, immunohistochemical analyses revealed pronounced overexpression of A2/RA33 in synovial tissue of RA patients. Taken together, the presence of autoreactive Th1-like cells in RA patients in conjunction with synovial overexpression of A2/RA33 may indicate potential involvement of this autoantigen in the pathogenesis of RA.

Item does not contain fulltext We constructed miniaturized autoantigen arrays to perform large-scale multiplex characterization of autoantibody responses directed against structurally diverse autoantigens, using submicroliter quantities of clinical samples. Autoantigen microarrays were produced by attaching hundreds of proteins, peptides and other biomolecules to the surface of derivatized glass slides using a robotic arrayer. Arrays were incubated with patient serum, and spectrally resolvable fluorescent labels were used to detect autoantibody binding to specific autoantigens on the array. We describe and characterize arrays containing the major autoantigens in eight distinct human autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. This represents the first report of application of such technology to multiple human disease sera, and will enable validated detection of antibodies recognizing autoantigens including proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA and post-translationally modified antigens. Autoantigen microarrays represent a powerful tool to study the specificity and pathogenesis of autoantibody responses, and to identify and define relevant autoantigens in human autoimmune diseases.

Objective The characteristics of 3 patients with longstanding rheumatoid arthritis (RA) and consecutive evolution of limited cutaneous systemic sclerosis (lcSSc) were evaluated and compared with those of patients with lcSSc alone (n = 20) or with RA alone (n = 120). Methods Clinical features of the different patient populations were compared. Serologic analyses included tests for antinuclear antibodies (ANA) and ANA subsets, in particular anticentromere antibodies (ACA) and anti-heterogeneous nuclear RNP (hnRNP)-A2/RA33 (anti-A2/RA33). Results The 3 patients with RA developed lcSSc 11, 29, or 50 years after the onset of RA. Features of lcSSc were Raynaud's phenomenon, sclerodactyly, and telangiactasias in all 3 patients, and esophageal dysmotility in 1 patient. Rheumatoid factor (RF) and anti-A2/RA33 were each found in 2 patients, and 1 of these patients was seropositive for both RF and anti-A2/RA33. ACA titers were positive in all cases. However, similar to the development of RA prior to lcSSc, the occurrence of autoantibodies typical of RA preceded the occurrence of ACA, at least in 2 of the patients. Using affinity-purified antibodies, cross-reactivities between anti-centromere protein A (CENP-A) and anti-CENP-B antibodies with anti-A2/RA33 antigens were seen in the 2 anti-A2/RA33-positive patients. Such cross-reactivities were not found in lcSSc patients without concomitant RA. Epitope mapping revealed that both autoantibody specificities recognized the known major epitopes: anti-CENP-B reacted with the C-terminal region and anti-A2/RA33 with the second RNA binding domain in the N-terminal region of hnRNP-A2. Conclusion The RA-lcSSc overlap syndrome in these 3 patients with longstanding RA was characterized by an incomplete CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasias) syndrome. The study demonstrated the presence of autoantibodies typical of both diseases and cross-reactivity of ACA with hnRNP-A2/RA33 in the sera of these patients.

Objective. To assess the significance of autoantibodies to RA33, the A2 protein of the heterogeneous nuclear ribonucleoproteins (hnRNP), and to the related hnRNP proteins A1, B1, and B2 in rheumatic diseases. Methods. Using a partially purified preparation of hnRNP-A and hnRNP-B proteins, sera from 303 patients with various rheumatic diseases were investigated by immunoblotting. For the analysis of crossreactivities, autoantibodies were affinity purified by blot elution. Results. Anti-A2/RA33 was found in 35% of rheumatoid arthritis (RA) patients, 38% of mixed connective tissue disease (MCTD) patients, 23% of systemic lupus erythematosus (SLE) patients, and, apart from single exceptions, not in patients with other rheumatic diseases. All anti-A2/RA33-positive sera were also reactive with B1 and B2, and anti-A2/RA33 antibodies cross-reacted with both proteins. Antibodies to hnRNP-A1 were found less frequently; moreover, the majority of anti-A1-positive sera also contained anti-A2/RA33 antibodies. In anti-A1, anti-A2/RA33 doublepositive sera, cross-reactivity between the 2 antibodies was generally observed. In SLE patients, the presence of anti-A2/RA33 was correlated with the presence of anti-(U1) small nuclear RNP (snRNP) and anti-Sm (P < 0.0001 and P < 0.005, respectively), but there was no evidence for cross-reactivity between antibodies to hnRNP and antibodies to snRNP antigens. Conclusion. Since both hnRNPs and snRNPs are essential components of the spliceosome, the data show that the immune systems of patients with RA, SLE, and MCTD react to this functional complex. However, compared with MCTD and SLE patients, RA patients have a more restricted immune response to the spliceosome: they react to hnRNP proteins, particularly to hnRNP-A2/RA33, but not to snRNPs.

Background Antibodies (Ab) towards citrullinated (cit) peptides (ACPA) are specific serological markers for a subset of rheumatoid arthritis (RA) patients with distinct genetic and environmental associations [1,2]. On the autoantigen-derived peptide level, the ACPA responses in RA patients are heterogeneous. Better understanding of the relationships between immunity to defined peptides, and structural variants of HLA molecules may provide novel insights concerning HLA-driven pathology in RA. Objectives To investigate whether RA specific Ab towards cit peptides can detect subsets of patients with distinct associations to different HLA structures. Methods We used a multiplex peptide array [3] to detect reactivities in sera of patients (n=6400) and selected controls (n=12000) across cohorts from Sweden (EIRA), United Kingdom (WTCCC), and the United States (NARAC). Genetic data from the immunochip platform was used to investigate variation in the HLA region. First, we tested the association between AA-alleles of HLA molecules and Ab defined strata of RA patients. In order to quantify the similarity between different peptide reactivities we used permutation, scaled Jaccard distance metrics and hierarchical clustering. Next, we assessed genetic associations with antibody titers in RA cases, and estimated interactive effects on susceptibility to Ab-positive RA. Results Comparing 20 different ACPAs reactive with different cit peptides, we observed that reactivities to specific peptides are highly correlated across patients (>87% pairs co-occurs more than expected). The majority of Abs are highly co-expressed in patients who have high frequencies of HLA-alleles known to be associated to ACPA-positive RA. AA at positions 11/13 of HLA-DRB1 display the strongest susceptibility to 18 fine-specific ACPAs (omnibus p≤9.3x10 –64 ), the exceptions being reactivities towards cit Fibrinogen (Fib) β 62–81 with citrullination at position 72 or 74. The reactivities towards two Fib peptides are not highly co-occurring with other Ab; The expression of Ab against cit Fib α peptide 36–50 is independent of HLA variants and for Ab towards cit Fib β 62–81 (72) there is even a negative association to HLA-variants previously shown to associate with ACPA-positive RA. Finally, we observed several allelic combinations showing interactive effects on fine specific ACPAs, and not or weakly with CCP2. Conclusions RA patients often co-express many different RA specific Ab, and the expressions of most of these are associated to HLA variants linked with ACPA-positive RA. However we also identified ACPAs with distinct expression profiles and HLA associations. References Klareskog L et al. PMID: 18173373 Raychaudhuri S et al. PMID: 22286218 Hansson M et al. PMID: 23025688 Disclosure of Interest None declared