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The accelerated risk of cardiovascular disease (CVD) in Rheumatoid Arthritis (RA) requires further study of the underlying pathophysiology and determination of the at-risk RA phenotype. Our objectives were to describe the cardiac structure and function and arterial stiffness, and association with disease phenotype in patients with established) RA, in comparison to healthy controls, as measured by cardiovascular magnetic resonance imaging (CMR). 76 patients with established RA and no history of CVD/diabetes mellitus were assessed for RA and cardiovascular profile and underwent a non-contrast 3T-CMR, and compared to 26 healthy controls. A univariable analysis and multivariable linear regression model determined associations between baseline variables and CMR-measures. Ten-year cardiovascular risk scores were increased in RA compared with controls. Adjusting for age, sex and traditional cardiovascular risk factors, patients with RA had reduced left ventricular ejection fraction (mean difference − 2.86% (− 5.17, − 0.55) p = 0.016), reduced absolute values of mid systolic strain rate (p 2 (− 8.92, − 0.20), p = 0.041). CMR-measures predominantly associated with traditional cardiovascular risk factors; male sex and systolic blood pressure independently with increasing LVMI. Patients with established RA and no history of CVD have evidence of reduced LV systolic function and LVMI after adjustment for traditional cardiovascular risk factors; the latter suggesting cardiac pathology other than atherosclerosis in RA. Traditional cardiovascular risk factors, rather than RA disease phenotype, appear to be key determinants of subclinical CVD in RA potentially warranting more effective cardiovascular risk reduction programs.

The presence of a disease continuum in inflammatory arthritis (IA) is a recognised concept, with distinct stages from at-risk stage (presence of anti citrullinated-peptide autoantibody) to diagnosis of rheumatoid arthritis (RA), including therapy-induced remission. Despite T-cell dysregulation being a key feature of RA, there are few reports of T-cell phenotyping along the IA-continuum. We investigated the disturbances of naïve, regulatory and inflammation related cell (IRC) CD4+ T-cell subsets in 705 individuals across the IA-continuum, developing a simple risk-score (summing presence/absence of a risk-associated with a subset) to predict progression from one stage to the next. In 158 at-risk individuals, the 3 subsets had individual association with progression to IA and the risk-score was highly predictive (p

Background The genetic risk associated with rheumatoid arthritis (RA) includes genes regulating DNA methylation, one of the hallmarks of epigenetic re-programing, as well as many T-cell genes, with a strong MHC association, pointing to immunogenetic mechanisms as disease triggers leading to chronicity. The aim of our study was to explore DNA methylation in early, drug-naïve RA patients, towards a better understanding of early events in pathogenesis. Result Monocytes, naïve and memory CD4+ T-cells were sorted from 6 healthy controls and 10 RA patients. DNA methylation was assessed using a genome-wide Illumina 450K CpG promoter array. Differential methylation was confirmed using bisulfite sequencing for a specific gene promoter, ELISA for several cytokines and flow cytometry for cell surface markers. Differentially methylated (DM) CpGs were observed in 1047 genes in naïve CD4+ T-cells, 913 in memory cells and was minimal in monocytes with only 177 genes. Naive CD4+ T-cells were further investigated as presenting differential methylation in the promoter of > 500 genes associated with several disease-relevant pathways, including many cytokines and their receptors. We confirmed hypomethylation of a region of the TNF-alpha gene in early RA and differential expression of 3 cytokines (IL21, IL34 and RANKL). Using a bioinformatics package (DMRcate) and an in-house analysis based on differences in β values, we established lists of DM genes between health and RA. Publicly available gene expression data were interrogated to confirm differential expression of over 70 DM genes. The lists of DM genes were further investigated based on a functional relationship database analysis, which pointed to an IL6/JAK1/STAT3 node, related to TNF-signalling and engagement in Th17 cell differentiation amongst many pathways. Five DM genes for cell surface markers (CD4, IL6R, IL2RA/CD25, CD62L, CXCR4) were investigated towards identifying subpopulations of CD4+ T-cells undergoing these modifications and pointed to a subset of naïve T-cells, with high levels of CD4, IL2R, and CXCR4, but reduction and loss of IL6R and CD62L, respectively. Conclusion Our data provided novel conceptual advances in the understanding of early RA pathogenesis, with implications for early treatment and prevention.

Objective The diagnosis of RA patients remains a challenge, especially in ACPA-negative disease. Novel T-cell subsets, particularly Th17 may be useful, although data on Th17 frequency using flow cytometry in RA are conflicting. We investigated whether a novel epigenetic qPCR assay for the quantification of Th17 could differentiate patients with RA from those with symptoms evolving towards an alternative diagnosis. Methods We used a qPCR assay measuring the extent of the methylation at a key position in the IL-17 and CD4 genes. Assays were performed on whole blood from 49 healthy controls (HC) and 165 early arthritis clinic patients. Flow cytometry was further used to detect the expression of CXCR4 on Th17 cells. Results In 75 inflammatory arthritis patients who progressed to RA, the qPCR assays showed significantly fewer Th17 cells compared with 90 patients who did not (P Conclusion The results of the epigenetic qPCR assay showed that low levels of Th17 cells were predictive of developing RA, particularly in the ACPA-negative patients. This could have value for insights into pathogenesis and management. The results suggest the recruitment of Th17 to the inflammatory disease site, consistent with high CXCR4 expression.

Career situation of first and presenting author Student for a master or a PhD. Introduction Palindromic rheumatism (PR) is characterised by recurrent, episodic attacks of articular inflammation, which resolve completely without residual joint damage. Whether PR should be considered as a prodrome of rheumatoid arthritis (RA) or as a distinct syndrome remains unclear, over 70 years since first described.1 PR patients (non flare and during flare) were recruited in Leeds; RA and HC data was obtained from the RA-Map consortium. Differentially expressed genes (DEGs) were identified following a standard microarray analysis workflow. These findings reported the presence of distinct gene expression profiles within palindromic patients, compared to HC and those with drug naive, early RA.2 Objectives To validate a 10 gene signature as a discriminant between PR and RA in order to better classify patients suspected of having PR and to observe this signature as PR patients progress to early RA. Methods PBMCs were taken from non-flare PR (n=24); flare PR (n=16); PR progressors to RA (PR→RA, n=10); RA (n=16); and patients suspected of having PR without an official diagnosis (SpPR, n=31). All patients provided informed written consent. qPCR gene expression analysis was performed using 10 TaqMan assays and a house keeping gene. Results Functional analysis of the DEGs revealed significantly enriched gene pathways2, from which a group of 10 DEGs with high expression fold differences between PR and RA were selected and grouped into three signatures: PR signature (high RPL10, low IL10, CCR6, UBC, and SHCBP1); RA signature (high ALPL, JUN and IFTIM2); Non-Flare vs Flare signature (high EIF3E, low HSPA9). Comparing PR (non-flare and flare) with RA, 2 of the 5 genes (IL10, UBC) associated with PR were significantly different to RA (p Finally, comparing SpPR to PR and RA groups, we observed little differences in gene expression with PR; suggesting a correct diagnosis as no patients displayed a signature resembling that of RA. Nevertheless, one gene (JUN) was significantly higher in SpPR compared with PR, suggesting these patients may be in a more progressed disease state when presenting at the clinic. Conclusions There are distinct differences (at the level of gene expression) between PR and RA, and those patients who progress to RA from PR display a molecular signature that remains closer to PR than RA. References Hench P, et al. Arch Int Med 1944;73:293–321. Ajaib S, et al. EWRR-2018. P090. Annals of the Rheumatic Diseases 2018;77:A51–A52. Disclosure of Interest None declared.

Career situation of first and presenting author Post-doctoral fellow. Introduction RA is a heterogenous disease and there is a substantial evidence to indicate the contribution of type I interferon (IFN-I) in 20%–40% of RA patients with possibly more local IFNβ role; compared to systemic IFN-α action in SLE. Patients with RA have an increased risk of cardiovasular disease (CVD) equivalent to type 2 diabetes, predominantly driven by excess atherosclerosis (ATS). Pre-clinical and human data suggest IFN-I plays a key role in the development of ATS. IFN-I has been shown to underlie cardiovascular (CV) abnormalities in SLE. Objectives IFN-I is important at the initiation of the pathological processes in early RA and increased IFN-I activity is associated with CVD in RA. Methods The Vital group recently published a continuous 2-score IFN system,1 IFN scores A and B (as opposed to an often used categorical classification of IFN high/low). We applied this scoring system in a cohort of early (ERA n=75) and established RA (EstRA n=101) as well as in HC n=71. Next RA patients were stratified for CVD using multi-parametric cardiac MRI (CMR) evaluation that included aortic distensibility (vascular stiffness), LV mass/BSA (LV geometry) and Myocardial T1 (indicating myocardial fibrosis) as follows: (i) ‘ERA-no CVD’ n=37 (no abnormalities on CMR), (ii) ‘ERA-sub CVD’ n=37 (at least one of the three parameters abnormal), (iii) ‘EstRA-no CVD’, n=14 (iv) ‘EstRA-sub CVD’, n=54 (v) ‘RA-CVD’, n=32 (defined as per ‘Major Adverse Cardiovascular Event’(MACE)). Results We confirmed a higher IFN score B than A in RA patients, similar to the observation made in the original paper that developed the scoring system. Significantly higher IFN score A and B in was observed in ERA than in EstRA and HCs. There was no association between IFN scores and markers of inflmmation. Increase in expression of IFN score B was observed across a CVD continuum (i.e. from RA-no CVD to RA-sub CVD to RA-CVD) in EstRA but not in ERA. Conclusions IFN-I may play a particularly important pathological role at time of development of disease (ERA). CVD stratification suggests that genes included in IFN Scores A and B may be implicated in the progression along a CVD continuum; and appearing to associate with a pro-atherogenic role. This observation only in EstRA may reflect CVD burden over time. If confirmed, these data imply multiple organ specificities for the IFN scores. Further work is planned to interrogate IFN status in RA-CVD, towards improved risk stratification and tailored management of CVD co-morbidity. Reference El-Sherbiny YM, et al. Scientific Reports 2018;8(1):5793. Disclosure of Interest None declared

