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ObjectivesTo determine the relationship between synovial versus skin transcriptional/histological profiles in patients with active psoriatic arthritis (PsA) and explore mechanistic links between diseased tissue pathology and clinical outcomes.MethodsTwenty-seven active PsA patients were enrolled in an observational/open-label study and underwent biopsies of synovium and paired lesional/non-lesional skin before starting anti-tumour necrosis factor (TNF) (if biologic-naïve) or ustekinumab (if anti-TNF inadequate responders). Molecular analysis of 80-inflammation-related genes and protein levels for interleukin (IL)-23p40/IL-23p19/IL-23R were assessed by real-time-PCR and immunohistochemistry, respectively.ResultsAt baseline, all patients had persistent active disease as per inclusion criteria. At primary end-point (16-weeks post-treatment), skin responses favoured ustekinumab, while joint responses favoured anti-TNF therapies. Principal component analysis revealed distinct clustering of synovial tissue gene expression away from the matched skin. While IL12B, IL23A and IL23R were homogeneously expressed in lesional skin, their expression was extremely heterogeneous in paired synovial tissues. Here, IL-23 transcriptomic/protein expression was strongly linked to patients with high-grade synovitis who, however, were not distinguishable by conventional clinimetric measures.ConclusionsPsA synovial tissue shows a heterogeneous IL-23 axis profile when compared with matched skin. Synovial molecular pathology may help to identify among clinically indistinguishable patients those with a greater probability of responding to IL-23 inhibitors.

ObjectivesTo explore the relevance of T-follicular-helper (Tfh) and pathogenic peripheral-helper T-cells (Tph) in promoting ectopic lymphoid structures (ELS) and B-cell mucosa-associated lymphoid tissue (MALT) lymphomas (MALT-L) in Sjögren’s syndrome (SS) patients.MethodsSalivary gland (SG) biopsies with matched peripheral blood were collected from four centres across the European Union. Transcriptomic (microarray and quantitative PCR) analysis, FACS T-cell immunophenotyping with intracellular cytokine detection, multicolor immune-fluorescence microscopy and in situ hybridisation were performed to characterise lesional and circulating Tfh and Tph-cells. SG-organ cultures were used to investigate functionally the blockade of T-cell costimulatory pathways on key proinflammatory cytokine production.ResultsTranscriptomic analysis in SG identified Tfh-signature, interleukin-21 (IL-21) and the inducible T-cell co-stimulator (ICOS) costimulatory pathway as the most upregulated genes in ELS+SS patients, with parotid MALT-L displaying a 400-folds increase in IL-21 mRNA. Peripheral CD4+CXC-motif chemokine receptor 5 (CXCR5)+programmed cell death protein 1 (PD1)+ICOS+ Tfh-like cells were significantly expanded in ELS+SS patients, were the main producers of IL-21, and closely correlated with circulating IgG and reduced complement C4. In the SG, lesional CD4+CD45RO+ICOS+PD1+ cells selectively infiltrated ELS+ tissues and were aberrantly expanded in parotid MALT-L. In ELS+SG and MALT-L parotids, conventional CXCR5+CD4+PD1+ICOS+Foxp3- Tfh-cells and a uniquely expanded population of CXCR5-CD4+PD1hiICOS+Foxp3- Tph-cells displayed frequent IL-21/interferon-γ double-production but poor IL-17 expression. Finally, ICOS blockade in ex vivo SG-organ cultures significantly reduced the production of IL-21 and inflammatory cytokines IL-6, IL-8 and tumour necrosis factor-α (TNF-α).ConclusionsOverall, these findings highlight Tfh and Tph-cells, IL-21 and the ICOS costimulatory pathway as key pathogenic players in SS immunopathology and exploitable therapeutic targets in SS.

ObjectivesSyntenin-1, a novel endogenous ligand, was discovered to be enriched in rheumatoid arthritis (RA) specimens compared with osteoarthritis synovial fluid and normal synovial tissue (ST). However, the cellular origin, immunoregulation and molecular mechanism of syntenin-1 are undescribed in RA.MethodsRA patient myeloid and lymphoid cells, as well as preclinical models, were used to investigate the impact of syntenin-1/syndecan-1 on the inflammatory and metabolic landscape.ResultsSyntenin-1 and syndecan-1 (SDC-1) co-localise on RA ST macrophages (MΦs) and endothelial cells. Intriguingly, blood syntenin-1 and ST SDC-1 transcriptome are linked to cyclic citrullinated peptide, erythrocyte sedimentation rate, ST thickness and bone erosion. Metabolic CD14+CD86+GLUT1+MΦs reprogrammed by syntenin-1 exhibit a wide range of proinflammatory interferon transcription factors, monokines and glycolytic factors, along with reduced oxidative intermediates that are downregulated by blockade of SDC-1, glucose uptake and/or mTOR signalling. Inversely, IL-5R and PDZ1 inhibition are ineffective on RA MΦs-reprogrammed by syntenin-1. In syntenin-1-induced arthritis, F4/80+iNOS+RAPTOR+MΦs represent glycolytic RA MΦs, by amplifying the inflammatory and glycolytic networks. Those networks are abrogated in SDC-1-/-animals, while joint prorepair monokines are unaffected and the oxidative metabolites are moderately replenished. In RA cells and/or preclinical model, syntenin-1-induced arthritogenicity is dependent on mTOR-activated MΦ remodelling and its ability to cross-regulate Th1 cells via IL-12 and IL-18 induction. Moreover, RA and joint myeloid cells exposed to Syntenin-1 are primed to transform into osteoclasts via SDC-1 ligation and RANK, CTSK and NFATc1 transcriptional upregulation.ConclusionThe syntenin-1/SDC-1 pathway plays a critical role in the inflammatory and metabolic landscape of RA through glycolytic MΦ and Th1 cell cross-regulation (graphical abstract).

