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Abstract Objective Based on the recent evidence of IL-1 inhibition in patients with rheumatoid arthritis (RA) and concomitant type 2 diabetes (T2D), we evaluated the synovial tissue expression of IL-1 related genes in relationship to the ubiquitin–proteasome system and the effects of insulin on ubiquitinated proteins in fibroblast-like synoviocytes (FLSs). Methods The synovial expression of IL-1 pathway genes was compared in early (

Abstract Rheumatoid arthritis (RA) is an autoimmune disease involving antigen-specific T and B cells. Here, we perform single-cell RNA and repertoire sequencing on paired synovial tissue and blood samples from 12 seropositive RA patients. We identify clonally expanded CD4 + T cells, including CCL5+ cells and T peripheral helper (Tph) cells, which show a prominent transcriptomic signature of recent activation and effector function. CD8 + T cells show higher oligoclonality than CD4 + T cells, with the largest synovial clones enriched in GZMK+ cells. CD8 + T cells with possibly virus-reactive TCRs are distributed across transcriptomic clusters. In the B cell compartment, NR4A1+ activated B cells, and plasma cells are enriched in the synovium and demonstrate substantial clonal expansion. We identify synovial plasma cells that share BCRs with synovial ABC, memory, and activated B cells. Receptor-ligand analysis predicted IFNG and TNFRSF members as mediators of synovial Tph-B cell interactions. Together, these results reveal clonal relationships between functionally distinct lymphocyte populations that infiltrate the synovium of patients with RA.

Abstract Background Kinases are intracellular signalling mediators and key to sustaining the inflammatory process in rheumatoid arthritis (RA). Oral inhibitors of Janus Kinase family (JAKs) are widely used in RA, while inhibitors of other kinase families e.g. phosphoinositide 3-kinase (PI3K) are under development. Most current biomarker platforms quantify mRNA/protein levels, but give no direct information on whether proteins are active/inactive. Phosphoproteome analysis has the potential to measure specific enzyme activation status at tissue level. Methods We validated the feasibility of phosphoproteome and total proteome analysis on 8 pre-treatment synovial biopsies from treatment-naive RA patients using label-free mass spectrometry, to identify active cell signalling pathways in synovial tissue which might explain failure to respond to RA therapeutics. Results Differential expression analysis and functional enrichment revealed clear separation of phosphoproteome and proteome profiles between lymphoid and myeloid RA pathotypes. Abundance of specific phosphosites was associated with the degree of inflammatory state. The lymphoid pathotype was enriched with lymphoproliferative signalling phosphosites, including Mammalian Target Of Rapamycin (MTOR) signalling, whereas the myeloid pathotype was associated with Mitogen-Activated Protein Kinase (MAPK) and CDK mediated signalling. This analysis also highlighted novel kinases not previously linked to RA, such as Protein Kinase, DNA-Activated, Catalytic Subunit (PRKDC) in the myeloid pathotype. Several phosphosites correlated with clinical features, such as Disease-Activity-Score (DAS)-28, suggesting that phosphosite analysis has potential for identifying novel biomarkers at tissue-level of disease severity and prognosis. Conclusions Specific phosphoproteome/proteome signatures delineate RA pathotypes and may have clinical utility for stratifying patients for personalised medicine in RA.

