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Background: To evaluate the clinical and laboratory factors associated with long-term responses to rituximab therapy in patients with RA. Methods: One hundred fourteen RA patients received intravenous Rituximab between 2003-2016. Prior to treatment, arthroscopy and synovial biopsy was performed on a subgroup of this cohort who had active knee arthritis. Demographic, clinical, and outcome data were collected prospectively and immunohistology was performed on synovial tissue biopsies.Results: In the overall cohort, 89% of patients were seropositive for either RF (rheumatoid factor) or ACPA (anti-citrullinated protein antibodies). At baseline, median disease duration was 13.5 years. Seventy-four percent of patients had received a csDMARD and two thirds had received a bDMARD before rituximab. Rituximab monotherapy was used in 34 patients, while 80 patients received rituximab-csDMARD combination therapy. Twenty-six of the 68 patients in remission (38%) received rituximab monotherapy and 42/68 (62%) received combination therapy with a csDMARD. Twenty-four of 39 (62%) biologic naïve patients achieved remission on treatment with rituximab. Forty-four patients underwent an arthroscopy and synovial biopsy prior to treatment. Synovial tissue lymphoid aggregates (LA) were observed in 21 subjects, of which 17 (81%) showed complete or partial remission in response to treatment with rituximab. The presence of LA was significantly associated with rituximab-induced remission in these patients (p=0.007, OR=7.286 [1.737-30.555]). Conclusion: In this real world series, patients with RA demonstrate long-term, high response rates to rituximab therapy and synovial biopsy B-cell rich lymphoid aggregates are associated with a higher rate of good response.

© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ., Background: Synovial tissue research has become widely developed in several rheumatology centres, however, large discrepancies exist in the way synovial tissue is handled and, more specifically, how data pertaining to biopsy procedure, quality check and experimental results are reported in the literature. This heterogeneity hampers the progress of research in this rapidly expanding field. In that context, under the umbrella of European Alliance of Associations for Rheumatology, we aimed at proposing points to consider (PtC) for minimal reporting requirements in synovial tissue research. Methods: Twenty-five members from 10 countries across Europe and USA met virtually to define the key areas needing evaluation and formulating the research questions to inform a systematic literature review (SLR). The results were presented during a second virtual meeting where PtC were formulated and agreed. Results: Study design, biopsy procedures, tissue handling, tissue quality control and tissue outcomes (imaging, DNA/RNA analysis and disaggregation) were identified as important aspects for the quality of synovial tissue research. The SLR interrogated four databases, retrieved 7654 abstracts and included 26 manuscripts. Three OPs and nine PtC were formulated covering the following areas: description of biopsy procedure, overarching clinical design, patient characteristics, tissue handling and processing, quality control, histopathology, transcriptomic analyses and single-cell technologies. Conclusions: These PtC provide guidance on how research involving synovial tissue should be reported to ensure a better evaluation of results by readers, reviewers and the broader scientific community. We anticipate that these PtC will enable the field to progress in a robust and transparent manner over the coming years., This work was funded by EULAR (SCI020)

ObjectivesThis study investigates pathogenic and protective polyfunctional T-cell responses in patient with rheumatoid arthritis (RA), individuals at risk (IAR) and healthy control (HC) synovial-tissue biopsies and identifies the presence of a novel population of pathogenic polyfunctional T-cells that are enriched in the RA joint prior to the development of clinical inflammation.MethodsPathway enrichment analysis of previously obtained RNAseq data of synovial biopsies from RA (n=118), IAR (n=20) and HC (n=44) was performed. Single-cell synovial tissue suspensions from RA (n=10), IAR (n=7) and HC (n=7) and paired peripheral blood mononuclear cells (PBMC) were stimulated in vitro and polyfunctional synovial T-cell subsets examined by flow cytometric analysis, simplified presentation of incredibly complex evaluations (SPICE) and FlowSom clustering. Flow-imaging was utilised to confirm specific T-cell cluster identification. Fluorescent lifetime imaging microscopy (FLIM) was used to visualise metabolic status of sorted T-cell populations.ResultsIncreased plasticity of Tfh cells and CD4 T-cell polyfunctionality with enriched memory Treg cell responses was demonstrated in RA patient synovial tissue. Synovial-tissue RNAseq analysis reveals that enrichment in T-cell activation and differentiation pathways pre-dates the onset of RA. Switch from potentially protective IL-4 and granulocyte macrophage colony stimulating factor (GMCSF) dominated polyfunctional CD4 T-cell responses towards pathogenic polyfunctionality is evident in patient with IAR and RA synovial tissue. Cluster analysis reveals the accumulation of highly polyfunctional CD4+ CD8dim T-cells in IAR and RA but not HC synovial tissue. CD4+ CD8dim T-cells show increased utilisation of oxidative phosphorylation, a characteristic of metabolically primed memory T-cells. Frequency of synovial CD4+ CD8dim T-cells correlates with RA disease activity.ConclusionSwitch from potentially protective to pathogenic T-cell polyfunctionality pre-dates the onset of clinical inflammation and constitutes an opportunity for therapeutic intervention in RA.

BackgroundMicroRNAs (miRNAs) are small non-coding RNAs which have been implicated as potential biomarkers or therapeutic targets in autoimmune diseases. This study examines circulatory miRNAs in RA patients and further investigates if a serum miRNA signature precedes clinical manifestations of disease in arthralgia or “at-risk individuals”.MethodsSerum was collected from HC subjects (N = 20), RA patients (N = 50), and arthralgia subjects (N = 10), in addition to a subgroup of the RA patients post-methotrexate (MTX) (N = 18). The FirePlex miRNA Immunology-V2 panel was selected for multiplex analysis of 68 miRNAs in each sample. DNA intelligent analysis (DIANA)-mirPath and Ingenuity Pathway Analysis (IPA) software were used to predict pathways targeted by the dysregulated miRNAs.Results8 miRNA (miR-126-3p, let-7d-5p, miR-431-3p, miR-221-3p, miR-24-3p, miR-130a-3p, miR-339-5p, let-7i-5p) were significantly elevated in RA serum compared to HC (all p < 0.01) and 1 miRNA (miR-17-5p) was significantly lower in RA (p < 0.01). High specificity and sensitivity were determined by receiver operating characteristic (ROC) curve analysis. Both miR-339-5p and let-7i-5p were significantly reduced post-MTX (both p < 0.01). MiR-126-3p, let-7d-5p, miR-431-3p, miR-221-3p, miR-24-3p, miR-130a-3p were also significantly elevated in subjects “at risk” of developing RA (all p < 0.05) compared to HC. IPA analysis of this miRNA signature identified downstream targets including key transcription factors NF-κB, STAT-1, STAT-3, cytokines IL-1β, TNF-α, and matrix-metalloproteases all importantly associated with RA pathogenesis.ConclusionThis study identified six miRNAs that are altered in both RA and “at-risk individuals,” which potentially regulate key downstream pathways involved in regulating inflammation. These may have potential as predictive signature for disease onset and early progression.

Background Rituximab is a monoclonal chimeric antibody targeting the mature B cell molecule CD20, expressed on the cell surface. It is licenced and widely used in the treatment of autoimmune diseases, including the ANCA associated vasculitides and rheumatoid arthritis. The efficacy and safety of rituximab in rare diseases requires ongoing appraisal. Methods We interrogated the records of 62 consecutive patients treated with rituximab for small vessel systemic vasculitis at our unit over the last 3 years, using a contemporaneously maintained central database, as well as individual electronic patient records. Results The patient demographic data, and main results are presented in Table 1. 31 (50%) of the patients treated with rituximab were female, and the indication for treatment in most cases was ANCA associated vasculitis (77.4%). After a mean follow up period of 2.04 years, the mean reduction in BVAS was 1.64 (3.83). 5 severe adverse events were recorded, defined as death or hospitalisation potentially related to treatment with rituximab. 3 patients died of respiratory sepsis, 1 experienced a wound infection, and 1 experienced an ischaemic colon. Four patients were noted to have low immunoglobulins. Conclusion Rituximab is an effective treatment for many patients with systemic, small vessel vasculitis. Understanding the safety profile of this drug in the context of our cohort is challenging, given the significant morbidity associated with the underlying disease, as well as the age profile and concomitant immunosuppressive medications. Continued surveillance regarding efficacy and safety of rituximab in the small vessel systemic vasculitides remains invaluable. Disclosures C. Orr None. A. Verdiyeva None. N. Ahmad None. H. Ariff None. R. Luqmani None.

