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ObjectivesMacrophage subsets, activated by T cells, are increasingly recognised to play a central role in rheumatoid arthritis (RA) pathogenesis. Janus kinase (JAK) inhibitors have proven beneficial clinical effects in RA. In this study, we investigated the effect of JAK inhibitors on the generation of cytokine-activated T (Tck) cells and the production of cytokines and chemokines induced by Tck cell/macrophage interactions.MethodsCD14+monocytes and CD4+T cells were purified from peripheral blood mononuclear cells from buffy coats of healthy donors. As representative JAK inhibitors, tofacitinib or ruxolitinib were added during Tck cell differentiation. Previously validated protocols were used to generate macrophages and Tck cells from monocytes and CD4+T cells, respectively. Cytokine and chemokine including TNF, IL-6, IL-15, IL-RA, IL-10, MIP1α, MIP1β and IP10 were measured by ELISA.ResultsJAK inhibitors prevented cytokine-induced maturation of Tck cells and decreased the production of proinflammatory cytokines TNF, IL-6, IL-15, IL-1RA and the chemokines IL-10, MIP1α, MIP1β, IP10 by Tck cell-activated macrophages in vitro (pConclusionsOur findings show that JAK inhibition disrupts T cell-induced macrophage activation and reduces downstream proinflammatory cytokine and chemokine responses, suggesting that suppressing the T cell-macrophage interaction contributes to the therapeutic effect of JAK inhibitors.

Background:Patients who achieve remission promptly could have a specific genetic risk profile that supports regaining immune tolerance. The identification of these genes could provide novel drug targets.Objectives:To test the association between RA genetic risk variants with achieving remission at 6 months.Methods:We computed genetic risk scores (GRS) comprising of the RA susceptibility variants1 and HLA-SE status separately in 4425 patients across eight datasets from inception cohorts. Remission was defined as DAS28CRPResults:Evaluation of the complete dataset, baseline clinical variables did not differ between patients achieving remission and those who did not (Table 1). Distribution of GRS was consistent between datasets. Neither GRS nor HLA-SE was associated with baseline DAS2DAS (OR1.01; 95% CI 0.99-1.04). A fixed effect meta-analysis (Figure 1.) showed no significant effect of the GRS (OR 0.99; 95% CI 0.94-1.03) or HLA-SE (OR 0.8CRP87; 95% CI 0.75-1.01) on remission at 6 months.Table 1.Summary of the data separated by disease activity after 6 months.allRemission at 6 monthsNo remission at 6 monthsN4425*15582430Age, mean (sd)55.38 (13.87)5517 (14.09)55.62 (13.59)Female %68.98%65.43%70.73%ACPA+ %61.94%63.53%61.67%Baseline DAS28, mean (sd)4.76 (1.22)4.47 (1.23)5.1 (1.15)*not all patients had 6 months dataConclusion:In these combined cohorts, RA genetics risk variants are not associated with early disease remission. At baseline there was no difference in genetic risk between patients achieving remission or not. Studies encompassing other genetic variants are needed to elucidate the genetics of RA remission.References:[1]Knevel R et al. Sci Transl Med. 2020;12(545):eaay1548.Acknowledgements:This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 777357, RTCure.This project has received funding from Pfizer Inc.Disclosure of Interests:Samantha Jurado Zapata: None declared, Marc Maurits: None declared, Yann Abraham Employee of: Pfizer, Erik van den Akker: None declared, Anne Barton: None declared, Philip Brown: None declared, Andrew Cope: None declared, Isidoro González-Álvaro: None declared, Carl Goodyear: None declared, Annette van der Helm - van Mil: None declared, Xinli Hu Employee of: Pfizer, Thomas Huizinga: None declared, Martina Johannesson: None declared, Lars Klareskog: None declared, Dennis Lendrem: None declared, Iain McInnes: None declared, Fraser Morton: None declared, Caron Paterson: None declared, Duncan Porter: None declared, Arthur Pratt: None declared, Luis Rodriguez Rodriguez: None declared, Daniela Sieghart: None declared, Paul Studenic: None declared, Suzanne Verstappen: None declared, Leonid Padyukov: None declared, Aaron Winkler Employee of: Pfizer, John D Isaacs: None declared, Rachel Knevel Grant/research support from: Pfizer