Abstract The TAM tyrosine kinases, Axl and MerTK, play an important role in rheumatoid arthritis (RA). Here, using a unique synovial tissue bioresource of patients with RA matched for disease stage and treatment exposure, we assessed how Axl and MerTK relate to synovial histopathology and disease activity, and their topographical expression and longitudinal modulation by targeted treatments. We show that in treatment-naive patients, high AXL levels are associated with pauci-immune histology and low disease activity and inversely correlate with the expression levels of pro-inflammatory genes. We define the location of Axl/MerTK in rheumatoid synovium using immunohistochemistry/fluorescence and digital spatial profiling and show that Axl is preferentially expressed in the lining layer. Moreover, its ectodomain, released in the synovial fluid, is associated with synovial histopathology. We also show that Toll-like-receptor 4-stimulated synovial fibroblasts from patients with RA modulate MerTK shedding by macrophages. Lastly, Axl/MerTK synovial expression is influenced by disease stage and therapeutic intervention, notably by IL-6 inhibition. These findings suggest that Axl/MerTK are a dynamic axis modulated by synovial cellular features, disease stage and treatment.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder which can lead to long-term joint damage and significantly reduced quality of life if not promptly diagnosed and adequately treated. Despite significant advances in treatment, about 40% of patients with RA do not respond to individual pharmacological agents and up to 20% do not respond to any of the available medications. To address this large unmet clinical need, several recent studies have focussed on an in-depth histological and molecular characterisation of the synovial tissue to drive the application of precision medicine to RA. Currently, RA patients are clinically divided into “seropositive” or “seronegative” RA, depending on the presence of routinely checked antibodies. Recent work has suggested that over the last two decades, long-term outcomes have improved significantly in seropositive RA but not in seronegative RA. Here, we present up-to-date differences in epidemiology, clinical features, and serological biomarkers in seronegative versus seropositive RA and discuss how histological and molecular synovial signatures, revealed by recent large synovial biopsy-based clinical trials, may be exploited to refine the classification of RA patients, especially in the seronegative group.

Abstract Background To determine whether gene-gene interaction network analysis of RNA sequencing (RNA-Seq) of synovial biopsies in early rheumatoid arthritis (RA) can inform our understanding of RA pathogenesis and yield improved treatment response prediction models. Methods We utilized four well curated pathway repositories obtaining 10,537 experimentally evaluated gene-gene interactions. We extracted specific gene-gene interaction networks in synovial RNA-Seq to characterize histologically defined pathotypes in early RA and leverage these synovial specific gene-gene networks to predict response to methotrexate-based disease-modifying anti-rheumatic drug (DMARD) therapy in the Pathobiology of Early Arthritis Cohort (PEAC). Differential interactions identified within each network were statistically evaluated through robust linear regression models. Ability to predict response to DMARD treatment was evaluated by receiver operating characteristic (ROC) curve analysis. Results Analysis comparing different histological pathotypes showed a coherent molecular signature matching the histological changes and highlighting novel pathotype-specific gene interactions and mechanisms. Analysis of responders vs non-responders revealed higher expression of apoptosis regulating gene-gene interactions in patients with good response to conventional synthetic DMARD. Detailed analysis of interactions between pairs of network-linked genes identified the SOCS2/STAT2 ratio as predictive of treatment success, improving ROC area under curve (AUC) from 0.62 to 0.78. We identified a key role for angiogenesis, observing significant statistical interactions between NOS3 (eNOS) and both CAMK1 and eNOS activator AKT3 when comparing responders and non-responders. The ratio of CAMKD2/NOS3 enhanced a prediction model of response improving ROC AUC from 0.63 to 0.73. Conclusions We demonstrate a novel, powerful method which harnesses gene interaction networks for leveraging biologically relevant gene-gene interactions leading to improved models for predicting treatment response.

Abstract We showed that the chemokine receptor C‐X‐C Motif Chemokine Receptor 2 (CXCR2) is essential for cartilage homeostasis. Here, we reveal that the CXCR2 ligand granulocyte chemotactic protein 2 (GCP‐2) was expressed, during embryonic development, within the prospective permanent articular cartilage, but not in the epiphyseal cartilage destined to be replaced by bone. GCP‐2 expression was retained in adult articular cartilage. GCP‐2 loss‐of‐function inhibited extracellular matrix production. GCP‐2 treatment promoted chondrogenesis in vitro and in human cartilage organoids implanted in nude mice in vivo. To exploit the chondrogenic activity of GCP‐2, we disrupted its chemotactic activity, by mutagenizing a glycosaminoglycan binding sequence, which we hypothesized to be required for the formation of a GCP‐2 haptotactic gradient on endothelia. This mutated version (GCP‐2‐T) had reduced capacity to induce transendothelial migration in vitro and in vivo, without affecting downstream receptor signaling through AKT, and chondrogenic activity. Intra‐articular adenoviral overexpression of GCP‐2‐T, but not wild‐type GCP‐2, reduced pain and cartilage loss in instability‐induced osteoarthritis in mice. We suggest that GCP‐2‐T may be used for disease modification in osteoarthritis.