The treatment of rheumatoid arthritis (RA) has been transformed with the introduction of biologic disease modifying anti-rheumatic drugs (bDMARD) and more recently, targeted synthetic DMARD (tsDMARD) therapies in the form of janus-kinase inhibitors. Nevertheless, response to these agents varies such that a trial and error approach is adopted; leading to poor patient quality of life, and long-term outcomes. There is thus an urgent need to identify effective biomarkers to guide treatment selection. A wealth of research has been invested in this field but with minimal progress. Increasingly recognized is the importance of evaluating synovial tissue, the primary site of RA, as opposed to peripheral blood-based investigation. In this mini-review, we summarize the literature supporting synovial tissue heterogeneity, the conceptual basis for stratified therapy. This includes recognition of distinct synovial pathobiological subtypes and associated molecular pathways. We also review synovial tissue studies that have been conducted to evaluate the effect of individual bDMARD and tsDMARD on the cellular and molecular characteristics, with a view to identifying tissue predictors of response. Initial observations are being brought into the clinical trial landscape with stratified biopsy trials to validate toward implementation. Furthermore, development of tissue based omics technology holds still more promise in advancing our understanding of disease processes and guiding future drug selection.

Objectives In a cross-sectional study, we evaluated the prevalence of ‘multi-dimensional remission’ (MDR) and its component parameters, assessed using objective measures in a cohort of RA patients in treatment-induced DAS28-remission, and their relationship with patient-reported outcome measures. We sought to confirm the feasibility and face validity of the MDR construct, providing a platform for future longitudinal studies in which its clinical utility might be further established. Methods 605 patients were selected from an inflammatory arthritis register using DAS28(CRP) Results Overall, only 53% (321/605) of the patients achieved clinical parameters, failures being mainly due to raised CRP (52%), TJC (28)>1 (37%) or SJC (28)>1 (16%). 211/364 (58%) of patients achieved ultrasound remission and 193/297 (65%) patients showed T-cell remission. Complete data were available for 231 patients. MDR was observed in only 35% and was associated with the best (lower) PRO scores (all P ⩽ 0.05 vs non-MDR) when compared with the other definitions of remission assessed. The MDR rate was similar in early and established RA patients on b-DMARDs; however, it was lower in established RA patients who received multiple cs-DMARDs (P = 0.011). Conclusions In this study, MDR, which may represent a state closer to normality, was found to occur in about a third of DAS28-remission patients and was associated with better patient-reported outcome measures. MDR could be a novel optimal treatment target, notably from a patient’s perspective. The relevance of these findings needs further assessment.

Background:Interferon (IFN) is known to play a role in the pathogenesis of many autoimmune diseases, among them, rheumatoid arthritis (RA). A study showed that two interferon-stimulated gene expression scores (IFN-Score-A and IFN-Score-B) can be used to predict progression to connective tissue disease (CTD) in at-risk individuals, positive for anti-nuclear antibodies (ANA+) [1]. This validated score could potentially be applied to individuals at-risk of RA.Objectives:To investigate the role of type I IFN in patients at-risk of RA and assess its potential role as a biomarker to predict progression to inflammatory arthritis (IA).Methods:PBMC samples were taken from 36 at-risk individuals positive for anti-citrullinated protein antibodies (ACPA+), with a non-specific musculoskeletal complaint but no clinical synovitis and a normal ultrasound scan at baseline (BL). 17 of them developed IA later on, and had a second sample taken at the moment of progression. The other 19 did not progress and had a second sample taken after 1 year. Expression of IFN stimulated genes was assessed using TaqMan. T-test was used to compare the expression of the genes at the two time points of each individual. Binary logistic regression was used to assess BL predictors of progression. Multivariable analysis was adjusted for confounders such as C-reactive protein (CRP) rheumatoid factor (RF) and ACPA titre.Results:Table 1 shows the list of tested genes. There were no differences in the gene expression of progressors at BL and at the moment of IA diagnosis. Similarly, no differences were found between the non-progressors at BL and after 1 year. Comparing BL samples from progressors and non-progressors, there was a trend to higher expression of IFN-score-B in the progressors (Mann Whitney p=0.055), whereas IFN-score-A was similarly expressed in both groups. This is similar to the results found in a cohort of ANA+ individuals at risk of progressing to CTD [1]. Multivariable analysis showed that IFN-score-B and RF titre were predictive for IA progression with an odds ratio (OR) of 2.18 (p=0.048) and OR 1.01 (p=0.035).Table 1.Interferon stimulated genes and scores A and BGENEIFN scoreGENEIFN scoreCCL8BST2CEACAM1IFI16CXCL10IFIH1GBP1LAMP3IFI27NT5C3BIFI44PHF11IFI44LASERPING1BIFIT1SOCS1IRF7SP100ISG15STAT1RSAD2TAP1XAF1TRIM38UBE2L6UNC93B1Table 2.Baseline characteristics and predictors of progressionConclusion:These exploratory results suggest that IFN changes in IA progressors precede subclinical inflammation on ultrasound. A preliminary risk model shows that IFN-score-B could be useful to predict progression to IA in at-risk of RA individuals; however, these results require validation in a larger at-risk cohort.References:[1]Md Yusof MY. Ann. Rheum. Dis. 2018;77(10):1432-9.Disclosure of Interests:Leticia Garcia-Montoya: None declared, Zoe Wigston: None declared, Agata Burska: None declared, Kulveer Mankia: None declared, Edward Vital Grant/research support from: AstraZeneca, Roche/Genentech, and Sandoz, Consultant of: AstraZeneca, GSK, Roche/Genentech, and Sandoz, Speakers bureau: Becton Dickinson and GSK, Paul Emery Grant/research support from: AbbVie, Bristol-Myers Squibb, Merck Sharp & Dohme, Pfizer, Roche (all paid to employer), Consultant of: AbbVie (consultant, clinical trials, advisor), Bristol-Myers Squibb (consultant, clinical trials, advisor), Lilly (clinical trials, advisor), Merck Sharp & Dohme (consultant, clinical trials, advisor), Novartis (consultant, clinical trials, advisor), Pfizer (consultant, clinical trials, advisor), Roche (consultant, clinical trials, advisor), Samsung (clinical trials, advisor), Sandoz (clinical trials, advisor), UCB (consultant, clinical trials, advisor)

Background Increased cardiovascular (CV) risk due to excess atherosclerosis in rheumatoid arthritis (RA) is attributed to systemic inflammation. Effective disease modifying drugs have been associated with reduced CV burden. Pre-clinical models of atherosclerosis suggest atheroprotective IgM and atherogenic IgG B-cell populations; with reduced atherosclerosis in murine models treated with depleting anti-CD20 monoclonal antibody suggesting relative preservation of protective B-cell population. The specific impact of rituximab (RTX) on the development of CV disease in RA has not been evaluated thus far. Objectives To determine the incidence of major cardiovascular events (MACE) in RA patients treated with rituximab and factors associated with any increased risk. Methods This was a single-centre, cohort study of patients with RA treated with ≥1 RTX cycle recruited prospectively. MACE outcomes were retrospectively identified as myocardial infarction, cerebrovascular accident, or death due to CV disease. Patients with and without MACE were compared for age, sex, CV risk factors (diabetes mellitus, hypertension, hyperlipidaemia, smoking, prior CV disease), disease characteristics (disease duration, ACPA, RF, methotrexate use, DAS28) and immunoglobulin levels. Association between the proportion of MACE and these variables of interest was analysed using the Student T test or Chi squared test as appropriate. Results A total of 434 patients were studied (mean age 58 years [SD 13], 80% females). Total follow-up was 3211 patient-years (PY). Of these, 32/434 (7.4%) had a MACE (incidence rate 10 per 1000 PY). Forty-three deaths (any cause; 9.9%) were recorded, 6/43 and 26/391 deaths respectively in patients with and without MACE (14% vs 7%, p=0.114). Patients with MACE were older (64 [SD 9] vs 58 [SD 13] years, p=0.001), more likely to be diabetics (22% vs 6%, p=0.001), smokers (11% vs 5%, p=0.027), and were treated less frequently with methotrexate (4% vs 13%, por=0.002), but did not differ for hypertension, hyperlipidaemia, prior CV disease nor RA characteristics. Data on immunoglobulins at baseline and after the first cycle were available in the first instance in 287/434 patients. There were no significant differences in baseline immunoglobulin levels between patients with MACE and those without. However, the proportion of patients with MACE was significantly lower among those who had a reduction in their IgM levels from baseline (5% vs 21%, p=0.006), but not for IgG or IgA. No significant differences in the clinical profile of patients with decreased IgM and those without were observed. Conclusions These preliminary data suggest decrease of IgM is associated with decreased risk of MACE in RA patients treated with RTX. Planned analysis on serial Ig with cumulative RTX exposure will clarify this initial observation. Whether and how any such association is related to a specific RTX-mediated effect and/or the overall reduction in the inflammatory burden deserves further investigation. Disclosure of Interest None declared