Background:Rheumatoid arthritis (RA) is a chronic autoimmune disease with substantial immunopathogenic heterogeneity. It is well established that early diagnosis and initiation of effective therapy is crucial to prevent loss of function. Previously, various RA treatment trajectories have been identified, however there are currently no clinically validated biomarkers that can identify these trajectories at the start of treatment. Evaluation of the structural epigenome has revealed that chromosome conformation signatures (CCS) offer great potential as binary, informative biomarkers, and have been previously shown to predict early RA patient response to Methotrexate with 90% sensitivity (1). These signatures can also reveal highly regulated areas of the genome, which may be underpinning disease endotypes.Objectives:The objective of this study was to evaluate the structural epigenome in early RA over longitudinal samples to determine whether it is associated with treatment trajectories.Methods:Patient data and samples were from the Scottish Early Rheumatoid Arthritis (SERA) cohort; a pan-Scotland inception cohort. CDAI, DAS28 ESR and DAS28 CRP measurements were calculated at baseline, 6 months and 12 months to determine longitudinal treatment response. From 3 principal longitudinal response trajectories, 18 patients (who had equivalent disease activity at baseline) were chosen to investigate the structural epigenome. These 18 comprised of responders (R), non-responders (NR) and initial responders (IR; low disease activity/remission at 6 months but moderate/high disease activity at 12 months) with 6 patients per group at each time point. 20 pooled healthy samples were used as a comparator population. EpiSwitch libraries were probed on 180K Agilent SureSelect custom arrays that were designed using EpiSwitch propriety information and publicly available data from Walshet al(2). Microarray data was analysed using the Limma package within R studio.Results:EpiSwitch array analysis showed that there were >10,000 statistically significant differential chromosomal loops between R, NR and IR. Evaluation of the 3 trajectory groups (R, NR and IR), taking into account the healthy chromosomal conformation, revealed an RA-associated structural epigenome that comprised of 10,445 chromosomal loops that were stable, over the three time points. Subsequent analysis of the distinct treatment trajectories demonstrated that 3683 of the stable, disease-associated chromosomal loops were shared by all 3. However, 4496 were associated with distinct response trajectories, with 1221, 2574 and 701 loops unique to R, NR and IR respectively.Conclusion:The stable chromosomal architecture unique to each treatment trajectory suggests that various underlying molecular endotypes may exist. Moreover, the stable loops common to all groups allude to a baseline level of dysregulation in RA and offers the potential to discover novel drivers of disease. This work provides the foundation to further our understanding of RA pathogenesis and the potential of finding a biomarker that would be of significant value in a clinical setting.References:[1] Carini, C., Hunter, E., Scottish Early Rheumatoid Arthritis Inception cohort Investigators, Ramadass, A. S., Green, J., Akoulitchev, A., et al. (2018). Chromosome conformation signatures define predictive markers of inadequate response to methotrexate in early rheumatoid arthritis.Journal of Translational Medicine,16(1), 18–11[2] Walsh, A. M., Whitaker, J. W., Huang, C. C., Cherkas, Y., Lamberth, S. L., Brodmerkel, C., et al. (2016). Integrative genomic deconvolution of rheumatoid arthritis GWAS loci into gene and cell type associations.Genome Biology,17(1), 2205Disclosure of Interests:Caitlin Duncan: None declared, Ewan Hunter: None declared, Christina Koutsothanasi: None declared, Matthew Salter: None declared, Alexandre Akoulitchev: None declared, Iain McInnes Grant/research support from: Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Janssen, and UCB, Consultant of: AbbVie, Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Gilead, Janssen, Novartis, Pfizer, and UCB, Carl Goodyear: None declared