BackgroundDespite the significant progress in the treatment of multiple myeloma (MM), the disease remains untreatable and its cure is still an unmet clinical need. Neoplastic transformation in MM is initiated in the germinal centers (GCs) of secondary lymphoid tissue (SLT) where B cells experience extensive somatic hypermutation induced by follicular dendritic cells (FDCs) and T-cell signals.ObjectiveWe reason that secreted protein acidic and rich in cysteine (SPARC), a common stromal motif expressed by FDCs at the origin (SLTs) and the destination (BM) of MM, plays a role in the pathogenesis of MM, and, here, we sought to investigate this role.MethodsThere were 107 BM biopsies from 57 MM patients (taken at different time points) together with 13 control specimens assessed for SPARC gene and protein expression and compared with tonsillar tissues. In addition, regulation of myeloma-promoting genes by SPARC-secreting FDCs was assessed in in vitro GC reactions (GCRs).ResultsSPARC gene expression was confirmed in both human primary (BM) and secondary (tonsils) lymphoid tissues, and the expression was significantly higher in the BM. Sparc was detectable in the BM and tonsillar lysates, co-localized with the FDC markers in both tissues, and stimulation of FDCs in vitro induced significantly higher levels of SPARC expression than unstimulated controls. In addition, SPARC inversely correlated with BM PC infiltration, ISS staging, and ECOG performance of the MM patients, and in vitro addition of FDCs to lymphocytes inhibited the expression of several oncogenes associated with malignant transformation of PCs.ConclusionFDC-SPARC inhibits several myelomagenic gene expression and inversely correlates with PC infiltration and MM progression. Therapeutic induction of SPARC expression through combinations of the current MM drugs, repositioning of non-MM drugs, or novel drug discovery could pave the way to better control MM in clinically severe and drug-resistant patients.

Follicular dendritic cells (FDCs) fundamentally contribute to the formation of synovial ectopic lymphoid-like structures in rheumatoid arthritis (RA) which is associated with poor clinical prognosis. Despite this critical role, regulation of FDC development in the RA synovium and its correlation with synovial pathotype differentiation remained largely unknown. Here, we demonstrate that CNA.42+ FDCs distinctively express the pericyte/fibroblast-associated markers PDGFR-β, NG2, and Thy-1 in the synovial perivascular space but not in established follicles. In addition, synovial RNA-Seq analysis revealed that expression of the perivascular FDC markers was strongly correlated with PDGF-BB and fibroid synovitis, whereas TNF-α/LT-β was significantly associated with lymphoid synovitis and expression of CR1, CR2, and FcγRIIB characteristic of mature FDCs in lymphoid follicles. Moreover, PDGF-BB induced CNA.42+ FDC differentiation and CXCL13 secretion from NG2+ synovial pericytes, and together with TNF-α/LT-β conversely regulated early and late FDC differentiation genes in unsorted RA synovial fibroblasts (RASF) and this was confirmed in flow sorted stromal cell subsets. Furthermore, RASF TNF-αR expression was upregulated by TNF-α/LT-β and PDGF-BB; and TNF-α/LT-β-activated RASF retained ICs and induced B cell activation in in vitro germinal center reactions typical of FDCs. Additionally, FDCs trapped peptidyl citrulline, and strongly correlated with IL-6 expression, and plasma cell, B cell, and T cell infiltration of the RA synovium. Moreover, synovial FDCs were significantly associated with RA disease activity and radiographic features of tissue damage. To the best of our knowledge, this is the first report describing the reciprocal interaction between PDGF-BB and TNF-α/LT-β in synovial FDC development and evolution of RA histological pathotypes. Selective targeting of this interplay could inhibit FDC differentiation and potentially ameliorate RA in clinically severe and drug-resistant patients.

Rheumatoid arthritis (RA) is a complex condition that displays heterogeneity in disease severity and response to standard treatments between patients. Failure rates for conventional, target synthetic, and biologic disease-modifying rheumatic drugs (DMARDs) are significant. Although there are models for predicting patient response, they have limited accuracy, require replication/validation, or for samples to be obtained through a synovial biopsy. Thus, currently, there are no prediction methods approved for routine clinical use. Previous research has shown that genetics and environmental factors alone cannot explain the differences in response between patients. Recent studies have demonstrated that deoxyribonucleic acid (DNA) methylation plays an important role in the pathogenesis and disease progression of RA. Importantly, specific DNA methylation profiles associated with response to conventional, target synthetic, and biologic DMARDs have been found in the blood of RA patients and could potentially function as predictive biomarkers. This review will summarize and evaluate the evidence for DNA methylation signatures in treatment response mainly in blood but also learn from the progress made in the diseased tissue in cancer in comparison to RA and autoimmune diseases. We will discuss the benefits and challenges of using DNA methylation signatures as predictive markers and the potential for future progress in this area.