Background Rheumatoid Arthritis (RA) patients have a prominent increase in cardiovascular disease (CVD) not fully explained by traditional risk factors 1 . Interferons type 1 (IFN-I) have been associated with premature CVD in SLE 2 and are implicated in several aspects of atherosclerosis and acute coronary syndromes 3 . A subpopulation of RA patients also display a peripheral blood IFN-I signature from 25% to 65% 4 , associated with clinical response to biologics 5 . The IFN-I signature association with CVD in RA remains unclear. Objectives To analyse expression of interferon type 1 response genes (ISGs) along a cardiac MRI (CMR) phenotyped CVD continuum in patients with RA. Methods PBMC samples from 94 RA patients and 21 healthy controls (HC) were obtained. RA patients were stratified based on CMR into: RA CMRneg (no CVD-RA, n=13), RA CMRpos (subclinical CVD-RA, n=54), and RA with clinical CVD (defined as history of cerebrovascular disease or ischaemic heart disease) (RA-CVD n=25). qPCR of ISGs was performed using TaqMan Gene Expression Assays on Biomark (Fluidigm) with compatible reagents. Factor analysis of Ct values from 51 genes was used to create scores by calculating median dCt for genes loaded by each factor. Results RA cohort median(IQR) age 63 (13.3)yrs, 69% female, disease duration 148.7 (215.3)mths; HC age 43 (16.5)y, 62% female. Three IFN-I factors (IFN Score 1, 2,3) were present in the dataset and were composed of 19, 21 and 7 genes respectively. The Jonckheere Tepstra test showed significant increases in expression of Score 2 across the 4 groups(p=0.002), consistent with a continuum, and multiplicity-corrected post-hoc analysis identified differences between HC and subclinical CVD (p=0.034), HC and RA-CVD (p=0.004); as well as between no CVD-RA and subclinical CVD-RA (p=0.034); and subclinical CVD-RA with RA-CVD (p=0.029). Scores 1 and 3 did not show consistent directional trends across all studied groups. In no CVD-RA expression of Score 1 and 2 positively correlated with CRP (rho=0.744, p=0.002 and rho=659, p=0.010 recspectively). Score 1 with Low-Density Lipoproteins (rho=0.527, p=0.044), Framingham 10y risk (rho=0.595, p=0.019) and Score 3 with Pulse Wave Velocity (rho=0.584, p=0.022). In subclinical CVD-RA score 3 expression negatively correlated with Left Ventricular mass (rho=−0.381, p=0.005), in RA-CVD score 3 expression positively correlated with Glucose (rho=0.724, p=0.042, Triglycerydes (rho=0.821, p=0.023) and Total Cholesterol/High-Density Lipoprotein ratio (rho=0.505, p=0.039). Conclusions An IFN-I score (Score 2) emerged as a possible factor characterising progression along a CVD continuum in RA patients, from no CVD to subclinical and clinical CVD; also distinguishing between HC and RA with subclinical and clinical CVD. IFN –I is involved in metabolic disturbancies associated with CVD development in RA. These results warrant further evaluation to confirm the findings in a larger cohort. References [1] Kaplan MJ. Curr. Opin. Rheumatol2006;18(3):289–297. [2] Somers EC, et al. PloS one2012;7(5):e37000. [3] de Winther MPJ. Arterioscler. Thromb. Vasc. Biol2016;36(2):217–218. [4] Rodriguez-Carrio J, et al. Front Immunol2018;8. [5] Thurlings RM, et al. Arthritis Rheum2010;62:3607–3614. Disclosure of Interest None declared

Background Rheumatoid Arthritis (RA) patients have increased cardiovascular risk due to accelerated atherosclerosis (ATS), which significantly contributes to excess mortality in RA1. The increased cardiovascular risk cannot be fully explained by traditional risk factors and systemic chronic inflammation appears to play a crucial role. Interestingly, IL-7, a proinflammatory cytokine involved in RA pathogenesis, appears to play a role also in atherosclerosis2 but its effect on cardiovascular disease (CVD) in RA has not been studied yet. Objectives To examine serum IL-7 levels and expression of IL-7, IL-7R, CD3 and CD20 in aortic adventitia of RA and non-RA patients with coronary artery disease (CAD) and to search for relationships between systemic IL-7 levels and expression of vascular markers, cardiovascular risk factors including metabolic and inflammatory markers. Methods We examined 19 RA patients and 20 non-RA patients undergoing coronary artery bypass graft surgery included in the Feiring Heart Biopsy Study. Serum IL-7 levels were measured by chemiluminescence (MSD). Biopsies from the adventitia of thoracic aorta from a subset of patients (12 RA and 14 non-RA) were stained for IL-7, IL-7R, CD3 and CD20 by immunohistochemistry and scored per mm2 of tissue. Results Non-RA patients had lower IL-7 serum levels than RA (3.4±3.3 vs. 6.7±3.5, p The number of IL-7 +and IL-7R+cells/mm2 in adventitia were significantly higher in RA (134.2±45.5 and 144±49.9 respectively) than non-RA patients (46.9±22.8 and 54.4±20.2, p Conclusions Among patients with CAD, those with RA had higher serum IL-7 and a greater expression of both IL-7/IL-7R is aortic adventitia. Systemic levels of IL-7 were related to its vascular expression. Thus, the IL-7/IL-7R axis may play a role in the accelerated atherogenesis observed in RA; further studies are needed to elucidate the precise role of IL-7 and impact of potential IL-7R blockade in CV risk in RA. References [1] Kaplan MJ. Curr. Opin. Rheumatol2006;18(3):289–297. [2] Damas JK, et al. Circulation2003;107(21):2670–2676. Disclosure of Interest None declared

Introduction Alterations in DNA methylation patterns have been related to several diseases, including Rheumatoid Arthritis (RA). Objectives To identify changes in DNA methylation pattern of naive and memory CD4 +T cells and monocytes in early, drug naive RA patients to help understand early event in disease pathology. Methods The methylation patterns of 480,000 CpGs were analysed in the 3 cell types from 6 healthy control (HC) and 10 RA patients using an Illumina genome-wide array. Standard t-tests were performed to associate p-value to individual CpG-probe. A scoring system was developed to select and prioritise differentially methylated CpGs with potential cumulative effect due to proximity with other significant CpGs. Rules for scoring were designed with respect to the significance of each CpGs and the distance (in bp) between them. Further filtering was applied to initially select the CpGs which highest significant (p-value≤0.0001) which have at least 2 proximal significant CpGs (p-value≤0.01). Rules were coded in R for systematic analysis. Lists of selected CpGs for all three cells for both hypo- and hyper-methylation were generated. Commonality was analysed using Venn diagram. Results Different changes in methylation patterns were observed between HC and RA in the 3 cell types and thresholds of significance were set at p-value 0.01, 0.001 and 0.0001. The total number of differentially methylated CpGs was enriched in naive T-cells (18,020) compared to memory (14,197) and monocytes (6,490) (p-value≤0.01). Using our designed rules, we were able to prioritise cluster of differentially methylated CpGs which were then associated to 420, 7, and 48 hypomethylated genes, and 420, 719 and 21 hypermethylated genes respectively in naive/memory T-cells and monocytes. Venn diagrams of hypermethylated gene showed only 1 genes (ABAT) in common to all 3 subsets. Hypomethylated gene showed no commonalities between the 3 cell subsets, and only 1 gene common between T-cell subsets (SLC43A2). Of note, the TNF gene was second on the priority list for naive T-cell hypomethylation while no difference was observed in memory/monocytes. The IL-17 gene was de-methylated only in memory T-cells, hypermethylated in RA, but fully methylated in naive/monocytes. Hypomethylation of socs3 was specific to monocytes. Conclusions These data suggest quite different types of changes in patterns of DNA methylation affecting the 3 subsets early in the RA disease process. Our scoring system to prioritise clusters of differential methylation highlighted genes known to be related to early RA pathogenesis. Further work remains to explore the relationship between these genes and the biological effect at the transcriptional/translational level resulting from these alterations in DNA methylation. Disclosure of interest None declared