Objective To examine the effects of tofacitinib on metabolic activity, mitochondrial function, and proinflammatory mechanisms in rheumatoid arthritis (RA). Methods Ex vivo RA synovial explants and primary RA synovial fibroblasts (RASFs) were cultured with 1 μM tofacitinib. RASF bioenergetics were assessed using an XF24 analyzer, and key metabolic genes were assessed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Mitochondrial function was assessed using specific cell fluorescent probes and by mitochondrial gene arrays. Mitochondrial mutagenesis was quantified using a mitochondrial random mutation capture assay, and lipid peroxidation was quantified by enzyme-linked immunosorbent assay (ELISA). The effect of tofacitinib on spontaneous release of proinflammatory mediators from RA whole tissue synovial explants was quantified by ELISAs/MSD multiplex assays, and metabolic markers were quantified by RT-PCR. Finally, RASF invasion, matrix degradation, and synovial outgrowths were assessed by transwell invasion/Matrigel outgrowth assays and ELISA. Results Tofacitinib significantly decreased mitochondrial membrane potential, mitochondrial mass, and reactive oxygen species production by RASFs and differentially regulated key mitochondrial genes. Tofacitinib significantly increased oxidative phosphorylation, ATP production, and the maximal respiratory capacity and the respiratory reserve in RASFs, an effect paralleled by a decrease in glycolysis and the genes for the key glycolytic enzymes hexokinase 2 (HK2), glycogen synthase kinase 3α (GSK-3α), lactate dehydrogenase A, and hypoxia-inducible factor 1α. Tofacitinib inhibited the effect of oncostatin M (OSM) on interleukin-6 (IL-6) and monocyte chemotactic protein 1 and reversed the effects of OSM on RASF cellular metabolism. Using RA whole tissue synovial explants, we found that tofacitinib inhibited the key metabolic genes for glucose transporter 1, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, 3'-phosphoinositide-dependent protein kinase 1, HK2, and GSK-3α, the proinflammatory mediators IL-6, IL-8, IL-1β, intercellular adhesion molecule 1, vascular endothelial growth factor, and TIE-2, and RASF outgrowth from synovial explants, RASF invasion, and matrix metalloproteinase 1 activity. Conclusion This study demonstrates that JAK/STAT signaling mediates the complex interplay between inflammation and cellular metabolism in RA pathogenesis.

Objective Serum anti–citrullinated peptide antibodies (ACPAs) may be present before the development of rheumatoid arthritis (RA) and may be predictive of more severe, erosive disease. This study was undertaken to examine the synovial tissue immunophenotype according to ACPA status in patients with RA, as well as the response to treatment and erosion status. Methods Consecutive RA patients were prospectively recruited and underwent clinical and serologic assessments before and after treatment. Radiologic assessment was performed at the time of clinical follow-up. Synovial tissue was immunostained for specific markers of B cells (CD19), T cells (CD3, CD4, and CD8), macrophages (CD68), and blood vessels (factor VIII). Serum CXCL13 levels were quantified by enzyme-linked immunosorbent assay. Synovial tissue sections were analyzed for immunophenotype according to ACPA status, using a validated semiquantitative scoring method, and also analyzed for the presence of lymphoid aggregates. Response to treatment with nonbiologic or biologic disease-modifying antirheumatic drugs was assessed using the European League Against Rheumatism (EULAR) response criteria. Results In total, 123 subjects (78 ACPA+) were included. Compared to ACPA– RA patients, synovium from ACPA+ RA patients was characterized by significantly higher levels of CD19+ B cells and CD3+ and CD8+ T cells (each P < 0.05), and CD19+ B cell levels were significantly higher in patients who were naive to treatment. The CD19+ B cell infiltrate level was higher in patients with erosions at follow-up (P = 0.0128). Levels of lymphoid aggregates of CD19+ B cells were significantly higher in ACPA+ patients (P < 0.05), and this was associated with increased serum CXCL13 levels. The EULAR response was significantly associated with the level of CD3+ T cell infiltrates (P < 0.05), while CD68+ macrophage and CD8+ T cell levels were predictive of the response to tumor necrosis factor inhibitors (P < 0.05). Conclusion The results of this prospective study demonstrate that the levels of synovial B cell infiltrates and lymphoid aggregates were significantly higher in ACPA+ RA patients, especially those who were naive to treatment. In addition, ACPA+ subjects developed more erosions during progression of the disease and had higher serum levels of CXCL13. The EULAR response to therapy in ACPA+ RA patients was associated with increased levels of T cell and macrophage markers.

Objective To examine the prevalence of α1-antitrypsin deficiency (AATD) in rheumatoid arthritis (RA), and to determine whether AATD is associated with higher levels of rheumatoid factor (RF), antinuclear antibodies (ANAs), and anti–citrullinated peptide autoantibodies (ACPAs). Methods RF, ANAs, and ACPAs were measured by standard immunoturbidimetry, immunofluorescence assay, and enzyme-linked immunosorbent assay, respectively. Characterization of AAT phenotypes was performed by isoelectric focusing and immunofixation. The chi-square test with Yates' correction and the Mann-Whitney U test were used to assess the prevalence of alleles associated with AATD in RA and to compare mean antibody titers, respectively. Results Of 246 patients with RA, 24 who were heterozygous for AATD were identified, with no statistically significant difference in the prevalence of AATD between RA patients and the general population (P = 0.39). A positive association between heterozygosity for AATD and the production of ACPAs was observed (P

The inflammatory CD40–CD40L pathway is implicated in various autoimmune diseases, but the activity status of this pathway in various stages of rheumatoid arthritis (RA) progression is unknown. In this study, we used gene signatures of CD40L stimulation derived from human immature dendritic cells and naive B cells to assess the expression of CD40-downstream genes in synovial tissues from anti-citrullinated protein Ab–positive arthralgia, undifferentiated arthritis (UA), early RA, and established RA cohorts in comparison with healthy donors. Interestingly, the expression of CD40LG and active full-length CD40 was increased in the disease tissues, whereas that of a dominant-negative CD40 isoform was decreased. Gene set variation analysis revealed that CD40L-responsive genes in immature dendritic cells and naive B cells were significantly enriched in synovial tissues from UA, early RA, and established RA patients. Additionally, CD40L-induced naive B cell genes were also significantly enriched in synovial tissues from arthralgia patients. In our efforts to characterize downstream mediators of CD40L signaling, we have identified GPR120 and KDM6B as novel components of the pathway. In conclusion, our data suggest that therapeutic CD40–CD40L blocking agents may prove efficacious not only in early and established RA, but also in inhibiting the progression of the disease from arthralgia or UA to RA.

Background: The aim of this global collaboration was to develop a consensual set of items for the analysis of synovial biopsies in clinical practice and translational research through the EULAR Synovitis Study Group (ESSG) and OMERACT Synovial Tissue Biopsy Group. Methods: Participants were consulted through a modified Delphi method. Three sequential rounds occurred over 12 months. Members were sent a written questionnaire containing items divided into two parts. Items were identified and formulated based on a scoping review. The first part of the questionnaire referred to synovial biopsies in clinical practice including five subsections, and the second part to translational research with six subsections. Every participant was asked to score each item on a 5-point Likert scale. Items with a median score above 3.5 and a ≥ 70% agreement were selected for the next round. The last round was conducted orally at EULAR in June 2017. Results: Twenty-seven participants from 19 centers were contacted by email. Twenty participants from 17 centers answered. Response rates for next rounds were 100%. For the first part relating to clinical practice, 20/44 items (45.5%) were selected. For the second part relating to translational research, 18/43 items (41.9%) were selected for the final set. Conclusions: We herein propose a consensual set of analysis items to be used for synovial biopsies conducted in clinical practice and translational research.

Despite their common use in treating psoriatic arthritis, there is little evidence supporting the use of conventional disease modifying agents such as methotrexate. Although treatment with inhibitors of TNF-α has brought significant benefit to certain patients with PsA, many do not respond. TNF-α inhibitors have also demonstrably failed to prevent new bone formation, a critical aspect to the changes in PsA that ultimately leads to joint destruction and disability. The identification of several new targets in PsA, and the advent of recently approved compounds inhibiting these targets, heralds a new dawn for PsA. The differential relevance of targets in rheumatoid arthritis and PsA underlines the need for a paradigm shift in how we name, describe and categorize rheumatic diseases.