ObjectivesTo develop evidence-based Points to Consider (PtC) for the use of imaging modalities to guide interventional procedures in patients with rheumatic and musculoskeletal diseases (RMDs).MethodsEuropean Alliance of Associations for Rheumatology (EULAR) standardised operating procedures were followed. A systematic literature review was conducted to retrieve data on the role of imaging modalities including ultrasound (US), fluoroscopy, MRI, CT and fusion imaging to guide interventional procedures. Based on evidence and expert opinion, the task force (25 participants consisting of physicians, healthcare professionals and patients from 11 countries) developed PtC, with consensus obtained through voting. The final level of agreement was provided anonymously.ResultsA total of three overarching principles and six specific PtC were formulated. The task force recommends preference of imaging over palpation to guide targeted interventional procedures at peripheral joints, periarticular musculoskeletal structures, nerves and the spine. While US is the favoured imaging technique for peripheral joints and nerves, the choice of the imaging method for the spine and sacroiliac joints has to be individualised according to the target, procedure, expertise, availability and radiation exposure. All imaging guided interventions should be performed by a trained specialist using appropriate operational procedures, settings and assistance by technical personnel.ConclusionThese are the first EULAR PtC to provide guidance on the role of imaging to guide interventional procedures in patients with RMDs.

Objective: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that mainly targets joints. Monocytes and macrophages are critical in RA pathogenesis and contribute to inflammatory lesions. These extremely plastic cells respond to extracellular signals which cause epigenomic changes that define their pathogenic phenotype. Here, we interrogated how DNA methylation alterations in RA monocytes are determined by extracellular signals. Methods: High-throughput DNA methylation analyses of patients with RA and controls and in vitro cytokine stimulation were used to investigate the underlying mechanisms behind DNA methylation alterations in RA as well as their relationship with clinical parameters, including RA disease activity. Results: The DNA methylomes of peripheral blood monocytes displayed significant changes and increased variability in patients with RA with respect to healthy controls. Changes in the monocyte methylome correlate with DAS28, in which high-activity patients are divergent from healthy controls in contrast to remission patients whose methylome is virtually identical to healthy controls. Indeed, the notion of a changing monocyte methylome is supported after comparing the profiles of same individuals at different stages of activity. We show how these changes are mediated by an increase in disease activity-associated cytokines, such as tumour necrosis factor alpha and interferons, as they recapitulate the DNA methylation changes observed in patients in vitro. Conclusion: We demonstrate a direct link between RA disease activity and the monocyte methylome through the action of inflammation-associated cytokines. Finally, we have obtained a DNA methylation-based mathematical formula that predicts inflammation-mediated disease activity for RA and other chronic immune-mediated inflammatory diseases., We thank CERCA Programme/Generalitat de Catalunya for institutional support. EB was funded by the Spanish Ministry of Economy and Competitiveness (MINECO; grant numbers SAF2014-55942-R and SAF2017-88086-R). JDC was funded by FIS grant (PI17/00993) from Institute of Health Carlos III (ISCIII). JDC, JM and EB are supported by RETICS network grant from ISCIII (RIER, RD16/0012/0013), FEDER 'Una manera de hacer Europa'

We would like to thank Professor Buch and colleagues for their interest and thoughtful comments regarding our manuscript,1 and we are grateful for the opportunity to provide further clarification on the clinical phenotype displayed by the rheumatoid arthritis (RA) patients with a pauci-immune pathotype analysed in our early arthritis cohort.2 We have segregated this group into anti-citrullinated peptide antibodies (ACPA) +ve and -ve subsets and analysed age distribution, disease activity score (DAS28) components, ultrasound (US) scores, joint biopsied and modified Sharp van der Heijde score (SHSS) components, as indicated by Buch and colleagues to evaluate whether significant differences in clinical parameters could be identified (table 1). …

Background:The study of synovial tissue in patients with Rheumatoid Arthritis (RA) has led to the identification of synovial patterns of immune cell infiltration and specific cellular subsets associated that have been disease activity and clinical outcomes(1–3). However, the relationship of circulating and synovial immune cell sub-sets with histopathological features and clinical outcomes remains to be defined.Objectives:To assess the relationship of peripheral blood and synovial immune cells with RA histopathology and clinical outcomes, by performing flow and digital cytometry in matched peripheral blood and synovial samples from patients with early RA.Methods:70 patients with early (1). Peripheral blood mononuclear cells (n=70) were analysed by flow cytometry. Matched synovial tissues (n=70) obtained by minimally invasive ultrasound-guided synovial biopsy underwent semi-quantitative scoring (0-4) of immune cell infiltration and classification into lympho-myeloid (LM), diffuse-myeloid (DM) and pauci-immune (PI) pathotypes, as previously described(1). 49 synovial and 36 matched peripheral blood samples underwent RNA-sequencing and were analysed by digital cytometry (Xcell) (4) and Singular Value Decomposition (SVD).Results:Circulating B cells and their subsets showed significant inverse correlations with inflammatory markers (ESR, CRP), disease activity (swollen joints, four components and two components(5) DAS28) and ultrasound scores (Fig 1A). Among T cell subsets, CXCR5-PD1hiICOS+CD4+ T cells (T peripheral helper cells, Tph) had strong positive correlations with inflammatory markers (ESR and CRP), disease activity (DAS28) and ultrasound scores (Fig 1B). Tph in the peripheral blood also correlated with immune cell infiltration in synovia (Fig 1C) and were significantly higher in patients with a LM pathotype (Fig 1D). Accordingly, circulating Tph were associated with synovial LM pathotype independently of clinical features such as DAS28, ACPA positivity, Body Mass Index (BMI) and age (AUC 0.821). By applying digital cytometry in matched synovial and peripheral blood samples, synovial B and T cells were significantly higher in patients with a LM pathotype, in line with the histological definition of the LM pathotype – rich in B and T cells. On the contrary, circulating B cells and total CD4 and CD8 T cells were significantly lower in patients with a synovial LM pathotype (Fig 1E). The Tph signature in synovia derived by Singular Value Decomposition (SVD) correlated with baseline ESR (R 0.38, pConclusion:By combining conventional flow cytometry in the peripheral blood and digital cytometry on matched synovial and peripheral blood samples, we highlight diverging associations of circulating immune cell subsets with synovial inflammation and pathotypes. Tph cells, in particular, emerge as predictors of lympho-myeloid synovial inflammation and disease progression.References:[1]F. Humby et al., Ann. Rheum. Dis. 78, 761–772 (2019), doi:10.1136/annrheumdis-2018-214539. [2]M. J. Lewis et al., Cell Rep. 28, 2455-2470.e5 (2019), doi:10.1016/j.celrep.2019.07.091.[3]D. a Rao et al., Nat. Publ. Gr. 542, 110–114 (2017), doi:10.1038/nature20810.[4]D. Aran et al., Genome Biol. 18, 220 (2017), doi:10.1186/s13059-017-1349-1.[5]E. M. A. Hensor et al., Rheumatology. 58, 1400–1409 (2019), doi:10.1093/rheumatology/kez049.Figure 1.Acknowledgements:The Pathobiology of Early Arthritis Cohort (PEAC) was supported by the MRC (grant 36661). Versus Arthritis provided funding infrastructure support (grant 20022). F. Rivellese is funded by an NIHR Transitional Research Fellowship (TRF-2018-11-ST2-002). We would like to thank the patients and the clinical and laboratory team (core team) at Queen Mary University of London.Disclosure of Interests:None declared