Introduction Rheumatoid Arthritis (RA) patients have increased cardiovascular risk (CV) due to accelerated atherosclerosis (ATS), which significantly contributes to excess mortality in RA.1 The increased CV risk cannot be fully explained by traditional risk factors and systemic chronic inflammation appears to play a crucial role. Interestingly, IL-7, a proinflammatory cytokine involved in RA pathogenesis, appears to play a role also in ATS2 but its effect on cardiovascular disease (CVD) in RA has not been studied yet. Objectives To examine serum IL-7 levels and expression of IL-7, IL-7R, CD3 and CD20 in aortic adventitia of RA and non-RA patients with coronary artery disease (CAD) and to search for relationships between systemic IL-7 levels and expression of vascular markers, cardiovascular risk factors including metabolic and inflammatory parameters. Methods We examined 19 RA and 20 non-RA patients undergoing coronary artery bypass graft surgery included in the Feiring Heart Biopsy Study. Serum IL-7 levels were measured by chemiluminescence (MSD). Biopsies from the adventitia of thoracic aorta from a subset of patients (12 RA and 14 non-RA) were stained for IL-7, IL-7R, CD3 and CD20 by immunohistochemistry and scored per mm2 of tissue. Results Non-RA patients had lower IL-7 serum levels than RA (3.4±3.3 vs. 6.7±3.5, p Conclusions Among patients with CAD, those with RA had higher serum IL-7 and a greater expression of both IL-7 and IL-7R is aortic adventitia. Systemic levels of IL-7 were related to its vascular expression. Thus, the IL-7/IL-7R axis may play a role in the accelerated ATS observed in RA; further studies are needed to elucidate the precise role of IL-7 in CV risk in RA. References . Kaplan MJ. Curr. Opin. Rheumatol2006;18(3):289–297. . Damas JK, et al. Circulation2003;107(21):2670–2676. Acknowledgements None. Disclosure of interest None declared

Introduction Rheumatoid Arthritis (RA) patients have a prominent increase in cardiovascular (CV) comorbidity not fully explained by traditional risk factors.1 Interferons type 1 (IFN-1) have been associated with premature CV disease (CVD) in SLE2 and are implicated in several aspects of atherosclerosis and acute coronary syndromes.3 In RA a subpopulation of patients also display an IFN-1 signature in the blood,4 which is associated with response to biologics. The IFN- 1 signature association with CVD in RA remains unclear. Objectives To analyse expression of interferon type 1 response genes (ISGs) along a CVD continuum in patients with RA using a well phenotyped cohort of RA patients whereby cardiovascular changes were assessed by Cardiac Magnetic Resonance Imaging (CMR). Methods PBMCs samples from 60 RA patients (mean (90% CI)) age 62.4 (60.2, 64.6)y.o, sex F/M 42/18, disease duration 153.3 (121.3, 185.2) mths and n=18 healthy controls (HC); age 42 (37.8,46.0) y, F/M 12/6 were obtained. RA patients were stratified based on CMR into: no CVD (RA CMRneg n=13) and abnormal CMR (RA CMRpos n=26), and RA with clinical CVD (no CMR, defined as history of cerebrovascular disease or ischaemic heart disease) (RA CVD n=21). qPCR of ISGs was performed using TaqMan Gene Expression Assays on Biomark (Fluidigm). Factor scores were created using the set of 51 genes measured in 78 subjects. Results All routinely used biomarkers were associated with RA diagnosis (ACPA, RF, TJC, SJC, CRP, DAS28). Total cholesterol and LDL levels were higher in RA than in HC group (p=0.051 and p=0.015). Three IFN-1 scores were present in the dataset (IFN Score 1, 2 and 3) and were composed of 19, 21 and 7 genes respectively. IFN-1 score 2 showed significant differences between HC and RA CMRpos groups (mean difference and 90% CI: 0.52 (0.09, 0.96)) and HC and RA CVD (0.57 (0.10, 1.03)) as well as all RA patients with CVD (CMRpos and RA CVD) ((0.54 (0.16, 0.93); p Conclusions An IFN-1 score 2 emerged as a possible factor characterising progression along a CVD continuum in RA patients, from no CVD to subclinical and clinical CVD; also distinguishing between HC and RA with subclinical and clinical CVD. These results warrant further evaluation in a larger cohort to confirm the findings. References . Kaplan MJ. Curr. Opin. Rheumatol2006;18(3):289–297. . Somers EC, et al. PloS One2012;7(5):e37000. . de Winther MPJ. Arterioscler. Thromb. Vasc. Biol2016;36(2):217–218. . van der Pouw Kraan TC, et al. Ann. Rheum. Dis2007;66(8):1008–1014. Disclosure of interest None declared

Background Making the earliest diagnosis of rheumatoid arthritis (RA) is crucial to initiate treatment and prevent further disease progression and joint damage. Despite recent advances with the discovery and integration of anti–cyclic citrullinated protein antibody (ACPA) in classification criteria, there is still an unmet need for new diagnostic biomarkers, notably for ACPA-negative disease. Power Doppler ultrasound has been shown to identify poor prognosis disease in ACPA negative patients. Objectives The receptor-activator-nuclear-factor-κB axis (RANK/RANKL) is known to regulate bone homeostasis. The aim of this pilot study is to establish whether serum RANKL levels in people with early inflammatory arthritis are associated with RA diagnosis at follow-up and to evaluate the added value of RANKL with ultrasound for early RA diagnosis. Methods Serum from 298 subjects (95/204 Male/Female) was collected at the baseline participant visit to the Leeds Early Arthritis clinic. Demographic (age, gender symptom duration) and clinical data (swollen and tender joint counts (SJC, TJC), CRP, DAS28, rheumatoid factor (RF) and ACPA, shared epitope (SE)) were collected. A commercial ELISA (BioVENDOR) was used to measure RANKL. Ultrasound of 26 joints (bilateral elbows, wrists, MCP 2–3, PIP 2–3, knees, ankles and MTP 1–5) was performed at baseline recording summative scores for power Doppler (PD), grey scale hypertrophy (GS) and erosions (ERO). Results At 1 year follow-up, 151 patients had a confirmed diagnosis of RA (EULAR 2010 criteria) and 147 were classified as non-RA (undifferentiated arthritis, other inflammatory diagnoses or non-persistent inflammation). All routinely used biomarkers were associated with RA diagnosis (ACPA, RF, SE, TJC, SJC, CRP, DAS28, p 700, age>62, TPD>3, SJC>4). This score predicted RA with an AUROC of 0.782 ((0.23–0.840), p Conclusions A score incorporating RANKL, age, SJC and PD showed good predictive value for non-RA when low and for RA when high. Furthermore, the analysis redone in ACPA-negative patients performed particularly well for predicting RA with a good AUROC value. Disclosure of Interest None declared

Background/objectives The earliest diagnosis of rheumatoid arthritis (RA) is crucial to initiate treatment and prevent further disease progression and the accumulation of irreversible damages to the joints. Despite recent advances with the discovery and integration of anti–citrullinated protein antibody (ACPA) in diagnostic criteria, there is still an unmet need for new diagnostic biomarkers, notably for ACPA-negative disease. The receptor-activator-nuclear-factor-κB axis (RANK/RANKL) is known to regulate bone homeostasis. The aim of this pilot study is to establish whether serum RANKL levels in people with early inflammatory arthritis, are associated with RA diagnosis at follow-up and to evaluate the added value of RANKL for early RA diagnosis. Materials/methods Serum from 298 subjects (95/204 Male/Female) was collected at the baseline participant visit to the Leeds early arthritis clinic. Demographic (age, symptom duration) and clinical data (joint counts swollen and tender (SJC, TJC), CRP, DAS28, rheumatoid factor (RF) and ACPA, shared epitope (SE)) were collected. A commercial ELISA (BioVENDOR) was used to measure RANKL. Ultrasound of 26 joints (bilateral elbows, wrists, MCP 2–3, PIP 2–3, knees, ankles and MTP 1–5) was performed at baseline recording summative scores for power Doppler (PD), grey scale hypertrophy (GS) and synovitis (SYN). Results At follow-up, 151 patients had a confirmed diagnosis of RA (EULAR-2010 criteria) and 147 were classified as non-RA (undifferentiated arthritis, other inflammatory or non-inflammatory diagnosis). All routinely-used biomarkers were associated with RA diagnosis (ACPA, RF, SE, TJC, SJC, CRP, DAS28, p 700, age >62, TPD >3, SJC>4). This score predicted RA with an AUROC of 0.782 ((0.23–0.840), p Conclusions A score including RANKL, age, SJC and PD showed good predictive value for non-RA when low and for RA when high. Furthermore, the regression redone in ACPA-negative only patients performed particularly well for (77.4% accurate).