Introduction: Identifying and quantifying inflammatory disease activity in rheumatoid arthritis remains a challenge. Many studies have suggested that a large proportion of patients may have active inflammation, but normal inflammatory markers. Although various disease activity scores have been validated, most rely to a large degree on biomarkers such as CRP and ESR. In this study, we examine the utility and limitations of these biomarkers, as well as the DAS28-CRP in appraising disease activity in RA.\ud \ud Methods: Two hundred and twenty three consecutive rheumatoid arthritis reporting knee arthralgia underwent synovial sampling of the affected knee via needle arthroscopy. The synovium was examined by microscopy with H+E staining as well as immunohistochemistry, and related to the ESR, CRP and DAS28-CRP on blood samples taken immediately before arthroscopy.\ud \ud Results: Although a statistically significant positive correlation was observed between CRP and the level of inflammation in the biopsy retrieved (n = 197, rho = 0.43, CI 0.30–0.54, p < 0.0001), there was histological evidence of inflammation in the synovium in 49.4% of the patients who had a normal CRP. A positive correlation was also observed between ESR and the level of inflammation in the biopsy retrieved (n = 188, rho = 0.29, CI 0.15–0.42 p < 0.0001). A statistically significant but weak positive correlation was observed between the DAS28-CRP and synovial inflammation (n = 189, rho = 0.23, CI 0.09–0.37, p = 0.0011). Only the CD19 infiltrate in the synovium correlated with serum CRP (n = 70, rho = 0.32, CI 0.08–0.52, p = 0.0068).\ud \ud Conclusion: CRP has a moderately strong relationship with disease activity, but there are significant pitfalls in the use of this biomarker in RA, and therefore a need interpret CRP results judiciously. The results of this study underline the heterogeneity of RA, and the need to develop improved panels of biomarkers, to better stratify RA, and to identify the cohort for whom inflammatory activity cannot be measured accurately with CRP.

Background Synovial biopsies are increasingly performed in both clinical setting and translational research.1 An unmet need in the field is the standardisation of synovial biopsies handling and analysis procedures. Objectives The aim of this collaborative work was to create a consensual set of points to consider for handling and analysis of synovial biopsies in clinical practice and translational research through EULAR Synovitis Study Group (ESSG). Methods The items were identified and formulated based on a comprehensive literature review.1 A task force (TF) of EULAR Synovitis Study Group (ESSG) members were constituted and TF members were consulted through a 2-stage eDelphi process. the 2 sequential rounds occurred in 9 months. The first written round occurred in June 2016. Members were sent a written questionnaire containing items divided in 2 parts. The items were identified and formulated based on a comprehensive literature review. The first part of the questionnaire referred to clinical practice containing 5 subsections: biopsy sampling, biopsy handling, histological analysis, staining and immunohistochemistry (IHC), biopsy analysis and pathologist’s report. The second part referred to translational research and contained 6 subsections (same 5 plus RNA analysis). Every participant was asked to score each item with a 5 points Likert (0: strongly disagree, 5: strongly agree), comments were allowed for each item. Items with a median score above 3.5 on 5 and a percentage of agreement above 70% were for the next round. Items with lower score were either suppressed of modified according to participants’ comments. Anonymized detailed results were circulating through participants between each round. The face to face meeting occurred at ESSG meeting in June 2017. Results 27 ESSG members from 19 centres were contacted by email. 20 participants from 17 centres answered (response rate of 74%). Response rates for next rounds were 100%. First questionnaire contained 44 items for Part 1 Clinical practice. 52.3% (23items/44) were selected for the second round based on their score and agreement percentage. 83% (19items/23) were selected for the third round. First questionnaire contained 43 items for the second part about translational research. 44% (19 items/43) were selected for second round based on their score and agreement percentage. 95% (18 items/19) were selected for third round (figure 1). Third oral round allowed to obtain a final set of items unanimously (table 1). Conclusions We hereby propose a set of consensual points to consider on analysis of synovial biopsies in clinical practice and translational research. This standardisation initiative was conducted through ESSG members using a validated consensus method. Reference [1] Orr C, Sousa E, Boyle DL, Buch MH, Buckley CD, Canete JD, et al. Synovial tissue research: a state-of-the-art review. Nat Rev Rheumatol2017;13(8):463–75. Disclosure of Interest None declared

Objectives: Ultrasonography (US) is a fast, available and low-cost imaging tool used for detecting knee synovitis. Our aims were to assess the relationship between US and histology findings in appraising levels of inflammation and vascularity in the knee joint in subjects with inflammatory arthropathies; to determine whether differences exist in the appraisal between varying knee compartments and to compare US performances compared with gold standard histology for knee synovitis detection.\ud \ud Methods: Subjects with actively inflamed knee joint having crystal arthropathies, rheumatoid arthritis, psoriatic arthritis or ostoearthritis were prospectively recruited from rheumatology clinics after giving their written consent between May and October 2015. Study was approved by the institutional ethics committee. The knee was divided into three compartments (medial, lateral, superior). Patients had a knee US followed by a knee arthroscopy with biopsies retrieval from each compartment. Biopsies were blindly scored for lining layer hyperplasia, inflammation, vascularity, CD68 and factor VIII staining. Correlation was determined using the Spearman’s correlation test.\ud \ud Results: 26 patients with active arthritis in a knee joint and recent onset of disease were prospectively included. Strong correlations were observed between US synovitis grade and histological inflammation score (r=0.63; P=0.002), US Doppler grade and histological score for vascularity (r=0.68; P

Background In this study, we examined the effect of oxidative stress on cellular energy metabolism and pro-angiogenic/pro-inflammatory mechanisms of primary rheumatoid arthritis synovial fibroblast cells (RASFC) and human umbilical vein endothelial cells (HUVEC). Methods Primary RASFC and HUVEC were cultured with the oxidative stress inducer 4-hydroxy-2-nonenal (4-HNE), and extracellular acidification rate, oxygen consumption rate, mitochondrial function and pro-angiogenic/pro-inflammatory mechanisms were assessed using the Seahorse analyser, complex I–V activity assays, random mutation mitochondrial capture assays, enzyme-linked immunosorbent assays and functional assays, including angiogenic tube formation, migration and invasion. Expression of angiogenic growth factors in synovial tissue (ST) was assessed by IHC in patients with rheumatoid arthritis (RA) undergoing arthroscopy before and after administration of tumour necrosis factor inhibitors (TNFi). Results In RASFC and HUVEC, 4-HNE-induced oxidative stress reprogrammed energy metabolism by inhibiting mitochondrial basal, maximal and adenosine triphosphate-linked respiration and reserve capacity, coupled with the reduced enzymatic activity of oxidative phosphorylation complexes III and IV. In contrast, 4-HNE elevated basal glycolysis, glycolytic capacity and glycolytic reserve, paralleled by an increase in mitochondrial DNA mutations and reactive oxygen species. 4-HNE activated pro-angiogenic responses of RASFC, which subsequently altered HUVEC invasion and migration, angiogenic tube formation and the release of pro-angiogenic mediators. In vivo markers of angiogenesis (vascular endothelial growth factor, angiopoietin 2 [Ang2], tyrosine kinase receptor [Tie2]) were significantly associated with oxidative damage and oxygen metabolism in the inflamed synovium. Significant reduction in ST vascularity and Ang2/Tie2 expression was demonstrated in patients with RA before and after administration of TNFi. Conclusions Oxidative stress promotes metabolism in favour of glycolysis, an effect that may contribute to acceleration of inflammatory mechanisms and subsequent dysfunctional angiogenesis in RA. Electronic supplementary material The online version of this article (10.1186/s13075-018-1592-1) contains supplementary material, which is available to authorized users.