Background:Cartilage defects in the joints are reported in 61% of all arthroscopies1&2. The size of the cartilage repair market is estimated to be $2.195 million by 20253. Cartilage defects can evolve into osteoarthritis, in which abnormal load results in cartilage breakdown, joint pain and reduced mobility. Osteoarthritis is the leading cause of permanent disability and absenteeism and affects up to 1/3 of the people over 60yrs. In western countries osteoarthritis costs 1.5-2% of the GDP4. Joint replacement with a prosthesis restores some degree of independence but in up to 20% of patients it does not meet expectations 5 and has a limited life span. There is no pharmacological intervention that arrests or reverts the course of osteoarthritis, despite the desperate need.We previously published that agrin plays an important role in cartilage homeostasis6. The addition of agrin to chondrocytes in vivo resulted in enhanced cartilage formation, suggesting a potential role for agrin in cartilage repair.Objectives:Investigate the potential of agrin for use in cartilage repair.Methods:Critical size osteochondral defects were generated in mice and sheep and injected intraarticularly with type I collagen gel containing agrin or vehicle. Animals were monitored for 8 weeks or 6 months respectively. MicroCT, histological analysis, qPCR, linage tracking, reporter assays, chondrogenesis assay, immunohistochemistry were performed.Results:A single intraarticular administration of agrin induced regeneration of critical-size osteochondral defects in mice, restoring the tissue architecture and bone-cartilage interface. Agrin stem cells to the site of injury and, through simultaneous activation of CREB and suppression of canonical WNT signalling, induced GDF5 expression and differentiation into stable articular chondrocytes, forming stable articular cartilage. In sheep, agrin treatment resulted in regeneration of bone and cartilage, which promoted increased ambulatory activity.Conclusion:Agrin orchestrates repair morphogenesis at the joint surface by modulating multiple signalling pathways, supporting the therapeutic use of agrin for joint surface regeneration.References:[1]Curl, W. W. et al. Cartilage injuries: a review of 31,516 knee arthroscopies. Arthrosc. J. Arthrosc. Relat. Surg. Off. Publ. Arthrosc. Assoc. N. Am. Int. Arthrosc. Assoc. 13, 456–460 (1997).[2]Hjelle, K., Solheim, E., Strand, T., Muri, R. & Brittberg, M. Articular cartilage defects in 1,000 knee arthroscopies. Arthrosc. J. Arthrosc. Relat. Surg. Off. Publ. Arthrosc. Assoc. N. Am. Int. Arthrosc. Assoc. 18, 730–734 (2002).[3]Cartilage Repair Market Size, Share, Industry Analysis 2018-2025 | AMR. Allied Market Research https://www.alliedmarketresearch.com/cartilage-repair-market.[4]Hiligsmann, M. et al. Health economics in the field of osteoarthritis: an expert’s consensus paper from the European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis (ESCEO). Semin. Arthritis Rheum. 43, 303–313 (2013).[5]Dieppe, P., Lim, K. & Lohmander, S. Who should have knee joint replacement surgery for osteoarthritis? Int. J. Rheum. Dis. 14, 175–180 (2011).[6]Eldridge, S., et al. Agrin mediates chondrocyte homeostasis and requires both LRP4 and α-dystroglycan to enhance cartilage formation in vitro and in vivo. Annals of the rheumatic diseases 75 (6), 1228-1235 (2016).Acknowledgements:We thank the technical staff in the ARM Lab and Staff at the University of Aberdeen’s Animal Facility and Microscopy and Histology Facility for support. Funding: We gratefully acknowledge funding support of this work by the MRC (MR/L022893/1, MR/N010973/1,and MR/P026362/1), Versus Arthritis (19667, 21515, 20886, and 21621), Rosetrees Trust (A1205), the Medical College of St Bartholomew’s Hospital Trust, and the William Harvey Research Foundation.Disclosure of Interests:Suzanne Eldridge: None declared, Aida Barawi: None declared, Hui Wang: None declared, Anke Roelofs: None declared, Magdalena Kaneva: None declared, Zeyu Guan: None declared, Helen Lydon: None declared, Bethan Thomas: None declared, Anne-Sophie Thorup: None declared, Beatriz F Fernandez: None declared, Sara Caxaria: None declared, Danielle Strachan: None declared, Ahmed Ali: None declared, Kanatheepan Shanmuganathan: None declared, Costantino Pitzalis: None declared, James Whiteford: None declared, Fran Henson: None declared, Andrew McCaskie: None declared, Cosimo De Bari: None declared, Francesco Dell’Accio Consultant of: F.D. has received consultancy fees from Samumed and UCB.