Background In rheumatoid arthritis (RA), several studies demonstrate functionally distinct peripheral B-cell subsets relate to disease duration and response to B-cell depleting rituximab therapy.1–3 Changes following treatment with tumour necrosis factor (TNF)-inhibitor (TNFi) in peripheral blood and tonsillar tissue as well as synovial tissue have been reported.4,5 We have previously noted decreased peripheral memory B-cell and increased plasmablast numbers in active RA on no disease modifying anti-rheumatic drug (DMARD) compared with healthy controls (unpublished observations). Whilst methotrexate (MTX)-induced remission was associated with lower plasmablasts, TNFi (adalimumab+etanercept)-induced remission led to high peripheral plasmablasts. The aim of this study was to investigate further changes in peripheral B-cell subset populations with first-line conventional and TNF-inhibitor-induced suppression of inflammation. Materials and methods Highly sensitive flow cytometry was performed on peripheral blood of 120 patients with early, treatment-naive RA, randomised to either etanercept/MTX or MTX +/- additional conventional DMARD, switching to etanercept/MTX at 6 months if remission was not achieved.6 Preliminary baseline-week 24 findings are reported. Results Baseline: No significant differences in absolute number of or percentage B-cell populations were observed between groups 1 and 2. Week 0–24 changes: ETN/MTX: Total number of B-cells increased from week 0–24, (0.1083–0.1557); specifically, significant memory cell (0.0226–0.0338, p MTX/conventional DMARD: All changes following treatment were non-significant. Numerical increase in absolute naive (0.0952–0.0964) and memory B-cells (0.0272–0.0321); with decrease in plasmablast (0.0027–0.0012) numbers was observed. However, non-significant percentage naive population decrease (76.88–73.78) and plasmablast (2.29–1.50); with percentage increase in memory cells (20.78–24.73) was observed. Conclusions Initial analysis demonstrates more marked alteration in B-cell population composition following first-line ETN/MTX compared to conventional DMARD alone, with absolute increase in memory but percentage decrease in plasmablast cells. This suggests more efficient B-cell homeostasis with ETN/MTX combination in early RA (compared to established). More effective (synovial) inflammation suppression, additional role of MTX +/- etanercept also targeting lymphotoxin may underlie these observations. Planned detailed analysis will clarify the relationships further, including disease activity state and response status. References Daien CI, et al. Arthritis Research & Therapy2014, 16:R95 Vital E, et al. Arthritis Rheum2010. 62(5) Fedele AL, et al. BMC Immunology2014, 15:28 Souto-Carneiro MM, et al. Arthritis Research & Therapy2009, 11:R84 Anolik JH, et al. J Immunol2008, 180:688-692 Dumitru RB, et al. BMC MSK2016, 17:61

OBJECTIVES: To determine the change in established biomarkers of cardiovascular (CV) risk, namely, total cholesterol/high-density lipoprotein cholesterol ratio (TC/HDL-C), N-terminal pro-brain natriuretic peptide (NT-proBNP) and insulin resistance (IR) in patients with early RA treated with two different treat-to-target strategies.METHODS: Fasting glucose, lipids, insulin and NT-proBNP were measured at baseline, weeks 26 and 78 in 79 DMARD-naïve RA patients, free of CV disease, as part of a double-blind randomized controlled trial of MTX with either infliximab (IFX) or methylprednisolone as induction therapy. Homeostasis model assessment-estimated IR (HOMA-IR) (glucose*insulin/405) was used to measure IR. Multiple imputation was employed, and linear regression analyses were adjusted for baseline values.RESULTS: Changes in DAS44-CRP did not differ between the treatment arms at weeks 26 and 78. Mean TC/HDL-C, HOMA-IR and NT-proBNP improved in both groups at weeks 26 and 78, although change in NT-proBNP was not statistically significant at week 78. Changes in TC/HDL-C and NT-proBNP were similar between treatment arms, but HOMA-IR values in the IFX + MTX arm were 42% lower than those treated with MTX + methylprednisolone at week 78 (P = 0.003); the difference remained significant after adjustment for baseline BMI, ACPA positivity, smoking status and intramuscular glucocorticoid use (P = 0.007).CONCLUSION: When implementing a treat-to-target approach, treatment of early RA was associated with improvement in TC/HDL-C, HOMA-IR and NT-proBNP, and a greater long-term improvement in HOMA-IR was seen in those treated with IFX.TRIAL REGISTRATION: EU Clinical Trials Register, http://www.clinicaltrialsregister.eu, Eudract-2005-005013-37; ISRTCNregisrty, http://www.isrctn.com, ISRCTN48638981.

The therapeutic goal for patients with rheumatoid arthritis (RA) is clinical remission. This is best achieved by early diagnosis and appropriate therapeutic intervention. RA is associated with dysregulation of T-cell subsets (naïve, regulatory (Treg) and inflammation-related cells (IRC)) early in the disease. Our aim was to test the hypothesis that T-cell subset quantification can predict the achievement of clinical remission with early treatment in RA.T-cell subsets were quantified in 108 drug-naïve, early RA patients commencing methotrexate (MTX) or MTX+antitumor necrosis factor (anti-TNF) and in 105 healthy controls (HC). The primary outcome assessed was remission (DAS282.6). A pilot study used frozen cells (38 patients and 35 HCs, see online supplementary material) and was validated with fresh blood (70 patients and 70 HCs).Immune dysregulation in early RA was confirmed with an association between age and reduced naïve cells compared with HCs (p=0.006), a lower age-adjusted Treg and higher IRC frequency (p=0.001). Anticitrullinated peptide antibody (ACPA) positivity was associated with lower naïve (p=0.031) and Treg frequencies (p=0.039). In 50 patients treated with MTX, ACPA/age-adjusted analysis demonstrated that higher naïve cell frequency (relative to HC) was associated with remission (OR 5.90 (1.66 to 20.98), p=0.006, sensitivity/specificity 62%/79%, Positive Predictive Value (PPV)/Negative Predictive Value (NPV) 66%/76%). Remission with MTX+anti-TNF (n=20) was not found to be associated with naïve cell frequency, and for patients with reduced naïve cells the remission rate increased from 24% (MTX) to 42% (MTX+anti-TNF).Baseline T-cell subset analysis has a value in predicting early RA remission with first therapy with MTX. Immunological analysis could be used in conjunction with clinical/serological features to predict response to MTX and help select the most appropriate therapy at disease presentation.

Autoantibodies have been associated with human pathologies for a long time, particularly with autoimmune diseases (AIDs). Rheumatoid factor (RF) is known since the late 1930s to be associated with rheumatoid arthritis (RA). The discovery of anticitrullinated protein antibodies in the last century has changed this and other posttranslational modifications (PTM) relevant to RA have since been described. Such PTM introduce neoepitopes in proteins that can generate novel autoantibody specificities. The recent recognition of these novel specificities in RA provides a unique opportunity to understand human B-cell developmentin vivo. In this paper, we will review the three of the main classes of PTMs already associated with RA: citrullination, carbamylation, and oxidation. With the advancement of research methodologies it should be expected that other autoantibodies against PTM proteins could be discovered in patients with autoimmune diseases. Many of such autoantibodies may provide significant biomarker potential.

Rapid advances in genotyping technology, analytical methods, and the establishment of large cohorts for population genetic studies have resulted in a large new body of information about the genetic basis of human rheumatoid arthritis (RA). Improved understanding of the root pathogenesis of the disease holds the promise of improved diagnostic and prognostic tools based upon this information. In this review, we summarize the nature of new genetic findings in human RA, including susceptibility loci and gene-gene and gene-environment interactions, as well as genetic loci associated with sub-groups of patients and those associated with response to therapy. Possible uses of these data are discussed, such as prediction of disease risk as well as personalized therapy and prediction of therapeutic response and risk of adverse events. While these applications are largely not refined to the point of clinical utility in RA, it seems likely that multi-parameter datasets including genetic, clinical, and biomarker data will be employed in the future care of RA patients.

Objective\ud \ud Beyond the joints, TNFi (tumour necrosis factor inhibitor) therapy may confer systemic benefits in rheumatoid arthritis (RA). Several studies have investigated the role of TNFi on insulin resistance/sensitivity (IR/IS). This question is of general interest given the emerging evidence linking inflammation and insulin resistance. The main aim of this review was to summarise the published data and to determine the effects of TNFi on IR/IS.\ud \ud Methods\ud \ud We searched the PubMed and ISI Web of Knowledge databases for studies which examined the effects of TNFi on IR/IS. The studies were assessed independently by two reviewers according to a pre-specified protocol. The data on Homeostatic Model Assessment for Insulin resistance (HOMA) and Quantitative Insulin Sensitivity Check Index (QUICKI) were pooled and reported as standard difference in means (SDM) with 95% confidence interval (CI) using a random-effects model.\ud \ud Results\ud \ud A total of eight studies with 260 subjects met the selection criteria. The duration of the studies was from 8 weeks to 12 months. There was statistically significant reduction in HOMA index in six out of eight studies and four reported significant increment in QUICKI. The pooled analysis revealed significant reduction in HOMA [SDM-0.148, 95%CI[-0.278 to -0.017], p=0.026] and increment in QUICKI [SDM 0.312, 95%CI[0.019 to 0.606], p=0.037] with TNFi.\ud \ud Conclusion\ud \ud There is emerging evidence to support that TNFi therapy improves IS and reduces IR in RA. Further, well conducted trials are needed to determine if such effects translate to lower incidence of diabetes in RA or other autoimmune conditions on biologic therapy.

"A wide range of novel therapies are effective at the patient population level in rheumatoid arthritis and are widely used in clinical practice. However, for each of these, it has been clearly shown that only a subset of patients respond clinically, and of these an even smaller subset achieves complete remission."