Abstract Since the first report of SARS-CoV-2 in China in 2019, there has been a huge debate about the origin. In this work, using a different method we aimed to strengthen the observation that no evidence of genetic manipulation has been found by (1) detecting classical restriction site (RS) sequence in human SARS-CoV-2 genomes and (2) comparing them with other recombinant SARS-CoV-like virus created for experimental purposes. Finally, we propose a novel approach consisting in the generation of a restriction endonucleases site map of SARS-CoV-2 and other related coronavirus genomes to be used as a fingerprint to trace the virus evolution.

Background: Although biological therapies have transformed the outlook for those with rheumatoid arthritis, there is a lack of any meaningful response in approximately 40% of patients. The role of B cells in rheumatoid arthritis pathogenesis is well recognised and is supported by the clinical efficacy of the B-cell-depleting agent rituximab (MabThera, F. Hoffman La-Roche Ltd, Basel, Switzerland). Rituximab is licensed for use in rheumatoid arthritis following failure of conventional synthetic disease-modifying antirheumatic drugs and tumour necrosis factor inhibitor therapy. However, over 50% of patients show low/absent synovial B-cell infiltration, suggesting that, in these patients, inflammation is driven by alternative cell types. This prompted us to test the hypothesis that, in synovial biopsy B-cell-poor patients, tocilizumab (RoActemra, F. Hoffman La-Roche Ltd, Basel, Switzerland) (targeting interleukin 6) is superior to rituximab (targeting CD20+/B cells). Design: The R4–RA (A Randomised, open-labelled study in anti-TNFalpha inadequate responders to investigate the mechanisms for Response, Resistance to Rituximab versus Tocilizumab in Rheumatoid Arthritis patients) trial is a 48-week Phase IV, open-label, randomised controlled trial conducted in 19 European centres that recruited patients failing on or intolerant to conventional synthetic disease-modifying antirheumatic drug therapy and at least one tumour necrosis factor inhibitor. Participants: Synovial tissue was obtained at trial entry and classified histologically as B-cell rich or B-cell poor to inform balanced stratification. Patients were randomised on a 1 : 1 basis to receive standard therapy with rituximab or tocilizumab. B-cell-poor/-rich molecular classification was also carried out. The study was powered to test the superiority of tocilizumab over rituximab at 16 weeks in the B-cell-poor population. Main outcome measures: The primary end point was defined as an improvement in the Clinical Disease Activity Index (CDAI) score of ≥ 50% from baseline. In addition, patients were considered to be non-responders if they did not reach an improvement in CDAI score of ≥ 50% and a CDAI score of

Abstract WNT ligands can activate several signalling cascades of pivotal importance during development and regenerative processes. Their de-regulation has been associated with the onset of different diseases. Here we investigated the role of the WNT/Calcium Calmodulin Kinase II (CaMKII) pathway in osteoarthritis. We identified Heme Oxygenase I (HMOX1) and Sox-9 as specific markers of the WNT/CaMKII signalling in articular chondrocytes through a microarray analysis. We showed that the expression of the activated form of CaMKII, phospho-CaMKII, was increased in human and murine osteoarthritis and the expression of HMOX1 was accordingly reduced, demonstrating the activation of the pathway during disease progression. To elucidate its function, we administered the CaMKII inhibitor KN93 to mice in which osteoarthritis was induced by resection of the anterior horn of the medial meniscus and of the medial collateral ligament in the knee joint. Pharmacological blockade of CaMKII exacerbated cartilage damage and bone remodelling. Finally, we showed that CaMKII inhibition in articular chondrocytes upregulated the expression of matrix remodelling enzymes alone and in combination with Interleukin 1. These results suggest an important homeostatic role of the WNT/CaMKII signalling in osteoarthritis which could be exploited in the future for therapeutic purposes.

