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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: Retrospective studies have shown that anti-citrullinated protein antibodies (ACPA) are a risk factor for the development of clinical arthritis. Objectives: We aimed to investigate in a prospective setting if ACPA and other biomarkers could predict development of ultrasound detected arthritis. Methods: Subjects with positive ACPA-test referred from primary care to the Rheumatology clinic that lacked arthritis in hands, feet and any other symptomatic joints by clinical and ultrasound examination (according to EULAR-OMERACT definition), were recruited into the Risk-RA research program during 2015-2016 and were followed up to the end of 2017. At inclusion a detailed clinical examination was performed and blood samples were analyzed for 13 specific ACPA reactivities (using a custom made peptide microarray) as well as 92 inflammation-associated protein biomarkers (using a multiplex immunoassay with Olink proximity extension technology). Presence of HLA-SE was analyzed using DR low-resolution kit. Univariate and multivariate analysis were used to investigate the association between clinical and laboratory parameters and development of ultrasound detected arthritis adjusting for the follow-up time. Results: 42% (27 out of 65) of the Risk RA subjects developed ultrasound detectable arthritis during a median follow up of 8 months. The remaining 58% (38 out of 65) were followed for a median of 25 months (range 12-44) without any signs of ultrasound detectable arthritis. Subjects developing arthritis had higher prevalence of HLA-SE (89% vs 56%) and increased occurrence of ultrasound detected tenosynovitis (44% vs 5%), as compared to those not developing arthritis. ACPA reactivities to citrullinated vimentin peptides (cit vim 2-17: 22% vs 6%; and cit vim 60-75: 70% vs 43%) and citrullinated histone peptides (cit H4 31-50: 89% vs 49%; and cit H3 21-44: 48% vs 23%) were a more common occurrence in subjects developing ultrasound detectable arthritis. Backward selection in a Cox regression model showed that ultrasound detectable arthritis could be predicted in a model including HLA-SE, tenosynovitis and ACPA reactivity to cit H4 31-50. Hazard ratio (HR) for arthritis development were 3.4 (95% CI 1.0-12, p 0.06) for HLA-SE carriers, 2.9 (95% CI 1.3-6.7, p 0.01) for tenosynovitis and 4.1 (95% CI 1.2-14, p 0.02) for Anti-citrullinated H4 31-50 positivity. Only modest differences were observed for few of the tested inflammatory markers in those developing as compared to those not developing ultrasound detectable arthritis: Interleukin- 6 (3.9 vs 3.3 AU/ML), Programmed death-ligand 1 (4.9 vs 5.2 AU/ML) and Chemokine (C-X-C motif) ligand 6 (9.2 vs 9.5 AU/ML). Conclusion: Certain ACPA fine specificities, HLA-SE and tenosynovitis predict the development of ultrasound detectable arthritis in seropositive individuals with musculoskeletal symptom who are at risk for RA. Disclosure of Interests: Aase Hensvold: None declared, Yogan Kisten: None declared, Alexandra Circiumaru: None declared, Monika Hansson: None declared, Meng Sun Grant/research support from: Yes, but not for presented project., Guozhong Fei: None declared, Nancy Vivar: None declared, Erik Af Klint: None declared, Hamed Rezaei: None declared, Lars Klareskog Grant/research support from: Yes, but not for the presented study., Aleksandra Antovic: None declared, Anca Catrina Grant/research support from: Yes, but not for the presented study.

Introduction Anti-citrullinated protein antibodies (ACPA) – a hallmark of rheumatoid arthritis – antedate joint inflammation. Based on the epidemiological association between RA and periodontal disease (PD),1 it has been suggested that break of tolerance to citrullinated proteins may occur in the gum mucosa. This hypothesis is primarily based on the unique feature of Porphyromonas gingivalis (P.g) – a keystone pathogen in PD – to express a bacterial version of the peptidyl arginine deiminase enzyme (denoted P.PAD), capable of autocitrullination.2 Objectives In the present study, we have investigated the antibody response against CPP3, a citrullinated peptide derived from P.PAD, in order to address the hypothesis that P.g may drive ACPA-production. Methods This study included 2,859 RA cases and 4864 controls from the Epidemiological Investigation of RA (EIRA) cohort; 65 PD patients and 59 periodontally healthy individuals; and 218 monoclonal antibodies, derived from RA B cells. Reactivity with citrullinated P.g and human antigens was assayed by ELISA and/or multiplex. Associations with genetic risk factors and smoking were determined by logistic regression. Monoclonal antibody mutations were analysed by IgBLAST comparison. Results Anti-CPP3 IgG was detected in 11% of RA, 10% of PD and Conclusions Based on these data, we propose that P.g infection triggers an antibody response to CPP3, which cross-reacts with citrullinated human proteins by mechanisms of molecular mimicry. Future studies should address whether anti-CPP3 IgG could serve as a biomarker to identify individuals with PD at increased risk for RA. References . Fuggle NR, Smith TO, et al. Hand to mouth: A systematic review and meta-analysis of the association between rheumatoid arthritis and periodontitis. Frontiers in Immunology2016;7:80. . Quirke AM, et al. Heightened immune response to autocitrullinated porphyromonas gingivalis peptidylarginine deiminase: A potential mechanism for breaching immunologic tolerance in rheumatoid arthritis. Annals of the Rheumatic Diseases2014;73(1):263–269. Disclosure of interest None declared