Background:The American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) individually and collaboratively have produced/recommended diagnostic classification, response and functional status criteria for a range of different rheumatic diseases. While there are a number of different resources available for performing these calculations individually, currently there are no tools available that we are aware of to easily calculate these values for whole patient cohorts.Objectives:To develop a new software tool, which will enable both data analysts and also researchers and clinicians without programming skills to calculate ACR/EULAR related measures for a number of different rheumatic diseases.Methods:Criteria that had been developed by ACR and/or EULAR that had been approved for the diagnostic classification, measurement of treatment response and functional status in patients with rheumatoid arthritis were identified. Methods were created using the R programming language to allow the calculation of these criteria, which were incorporated into an R package. Additionally, an R/Shiny web application was developed to enable the calculations to be performed via a web browser using data presented as CSV or Microsoft Excel files.Results:acreular is a freely available, open source R package (downloadable fromhttps://github.com/fragla/acreular) that facilitates the calculation of ACR/EULAR related RA measures for whole patient cohorts. Measures, such as the ACR/EULAR (2010) RA classification criteria, can be determined using precalculated values for each component (small/large joint counts, duration in days, normal/abnormal acute-phase reactants, negative/low/high serology classification) or by providing “raw” data (small/large joint counts, onset/assessment dates, ESR/CRP and CCP/RF laboratory values). Other measures, including EULAR response and ACR20/50/70 response, can also be calculated by providing the required information. The accompanying web application is included as part of the R package but is also externally hosted athttps://fragla.shinyapps.io/shiny-acreular. This enables researchers and clinicians without any programming skills to easily calculate these measures by uploading either a Microsoft Excel or CSV file containing their data. Furthermore, the web application allows the incorporation of additional study covariates, enabling the automatic calculation of multigroup comparative statistics and the visualisation of the data through a number of different plots, both of which can be downloaded.Figure 1.The Data tab following the upload of data. Criteria are calculated by the selecting the appropriate checkbox.Figure 2.A density plot of DAS28 scores grouped by ACR/EULAR 2010 RA classification. Statistical analysis has been performed and shows a significant difference in DAS28 score between the two groups.Conclusion:The acreular R package facilitates the easy calculation of ACR/EULAR RA related disease measures for whole patient cohorts. Calculations can be performed either from within R or by using the accompanying web application, which also enables the graphical visualisation of data and the calculation of comparative statistics. We plan to further develop the package by adding additional RA related criteria and by adding ACR/EULAR related measures for other rheumatic disorders.Disclosure of Interests:Fraser Morton: None declared, Jagtar Nijjar Shareholder of: GlaxoSmithKline plc, Consultant of: Janssen Pharmaceuticals UK, Employee of: GlaxoSmithKline plc, Paid instructor for: Janssen Pharmaceuticals UK, Speakers bureau: Janssen Pharmaceuticals UK, AbbVie, Carl Goodyear: None declared, Duncan Porter: None declared

Background Extracellular vesicles (EVs) like microvesicles (MVs) and exosomes (EXOs) are released in an evolutionary conserved manner by cells. EVs mediate intercellular communication with the transmission of molecules from their parent cell to their targets. Objectives Our objective was to investigate the effect of EVs on human in vitro osteoclastogenesis in healthy donors, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients. Methods Blood samples of healthy volunteers, RA patients and PsA patients with peripheral arthritis were collected. EVs were isolated by filtration and differential centrifugation. CD14+ cells were isolated from PBMCs by using positive selection, and the cells were stimulated with 50 ng/ml recombinant human M-CSF for 24 hrs. Then the samples were treated with 50 ng/ml recombinant human RANKL and blood-derived microvesicles or exosomes. After 7 days, the cells were fixed and stained for tartarate resistant acid phosphatase (TRAP). TRAP-positive cells with at least 3 nuclei were considered as osteoclasts and counted by using the ImageJ software. Results Healthy and RA-derived exosomes significantly (p Conclusions Our data suggest that EXOs profoundly regulate human osteoclastogenesis. PsA-derived EXOs stimulate, while healthy and RA-derived EXOs inhibit the osteoclast differentiation. Disclosure of Interest None declared