Background Psoriatic arthritis (PsA) is a chronic inflammatory disease, characterised by synovitis and destruction of articular cartilage/bone. Janus-kinase and signal transducer and activator of transcription (JAK-STAT) signalling pathway is implicated in the pathogenesis of PsA. Objectives To examine the effect of tofacitinib (JAK inhibitor) on proinflammatory mechanisms in PsA. Methods Primary PsA synovial fibroblasts (PsAFLS) and ex vivo PsA synovial explants were cultured with tofacitinib (1 µM). PhosphoSTAT3 (pSTAT3), phosphoSTAT1 (pSTAT1), suppressor of cytokine signaling-3 (SOCS3), protein inhibitor of activated Stat3 (PIAS3) and nuclear factor kappa B cells (NFκBp65) were quantified by western blot. The effect of tofacitinib on PsAFLS migration, invasion, Matrigel network formation and matrix metallopeptidase (MMP)2/9 was quantified by invasion/migration assays and zymography. Interleukin (IL)-6, IL-8, IFN-gamma-inducible protein 10 (IP-10) monocyte chemoattractant protein (MCP)-1, IL-17, IL-10, MMP3 and tissue inhibitor of metalloproteinases 3 (TIMP3) were assessed by ELISA. Results Tofacitinib significantly decreased pSTAT3, pSTAT1, NFκBp65 and induced SOCS3 and PIAS3 expression in PsAFLS and synovial explant cultures (p

In autoimmune diseases such as rheumatoid arthritis (RA), regulatory T cells (Tregs) fail to constrain autoimmune inflammation; however, the reasons for this are unclear. We investigated T cell regulation in the RA joint. Tregs from RA synovial fluid suppressed autologous responder T cells; however, when compared with Tregs from healthy control peripheral blood, they were significantly less suppressive. Despite their reduced suppressive activity, Tregs in the RA joint were highly proliferative and expressed FOXP3, CD39, and CTLA-4, which are markers of functional Tregs. This suggested that the reduced suppression is due to resistance of RA synovial fluid responder T cells to Treg inhibition. CD161+ Th17 lineage cells were significantly enriched in the RA joint; we therefore investigated their relative susceptibility to Treg-mediated suppression. Peripheral blood CD161+ Th cells from healthy controls were significantly more resistant to Treg-mediated suppression, when compared with CD161- Th cells, and this was mediated through a STAT3-dependant mechanism. Furthermore, depletion of CD161+ Th cells from the responder T cell population in RA synovial fluid restored Treg-mediated suppression. In addition, CD161+ Th cells exhibited pathogenic features, including polyfunctional proinflammatory cytokine production, an ability to activate synovial fibroblasts, and to survive and persist in the inflamed and hypoxic joint. Because CD161+ Th cells are known to be enriched at sites of autoinflammation, our finding that they are highly proinflammatory and resistant to Treg-mediated suppression suggests an important pathogenic role in RA and other autoimmune diseases.

We thank Drs Hutchinson and Eggleton for their interest in our report and for raising some interesting points. While we believe there is some merit in the issues they raise, we do not believe these invalidate the data we have presented or the conclusions we made in our initial report. This article is protected by copyright. All rights reserved.

Copyright © 2017, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., The synovium is the major target tissue of inflammatory arthritides such as rheumatoid arthritis. The study of synovial tissue has advanced considerably throughout the past few decades from arthroplasty and blind needle biopsy to the use of arthroscopic and ultrasonographic technologies that enable easier visualization and improve the reliability of synovial biopsies. Rapid progress has been made in using synovial tissue to study disease pathogenesis, to stratify patients, to discover biomarkers and novel targets, and to validate therapies, and this progress has been facilitated by increasingly diverse and sophisticated analytical and technological approaches. In this Review, we describe these approaches, and summarize how their use in synovial tissue research has improved our understanding of rheumatoid arthritis and identified candidate biomarkers that could be used in disease diagnosis and stratification, as well as in predicting disease course and treatment response.

Septic arthritis represents 8-27% of cases of monoarthritis presenting to the emergency department. Tocilizumab is an interleukin-6 blocking monoclonal antibody with the mechanistic potential to interfere with the body's normal inflammatory response to an infectious insult. We present a case of septic arthritis with a normal white cell count, C-reactive protein, erythrocyte sedimentation rate and fibrinogen in a patient treated with tocilizumab.

Background Deep profiling of synovial tissue samples from rheumatoid arthritis (RA) patients may reveal the molecular underpinnings of phases of RA progression and provide new therapeutic targets to intervene earlier in disease pathogenesis. Objectives We sought to identify the molecular pathways expressed in different stages of disease (from seropositive subjects without clinically apparent synovitis to those with established disease) in synovial tissue compared to non-RA controls. Methods Transcriptomics profiling was performed on RNA isolated from synovial tissue biopsies. Normal synovium was collected from subjects with knee pain and without diagnosis of OA or RA (n=28). Arthralgia tissue was collected from ACPA-positive subjects without synovitis (n=10). Early RA tissue was collected from patients recently diagnosed ( 1 year of disease duration (n=95). Protein expression was confirmed on infiltrating immune cells from synovial biopsy cell suspensions by flow cytometry in separate RA subjects. Results Several pathways previously identified as important for RA pathogenesis (e.g., lymphocyte activation, osteoclast differentiation, NF-kappa B signaling) were enriched in differentially expressed genes in disease synovial biopsies compared to normal tissue samples. Interestingly, several genes known to function in T cell activation as signal 2 co-stimulatory or co-inhibitory molecules were differentially expressed, even in arthralgia and early RA subjects. 66 of 81 known co-stimulatory or co-inhibitory genes profiled were differentially expressed (FDR 2) in disease samples from at least one cohort. The genes encoding co-stimulatory proteins that were increased compared to normal included CD28, CD40LG, CD40 and ICOS. Interestingly, some of the genes encoding co-inhibitory proteins were increased (PDCD1/PD-1, CD274/PD-L1, HAVCR2/TIM3, TIGIT, BTLA), whereas others showed decreased expression (C10orf54/VISTA and LAG3) compared to normal controls. We focused on CD28 expression, which is elevated in “pre-RA” arthralgia samples, proposing that anti-CD28 therapeutics could be candidates for RA disease prevention. By flow cytometry we demonstrated that a majority of CD4+ (>90%) and CD8+ (>60%) T cells from RA synovial biopsy cell suspensions (n=4) showed surface expression of CD28. Conclusions We have generated a unique dataset from different phases of RA progression. Our results provide guidance for the selection of co-signaling molecules as therapeutic targets as well as for preventing the progression to RA. In particular, CD28 expression was elevated in synovial tissue biopsies before the development of RA and it was also expressed on the majority of synovial tissue infiltrating CD4+ and CD8+ T cells from RA patients. These observations provide rationale to target CD28 for both RA treatment and disease interception. Disclosure of Interest A. Walsh Shareholder of: Johnson & Johnson, Employee of: Johnson & Johnson, M. Canavan: None declared, Y. Guo Shareholder of: Johnson & Johnson, Employee of: Johnson & Johnson, T. McGarry: None declared, X. Yin Shareholder of: Johnson & Johnson, Employee of: Johnson & Johnson, M. Wechalekar Grant/research support from: Johnson & Johnson, M. Smith Consultant for: Johnson & Johnson, S. Proudman: None declared, C. Orr: None declared, S. Kelly: None declared, C. Pitzalis Grant/research support from: Johnson & Johnson, Consultant for: Abbott, Astellas, MedImmune, BMS, Celgene, Grunenthal, GSK, MSD, Pfizer, Sanofi, Roche, UCB, D. Veale: None declared, U. Fearon: None declared, S. Nagpal Shareholder of: Johnson & Johnson, Employee of: Johnson & Johnson

CD141+ DC are implicated in antiviral and antitumor immunity. However, mechanistic studies in autoimmune disease are limited. This is the first study to our knowledge examining CD141+ DC in autoimmune disease, specifically inflammatory arthritis (IA). We identified significant enrichment of CD141+ DC in the inflamed synovial joint, which were transcriptionally distinct from IA and healthy control (HC) blood CD141+ DC and significantly more activated, and they exhibited increased responsiveness to TLR3. Synovial CD141+ DC represent a bone fide CD141+ DC population that is distinct from CD1c+ DC. Synovial CD141+ DC induced higher levels of CD4+ and CD8+ T cell activation compared with their peripheral blood counterparts, as made evident by expression of IFN-γ, TNF-α, and granulocyte-macrophage CSF (GMCSF). Autologous synovial CD141+ DC cocultures also induce higher levels of these cytokines, further highlighting their contribution to synovial inflammation. Synovial CD141+ DC-T cell interactions had the ability to further activate synovial fibroblasts, inducing adhesive and invasive pathogenic mechanisms. Furthermore, we identify a mechanism in which synovial CD141+ DC are activated, via ligation of the hypoxia-inducible immune-amplification receptor TREM-1, which increased synovial CD141+ DC activation, migratory capacity, and proinflammatory cytokines. Thus, synovial CD141+ DC display unique mechanistic and transcriptomic signatures, which are distinguishable from blood CD141+ DC and can contribute to synovial joint inflammation.