Introduction Interleukin-7 (IL-7) is a cytokine essential for T-cell lymphopoiesis, survival and polarization with an emerging role in autoimmunity. We previously demonstrated reduced levels of circulating IL-7 in rheumatoid arthritis (RA), although high amounts are expressed in joints, suggesting differences between systemic and synovial effects. We observed healthy levels of IL-7 in 48% of RA patients in clinical remission (CR) and aimed to investigate the consequences of IL-7 deficiency on T-cell responses. Methods We used RA patients with active disease and in CR presenting various levels of IL-7, to investigate its modulatory effects on T cells by analysing responses to phyto-haemagglutinin (PHA), expression of polarization or survival factors, or suppression by regulatory T cells (Tregs). Results IL-7 levels were normal (>10 pg/ml) in 48% of RA patients in CR. Amongst 63 CR patients followed up for 18 months, lack of IL-7 recovery was observed in 13 out of 15 (86%) patients experiencing relapse but only 11 out of 48 (23%) of those who did not (P = 0.0002). Binary regressions showed high significance for below normal IL-7 levels for self-reported maternal family history of arthritis (odds ratio (OR): 7.66, P = 0.006) and a trend for smoking (OR: 3.33, P = 0.068) with no further demographic or clinical associations. Serum IL-7 correlated with restored CD4+T-cell response to PHA (rho = 0.879); this was not related to an increase in T-cell proliferation capacity or expression of survival factors B-cell lymphoma 2 (BCL2) and BCL2-associated protein X (BAX). Expression of Th1 polarization factor (TBET) was also dependent on exposure to IL-7 in vivo (rho = 0.600). In contrast CD25highTregs’ response to PHA was not affected by in vivo IL-7, but their suppression capabilities were related to circulating IL-7 (rho = 0.589). Co-stimulation with IL-7 (mimicking the joint environment) increased responsiveness of CD4+T-cells to PHA, lowering the ability of CD25highTregs to suppress them. Conclusions Our data demonstrate that IL-7 has a critical role in modulating T-cell function in vivo, possibly explaining opposing effects observed systemically and in the joint. Lack of IL-7 recovery in CR by maintaining a suppressed immune system may be a determinant factor in the occurrence of relapse. Electronic supplementary material The online version of this article (doi:10.1186/s13075-014-0511-3) contains supplementary material, which is available to authorized users.

RA is a complex disease that develops as a series of events often referred to as disease continuum. RA would benefit from novel biomarker development for diagnosis where new biomarkers are still needed (even if progresses have been made with the inclusion of ACPA into the ACR/EULAR 2010 diagnostic criteria) and for prognostic notably in at risk of evolution patients with autoantibody-positive arthralgia. Risk biomarkers for rapid evolution or cardiovascular complications are also highly desirable. Monitoring biomarkers would be useful in predicting relapse. Finally, predictive biomarkers for therapy outcome would allow tailoring therapy to the individual. Increasing numbers of cytokines have been involved in RA pathology. Many have the potential as biomarkers in RA especially as their clinical utility is already established in other diseases and could be easily transferable to rheumatology. We will review the current knowledge’s relation to cytokine used as biomarker in RA. However, given the complexity and heterogeneous nature of RA, it is unlikely that a single cytokine may provide sufficient discrimination; therefore multiple biomarker signatures may represent more realistic approach for the future of personalised medicine in RA.

Contains fulltext : 138077.pdf (Publisher’s version ) (Open Access) Gene expression has recently been at the forefront of advance in personalized medicine, notably in the field of cancer and transplantation, providing a rational for a similar approach in rheumatoid arthritis (RA). RA is a prototypic inflammatory autoimmune disease with a poorly understood etiopathogenesis. Inflammation is the main feature of RA; however, many biological processes are involved at different stages of the disease. Gene expression signatures offer management tools to meet the current needs for personalization of RA patient's care. This review analyses currently available information with respect to RA diagnostic, prognostic and prediction of response to therapy with a view to highlight the abundance of data, whose comparison is often inconclusive due to the mixed use of material source, experimental methodologies and analysis tools, reinforcing the need for harmonization if gene expression signatures are to become a useful clinical tool in personalized medicine for RA patients.

Background Evidence suggests cardiovascular disease (CVD) risk in patients (pt, pts) with Inflammatory arthritis (IA), Rheumatoid arthritis (RA) is equivalent to that of type II diabetes mellitus 1 . The accepted guidance is lifestyle factor modifications should be addressed in IA 2 , but little is known of the impact of CVD risk reduction pt education programmes delivered to pts with IA within a Rheumatology service setting. Objectives To describe the utility of a comprehensive CVD risk reduction pt education programme in pts with newly diagnosed IA following a treat to target standard therapeutic regime. Methods 387 pts with IA (RA and Undifferentiated Inflammatory Arthritis: UIA) were assessed at baseline (BL) for CV risk factors (RFs) (see Table 1) Pts were; RA; pts (1987 or 2010 ACR criteria) (n=237) and UIA; pts not meeting ACR RA criteria (n=150). A standardized co-morbidity pt education programme, which included CVD risk reduction, was delivered throughout the first 12 months (M). The impact on modifiable CVD RFs was assessed at 12M. Results 108 RA, 34 UIA pts had complete datasets (see Table 1). Of those smoked at BL, 15% reported being ex-smokers at 12M and the number smoked had decreased in 44%. 10% who reported being ex-smokers at BL altered their status to never having smoked by 12M and their reported number previously smoked reduced by 43% at 12M. Alcohol use reduced in 39% and increased in 24% (overall decrease, p=0.033). At BL, 56% of pts reported doing no moderate exercise at all; at 12M around a third of the pts had either increased or decreased the amount of exercise (no overall change). Disease activity generally decreased (p Conclusions A significant reduction in alcohol units and improvement in smoking habits were seen. However, despite significant improvements in IA disease activity and mobility, there was no improvement in exercise from the low level reported at BL. The need to address modifiable CVD RFs in IA is widely accepted by Rheumatologists but effecting lifestyle change is challenging. Behavioural change in exercise is complex, particularly in chronic disease. Further studies are required to optimise the approach in IA. References Ann Rheum Dis 2009; 68:1395–1400 ARD 2010; 69:325–331 Disclosure of Interest None declared

Background An increased risk of heart failure in RA exists 1 . Aetiology is unclear with some evidence that cardiomyopathy may occur early in RA, from pathology distinct to atherosclerosis 3 . Large well characterised CMR-RA studies to investigate pathogenesis are few 2 . Objectives To evaluate sub-clinical CVD in an asymptomatic-CVD established RA cohort compared to healthy controls (HC), using reference standard CMR-measured outcomes & novel CMR-measures of carotid artery. Methods 76 ACR1987 RA patients (pts) with dis.>5yrs, no CVD/diabetes, assessed for CV traditional risk factor (TRFs)/RA profile, pulse wave velocity (PWV), non-contrast 3T CMR (heart & carotids) reported by CMR-cardiologists, compared to 26 HC. Carotid measures inc. mean (MWT) & maximum (MxWT) wall thickness, wall (WVol) & luminal (LVol) volume & WVol-index (WVol-I, WVol/(WVol+LVol)). UVA variables (var.)=TRFs, BMI, waist/hip ratio (WHR), HOMA-IR, NTproBNP, dis. duration (ddur), 3vDAS28, ACPA, HAQ-DI, joint surgery hx, biologic use & PWV. MVA var.=age/sex/known associated (ass.) var./UVA r>0.3. Results Mean (SD) age (yrs) of RA pts; 60 (9.2), 74% female; HC 52 (11.4), 54% female. Median (IQR) ddur 16.5 (10.7, 25.7)yrs, 81% ACPA+ve, DAS28CRP 2.59 (1.30, 3.33), 67% on biologic. There were no significant differences in lipids/glucose/HOMA-IR between RA/HC. Trend for higher NT-proBNP in RA seen. In MVA (inc. age/sex), increasing ddur ass. with NTproBNP (0.5% rise in NTproBNP per 1% increase in ddur, p0.034). RA pts (after adj. for age/sex/TRFs) had reduced LVmass/BSA, LVEF, native T1 values & increased strain (Mid-S9) vs HC; no difference seen for LVEDV, LVESV, SV, LVmass/EDV, torsion or arterial stiffness (PWV/distensibility). No significant differences in carotid measures between RA/HC seen. In RA pts, 10yr JBS2 CV risk score correlated with WVol (r0.377 p=0.004) & LVol (r0.441 p=0.001). TRFs ass. with cardiac & carotid measures in RA. Conclusions CMR evidence for reduced LV mass in RA, suggesting pathology other than atherosclerosis as the cause of heart failure; perhaps microvascular dysfunction. TRFs, not RA features, are important determinants of CMR measures of subclinical CVD in RA. References ArthRheum 2005;52:412–20 EULAR 2015 OP0163 A&R 2010:62v4 Disclosure of Interest None declared

Background and Objectives Ankylosing spondylitis (AS) is a debilitating chronic inflammatory condition characterised by sacroiliac/axial joint inflammation and peripheral/extra-articular manifestations in some cases. Anti-TNF treatment has revolutionised disease control, however, not all patients respond, creating a significant need for biomarkers to tailor biological treatments to appropriate patients. Interferon (IFN)-related genes (IRGs) and gene expression signatures have been associated with a number of inflammatory autoimmune arthritides, including AS. Therefore, the aims of this study were to explore a large panel of IRGs to select possible candidate for further work developing a signature. Materials and Methods 12 AS patients treated with infliximab (4 infusions of 6 mg/kg over 22 weeks) were selected from our tissue bank. Response was defined as a decrease in Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) of > 2 points or > 50% at 6 months post treatment. cDNA was obtained from baseline peripheral blood mononuclear cells and was analysed for the expression of 75 IRGs using custom TaqMan Microfluidics Cards. Differences in gene expression between response groups was then evaluated using Mann-Whitney U tests, box- plots and unsupervised hierarchical clustering. Results Overall 6 patients responded to treatment (R) and 6 did not (NR). Although the study was exploratory and not powered for statistical significance, between the two groups, a trend (P Conclusions In this pilot study, we demonstrate the potential for IRGs to be associated with response to anti-TNF treatment in AS. Furthermore, certain genes may be unique to AS differentiating it from other autoimmune inflammatory diseases. However, due to small sample numbers, statistical significance could not be reliably determined. Selected genes will now be validated in a larger cohort to determine reproducibility and statistical significance.