ObjectiveELR+ CXC chemokines are heparin-binding cytokines signalling through the CXCR1 and CXCR2 receptors. ELR+ CXC chemokines have been associated with inflammatory arthritis due to their capacity to attract inflammatory cells. Here, we describe an unsuspected physiological function of these molecules in articular cartilage homeostasis.MethodsChemokine receptors and ligands were detected by immunohistochemistry, western blotting and RT-PCR. Osteoarthritis was induced in wild-type and CXCR2−/− mice by destabilisation of the medial meniscus (DMM). CXCR1/2 signalling was inhibited in vitro using blocking antibodies or siRNA. Chondrocyte phenotype was analysed using Alcian blue staining, RT-PCR and western blotting. AKT phosphorylation and SOX9 expression were upregulated using constitutively active AKT or SOX9 plasmids. Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay.ResultsCXCL6 was expressed in healthy cartilage and was retained through binding to heparan sulfate proteoglycans. CXCR2−/− mice developed more severe osteoarthritis than wild types following DMM, with increased chondrocyte apoptosis. Disruption of CXCR1/2 in human and CXCR2 signalling in mouse chondrocytes led to a decrease in extracellular matrix production, reduced expression of chondrocyte differentiation markers and increased chondrocyte apoptosis. CXCR2-dependent chondrocyte homeostasis was mediated by AKT signalling since forced expression of constitutively active AKT rescued the expression of phenotypic markers and the apoptosis induced by CXCR2 blockade.ConclusionsOur study demonstrates an important physiological role for CXCR1/2 signalling in maintaining cartilage homeostasis and suggests that the loss of ELR+ CXC chemokines during cartilage breakdown in osteoarthritis contributes to the characteristic loss of chondrocyte phenotypic stability.

The use of ultrasound (US) has been at the forefront of aiding diagnosis of inflammatory arthritis since its introduction. More recently, US imaging in patients with clinical remission appears to provide prognostic information with respect to disease flares and progression.1 2 Ultrasonographic scoring typically uses a semiquantitative scale of 0–3 for synovial hypertrophy (SH) and power Doppler (PD). This system works very well in smaller joints or those with single articulations. However, in complex joints such as the wrist with many articulations, the final grading can be difficult. Figure 1A illustrates three potential positions used to score wrist synovitis. As a consequence, there are a number of possible permutations for the wrist US score. Figure 1 (A) Illustration of wrist US probe positions and corresponding US images, used for US wrist assessment. (B) Fisher’s exact statistical testing for association between high and low SH/PD groups compared with high and low Krenn synovitis scores. The high US SH score is denoted by …

Objectives EuroQoL-5 dimension (EQ-5D) is a standardised preference-based tool for measurement of health-related quality of life and EQ-5D utility values can be converted to quality-adjusted life years (QALYs) to aid cost-utility analysis. This study aimed to evaluate the EQ-5D utility values of 639 patients with primary Sjogren9s syndrome (PSS) in the UK. Methods Prospective data collected using a standardised pro forma were compared with UK normative data. Relationships between utility values and the clinical and laboratory features of PSS were explored. Results The proportion of patients with PSS reporting any problem in mobility, self-care, usual activities, pain/discomfort and anxiety/depression were 42.2%, 16.7%, 56.6%, 80.6% and 49.4%, respectively, compared with 5.4%, 1.6%, 7.9%, 30.2% and 15.7% for the UK general population. The median EQ-5D utility value was 0.691 (IQR 0.587–0.796, range −0.239 to 1.000) with a bimodal distribution. Bivariate correlation analysis revealed significant correlations between EQ-5D utility values and many clinical features of PSS, but most strongly with pain, depression and fatigue (R values>0.5). After adjusting for age and sex differences, multiple regression analysis identified pain and depression as the two most important predictors of EQ-5D utility values, accounting for 48% of the variability. Anxiety, fatigue and body mass index were other statistically significant predictors, but they accounted for Conclusions This is the first report on the EQ-5D utility values of patients with PSS. These patients have significantly impaired utility values compared with the UK general population. EQ-5D utility values are significantly related to pain and depression scores in PSS.

Objectives The synovial endothelium targeting peptide (SyETP) CKSTHDRLC has been identified previously and was shown to preferentially localise to synovial xenografts in the human/severe combined immunodeficient (SCID) mouse chimera model of rheumatoid arthritis (RA). The objective of the current work was to generate SyETP-anti-inflammatory-cytokine fusion proteins that would deliver bioactive cytokines specifically to human synovial tissue. Methods Fusion proteins consisting of human interleukin (IL)-4 linked via a matrix metalloproteinase (MMP)-cleavable sequence to multiple copies of either SyETP or scrambled control peptide were expressed in insect cells, purified by Ni-chelate chromatography and bioactivity tested in vitro. The ability of SyETP to retain bioactive cytokine in synovial but not control skin xenografts in SCID mice was determined by in vivo imaging using nano-single-photon emission computed tomography-computed tomography (nano-SPECT-CT) and measuring signal transducer and activator of transcription 6 (STAT6) phosphorylation in synovial grafts following intravenous administration of the fusion protein. Results In vitro assays confirmed that IL-4 and the MMP-cleavable sequence were functional. IL-4-SyETP augmented production of IL-1 receptor antagonist (IL-1ra) by fibroblast-like synoviocytes (FLS) stimulated with IL-1β in a dose-dependent manner. In vivo imaging showed that IL-4-SyETP was retained in synovial but not in skin tissue grafts and the period of retention was significantly enhanced through increasing the number of SyETP copies from one to three. Finally, retention correlated with increased bioactivity of the cytokine as quantified by STAT6 phosphorylation in synovial grafts. Conclusions The present work demonstrates that SyETP specifically delivers fused IL-4 to human rheumatoid synovium transplanted into SCID mice, thus providing a proof of concept for peptide-targeted tissue-specific immunotherapy in RA. This technology is potentially applicable to other biological treatments providing enhanced potency to inflammatory sites and reducing systemic toxicity.