Summary: Background: Rheumatoid arthritis (RA) is a progressive degenerative disorder that leads to joint destruction. Available treatments only target the inflammatory component with minimal impact on joint repair. We recently uncovered a previously unappreciated family of pro-resolving mediators, the maresin conjugate in tissue regeneration (MCTR), that display both immunoregulatory and tissue-protective activities. Thus, we queried whether the production of these autacoids is disrupted in RA patients and whether they can be useful in treating joint inflammation and promoting joint repair. Methods: Using a highly phenotyped RA cohort we evaluated plasma MCTR concentrations and correlated these to clinical markers of disease activity. To evaluate the immunoregulatory and tissue reparative activities we employed both in vivo models of arthritis and organ culture models. Findings: Herein, we observed that plasma MCTR3 concentrations were negatively correlated with joint disease activity and severity in RA patients. Evaluation of the mechanisms engaged by this mediator in arthritic mice demonstrated that MCTR3 reprograms monocytes to confer enduring joint protective properties. Single cell transcriptomic profiling and flow cytometric evaluation of macrophages from mice treated with MCTR3-reprogrammed monocytes revealed a role for Arginase-1 (Arg-1) in mediating their joint reparative and pro-resolving activities. Arg-1 inhibition reversed both the anti-arthritic and tissue reparative actions of MCTR3-reprogrammed monocytes. Interpretation: Our findings demonstrate that circulating MCTR3 levels are negatively correlated with disease in RA. When administered to mice in vivo, MCTR3 displayed both anti-inflammatory and joint reparative activities, protecting both cartilage and bone in murine arthritis. These activities were, at least in part, mediated via the reprogramming of mononuclear phagocyte responses. Funding: This work was supported by funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant no: 677542) and the Barts Charity (grant no: MGU0343) to J.D. J.D. is also supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (grant 107613/Z/15/Z).

Summary: Ectopic lymphoid structures (ELS) can develop in rheumatoid arthritis (RA) synovial tissue, but the precise pathways of B cell activation and selection are not well understood. Here, we identify a synovial B cell population characterized by co-expression of a family of orphan nuclear receptors (NR4A1-3), which is highly enriched in RA synovial tissue. A transcriptomic profile of NR4A synovial B cells significantly overlaps with germinal center light zone B cells and an accrual of somatic hypermutation that correlates with loss of naive B cell state. NR4A B cells co-express lymphotoxins α and β and IL-6, supporting functions in ELS promotion. Expanded and shared clones between synovial NR4A B cells and plasma cells and the rapid upregulation with BCR stimulation point to in situ differentiation. Together, we identify a dynamic progression of B cell activation in RA synovial ELS, with NR4A transcription factors having an important role in local adaptive immune responses.

Abstract Biomarkers are needed for predicting the effectiveness of disease modifying antirheumatic drugs (DMARDs). Here, using functional lipid mediator profiling and deeply phenotyped patients with early rheumatoid arthritis (RA), we observe that peripheral blood specialized pro-resolving mediator (SPM) concentrations are linked with both DMARD responsiveness and disease pathotype. Machine learning analysis demonstrates that baseline plasma concentrations of resolvin D4, 10S, 17S-dihydroxy-docosapentaenoic acid, 15R-Lipoxin (LX)A4 and n-3 docosapentaenoic-derived Maresin 1 are predictive of DMARD responsiveness at 6 months. Assessment of circulating SPM concentrations 6-months after treatment initiation establishes that differences between responders and non-responders are maintained, with a decrease in SPM concentrations in patients resistant to DMARD therapy. These findings elucidate the potential utility of plasma SPM concentrations as biomarkers of DMARD responsiveness in RA.