Introduction Extracellular vesicles (EV) are subcellular sized intercellular messengers, which are present in various biological fluids. EVs carry a wide variety of biomolecules and they may alter the recipient cells’ functions. The microvesicles are plasma membrane derived EVs; the exosomes, the smallest vesicles, originate from the endosome. Our objectives were to investigate the effect of EVs on the human in vitro osteoclastogenesis, and to characterise the serum EV profile of healthy donors, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients. Methods Blood samples of healthy volunteers, RA patients and PsA patients with peripheral arthritis were collected into acid citrate dextrose vacutainer tubes. EVs were isolated by filtration and centrifugation. EV samples were stained with fluorescent anti-CD3, CD14, CD15, CD19, CD42b, CD235a antibodies (BioLegend) and detected by flow cytometry. CD14 + cells were extracted from PBMCs by using positive selection method (StemCell), and the cells were stimulated with 50 ng/ml recombinant human M-CSF for 24 hrs (PeproTech). Then the samples were treated with 50–50 ng/ml recombinant human M-CSF and RANKL (PeproTech) and with or without blood derived microvesicles or exosomes. After 7 days, the cells were fixed and stained for tartrate resistant acid phosphatase (TRAP) using a commercially available kit (Sigma) . TRAP-positive cells with at least 3 nuclei were considered as osteoclasts and counted by using the ImageJ software. Results RA and PsA EV samples showed increased expression of lymphocyte markers (CD3, CD19) compared to healthy donor-derived EVs (n = 6). Microvesicles did not alter the number of mature osteoclasts. By contrast, exosomes significantly (p Conclusion Our present data suggest that the plasma profile of EVs might be altered in active arthritis compared to physiological condition, and exosomes or their cargo can regulate the human osteoclastogenesis.

Background and objectives Destabilisation of the medial meniscus (DMM) and section of the anterior cruciate ligament (ACL) are commonly used in vivo models of osteoarthritis (OA). Using explant culture, the secretory profile of synovium and patellar ligament was evaluated in DMM and ACL models. Material and methods All murine procedures were Home Office (UK) approved. OA was induced in 10–12 week old C57BL/6 male mice by either DMM or ACL (1). SHAM and naive animals were used as controls. Knee joint diameter (KJD) was assessed prior to induction of arthritis and every three days thereafter. At 4 and 12 weeks post-surgery, synovium and patellar ligament were harvested and used for explant culture. Supernatants were collected after 4 h and analysed by Luminex. In parallel experiments, joints were collected for histological analysis ofF4/80+, IL-33+ and CCR4+ cells in synovium and ligaments. Freund’s complete adjuvant (FCA) monoarthritis model was used as a positive control of inflammatory joint disease. Data were expressed as mean values ±SEM (n = 6) and analysed using a 1 or 2 way ANOVA, with p Results At 4 weeks post-surgery no change in KJD was observed in either DMM or ACL. Nonetheless, secretion of MIP-1β/CCL4 was significantly increased in explant cultures from DMM and ACL compared to naive controls (p Conclusion This study characterised the secretory profile of DMM and ACL joint tissue at 4 and 12 weeks post-surgery. This profile alters with time, with reduced IL-33 and an absence of CCL4 at 12 weeks. The potential role of IL-33 and CCL4 in the early and late stage pathogenesis of these OA models merits further investigation. Reference Glasson SS, et al. The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthritis Cartilage. 2007;15(9):1061–1069