ObjectivesTo dissect the role of toll-like receptor (TLR) signalling and B cell survival/proliferating factors in the crosstalk between rheumatoid arthritis synovial fibroblasts (RASF) and B cells.MethodsRASF, rheumatoid arthritis dermal fibroblasts (RADF) and osteoarthritis synovial fibroblasts (OASF) were analysed for the expression of B cell survival/proliferating factors BAFF and APRIL in resting conditions and upon stimulation with TLR2/TLR3/TLR4 ligands. Unswitched IgD+ B cells were co-cultured with RASF/OASF/RADF in the presence/absence of TLR ligands and with/without BAFF/APRIL blocking antibodies. Activation-induced cytidine deaminase (AID) mRNA expression, Iγ-Cμ and Iα-Cμ circular transcripts (CTs; markers of ongoing class-switching to IgG and IgA) and IgM/A/G production were measured to assess functional activation of B cells.ResultsTLR3 and to a lesser extent TLR4, but not TLR2 stimulation, induced up to ∼1000-fold BAFF mRNA and increased soluble BAFF release. APRIL was less significantly regulated by TLR3. Resting and TLR3-stimulated RASF released higher levels of BAFF/APRIL compared with RADF. TLR3 stimulation of RASF but not RADF in co-culture with B cells strongly enhanced AID expression, Iγ-Cμ and Iα-Cμ CTs and class-switching to IgG/IgA. Blockade of BAFF/APRIL signalling completely inhibited TLR3-induced, RASF-dependent expression of AID, CTs and the secretion of IgG/IgA.ConclusionsRASF produce high levels of BAFF and APRIL constitutively and in response to TLR3 stimulation. These factors are critical in directly modulating AID expression, class-switch recombination and IgG/IgA production in IgD+ B cells. Overall, this work highlights a novel and fundamental role for the TLR3/B cell survival factor axis in sustaining B cell activation in the rheumatoid arthritis synovium.

We thank Mossel and colleagues for their interest in our work.1 2 They raise a number of limitations that we acknowledge and have discussed. In addition, we agree that an understanding of the pathology underlying the abnormal salivary gland ultrasound findings in Sjogren’s syndrome is an important objective to help establish the validity of this tool as a potential outcome measure in clinical trials. As both we and Mossel and colleagues discuss, we did not observe a difference in hypoechoic areas between rituximab and placebo. Similar hypoechoic areas can be observed in long-standing postradiotherapy salivary glands where inflammatory cell infiltrate is variable and of a differing pattern.3–5 Furthermore, the pattern of hypoechoic areas on ultrasound in Sjogren’s syndrome is reminiscent of sialography findings which are characterised by contrast-defined changes …

ObjectivesOsteoarthritis (OA) is a leading cause of disability for which there is no cure. The identification of molecules supporting cartilage homeostasis and regeneration is therefore a major pursuit in musculoskeletal medicine. Agrin is a heparan sulfate proteoglycan which, through binding to low-density lipoprotein receptor-related protein 4 (LRP4), is required for neuromuscular synapse formation. In other tissues, it connects the cytoskeleton to the basement membrane through binding to α-dystroglycan. Prompted by an unexpected expression pattern, we investigated the role and receptor usage of agrin in cartilage.MethodsAgrin expression pattern was investigated in human osteoarthritic cartilage and following destabilisation of the medial meniscus in mice. Extracellular matrix (ECM) formation and chondrocyte differentiation was studied in gain and loss of function experiments in vitro in three-dimensional cultures and gain of function in vivo, using an ectopic cartilage formation assay in nude mice. Receptor usage was investigated by disrupting LRP4 and α-dystroglycan by siRNA and blocking antibodies respectively.ResultsAgrin was detected in normal cartilage but was progressively lost in OA. In vitro, agrin knockdown resulted in reduced glycosaminoglycan content, downregulation of the cartilage transcription factor SOX9 and other cartilage-specific ECM molecules. Conversely, exogenous agrin supported cartilage differentiation in vitro and ectopic cartilage formation in vivo. In the context of cartilage differentiation, agrin used an unusual receptor repertoire requiring both LRP4 and α-dystroglycan.ConclusionsWe have discovered that agrin strongly promotes chondrocyte differentiation and cartilage formation in vivo. Our results identify agrin as a novel potent anabolic growth factor with strong therapeutic potential in cartilage regeneration.

OBJECTIVES: The aim of this 12-week Phase IIb study was to assess the efficacy and safety of olokizumab (OKZ), a humanised anti-IL6 monoclonal antibody, in patients with rheumatoid arthritis (RA) with moderate-to-severe disease activity who had previously failed tumour necrosis factor (TNF) inhibitor therapy. The dose-exposure-response relationship for OKZ was also investigated. METHODS: Patients were randomised to one of nine treatment arms receiving placebo (PBO) or OKZ (60, 120 or 240 mg) every 4 weeks (Q4W) or every 2 weeks (Q2W), or 8 mg/kg tocilizumab (TCZ) Q4W. The primary endpoint was change from baseline in DAS28(C-reactive protein, CRP) at Week 12. Secondary efficacy endpoints were American College of Rheumatology 20 (ACR20), ACR50 and ACR70 response rates at Week 12. Exploratory analyses included comparisons of OKZ efficacy with TCZ. RESULTS: Across 221 randomised patients, OKZ treatment produced significantly greater reductions in DAS28(CRP) from baseline levels at Week 12, compared to PBO (p

ObjectivesTo establish the role and effect of glucocorticoids and the endogenous annexin A1 (AnxA1) pathway in inflammatory arthritis.MethodsAnkle joint mRNA and protein expression of AnxA1 and its receptors were analysed in naive and arthritic mice by real-time PCR and immunohistochemistry. Inflammatory arthritis was induced with the K/BxN arthritogenic serum in AnxA1+/+ and AnxA1−/− mice; in some experiments, animals were treated with dexamethasone (Dex) or with human recombinant AnxA1 or a protease-resistant mutant (termed SuperAnxA1). Readouts were arthritic score, disease incidence, paw oedema and histopathology, together with pro-inflammatory gene expression.ResultsAll elements of the AnxA1 pathway could be detected in naive joints, with augmentation during ongoing disease, due to the infiltration of immune cells. No difference in arthritis intensity of profile could be observed between AnxA1+/+ and AnxA1−/− mice. Treatment of mice with Dex (10 µg intraperitoneally daily from day 2) afforded potent antiarthritic effects highly attenuated in the knockouts: macroscopic changes were mirrored by histopathological findings and pro-inflammatory gene (eg, Nos2) expression. Presence of proteinase 3 mRNA in the arthritic joints led the authors to test AnxA1 and the mutant SuperAnxA1 (1 µg intraperitoneally daily in both cases from day 2), with the latter one being able to accelerate the resolving phase of the disease.ConclusionAnxA1 is an endogenous determinant for the therapeutic efficacy of Dex in inflammatory arthritis. Such an effect can be partially mimicked by application of SuperAnxA1 which may represent the starting point for novel antiarthritic therapeutic strategies.