Synovial immunopathology in rheumatoid arthritis is complex involving both resident and infiltrating cells. The synovial tissue undergoes significant neovascularization, facilitating an influx of lymphocytes and monocytes that transform a typically acellular loose areolar membrane into an invasive tumour-like pannus. The microvasculature proliferates to form straight regularly-branching vessels; however, they are highly dysfunctional resulting in reduced oxygen supply and a hypoxic microenvironment. Autoantibodies such as rheumatoid factor and anti-citrullinated protein antibodies are found at an early stage, often before arthritis has developed, and they have been implicated in the pathogenesis of RA. Abnormal cellular metabolism and mitochondrial dysfunction thus ensue and, in turn, through the increased production of reactive oxygen species actively induce inflammation. Key pro-inflammatory cytokines, chemokines and growth factors and their signalling pathways, including nuclear factor κB, Janus kinase-signal transducer, are highly activated when immune cells are exposed to hypoxia in the inflamed rheumatoid joint show adaptive survival reactions by activating. This review attempts to highlight those aberrations in the innate and adaptive immune systems including the role of genetic and environmental factors, autoantibodies, cellular alterations, signalling pathways and metabolism that are implicated in the pathogenesis of RA and may therefore provide an opportunity for therapeutic intervention.

Background Dendritic cell (DC) are a heterogeneous group of antigen presenting cells that can be subdivided into CD1c and CD141+DC. CD141+DC were first discovered in 2010 and little is known about their function in health or disease. No previous studies on CD141+DC in inflammatory arthritis (IA) have been performed therefore our aim was to examine if these newly described DC play a pathogenic role in IA. Methods CD141+DC were purified from SFMC/PBMC, stimulated and stained with a panel of DC specific activation markers. CD141+DC were cocultured with T cells for 6d basally or in the presence of TREM-1 after which intracellular cytokine production was assessed by flow cytometry. Supernatants from DC-T cell cocultures were used to treat synovial fibroblasts and the expression of adhesion molecules, cytokines and MMPs was measured. CD141+DC were also identified in synovial-tissue by immunohistology/flow cytometry for CD141/CLEC9A/TREM-1. Finally using sorted populations of CD141+DC from SFMC and PBMC, RNA sequencing was performed and differentially expressed genes and interaction network analysis were identified using the DeSeq2 R package, Ingenuity® Pathway Analysis (IPA) and InnateDB and Cytoscape. Results SF-CD141+DC are significantly enriched compared to WB and express higher levels of activation markers CD80/CD86 and CD40. SF-CD141+DC induced both CD8+ and CD4+T cell proliferation, granzyme B production from CD8+ and TNFα, IFNγ and GMCSF from CD4+T cells. Supernatants from CD141+DC activated T cells subsequently induced synovial fibroblast expression of ICAM-1, IL-6, IL-8, MMP1 and MMP3. SF-CD141+DC expressed significantly higher levels of the the hypoxia marker TREM-1, activation of which further induces expression of CD80, CD86 and CD40. Coculture of these TREM-1 activated CD141+DC with CD3+ T cells increased IFNγ and IL-17a production. Finally RNASeq analysis revealed that there are 2089 differentially expressed genes between SF-CD141+DC and PBMC CD141+DC, with induction of a number of key network pathways identified in SF CD141+ DC, including energy metabolism, chemokine and cytokine signalling. Conclusion CD141+ DC are enriched in the IA joint in an active state. RNASeq analysis revealed they are distinct from blood CD141+DC and our in vitro data would support the hypothesis that these CD141+DC contribute to synovial inflammation and joint destruction.

Background Rheumatoid Arthritis (RA) is a progressive autoimmune disease characterised by synovial hyperplasia resulting in destruction of adjacent articular cartilage and bone. While new therapeutic strategies over the last 15 years represent an advance in treatment, a significant proportion of patients do not respond, have sub-optimal responses or suffer adverse effects. Therefore identification of baseline markers that can predict clinical response along with new therapies will allow for better treatment stratification for RA patients. Materials and methods Peripheral blood mononuclear cells were obtained from patients with RA at baseline. Patients were followed clinically and categorised into responders (n=10) vs non responders (n=9) based on clinical disease outcome. mRNA was isolated from PBMC and RNAseq performed. Ingenuity® Pathway Analysis (IPA) software was used to examine gene enrichment within differentially expressed genes. Interaction network analysis and identification of key hub nodes was performed using InnateDB and Cytoscape. Results Initial analysis identified differential expression of 155 genes between responders vs non responders. IPA pathway and Gene Ontology analysis demonstrated these were associated with several relevant pathways eg, antigen presentation, with top GO functions related to autoimmune disease, specifically RA. Differential gene expression was associated with leukocyte trafficking and alterations in extracellular matrix components, which was further confirmed by additional IPA network analysis of the RA dataset, identifying a key Inflammatory response/cell movement network. Potential upstream target regulators of these pathways incude TREM-1, IL-17A and HIF1A, which are known to be significantly involved in regulating synovial proliferation in the RA joint. Finally an interaction network analysis identifed CXCL5, ADAMTSL4, CSTB, PPBP, CBLB, ELANE, PTGDS, CXCL2, SP1, CXCL1 as key hub genes preferentially connected to the 155 differentially expressed genes. Conclusion These data demonstrate differential gene expression and gene networks in responders vs non responder patients, specifically associated with leukocyte trafficking and synovial extravasation. However, given the small sample size, these results need to be validated in independent cohorts. Understanding differences in responder vs nonresponder patients at baseline represents an opportunity to both better understand the mechanisms of current drugs and investigate new therapeutic targets in those who don’t respond to current treatment.

During inflammation, immune cells activated by toll-like receptors (TLRs) have the ability to undergo a bioenergetic switch towards glycolysis in a manner similar to that observed in tumour cells. While TLRs have been implicated in the pathogenesis of rheumatoid arthritis (RA), their role in regulating cellular metabolism in synovial cells, however, is still unknown. In this study, we investigated the effect of TLR2-activation on mitochondrial function and bioenergetics in primary RA-synovial fibroblast cells (RASFC), and further determined the role of glycolytic blockade on TLR2-induced inflammation in RASFC using glycolytic inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). We observed an increase in mitochondrial mutations, ROS and lipid peroxidation, paralleled by a decrease in the mitochondrial membrane potential in TLR2-stimulated RASFC. This was mirrored by differential regulation of key mitochondrial genes, coupled with alteration in mitochondrial morphology. TLR2-activation also regulated changes in the bioenergetic profile of RASFC, inducing PKM2 nuclear translocation, decreased mitochondrial respiration and ATP synthesis and increased glycolysis:respiration ratio, suggesting a metabolic switch. Finally, using 3PO, we demonstrated that glycolytic blockade reversed TLR2-induced pro-inflammatory mechanisms including invasion, migration, cytokine/chemokine secretion and signalling pathways. These findings support the concept of complex interplay between innate immunity, oxidative damage and oxygen metabolism in RA pathogenesis.

To examine the prevalence of αRF, ANAs, and ACPAs were measured by standard immunoturbidimetry, immunofluorescence assay, and enzyme-linked immunosorbent assay, respectively. Characterization of AAT phenotypes was performed by isoelectric focusing and immunofixation. The chi-square test with Yates' correction and the Mann-Whitney U test were used to assess the prevalence of alleles associated with AATD in RA and to compare mean antibody titers, respectively.Of 246 patients with RA, 24 who were heterozygous for AATD were identified, with no statistically significant difference in the prevalence of AATD between RA patients and the general population (P = 0.39). A positive association between heterozygosity for AATD and the production of ACPAs was observed (P 0.0001), with increased ACPA titers recorded in the AATD RA cohort compared with the general population (P = 0.01).AAT heterozygous status in RA is strongly associated with positive ACPAs and may define a distinct subset of patients with increased disease severity.

Th17 cells are an important therapeutic target in autoimmunity. However, it is known that Th17 cells exhibit considerable plasticity, particularly at sites of autoimmune inflammation. Th17 cells can switch to become ex-Th17 cells that no longer produce IL-17 but produce IFN-γ. These ex-Th17 cells are also called nonclassical Th1 cells because of their ability to produce IFN-γ, similar to Th1 cells; however, it is unclear whether they resemble Th1 or Th17 cells in terms of their function and regulation, and whether they have a pathogenic role in autoimmunity. We compared the phenotypic and functional features of human Th17, Th1, and ex-Th17 cell populations. Our data showed that despite their loss of IL-17 expression, ex-Th17 cells were more polyfunctional in terms of cytokine production than either Th1 or bona fide Th17 cells, and produced increased amounts of proinflammatory cytokines. The proliferative brake on Th17 cells appeared to be lifted because ex-Th17 cells proliferated more than Th17 cells after stimulation. In contrast with Th1 and Th17 cells, ex-Th17 cells were highly resistant to suppression of proliferation and cytokines by regulatory T cells. Finally, we showed that ex-Th17 cells accumulated in the joints of rheumatoid arthritis patients. Taken together, these data indicate that human ex-Th17 cells are functionally distinct from Th1 and Th17 cells, and suggest that they may play a pathogenic role at sites of autoimmunity, such as the rheumatoid arthritis joint where they accumulate. These findings have implications for therapeutic strategies that target IL-17, because these may not inhibit pathogenic ex-Th17 cells.