Background and objectives GM-CSF is a pivotal growth factor/cytokine that can drive aspects of RAimmunopathogenesis. In particular, GM-CSF can activate monocytes and induce their differentiation towards alternative phenotypes. The synovial environment also contains additional factors that can influence monocyte activation such as damage associated molecular patterns (DAMPs); many of which signal via TLR4. However, the impact of GM-CSF on monocytes in a synovial setting is not well understood and therefore we aimed to investigate the response of monocytes to GM-CSF in the context of synovial fluid (SF) co-stimulation. Materials and methods Monocytes isolated from human blood or murine bone marrow were cultured in the presence or absence of GM-CSF (1ng/ml) and co-stimulated with SF (2.5%/5%) from RA or OA patients. Supernatants were harvested after 24 h and chemokine and cytokine secretion was analysed by ELISA and/or MSD. Immune complexes were made by incubating human IgG and Staphylococcal Protein A at a ratio of 4:2 respectively. Results Monocytes from healthy individuals and RA patients, secreted substantial amounts of CCL17 upon stimulation with GM-CSF. In healthy monocytes, GM-CSF driven induction of CCL17 was significantly inhibited (P Conclusions GM-CSF induced production of CCL17 in monocytes is inhibited by RA-SF. The underlying mechanism is not known, however, our work shows that it is TLR-independent and downstream of STAT5 phosphorylation. Immune complexes also inhibit GM-CSF induction of CCL17 suggesting these may contribute to the phenomenon.

Background and objectives HLA-B27 expression is associated with spondyloarthropathy (SpA) and transgenic rats expressing human HLA-B27 and b2-microglobulin (B27 rats) display systemic inflammation and bone loss, which resembles human SpA. We have previously shown that these rats lack a dendritic cell population. However, the myeloid compartment has not been fully characterised. Here, we aimed to characterise monocyte and pre-osteoclast subsets in the bone marrow and blood of B27 rats, and examined the potential of different monocyte/pre-osteoclast subsets to generate mature osteoclasts. Materials and methods 14–16 week old control (B7) and B27 rats were bled and plasma CCL2 levels were measured by ELISA. Monocytes subsets were analysed and quantified in both the bone marrow and blood of B27, B7 and non-transgenic animals, by evaluation of surface markers (CD172a, CD43, and CD11b) and intracellular uptake of fluorescent M-CSF and CCL2 by flow cytometry. Monocyte populations were FACS sorted and cultured in pro-osteoclastogenic medium for 7 days to evaluate osteoclastogenic potential. Cultures were stained with tartrate-resistant acid phosphatase (TRAP) and mature osteoclasts (TRAP + and ≥3 nuclei) were quantified by light microscopy. Results A previously unidentified CD172a + CD43 lo CD11b - population of monocytes was observed in the bone marrow. These are bona fide monocytes, expressing CD115 and CCR2, as determined by uptake of fluorescent M-CSF and CCL2 respectively. Interestingly, this new monocyte population was significantly increased in B27 rats. Assessment of the osteoclastogenic potential of bone marrow monocyte subsets revealed that CD172a + CD43 lo CD11b - , but not CD172a + CD43 lo CD11b + monocytes,can differentiate into mature osteoclasts. Furthermore, although CD172a + CD43 lo CD11b - have osteoclastogenic potential, optimal osteoclastogenesis was observed only when all CD43 lo (CD172a + CD43 lo CD11b - and CD172a + CD43 lo CD11b + ) were present. No differences in osteoclastogenesis were observed between B27 and controls rats. Finally, evaluation of circulating monocytes demonstrated that all blood monocytes express CD11b and that the CD43 lo CD11b + population was increased in B27 rats. This corresponded with an increase in CCL2 plasma levels in the B27 rats. Conclusions We have identified a previously unreported CD11b - monocyte population in the bone marrow of rats, which can differentiate into mature osteoclasts. Along with the numbers of total CD43 lo monocytes, this population is significantly increased in the bone marrow and blood of rats over-expressing HLA-B27. The increase in these populations in the B27 rats may contribute to enhanced inflammation and bone loss.