IL-18 is a pleiotropic immunoregulatory cytokine that has been described and implicated in the pathogenesis of a variety of inflammatory diseases.1–4 Studies in murine models of arthritis and clinical studies suggest that dendritic cells, macrophages and synoviocites within the synovial membrane can produce IL-18.1 5–7 IL-18 expression has in turn been implicated in the reciprocal regulation of other pro-inflammatory cytokines, such as tumour necrosis factor alpha.8 Recent data clearly demonstrated that IL-18 serum levels were significantly elevated in adult-onset Still’s disease (AOSD) and correlated with disease activity and serum ferritin levels.2–4 AOSD is characterised by substantial and dysregulated …

This is the peer reviewed version of the following article: Next-Generation Sequencing Demonstrates Dynamic Recirculation of B Cell Clones in Ectopic Lymphoid Structures of Sjogren's Syndrome, which has been published in final form at 10.1136/annrheumdis-2015-eular.2047. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.

Background and Objectives Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated proteins. Moreover, RA patients are characterised by increased neutrophils in their synovial fluid, in particular at early stage of the disease. Recent evidence suggests a critical role of neutrophils in sustaining the inflammatory response in the RA joint. Around 40% of patients display synovial tertiary lymphoid structures (TLS) with functional B cell follicles supporting a germinal-centre response and local autoantibody production. However, the nature of the main (auto)antigenic reactivity of synovial B cells is unknown. Here we characterised the autoreactive B cell response of lesional B cells isolated from TLS + RA synovium. Materials and Methods Single CD19 + B cells were FACS sorted from synovial cell suspension of 4 TLS + RA patients. RNA was used to amplify Ig VH and VL genes and PCR products were cloned and expressed as recombinant monoclonal antibodies displaying identical specificity of the original B cells. Recombinant antibodies were then tested 1) to determine the frequency of polyreactive clones and 2) to define their immunoreactivity towards native and citrullinated antigens using a synovial antigen microarray platform. Results We obtained 139 individual VH sequences of which 33% were IgM, 40% IgG, 27% IgA and 175 VL sequences and we generated a total of 66 complete (H + L chains) recombinant monoclonal antibodies. Analysis of the VH gene somatic mutation rate showed evidence of antigen selection and intra-synovial clonal diversification. No skewed distribution of the VH and VL gene usage was observed. Around 30% of synovial monoclonal antibodies were reactive towards citrullinated histones in the antigen microarray, in particular citH2A and citH2B. This reactivity was confirmed by citH2A and citH2B ELISA. Moreover, when the synovial monoclonal antibodies were tested on neutrophil extracellular traps (NETs), which contained citrullinated histones, reactivity towards NETs proteins but not neutrophil nuclear antigens was observed. Conclusions Here we provided novel evidence that highly mutated, locally differentiated B cells within RA synovial germinal centre-like structures display a strong immunoreactive bias towards citrullinated histones which likely derived from neutrophil NETs that continuously form in the RA synovial fluid. This suggests that citrullinated histones are the main antigens driving in situ B cell activation and differentiation sustaining the humoral autoimmune response within the RA joints.

Background and Objectives Despite the obvious success of current biological agents for treatment of rheumatoid arthritis (RA), achievement of broader efficacy and improved safety profile remains an unmet need in RA therapy. There is significant evidence demonstrating molecular heterogeneity within the vascular endothelium of different tissues, conferring organ tropism to migrating lymphocytes subsets by interaction with specific homing receptors. We have previously reported the isolation of a single-chain Fv (scFvA7) antibody with specificity for synovial arthritic microvasculature, following an in vivo phage display selection on SCID mice model of RA. The aim of the present study was to develop a tissue specific therapeutic for Rheumatoid Arthritis. Materials and Methods The scFvA7 was subcloned in fusion with the CH2-CH3 domain of human IgG (A7 scFv-Fc) and coupled with the anti-TNF antibody Adalimumab via a monocistronic RNA approach, to form a bispecifc antibody (BsAb) using the Knobs-into-Holes technology (A7/Adalimumab). The reactivity in frozen and paraffin embedded tissue sections was investigated using immunohistochemistry and immunofluorescence analysis. In-vitro functionality and biological activity was assessed in Biacore, TNF-ELISA and TNF cytotoxicity assay on L-929 cell line. Results The scFv-Fc fusion protein of Adalimumab showed analogous anti-TNF properties with the parent antibody, EC50 0.04nM (Adalimumab 0.025nM), confirming the validity of the scFv format. The BsAb A7/Adalimumab achieved a high degree of efficient heterodimerisation showing only 3% Adalimumab homodimers and was able to selectively bind TNFα in vitro with similar efficacy to the TNF blocker, EC50 0.06nM, with a KD of 31pM. Despite the monovalency for the anti-TNF activity, the BsAb showed a dose dependent rescue of TNF induced cytotoxicity in L-929 cell line with a 0.5nM IC50 (Adalimumab 0.17nM), demonstrating the biological functionality of the construct. In addition, the A7/Adalimumab antibody proved to efficiently and specifically target the stromal compartment of RA synovial microvasculature, maintaining unaltered the organ tropism of the original scFvA7. Conclusions Our results demonstrate the development of a bispecific antibody showing unaltered TNF blocking capacity and optimal synovial specificity, suggesting that the target molecule for A7 may have applications as a biomarker and immunotherapeutic target, potentially allowing reduction in the dosage and/or administration frequency, with the ultimate goal to reduce the systemic exposure and achieve a better therapeutic index and decreasing health care costs.