Immune checkpoint blockade with therapeutic anti-cytotoxic T lymphocyte-associated antigen (CTLA)-4 (Ipilimumab) and anti-programmed death (PD)-1 (Nivolumab and Pembrolizumab) antibodies alone or in combination has shown remarkable efficacy in multiple cancer types, concomitant with immune-related adverse events, including arthralgia and inflammatory arthritis (IA) in some patients. Herein, using Nivolumab (anti-PD-1 antagonist)-responsive genes along with transcriptomics of synovial tissue from multiple stages of rheumatoid arthritis (RA) disease progression, we have interrogated the activity status of PD-1 pathway during RA development. We demonstrate that the expression of PD-1 was increased in early and established RA synovial tissue compared to normal and OA synovium, whereas that of its ligands, programmed death ligand-1 (PD-L1) and PD-L2, was increased at all the stages of RA disease progression, namely arthralgia, IA/undifferentiated arthritis, early RA and established RA. Further, we show that RA patients expressed PD-1 on a majority of synovial tissue infiltrating CD4+ and CD8+ T cells. Moreover, enrichment of Nivolumab gene signature was observed in IA and RA, indicating that the PD-1 pathway was downregulated during RA disease progression. Furthermore, serum soluble (s) PD-1 levels were increased in autoantibody positive early RA patients. Interestingly, most of the early RA synovium tissue sections showed negative PD-L1 staining by immunohistochemistry. Therefore, downregulation in PD-1 inhibitory signaling in RA could be attributed to increased serum sPD-1 and decreased synovial tissue PD-L1 levels. Taken together, these data suggest that agonistic PD1 antibody-based therapeutics may show efficacy in RA treatment and interception.

Introduction This study investigates the role of Toll-like receptor 2 (TLR2) in the regulation of migratory and invasive mechanisms in rheumatoid arthritis (RA). Methods Invasion, migration, matrix metalloproteinase (MMP)-1, -3 and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) expression, β-integrin binding, cytoskeletal rearrangement and Ras-related C3 botulinum toxin substrate 1 (Rac1) activation in response to a TLR2-ligand, Pam3CSK4 (1 μg/ml), in ex vivo RA synovial tissue explants, primary RA synovial fibroblasts (RASFC) and microvascular endothelial cells (HMVEC) were assessed by Transwell Matrigel™ invasion chambers, enzyme-linked immunosorbent assay (ELISA), multiplex adhesion binding assay, reverse transcription polymerase chain reaction (RT-PCR), F-actin immunofluorescent staining, matrigel synovial outgrowths, Rac1 pull-down assays/Western blot and zymography. β1-integrin expression in RA/control synovial tissue was assessed by immunohistology. The effect of Pam3CSK4 on cell migration, invasion, MMP-3 and Rac1 activation was examined in the presence or absence of anti-β1-integrin (10 μg/ml) or anti-IgG control (10 μg/ml). The effect of an anti-TLR-2 mAb (OPN301)(1 μg/ml) or immunoglobulin G (IgG) control (1 μg/ml) on RASFC migration and RA synovial tissue MMP activity was assessed by wound assays, ELISA and zymography. Results Pam3CSK4 significantly induced cell migration, invasion, MMP-1, MMP-3, MMP-2 and MMP-9 expression and induced the MMP-1/TIMP-3 and MMP-3/TIMP-3 ratio in RASFC and explants (p

Nature Reviews Rheumatology 13, 463–475 (2017) In the original version of this article the name of one of the authors, Elsa Vieira-Sousa, was incorrectly given as Elsa Sousa. This error has now been corrected in the PDF and HTML versions of the article.

The armoury of biological agents available to treat rheumatoid arthritis (RA) has expanded greatly over the last 15 years, but treatment options for psoriatic arthritis (PsA) have failed to keep pace. Therapy with disease modifying anti-rheumatic drugs (DMARDs), such as methotrexate and leflunomide, remain first-line agents despite a paucity of randomised controlled trial evidence.1 There is a good evidence base for the efficacy of TNFα inhibition (TNFi), both as monotherapy and in combination with methotrexate, from a number of studies.2–5 The use of TNFi has the additional benefit of efficacy in treating spinal symptoms where conventional DMARDs show little efficacy.6 Five TNFi agents are now approved for use in PsA patients in Europe, and all show equivalent efficacy in the treatment of joint disease.1 ,7–9 PsA is the second most common inflammatory arthropathy and represents a considerable disease burden for patients and the healthcare system. Therefore, the relative paucity of alternative agents in the treatment of PsA compared with RA represents a significant challenge. Moreover, PsA is not a benign disease, as evidenced by radiological progression and disability.10 In one study of PsA patients attending an early arthritis clinic, treatment with conventional DMARDs for 2 years achieved good disease control, however as many as 50% of PsA patients developed significant bone erosions in that time.11 Our understanding of the pathogenesis of PsA has significantly advanced in recent years, and this has led to the identification of various targets of the inflammatory process observed in PsA.12 It has been clear for some time that in psoriasis (PsO) and PsA, there is a complex interaction of environmental and genetic factors that must lead to a sustained and persistent inappropriate inflammatory response. The relationship between skin and joint disease has been the focus of a …

Background Psoriatic Arthritis (PsA) is a chronic immune-mediated inflammatory disease, characterised by proliferation of synovial tissue and destruction of articular cartilage/bone with associated psoriasis. Dysregulated angiogenesis is one of the key early pathogenic processes in PsA. Monocyte subpopulations are increasingly recognized contributors to angiogenesis. Circulating monocytes can differentiate into tissue resident macrophages, monocyte derived dendritic cells and osteoclasts, which orchestrate both pro-inflammatory and pro-angiogenic signalling. These processes may be governed by microRNA (miRNA), a class of evolutionary conserved short non-coding RNAs which function as post-transcriptional repressors of gene expression. On such miRNA is miR-125a-5p, which in cancer, has been previously associated with altered angiogenesis, invasion and migration. Objectives To examine the expression and angiogenic associations of miR-125a-5p in PsA. Methods Synovial tissue biopsies were obtained from patients with PsA and osteoarthritis (OA). In parallel, CD14+ monocytes and CD3+ T cells were isolated from peripheral blood mononuclear cells. MiR-125a-5p levels were analysed by real-time PCR. To examine possible factors involved in regulating miR-125a-5p expression in endothelial cells,microvascular endothelial cells (HMVEC) were cultured with candidate pro-inflammatory stimuli including; TLR ligands (PAM, PolyIC, LPS), pro-inflammatory cytokines (TNFa, IL-1b, IL-17) and growth factors (VEGF, Ang2). Matrigel tube formation assays were performed with pre- and anti- 125a treated HMVEC to elucidate angiogenic function. Immunoistochemical analysis of synovial vasculature was performed using factor VIII (DAKO).Cliical markers assertained at the time of arthroscopy, includding synovitis and vascularity were correlated with synovial miRNA expression. Results Expression of miR-125a-5p was significantly decreased in PsA synovial biopsies (n=8) compared to OA (n=4) (p Conclusions Our data demonstrates decreased expression of miR-125a in the joint and peripheral CD14+ monocytes in PsA patients. MiR-125a expression was associated with joint vascularity, a hallmark of PsA, suggesting its important role in mediating key pro-angiogenic and thereby pro-inflammatory mechanisms in the synovium. Correcting these microRNA deficiencies, either by conventional pharmacological agents or as novel targets, may provide a therapeutic benefit, especially in early disease stages. Disclosure of Interest None declared