Primary Sjögren’s syndrome (pSS) is a chronic autoimmune disease resulting from the inflammatory infiltration of exocrine glands, mainly salivary and lacrimal glands, leading to secretory dysfunction and serious complications including debilitating fatigue, systemic autoimmunity, and lymphoma. Like other autoimmune disorders, a strong interferon (IFN) signature is present among subsets of pSS patients, suggesting the involvement of innate immunity in pSS pathogenesis. NCR3/NKp30 is a natural killer (NK) cell-specific activating receptor regulating the cross talk between NK and dendritic cells including type II IFN secretion upon NK-cell activation. A genetic association between single-nucleotide polymorphisms (SNPs) in the NCR3/NKp30 promoter gene and a higher susceptibility for pSS has been previously described, with pSS patients most frequently carrying the major allele variant associated with a higher NKp30 transcript and IFN-γ release as a consequence of the receptor engagement. In the present study, we combined RNA-sequencing and histology from pSS salivary gland biopsies to better characterize NKp30 (NCR3) and its ligand B7/H6 (NCR3LG1) in pSS salivary gland tissues. Levels of NCR3/NKp30 were significantly increased both in salivary glands and in circulating NK cells of pSS patients compared with sicca controls, especially in salivary glands with organized ectopic lymphoid structures. In line with this observation, a strong correlation between NCR3/NKp30 levels and salivary gland infiltrating immune cells (CD3, CD20) was found. Furthermore, NCR3/NKp30 levels also correlated with higher IFN-γ, Perforin, and Granzyme-B expression in pSS SGs with organized ectopic lymphoid structures, suggesting an activation state of NK cells infiltrating SG tissue. Of note, NKp30+ NK cells accumulated at the border of the inflammatory foci, while the NKp30 ligand, B7/H6, is shown to be expressed mainly by ductal epithelial cells in pSS salivary glands. Finally, immunomodulatory treatment, such as the B-cell depleting agent rituximab, known to reduce the infiltration of immune cells in pSS SGs, prevented the upregulation of NCR3/NKp30 within the glands.

AimsTo determine the relationship between PTX3 systemic and synovial levels and the clinical features of rheumatoid arthritis (RA) in a cohort of early, treatment naïve patients and to explore the relevance of PTX3 expression in predicting response to conventional-synthetic (cs) Disease-Modifying-Anti-Rheumatic-Drugs (DMARDs) treatment.MethodsPTX3 expression was analyzed in 119 baseline serum samples from early naïve RA patients, 95 paired samples obtained 6-months following the initiation of cs-DMARDs treatment and 43 healthy donors. RNA-sequencing analysis and immunohistochemistry for PTX3 were performed on a subpopulation of 79 and 58 synovial samples, respectively, to assess PTX3 gene and protein expression. Immunofluorescence staining was performed to characterize PTX3 expressing cells within the synovium.ResultsCirculating levels of PTX3 were significantly higher in early RA compared to healthy donors and correlated with disease activity at baseline and with the degree of structural damages at 12-months. Six-months after commencing cs-DMARDs, a high level of PTX3, proportional to the baseline value, was still detectable in the serum of patients, regardless of their response status. RNA-seq analysis confirmed that synovial transcript levels of PTX3 correlated with disease activity and the presence of mediators of inflammation, tissue remodeling and bone destruction at baseline. PTX3 expression in the synovium was strongly linked to the degree of immune cell infiltration, the presence of ectopic lymphoid structures and seropositivity for autoantibodies. Accordingly, PTX3 was found to be expressed by numerous synovial cell types such as plasma cells, fibroblasts, vascular and lymphatic endothelial cells, macrophages, and neutrophils. The percentage of PTX3-positive synovial cells, although significantly reduced at 6-months post-treatment as a result of global decreased cellularity, was similar in cs-DMARDs responders and non-responders.ConclusionThis study demonstrates that, early in the disease and prior to treatment modification, the level of circulating PTX3 is a reliable marker of RA activity and predicts a high degree of structural damages at 12-months. In the joint, PTX3 associates with immune cell infiltration and the presence of ectopic lymphoid structures. High synovial and peripheral blood levels of PTX3 are associated with chronic inflammation characteristic of RA. Additional studies to determine the mechanistic link are required.