Background A considerable interest has risen about the interplay between immune suppression and polarisation particularly with Th17 cells. Although Th2 polarisation seems intact in rheumatoid arthritis (RA), Th1 has long been known to be perturbed with lack of appropriate epigenetic commitment. Defective polarisation could be a mechanism by which Th1/Th17 cells preferentially develop. There are conflicting results about Th17 cell frequency in the blood of RA patients due to different phenotypes being used to define Th17 by flow-cytometry. Here, we investigated whether a novel epigenetic assay for Th17 cell frequency quantification could be used to differentiate patients with early RA from patients with early inflammatory arthritis symptoms evolving towards an alternative diagnosis. Materials and methods We used an epigenetic Th17 qPCR assay that quantifies Th17 cells based on a cell-specific methylation pattern at a key position in the IL-17 gene/promoter (Th17 qPCR; ). CD4+ T-cell frequencies were measured using the same epigenetic assay format based on a CD4+ T-cell-specific biomarker and results were used to normalise Th17 frequency to CD4+ T-cells. The Th17/CD4+ T-cell epigenetic assays were performed in whole blood extracted from healthy controls (HC, n=45), stable undifferentiated arthritis (UA) over 12–24 months (n = 43), UA patients progressing to RA (n = 22) and newly diagnosed RA patients (n = 45), other inflammatory arthritis (n = 30) and non-persisting symptoms (n = 16), as well as palindromic arthritis (n = 15). Results There was no difference between groups for the frequencies of CD4+ T-cells. Data suggest that Th17/CD4+ T-cells are significantly less frequent in the blood of early, DMARDs naive RA patients compared to HC (p < 0.0001) and patients with long lasting UA (p < 0.0001). While HC and UA are not different (p = 0.513), patients starting with UA and progressing to RA (within 6–12 months) are different from both HC (p < 0.00001) and stable UA (p = 0.001), but not from RA (p = 0.475). This finding can be explained by Th17 cell recruitment to the inflammatory disease site from the circulation as recently reported1,2,3 thus resulting in lower Th17/CD4+ T-cell frequencies in the blood. A power calculation based on these results suggested the need to test a further 30 patients from an early arthritis clinic evolving towards alternative diagnoses (AS, PsA, gout, reactive), and 16 patients with non-persistent symptoms. 15 patients with palindromic arthritis were also included. The measurement showed that Th17 frequencies in RA and other forms of inflammatory arthritis are less significantly different (p = 0.01), although frequencies between RA and non-inflammatory diseases are (p < 0.00001). Palindromic arthritis was not distinguishable from RA. Conclusions While there are conflicting results with respect to Th17 cell frequency in blood, we demonstrated using an epigenetic qPCR assay that RA patients show robust differences in their blood Th17/CD4+ levels compared to the group comprising HC, UA or non-persistent diseases. There was less difference between RA and other forms of inflammatory arthritis including palindromic arthritis. Our results suggest that together with Treg, Th17 may leave the blood in early RA and be recruited to the site of inflammation. A certain degree of interplay and plasticity between Treg/Th17 cells may explain why Treg are not capable of suppressing responses at the disease site if Th17 are also present. References 1. Chalan P, et al . PLoS One; 2013;8(11):e79370 2. Arroyo-Villa I, et al . PLoS One; 2012;7(8):e42189 3. Hirota K, et al . J Ex Med; 2007;204(12):2803–2812

Background and objectives Despite the well-established value of currently used diagnostic criteria for RA there is a constant demand for novel biomarkers to further improve early diagnosis notably in ACPA-negative patients. Inteleukin7 (IL-7) has been reported as a candidate diagnostic biomarker based on ACR-1987 criteria for RA, within 6 months of symptom development. 1 The aim of the present study was to validate these results using EULAR-2010 criteria. Materials and methods 255 patients attending early arthritis clinic were recruited. Demographic (age, gender, symptom duration) and clinical data (joint counts swollen and tender (SJC, TJC), CRP, DAS28, autoantibodies (RF and ACPA), SE) were collected. A commercial ELISA (R&D Systems, Abingdon, UK) was used to measure IL-7. Univariate and regression analysis were used to model outcome. Results At follow-up over 24 months, 118 patients had a confirmed diagnosis of RA (EULAR-2010) while 137 were classified as non-RA (persistent UA, other diagnostic or non-inflammatory). The RA diagnosis was associated with ACPA, RF and SE positivity (p Conclusions This study validates previous results using the EULAR-2010 criteria for diagnosing RA (in UK population of patients), however the predictability associated with lower IL-7 is less significant than in the initial study using ACR-1987 criteria (in French cohort). IL-7 remains a potential biomarker for ACPA-negative RA although further validation with larger number of ACPA-negative RA is needed. Further replication studies are required to translate these results into clinical applicability. Reference Goeb VP, Aegerter R, Parmar P, et al . Progression to rheumatoid arthritis in early inflammatory arthritis is associated with low IL-7 serum levels. Ann Rheum Dis. 2013;72:1032–6

Objectives The current optimal therapeutic approach in early rheumatoid arthritis (RA) is to start Methotrexate (MTX) to target inflammation and induce remission. Prediction of MTX therapy response remains a key clinical need to enable the identification of patients who would benefit from an alternative, more aggressive treatment strategy. T-cell subset analysis has been shown to have biomarker value in predicting MTX remission in early RA. 1 Given the clinical importance of these findings, our aim was to validate the original data 1 using T-cell subset quantification this time performed solemnly by routine hospital services, in order to replicate these original findings and validate naive T-cell frequencies as biomarker of the the achievement of clinical remission with MTX treatment in early RA. Methods T-cell subsets were quantified in 70 drug-naive early RA patients, commencing MTX. The primary outcome was remission (DAS28 Results Remission was achieved in 37/70 patients. In univariate analysis, the achievement of remission was strongly associated with age-corrected naive CD4 + T-cell frequency and current not-smoking (p Conclusion This study confirms the ability of baseline naive T-cell subset analysis in predicting early RA remission with first-line MTX therapy, suggesting clinical utility for the selection of the most appropriate therapy at disease presentation. All data being acquired by hospital routine services, they should be transferable widely towards personalised medicine. Reference Ponchel F, Goeb V, Parmar R, et al . An immunological biomarker to predict MTX response in early RA. Ann Rheum Dis 2014;73(11):2047–53

Background Lack of accurate early diagnostic testing of Rheumatoid Arthritis (RA) remains the limiting factor for early treatment to achieve remission. We hypothesised that molecular pathways implicated in RA pathology in the joint can inform a biomarker discovery programme based on flow-cytometry phenotyping of blood cells. Based on published work, we selected 74 subset/phenotypes and used high-dimensional dataset analysis to established their potential value in discriminating patients from an early arthritis clinic with Methods 46 patients were enrolled. 6–8 colour flowcytometry was performed using standard protocols. We recorded% of positive cells when 2 populations could be distinguished, and levels of expression (MFI) for single populations. A random forest ordered predictors from these 74 subset/phenotypes according to importance. Then a classification tree was fitted based on the most important factors and finally the variables identified from the classification tree were fitted in a logistic regression. Results 23 of the 46 patients had RA, 11 non-persistent inflammation and 12 other rheumatic diseases (AS, SPA, gout, OA and reactive). 47 individual phenotypes were analysed and 36 additional ones using a combination of 2 markers. No difference in lineage representation was found; significant difference were observed for 11/47 subsets (P Due to the low number of patients, multivariable analysis was limited. We ran un-supervised cluster analysis which separated 2 groups (RA/non-RA) quite efficiently with 5-RA and 6-non-RA misclassified. A 3-node classification tree using 47 phenotypes classified 20/23 RA patients correctly. The 3 phenotypes showing best discrimination power were IL-6R+NKT-cells, naive CD4 + T-cells and CCR6+ monocytes. A logistic regression confirmed that these 3 phenotypes were highly significant phenotypes (P + T-cells phenotypes, 1 CD8 + T-cells, 2 B-cells, 1 monocyte and 1 NKT-cells have potential value for discriminating RA from non-RA patients based on accuracy. Conclusion Despite the small sample size, this analysis demonstrated the value of hypothesis driven marker analysis, with 12 of the 47 phenotypes predicted to have potential showing significant differences. By triangulating different analysis approaches, the robustness of the findings was improved giving confidence in the identification of relevant biomarkers.