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 An increase in the number of anti-citrullinated protein antibodies (ACPA) reactivities precede RA onset, and may be involved in the pathogenesis of the disease. The presence of ACPA is associated with radiographic progression in RA, and it has been suggested that ACPAs with different reactivities may be associated with different phenotypes of RA. Objectives To assess the prevalence of baseline ACPA reactivities in an inception cohort of early RA patients, including subgroups based on anti-CCP/RF status, and to compare the findings to healthy controls. Methods 217 DMARD-naive early RA patients from the ARCTIC trial (1) were analysed. Radiographs were scored according to van der Heijde Sharp (vdHS) score. Anti-CCP status was analysed by FEIA (pos. if ≥10 IU/mL) and RF by ELISA (pos. if ≥25 IU/mL). ACPA titres (AU/ml) were considered pos. if above the 98-perc. of values in 619 non-RA subjects. Analysis of 13 ACPA reactivities targeting citrullinated peptides from fibrinogen, alpha-1 enolase, vimentin, fillagrin and histone was performed at baseline in patients and 94 controls (blood donors matched for age/gender/smoking), using a multiplex chip-based assay (2). Results Baseline characteristics are presented in the table. The figure shows the prevalence of ACPA reactivities in the subgroups, with median [IQR] number of antibody reactivities in all patients 7 [3,10], compared to 0 [0,0] in controls (p Conclusions Prevalence of ACPA reactivities differed in subgroups of DMARD-naive early RA patients according to anti-CCP and RF status. All RA subgroups, including RF- and anti-CCP- patients, had higher prevalence of ACPA reactivities compared to healthy controls. References Haavardsholm et al BMJ 2016. Hansson et al Arthr Res Ther 2012. Disclosure of Interest M. Jonsson: None declared, A. Hensvold: None declared, M. Hansson: None declared, L. Mathsson-Alm Employee of: Thermo Fisher Scientific, A.-B. Aga: None declared, J. Sexton: None declared, B.-T. Fevang Grant/research support from: Novartis, S. Lillegraven: None declared, A. Catrina: None declared, E. Haavardsholm Grant/research support from: AbbVie, MSD, Pfizer, Roche, UCB

Acpa against different citrullinated peptides identify specific phenotypes of rheumatoid arthritis

Background There is today a paucity of prospective studies to describe the natural longitudinal history of anti-ccp positive individuals developing RA or not developing RA. Further no study to investigate the detailed ACPA reactivities in such a setting is currently available. Methods Individuals at risk of developing RA were included in a cohort at Karolinska University Hospital, Stockholm. Examinations of peripheral joints was repeated at one year follow-up visit or at any time the patients experienced worsening of their symptoms. Peripheral blood samples were available at inclusion (n=70). Serum was run on a microarray based on the ImmuoCAP ISAC system testing for ACPA reactivities toward 13 different citrullinated peptides (fillagrin, fibrinogen, alpha-enolase, vimentin, histone) (1). Results Individuals referred from primary care with musculoskeletal complaints and positive anti-ccp test were systematically investigated as part of routine care at our rheumatology clinic. Individuals lacking self-reported history of suspect arthritis, clinical arthritis according to rheumatologist and signs of synovitis on ultrasound examination were included in a clinical Risk-RA program with life-style coaching and personalized information on the risk of developing RA. Seventy individuals, with a mean age of 48 years (SD 15) and 86% females, were included in the program. Twenty (29%) individuals developed arthritis during a medium follow up time of 7 months (range 1–25 months). Number of ACPA reactivities at baseline was significant higher among those developing (in mean 6 reactivities) as compared to those not developing arthritis (in mean 4 reactivities). A increased proportion of individuals were showing reactivity towards citrullinated (cit) vimentin (vim) 60–75, fibrinogen (fib) 573 and enolase (eno) (CEP-1) among those developing arthritis (80% for anti-cit-vim 45% for anti-cit-fib and 60% for anti-cit eno) as compared to those not developing arthritis (41% for anti-cit-vim, 30% for anti-cit-fib and 52% for anti-cit-eno). Increased level of anti-cit-vim and anti-cit-eno antibodies was also observed at inclusion for those individuals developing arthritis as compared to those not developing arthritis. Conclusions We describe here the pattern of ACPA reactivities in anti-CCP positive individuals with non-specific musculoskeletal symptoms at risk of developing RA and without clinical and ultrasonograph signs of synovitis and report that 30% of these patients will develop arthritis during a short follow-up. Number, frequency and titers of specific ACPA reactivities appear to be enriched already at inclusion among those patients that developed arthritis during follow-up. References Hansson et al Arthritis Res Ther 2012. Disclosure of Interest None declared