Tertiary lymphoid structures (TLSs) are common features of chronic inflammatory diseases including Sjogren's syndrome (SS). We recently showed that these ectopic structures acquire secondary lymphoid organs properties and are capable of supporting B cell activation and autoantibody production including expression of activation-induced cytidine deaminase (AID) and Ig class switching. Dissecting TLSs dynamics in humans is technically and ethically challenging. Thus, we used the NOD mouse, a spontaneous model of autoimmune sialoadenitis, to characterise the cellular and molecular basis of autoreactive B cell activation and …

Objectives Rheumatoid arthritis (RA) is associated with an increased Epstein-Barr virus (EBV) blood DNA load, a robust immune response to EBV and cross-reactive circulating antibodies for viral and self-antigens. However, the role of EBV in RA pathogenesis remains elusive. Here we investigated the relationship between synovial EBV infection, ectopic lymphoid structures (ELS) and immunity to citrullinated self and EBV proteins. Methods Latent and lytic EBV infection was investigated in 43 RA synovial tissues characterised for presence/absence of ELS and 11 OA samples by RT-PCR, in situ hybridisation and immunohistochemistry/immunofluorescence. Synovial production of anti-citrullinated proteins (ACPA) and anti-citrullinated EBV peptides (VCP1/VCP2) antibodies was investigated in situ or in vivo in the SCID/RA chimeric model. Results EBV dysregulation was observed exclusively in ELS+ RA, but not OA, synovia as revealed by presence of EBV latent [LMP2A, EBV-encoded small RNA (EBER)] transcripts and EBER+ cells and immunoreactivity for EBV latent (LMP1, LMP2A) and lytic (BFRF1) antigens in ELS-associated B cells and plasma cells, respectively. Importantly, ~20% of synovial plasma cells producing ACPA were infected with EBV. Furthermore, ELS-containing RA synovia transplanted into SCID mice supported production of ACPA and anti-VCP1/VCP2 antibodies cross-recognised by ACPA. Analysis of CD4+ and CD8+ T-cell localisation and granzyme B expression suggests that EBV persistence in ELS-containing synovia is favoured by exclusion of CD8+ T cells from B-cell follicles and impaired CD8-mediated cytotoxicity. Conclusions We demonstrated active EBV infection within ELS in the RA synovium that appears to contribute to local growth and differentiation of ACPA-reactive B cells.

Background and Objectives Sjogren’s syndrome (SS) is an autoimmune disease characterised by high affinity circulating autoantibodies and peripheral B cell disturbances with predominance of naive and reduction of memory B cells. The stage at which errors in B cell tolerance checkpoints accumulate in SS is unknown. Here we determined the frequency of self- and poly-reactive B cells in the circulating naive compartment of SS patients. Materials and Methods Single CD27-IgD+ B cells were sorted by FACS from peripheral blood of SS patients and healthy donors (HD). RNA was used to amplify Ig VH and VL genes and PCR products were cloned and expressed as recombinant monoclonal antibodies displaying identical specificity of the original B cells. Recombinant antibodies were tested towards different antigens to determine the frequency of autoreactive and polyreactive clones. Results 66 recombinant antibodies were generated from naive B cells of 4 SS patients and compared to 45 clones from 2 HD. Analysis of the VH and VL gene usage showed no significant differences between SS and HD. Conversely, we observed accumulation of circulating autoreactive naive B cells in SS as demonstrated by increased reactivity towards Hep2 cells (43.1% SS versus 25% HD) and ENA (19.6% SS clones versus none). Among ENA+ clones, 6 displayed reactivity towards Ro/SSA and/or La/SSB. Conclusions Here using an efficient strategy to express recombinant monoclonal antibodies from single B cells we demonstrated an elevated frequency of autoreactive naive B cells in the circulation of SS patients supporting the existence of early defects in B cell tolerance checkpoints in SS.

OBJECTIVE--To investigate the role of cytokines and cell adhesion molecules in the pathogenesis of Sjogren's syndrome (SS). METHODS--Using an indirect immunoperoxidase technique we assessed the expression of the cytokines interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-8 (IL-8), transforming growth factor beta (TGF beta) and granulocyte macrophage colony stimulating factor (GM-CSF), of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), lymphocyte function associated antigen-1 (LFA-1), the activated molecular form of LFA-1 (NKI-L16), CD2, and LFA-3, and of a panel of cellular markers in the minor salivary glands. RESULTS--In SS and chronic sialoadenitis (CS), the ductal epithelial cells and acini expressed all the cytokines examined. The percentage of glandular mononuclear cells which stained positive for cytokines did not differ significantly between SS and CS. NKI-L16 was detected on 33.6 (SD 10.1)% and 15.3 (4.3)% of LFA-1 cells in SS and CS, respectively (p < 0.002). CONCLUSION--SS and CS did not differ in the pattern of cytokines examined. The characteristic cell clustering seen in the salivary glands in SS may be caused by the upregulation of NKI-L16.

Backgroundand objectives Follicular dendritic cells (FDCs) are characteristic components of organised tertiary lymphoid structures (TLS) containing germinal centres (GCs) in chronically inflamed tissues. However, the role of FDCs in initiation, maintenance and regulation of autoreactive GCs and auto-antibody (Ab) production has not been analysed. The authors hypothesised that auto-immune complex (IC) retention on FDCs critically regulates auto-Ab production and that FDC-IC unloading would significantly inhibit auto-Ab levels. Materials and methods To test our hypothesis, purified human FDCs, B cells, T cells, complement, fibrinogen (Fib) and anti-Fib from high anti-citrullinated peptide protein Ab (ACPA) sera were used to set up controlled in vitro autoreactive GC reactions. The reactions were differentially reconstituted by the cellular and molecular components of GCs. The immunoglobulin G (IgG) specific Endoglycosidase S (EndoS) was used to inhibit Fib-IC retention on FDCs and Fib-specific auto-Ab production. Moreover, the ability of IC retention on FDCs to break B cell tolerance and induce auto-Ab production in tolerant animal models was verified. Results Our in vitro studies indicated that: (1) In the presence of Fib-IC loaded FDCs alone, significant amounts of Fib-specific IgM are produced; (2) In the presence of FDCs alone or T cells alone, Fib-specific IgG is insignificant; (3) The co-stimulatory effect of both FDCs and T cells is required for induction of significantly high levels of Fib-specific IgG; (4) EndoS significantly inhibited Fib-specific IgG production in FDC-T cell-assisted cultures via mechanisms involving interference with FDC-IC retention, inhibition of complement activation, and, possibly, IgG B cell receptor deglycosylation. In vivo, FDC-ICs induced autoreactive GCs and auto-Ab production in well-characterised animal models tolerant to hen egg lysozyme (HEL) as well as targets of therapeutic importance including TNF-α, IgE, and the extracellular domain two of the neu antigen (ECDII). However, safe termination of these induced auto-Ab responses was challenged by the non specific activity of EndoS as indicated by its inhibitory effect on protective immune responses to the foreign antigen ovalbumin (OVA). To avoid the non-selective activity of EndoS, the authors designed bait and pray ‘Split-EndoS conditional re-functionalisation’ system that selectively deglycosylates target Abs (pray) engaging their antigens (baits) on the EndoS molecules. The system was successfully tested as indicated by the ability of biotin-baited EndoS to selectively deglycosylate anti-biotin IgG. Conclusion This is the first report analysing the critical role of FDC-ICs in auto-Ab production in in vitro autoreactive germinal centre reactions, with potential therapeutic targeting of clinically important antigens and safe termination using EndoS.