Background and Objectives ACPA+ individuals with non-specific musculoskeletal symptoms are at high risk of developing rheumatoid arthritis (RA). We previously demonstrated dys-regulation of T-cell subsets with loss of naive and regulatory T-cells (Treg) in early disease. The aim of the current study is to demonstrate the predictive value of T-cell subset analysis for progression towards symptom onset in ACPA+ individuals. Materials and Methods 84 ACPA+ individuals without clinical synovitis at recruitment were followed. 95 healthy controls (HC) provided a reference group. At baseline T-cell subset analyses were performed using 6-colour flowcytometry for naive T-cells (CD4+ CD45RB + CD45RA+ CD62L+), Treg (CD4+ CD25 high Foxp3 + CD127 low ) and inflammation related cells (IRC: CD4+ CD45RB + CD45RA+ CD62L-). The relationship between naive cell frequency and age was established in HC and used to age-correct values in ACPA+ . ROC curve analysis was used to identify 2 T-cell cut-offs predicting progression to IA at any time; one which maximised the Youden index (sensitivity + specificity-1), and one which prioritised specificity over sensitivity. Results 42/84 (50%) of patients developed clinical synovitis within a median follow-up of 6.0 months (range 1 week-46 months). For age-corrected naive T-cells area under the ROC curve (AUC) was 0.67 (95% CI 0.55, 0.79; n = 84, p = 0.007), for IRC 0.70 (0.59, 0.81; n = 81, p = 0.002) and for Treg 0.67 (0.53, 0.80; n = 65, p = 0.021). For each of the three subsets, the Youden index cut-off correctly classified >65% of patients (Table 1). Cut-offs prioritising specificity were identified which did not greatly reduce overall classification success. The confidence intervals for these estimates remain wide and our sample size may still be limited for running such analysis. Abstract A1.33 Table 1 Sensitivity and specificity of T-cell subset frequencies for progression to IA, using two different cut-off values for each subset; one where the Youden index was maximised and another that prioritised specificity over sensitivity. Conclusions T-cell dys-regulation in ACPA+ individuals with non-specific musculoskeletal pain may be useful in predicting progression to inflammatory arthritis. Multivariable modelling in larger cohorts is needed to quantify the utility of T-cell subsets in predicting progression to IA.

Background Patients with rheumatoid arthritis (RA) are at higher risk of cardiovascular (CV) disease. Emerging evidence suggests that this accelerated risk is present in the early stages of RA. Objectives Our objective was to describe the CV profile of those with a new diagnosis of inflammatory arthritis (IA). Methods 339 patients (pts) with IA enrolled into the Inflammatory Arthritis disease CONtinuum (IACON) registry in Leeds were assessed for known CV disease (CVD) and CV risk factors (RFs) including; hypertension, diabetes, smoking, family history of CVD, blood pressure, body mass index, waist/hip circumference and lipid profile. In a sub-group of those meeting ACR RA criteria (n=92); insulin resistance (homeostasis model assessment of insulin resistance (HOMA-IR) and quantitative insulin sensitivity check index (QUICKI)) and NT-proBNP were measured. Pts were split into 3 groups; RA1; pts meeting 1987 ACR RA criteria (n=143, 42%), RA2; pts meeting 2010 ACR RA criteria (n=55, 16%) and UIA; those not meeting either ACR criteria for RA i.e. undifferentiated IA (n=141, 42%). Descriptive statistics and analysis for correlation, chi-squared and ANOVA tests were performed. Results Positivity for RF (p No differences in the prevalence of traditional CV RFs, overt CVD or metabolic syndrome (according to WHO and IDF criteria) were seen. Hypertension was prevalent across all groups (28, 27 and 25% respectively). There was a trend to significance in the prevalence of hypercholesterolaemia (26, 13 and 17% respectively, p=0.067). Similar proportions of pts within the groups were receiving anti-hypertensive, anti-platelet and lipid-lowering (LLT) therapies. Total cholesterol (TC) was lower in RA1 vs. RA2 (p=0.041) but this lost significance when results were adjusted for CRP. There was no significant difference in TC/HDL ratio. In RA1 and RA2, TC and LDL negatively correlated with CRP (p=0.003, rho=-0.402 and p=0.041, rho=-0.28 respectively), and TC/HDL ratio and HDL showed no association with CRP. Pts ACPA+ve were more likely to be smokers (27 vs. 19% ACPA–ve, p=0.016), have a past history of hypercholesterolaemia (25 vs. 15% ACPA-ve, p=0.029) and be receiving LLT (21 vs. 12% ACPA-ve, p=0.042). However, there was no significant difference in the lipid profile, even with adjustment for CRP and pts receiving LLT excluded. There was no significant difference in HOMA-IR, QUICKI and NT-proBNP between RA1 and RA2 or according to ACPA status. Conclusions Within this cohort of early IA CV RFs are prevalent, correlating with inflammation and not just a specific phenotype of RA. This supports the hypothesis that inflammation plays a key role in the known accelerated CV risk in RA, and effective treatment across the continuum is important. There may be an association between ACPA and lipid metabolism. Long-term follow-up is required to evaluate potential change in risk profile with effective disease modifying treatment. Disclosure of Interest L.-A. Bissell: None Declared, A. Burska: None Declared, S. Horton: None Declared, H. Donica: None Declared, J. Freeston: None Declared, M. Buch Consultant for: Abbot, Pfizer, Roche, Chugai, BMS, UCB, P. Emery Consultant for: Abbot, Pfizer, Roche, Chugai, BMS, UCB, MSD, J. Andrews: None Declared

Background Patients with rheumatoid arthritis (RA) have an elevated risk of cardiovascular disease (CVD), predominantly ischaemic heart disease (IHD). Systemic inflammation is thought to be a key contributor to this risk and it is hoped early therapeutic suppression of inflammation will reduce cardiovascular (CV) events in the long term. Insulin resistance, associated with metabolic syndrome, N-terminal pro-brain natriuretic peptide (NT-proBNP) and atherogenic lipid profiles have been proposed as potential surrogate measures of atherosclerosis in RA. Each has been shown to correlate with systemic inflammation, but their utility in RA above measurement of the acute phase response remains unknown. Objectives To determine whether suppression of inflammation in early RA influences surrogate measures of atherosclerosis, including the homeostasis model assessment of insulin resistance (HOMA-IR), NT-proBNP and dyslipidaemia. Methods CRP, fasting glucose and lipids, and in a sub-group, NT-proBNP and fasting insulin, were measured at baseline and week 26 in DMARD naive RA patients, with 3-12 months symptoms, recruited into the Infliximab as Induction therapy for Early rheumatoid Arthritis (IDEA) multicentre double-blind randomised controlled trial. Patients met the 1987 ACR criteria. Treatment was randomised to IFX +MTX or MTX with 250mg IV methylprednisolone as induction therapy. 120mg IM methylprednisolone was given if DAS>2.4 at 3 time points within the first 26 weeks. HOMA-IR was calculated at each time point. Descriptive statistics, Wilcoxon and paired t-test, and Spearman’s correlation were performed. Results Data from 112 patients (69% female, mean age 53 years, 55% RF and 70% anti-CCP positive) were analysed. At baseline, 9% were on a statin and 14% an anti-hypertensive agent. 4 patients had known IHD and were excluded from the subsequent analysis. The median (range) CRP was 13mg/l (0-30) with CRP normal ( 1 . In the subgroup of 79 patients with data on NT-proBNP and fasting insulin, 17% and 38% of patients had raised levels of NT-proBNP and HOMA-IR, respectively. Comparing week 26 to baseline, there was a significant fall in CRP (p Conclusions In this study, suppression of inflammation was associated with improvement in insulin resistance and increased HDL, but no significant change in NT-proBNP, TG or LDL following 26 weeks intensive RA treatment. Further analyses of biomarkers at week 78 and drug specific effects are underway. Long term follow up is being performed to determine if those patients with persistently abnormal CV biomarkers develop subclinical or overt CVD in the future despite intensive early treatment for RA. References JAMA. 2001;285(19):2486-97 Disclosure of Interest L.-A. Bissell: None Declared, S. Mackie: None Declared, L. Kozera: None Declared, J. Nam: None Declared, A. Burska: None Declared, E. Hensor: None Declared, H. Keen: None Declared, E. Villeneuve: None Declared, H. Donica: None Declared, P. Conaghan: None Declared, J. Andrews: None Declared, P. Emery: None Declared, A. Morgan Grant/Research support from: Supported in part by a research grant from Investigator-Initiated Studies Program of Merck Sharp & Dohme Limited. The opinions expressed in this paper are those of the authors and do not necessarily represent those of Merck Sharp & Dohme Limited

Background and Objectives The therapeutic goal for patients with rheumatoid arthritis (RA) is clinical remission. This is best achieved by early diagnosis followed by appropriate therapeutic intervention. RA is associated with dys-regulation of T-cell subsets early in the disease with naive cells and regulatory T-cell losses and acquisition of abnormal subset in realtion with inflammation (IRC). Our aim was to test the hypothesis that T-cell subset quantification can predict the achievement of clinical remission in patients with early RA. Materials and Methods T-cell subsets (naive, Treg and IRC) were quantified using flow cytometry in 108 DMARDs-naive, early RA patients ( Results In the pilot study, T-cell subset analysis in early RA confirmed immune dysregulation compared to HC with reduced frequency of naive CD4+ T-cells and Treg and increased IRC (all p Conclusions These data show that baseline naive T-cell subset analysis has a value in predicting early RA MTX treatment outcome. Immunological analysis could be used in conjunction with clinical/serological features to predict response to MTX and select the most appropriate therapy at disease presentation.