Background Arthroscopy, by allowing direct visualization of the synovial membrane, is the gold standard for the assessment of knee synovitis. Given the fact that it is an invasive procedure, other reliable ways to assess synovitis has developed over the past few years. Ultrasonography (US) is a fast, available and low cost imaging tool and has been validated for detecting knee synovitis (1). Ultrasonographic and arthroscopic findings have been shown to correlate (2). However, few studies have yet assessed the correlation between ultrasonographic and histological findings. Objectives The aim of this study was to assess the correlation between ultrasonographic scores, arthroscopic scores and histological scores for inflammation and vascularity in knee joint synovitis. Methods Patients with knee pain and/or effusion were prospectively included. Knee was divided in three compartments (medial, lateral and superior). 1 operator (AN) performed a knee ultrasonography (B mode and Doppler mode) prior to the arthroscopy with a semi-quantitative assessment for degree of synovitis. 1 operator then performed a knee arthroscopy (CO, DJV) and scored macroscopic aspects with a 100-mm visual analogic scale (VAS) blindly from US findings and retrieved biopsies from each compartment. Biopsies were analyzed by 1 operator (UF) blindly from both US and arthroscopic findings and scored for: lining layer hyperplasia, inflammation and vascularity. Statistical analysis was made with non parametric Spearman correlation test. Results 23 patients were included. 16 had rheumatoid arthritis (RA) (ACR/EULAR 2010 criteria). 3 patients had psoriatic arthritis (PsA) (CASPAR criteria), 3 had osteoarthritis (OA) and 1 had gout. Mean ultrasonographic, arthroscopic and histological scores for both synovitis and vascularity were calculated for each patient. A strong correlation was observed between: US synovitis grade and histological inflammation score (r=0.71; p=0.001), US Doppler grade and histological score for vascularity (r=0.6; p=0,0003); US measured synovial thickness and lining layer hyperplasia (r=0.61; p=0,002). Moreover, high positive correlations were observed for both histological lining layer hyperplasia and inflammation between lateral and medial compartments (r=0,90; p=0,00005 and r=0,66; p=0,014 respectively), lateral and superior compartments (r=0,75; p=0,001 and r=0,51; p=0,048, respectively) and medial et superior compartments (r=0,61; p=0,012 and r=0,62; p=0,010, respectively). Conclusions B mode and Power Doppler US findings strongly correlate with histological inflammation and vascularity scores in actively inflamed knee joints. Ultrasound is therefore a reliable tool for knee joint assessment of the four most common rheumatological diagnoses - RA, PsA, OA and gout -. Moreover, the histological inflammation and hyperplasia scores highly correlate between the superior, medial and lateral compartments of the knee, highlighting the homogeneity of synovial membrane features within the same joint. References Karim Z, Wakefield RJ, Quinn M, Conaghan PG, Brown AK, Veale DJ et al. Validation and reproducibility of ultrasonography in the detection of synovitis in the knee: a comparison with arthroscopy and clinical examination. Arthritis Rheum. 2004;50(2):387–94. Rubaltelli JL, Fiocco U, Cozzi L, Baldovin M, Rigon C, Bortoletto P et al. Ultrasound Med. 1994;13(11):855–62. Disclosure of Interest None declared

Background and aims Psoriatic Arthritis (PsA) is a chronic immune-mediated inflammatory disease, characterised by proliferation of synovial tissue and destruction of articular cartilage/bone with associated psoriasis. Dysregulated angiogenesis is one of the key early pathogenic processes in PsA. These processes may be governed by microRNA (miRNA), a class of evolutionary conserved short non-coding RNAs which function as post-transcriptional repressors of gene expression. On such miRNA is miR-125a-5p which, in cancer, has been previously associated with altered angiogenesis, invasion and migration. Objectives To examine the expression and angiogenic associations of miR-125a-5p in PsA. Methods Synovial tissue biopsies and peripheral blood mononuclear cells were obtained from patients with PsA, osteoarthritis (OA) and/or healthy controls (HC). MiR-125a-5p levels were analysed by real-time PCR using RNU48 as an endogenous control. To examine possible factors involved in regulating miR125a-5p expression, primary synovial fibroblasts (SFC) and microvascular endothelial cells (HMVEC) microvascular endothelial cells (HMVEC) were cultured with candidate pro-inflammatory stimuli including; TLR ligands (PAM, PolyIC, LPS), pro-inflammatory cytokines (TNFa, IL-1b, IL-17) and growth factors (VEGF, Ang2). Immunoistochemical analysis was performed using factor VIII (DAKO) and appropriate IgG controls, on cryostat synovial tissue sections. Clinical markers, including synovitis, vascularity, ESR, CRP, DAS28, ascertained at the time of arthroscopy were correlated with synovial miRNA expression. Results Expression of miR-125a-5p was significantly decreased in PsA synovial biopsies (n = 8) compared to OA (n = 4) (p Conclusion Decreased expression of miR-125a-5p in the joint and it’s association with vascularity, which is a hallmark of PsA, suggests it may be important in mediating key pro-angiogenic/pro-inflammatory mechanisms in the synovium. Correcting these microRNA deficiencies, either by conventional pharmacological agents or as novel targets, may provide a therapeutic benefit, especially in early disease stages.

Background and objectives Rheumatoid Arthritis (RA) is a chronic inflammatory autoimmune disease which leads to progressive joint destruction. RA is characterised by synovial hyperplasia and invasion of the synovium into the adjacent bone and cartilage. The precise aetiology of RA is not known, but both genetic and environmental influences play a role. Methotrexate (MTX) is the most commonly used disease modifying anti-rheumatic drug in the management of RA, however its mechanism of action remains poorly understood. In this study, we aim to identify the functional effects of MTX on primary RA synovial fibroblasts (RASFC) and to determine if the epigenetic signature of RASFC/CD14 + monocytes can predict clinical response to MTX. Materials and methods Primary RASFC were isolated from synovial biopsies obtained from patients undergoing arthroscopic examination. RASFC were cultured in the presence or absence of MTX (10–100 μM). Cell migration, invasion, viability/proliferation, adhesion and pro-inflammatory cytokine expression were assessed by: wound repairs assays, transwell matrigel TM invasion chambers, MTT/ Crystal violet cell growth assay, xCELLigence Real Time Cell Analyser and ELISA respectively. In parallel, CD14 + monocytes were isolated from peripheral blood mononuclear cells from RA patients both pre- and 12 weeks post-MTX therapy and global methylation was assessed. Results MTX (10 μM) inhibited RASFC repopulation of the wound margins in comparison to vehicle control where migration across the wound was clearly evident (n = 7, p = 0.03). In parallel, MTX (100 μM)significantly decreased RASFC invasion (n = 7, p = 0.01) and altered cytoskeletal dynamics through inhibition of lamellopodia and filopodia formation, which are indicative of cell movement. MTX had no inhibitory effect on the expression of IL-6, IL-8 and RANTES. Importantly, we determined that the effect of MTX on RASFC migration and invasion were independent of both cell viability (p = 0.37) and proliferation (p = 0.18). Finally preliminary results demonstrated altered DNA methylation in CD14 + monocytes from RA patients (n = 11) at 3 months post MTX treatment. Conclusions MTX inhibits RASFC migration, invasion and cytoskeletal re-arrangement, mechanisms which are critically involved in the pathogenesis of RA. Further preliminary data suggests that the effect of MTX on pro-inflammatory mechanisms in RA, maybe be mediated through alterations in epigenome.