A pivotal role for tertiary lymphoid structures (TLS) in promoting antigen-specific humoral responses during chronic inflammation is emerging in several autoimmune conditions, including rheumatoid arthritis, Sjogren9s syndrome and autoimmune thyroiditis. However, there is limited evidence on the cellular and molecular mechanisms underlying TLS formation and their contribution to autoimmunity in the pancreas during autoimmune insulitis. Here the authors performed a detailed and comprehensive assessment of the evolution of TLS during autoimmune insulitis in 126 female non-obese diabetic (NOD) mice from 4 to 38 weeks of age. The authors demonstrated that during progression from peri-insulitis to intrainsulitis in early diabetic mice, T and B cell infiltration follows a highly regulated process with the formation of lymphoid aggregates characterised by T/B cell segregation, follicular dendritic cells networks and differentiation of germinal centre B cells. This process is preceded by local upregulation of lymphotoxins (Lt)α/β and lymphoid chemokines (CKs) CXCL13 and CCL19 and is associated with infiltration of B220/IgD+/CD23/CD21 follicular B cells expressing CXCR5. Despite a similar incidence of insulitis, late diabetic mice displayed a significantly reduced incidence of fully organised TLS and reduced levels of Lt/CKs. Upon development, TLS were fully functional in supporting in situ autoreactive B cell differentiation, as demonstrated by the expression of activation-induced cytidine deaminase, the enzyme required for Ig affinity maturation and class-switching, and the presence of CD138 plasma cells displaying anti-insulin reactivity. Overall, this work provides direct evidence that TLS are of critical relevance in promoting autoimmunity and chronic inflammation during autoimmune insulitis and diabetes in NOD mice.

Background and objectives Development of functional B cell niches in peripheral tissues has been associated to chronicity and pathologic severity in different human conditions such as kidney allograft rejection and multiple sclerosis. Ex vivo studies in the rheumatoid arthritis (RA)-severe combined immunodeficiency mouse chimera model advocate a role for the synovial B cell compartment in enhancing local immune responses though production of auto-antibodies and by promoting local T cell activation. The aim of this study was to investigate the relationship between the degree of B cell infiltration and activation in the joint and clinico-pathologic variability in patients with RA. Methods Synovial tissues from 66 RA patients were evaluated by immunohistochemistry (IHC). 25 paired paraffin and RNA samples were used for comparative IHC and quantitative PCR. B cell infiltration was assessed semi-quantitatively (0–3) based on the size and density distribution of CD20 B cell aggregates and through quantitative evaluation of mRNA expression of activation-induced cytidine deaminase (AID), the enzyme required for immunoglobulin affinity maturation and class switching. B cell scores and AID expression levels were correlated to clinical and bio-molecular parameters of immune cell activation, disease activity and bone remodelling. Results The IHC CD20 B cell score was significantly related to the expression levels of CXCL13 (p=0.001) and lymphotoxin β (p=0.004). The degree of B cell aggregation tightly associated to progressive increase in CD3 lymphocyte and CD138 plasma cell infiltration as well as to in situ expression of AID (p=0.0006), interferon γ (p=0.02) and interleukin 2 (p=0.001). Despite signs of lymphoid cell activation, no significant variability in local and systemic markers of disease activity was found. Rather, increasing degrees of B cell aggregation and AID transcripts associated to progressive increase in the receptor activator of nuclear factor-κB ligand/osteoprotegerin (OPG) ratio primarily due to significantly reduced OPG levels (p

Background and objectives Autoimmune disorders frequently display autoreactive germinal centres (GCs) with immune complex (IC)-bearing follicular dendritic cells (FDCs) suggesting that FDCs are involved in the pathogenesis of autoimmune diseases. The authors recently described induction of T cell-independent (TI) antibody (Ab) responses to T dependent (TD) antigens (Ags) by presenting the TD Ags on FDCs. Rather than presenting peptide fragments, FDCs multimerize monomeric Ags by trapping them in ICs via FDC-FcγRIIB and then present them polyvalently with a characteristic periodic spacing between epitopes of 200–500 A. This allows extensive B cell receptor (BCR) cross-linking that induces TI B cell activation, GC formation, and Ag-specific Ig secretion in less than 48 h in the presence of FDC-derived co-stimulatory signals. These correlations prompted the hypothesis that multimerized self/neo-Ags on FDCs would break B cell tolerance; induce autoreactive GC formation, and auto-Ab responses. Materials and methods ICs of autologous tumour necrosis factor α or IgE, were injected into BALB/c mice. In addition, soluble hen egg lysozyme transgenic (sHEL-Tg) and rat neu -Tg FVBN202 mice were challenged with HEL or the subdomain II of the extracellular domain of rat neu (ECDII), respectively, in the form of ICs. These auto/neo Ags were selected because Abs reactive with these Ags are used clinically or, in the case of sHEL-Tg mice, the profound tolerance to sHEL has been well verified. Results Remarkably, in all cases, the ICs were trapped by FDCs; and autoreactive GCs, plasmablast differentiation and strong auto-Ab responses were induced within 48 h. In marked contrast, free Ag that would have unfettered access to BCRs did not load on FDCs and did not induce detectable auto-Abs. Conclusions This is the first report of deliberately inducing auto-Abs by self/neo-Ags on FDCs. The data provide a mechanism by which FDCs may contribute to autoimmunity, and suggest a novel approach for targeting mediators of chronic inflammation, hypersensitivity and cancer. Therapeutic induction of auto-Abs by FDC-ICs may provide self-regulated high affinity Abs efficient in long-term disease control.