Background and purpose Psoriatic Arthritis (PsA) is amultifaceted disease associated with Psoriasis. Key pathogeneic mechanisms seen at all disease sites include angiogenesis, invasion, migration, proliferation and more specifically to joint involvement, osteoclastogenesis. These aberrant processes have been previously associated with dysregulated miRNA expression, a class of small non-coding RNAs which exert their function through suppression of specific target genes. More specifically, the miR-23a Cluster (miR-23a, miR-27a and miR-24–2) as in other chronic conditions, such as cancer. Methods Synovial tissue biopsies and/or peripheral blood mononuclear cells (PBMC) were obtained from PsA (n = 8), osteoarthritis (OA) (n = 7), and healthy controls (n = 8). To determine factors involved in regulating miRNA expression, primary PsA synovial fibroblasts (SFC) were isolated and cultured with candidate pro-inflammatory stimuli including TLR ligands: Pam3CSK4 (1 µg/ml), LPS (1 µg/ml), polyIC (10 µg/ml) and pro-inflammatory cytokines: IL-1β (1 ng/ml), TNFα (10 ng/ml) and IL-17 (20ng/ml). MiR-23a, miR-27a and miR-24–2 expression was quantified by RT-PCR using RNU48 as an endogenous control. Clinical demographics such as synovitis, vascularity, DAS-28 CRP, TJC, SJC and patient global were also assessed. Results All members of the miR-23a cluster, miR-23a, miR-27a and miR-24–2, were significantly decreased PsA synovial tissue versus OA (p = 0.0172). Synovial miR-23a expression negatively correlated with matched PBMC miR-23a (r=-1.00, p = 0.0167), demonstrating a dissociation in miR-23a expression between systemic and local inflammation. In addition, miR-23a expression in PsA synovial biopsies inversely correlated with DAS28-CRP (r= -0.5294, p = 0.035) and distinguished patient synovial vascularity and synovitis into high versus low groupings (all via PolyIC (TLR3) and LPS (TLR4), but not Pam3CSK4 (TLR2), significantly decreased miR-23a expression in PsA SFC (all p in silico analysis identified putative targets for miR-23a, including PDE4B and PTK2B, which are known mediators in immune pathways, osteoclast function and angiogenic mechanisms. Conclusion Our data provides evidence that the miR-23a cluster is significantly decreased in PsAsynovium. Synovial miR-23a levels, regulated by TLRs, correlate and distinguish clinical markers. Both previously confirmed and new potential targets identified here may have putative pathogenic implications for PsA pathogenesis. Thereby, the miR-23a cluster may represent a potential novel biomarker for disease activity or elucidate new therapeutic strategies.

Introduction Dendritic cells (DC) are a heterogeneous population of professional antigen presenting cells. Currently their classification within blood and skin has been well characterised however their identification within other tissues, in particular in the context of autoimmunity is limited. This work aims to identify DC within the inflamed synovium and elucidate how DC are altered by the microenvironment within the joint. Methods DC immunophenotyping was assessed in whole blood (WB), synovial fluid (SF) and synovial tissue (ST) using 9 colour flow cytometry. CD141 + DC were isolated from SF, cultured overnight +/- TLR3 ligands and stained with a panel of antibodies. Monocyte derived DC were cultured in the presence of explant conditioned media (ECM) or SF. MoDC treated with SF were also cocultured with CD4 + T cells for 5 days, after which T cell proliferation and intracellular cytokines were examined. Results RA patients have a significant decrease (p + HLADR + myeloid cells are increased in the ST compared to WB, express higher levels of CD80/CD40 and express CD64, CD14 and CD16. These cells may represent a myeloid subset of cells unique to inflammatory tissues. DC in SF are more mature than PB DC (increased CD80/CD40; p + CLEC9A + DC compared to PB (p + DC express TREM1 while blood CD141 + DC do not. SF CD141 + DC express higher levels of CD80/CD86 and CD40 compared to blood CD141 + DC. To examine the effect of the synovial microenvironment on DC, MoDC were cultured in the presence of ECM or SF. Both induced a significant increase in the expression of CD80 (p + T cells have enhanced IFNγ production and T cell proliferation. Conclusion Unique DC subsets are found in both ST and SF within the joint. These cells display a more activated phenotype than DC in circulation and the inflammatory nature of the joint contributes to this activation. These uniquely activated DC induce T cell proliferation and cytokine production.

Background Rheumatoid Arthritis (RA) is a common, chronic immune-mediated inflammatory disease, characterised by synovitis and progressive destruction of articular cartilage and bone. Janus Kinase and Signal Transducer and Activator of Transcription (JAK/STAT), a critical signalling pathway involved in inflammatory mechanisms, has been implicated in the pathogenesis of RA. Objectives In this study we examine the effect of Tofacitinib (JAK inhibitor CP-690,550) on pro-inflammatory pathways in RA using in vitro and ex-vivo culture models. Methods Primary RA synovial fibroblasts (RASFC) and whole tissue ex vivo RA synovial explant cultures were established from RA biopsies obtained at arthroscopy. Phospho-STAT3 (p-STAT3), phospho-STAT1 (p-STAT1), Suppressor of Cytokine Signaling 3 (SOCS3) and Protein Inhibitor of Activated Stat 3 (PIAS3) expression were quantified by Western Blot in RASFC following culture in the presence of IL-17, OSM and IL-6. RASFC were cultured with IL-17, OSM and IL-6 in the presence or absence of Tofacitinib (1uM) and cytokine secretion in supernatants were measured by ELISA. Ex-vivo RA synovial explants were cultured with Tofacitinib (1uM) and spontaneous secretion of IL-6, IL-8, MMP3, Tie2, PIGF and bFGF were quantified in supernatants by ELISA or MSD multiplex. Finally conditioned media from control and Tofacitinib treated RA explants were cultured with RASFC and cell invasion assessed by Transwell Matrigel™ invasion chambers. Results IL-17, OSM and IL-6 induced p-STAT3, p-STAT1 expression and inhibited SOCS3 and PIAS3 expression in RASFC. Tofacitinib decreased IL-17, OSM and IL-6 induced cytokine production from RASFC. Tofacitinib inhibited spontaneous release of IL-6, IL-8, MMP3 and soluble Tie2 receptor from RA synovial explant cultures, with no effect observed for PIGF and bFGF. Finally, conditioned media from Tofacitinib treated RA explants inhibited RASFC invasion. Conclusions This study demonstrates inverse expression of pSTATs and their negative regulators SOCS3/PIAS3 in response to pro-inflammatory mediators in RA. In addition Tofacitinib inhibits pro-inflammatory mediators and RASFC invasion, further confirming a role for JAK-STAT inhibition in the treatment of RA. Disclosure of Interest None declared

Background There is an absence of literature on the merits or problems of repeat serology testing in patients with RA. The auto-antibodies RF and anti-CCP have significance far outreaching assistance in diagnosis, and clearly have prognostic relevance, in some cases predicting response to specific therapies.1,2 A complete phenotype of a given patients9 disease is only clear when both these sets of antibodies are known. There are indications from other specialities that serology testing is often repeated in as much as 15% of tests.3 Repeated testing is often unnecessary and expensive, but it is unclear to what extent this is a problem in RA. Objectives We sought to determine how often serology is repeated in an RA cohort. In those that had serology repeated, we sought to determine how many times they had been tested, and for RF, how often the result changed. Methods We analysed how many times each patient in a cohort comprising consecutive patients attending clinic in July 2014 had been tested for each auto-antibody. Equivocal results were excluded; only clear positives and negatives were included. To determine how often patients with positive index RF were retested, we examined those separately. Results 100 patients were included. 73 (73%) had RF tested more than once. 29 (29%) had RF tested 4 times or more. 65 (65%) were positive for RF at index testing. Of these patients, 50 (78.1%) had RF tested more than once and 22 (34.4%) had RF tested 4 times or more. 1 positive RF at index measured negative but positive again on third testing. 1 negative RF at index became positive at second testing. Anti-CCP status was known for 85 patients. 21 (24.7%) were tested more than once, and 4 (4.7%) were tested 4 times or more. 59 (69.4%) were positive for anti-CCP at index testing. Of these patients, 11 (18.6%) had anti-CCP tested more than once and 1 (1.7%) were tested 4 times or more. Conclusions Inappropriate repeat testing of RF is common, and the results only rarely change. Patients who have clearly tested positive for RF or anti-CCP should not be retested routinely. This study does not change the merits of repeat testing in equivocal cases.4 There is evidence that automated alerts from the laboratory identifying repeat testing can reduce this burden on laboratory services.3 It is likely that a great deal of repeat testing has been ordered by primary care physicians and it may therefore be worthwhile educating physicians that RF and anti-CCP have no role in assessing disease activity. References Gardette A, et al. High anti-CCP antibody titres predict good response to rituximab in patients with active rheumatoid arthritis. Joint Bone Spine. 2014;81(5):416-20. Masi AT, et al. Prospective study of the early course of rheumatoid arthritis in young adults: comparison of patients with and without rheumatoid factor positivity at entry and identification of variables correlating with outcome. Semin Arthritis Rheum. 1976;4(4):299. Nies J1, et al. Effects of automated alerts on unnecessarily repeated serology tests in a cardiovascular surgery department: a time series analysis. BMC Health Serv Res. 2010 Mar 19;10:70. doi: 10.1186/1472-6963-10-70. Wiik AS1, et al. IUIS/WHO/AF/CDC Committee for the Standardization of Autoantibodies in Rheumatic and Related Diseases. Cutting edge diagnostics in rheumatology: the role of patients, clinicians, and laboratory scientists in optimizing the use of autoimmune serology. Arthritis Rheum. 2004 Apr 15;51(2):291-8. Disclosure of Interest None declared