Objective To evaluate the effects of targeting Ikaros and Aiolos by cereblon modulator iberdomide on the activation and differentiation of B-cells from patients with systemic lupus erythematosus (SLE).Methods CD19+ B-cells isolated from the peripheral blood of patients with SLE (n=41) were cultured with TLR7 ligand resiquimod ±IFNα together with iberdomide or control from day 0 (n=16). Additionally, in vitro B-cell differentiation was induced by stimulation with IL-2/IL-10/IL-15/CD40L/resiquimod with iberdomide or control, given at day 0 or at day 4. At day 5, immunoglobulins were measured by ELISA and cells analysed by flow cytometry. RNA-Seq was performed on fluorescence-activated cell-sorted CD27-IgD+ naïve-B-cells and CD20lowCD27+CD38+ plasmablasts to investigate the transcriptional consequences of iberdomide.Results Iberdomide significantly inhibited the TLR7 and IFNα-mediated production of immunoglobulins from SLE B-cells and the production of antinuclear antibodies as well as significantly reducing the number of CD27+CD38+ plasmablasts (0.3±0.18, vehicle 1.01±0.56, p=0.011) and CD138+ plasma cells (0.12±0.06, vehicle 0.28±0.02, p=0.03). Additionally, treatment with iberdomide from day 0 significantly inhibited the differentiation of SLE B-cells into plasmablasts (6.4±13.5 vs vehicle 34.9±20.1, p=0.013) and antibody production. When given at later stages of differentiation, iberdomide did not affect the numbers of plasmablasts or the production of antibodies; however, it induced a significant modulation of gene expression involving IKZF1 and IKZF3 transcriptional programmes in both naïve B-cells and plasmablasts (400 and 461 differentially modulated genes, respectively, false discovery rate

Abstract Cartilage defects repair poorly. Recent genetic studies suggest that WNT3a may contribute to cartilage regeneration, however the dense, avascular cartilage extracellular matrix limits its penetration and signalling to chondrocytes. Extracellular vesicles actively penetrate intact cartilage. This study investigates the effect of delivering WNT3a into large cartilage defects in vivo using exosomes as a delivery vehicle. Exosomes were purified by ultracentrifugation from conditioned medium of either L‐cells overexpressing WNT3a or control un‐transduced L‐cells, and characterized by electron microscopy, nanoparticle tracking analysis and marker profiling. WNT3a loaded on exosomes was quantified by western blotting and functionally characterized in vitro using the SUPER8TOPFlash reporter assay and other established readouts including proliferation and proteoglycan content. In vivo pathway activation was assessed using TCF/Lef:H2B‐GFP reporter mice. Wnt3a loaded exosomes were injected into the knees of mice, in which large osteochondral defects were surgically generated. The degree of repair was histologically scored after 8 weeks. WNT3a was successfully loaded on exosomes and resulted in activation of WNT signalling in vitro. In vivo, recombinant WNT3a failed to activate WNT signalling in cartilage, whereas a single administration of WNT3a loaded exosomes activated canonical WNT signalling for at least one week, and eight weeks later, improved the repair of osteochondral defects. WNT3a assembled on exosomes, is efficiently delivered into cartilage and contributes to the healing of osteochondral defects.

Programmed cell death protein 1 (PD-1) is expressed on T cells upon T cell receptor (TCR) stimulation. PD-1 ligand 1 (PD-L1) is expressed in most tumor environments, and its binding to PD-1 on T cells drives them to apoptosis or into a regulatory phenotype. The fact that PD-L1 itself is also expressed on T cells upon activation has been largely neglected. Here, we demonstrate that PD-L1 ligation on human CD25-depleted CD4+ T cells, combined with CD3/TCR stimulation, induces their conversion into highly suppressive T cells. Furthermore, this effect was most prominent in memory (CD45RA-CD45RO+) T cells. PD-L1 engagement on T cells resulted in reduced ERK phosphorylation and decreased AKT/mTOR/S6 signaling. Importantly, T cells from rheumatoid arthritis patients exhibited high basal levels of phosphorylated ERK and following PD-L1 cross-linking both ERK signaling and the AKT/mTOR/S6 pathway failed to be down modulated, making them refractory to the acquisition of a regulatory phenotype. Altogether, our results suggest that PD-L1 signaling on memory T cells could play an important role in resolving inflammatory responses; maintaining a tolerogenic environment and its failure could contribute to ongoing autoimmunity.

Biologic drugs, especially anti-TNF, are considered as the gold standard therapy in rheumatoid arthritis. However, non-uniform efficacy, incidence of infections, and high costs are major concerns. Novel tissue-specific agents may overcome the current limitations of systemic administration, providing improved potency, and safety. We developed a bispecific antibody (BsAb), combining human arthritic joint targeting, via the synovial-specific single-chain variable fragment (scFv)-A7 antibody, and TNFα neutralization, via the scFv-anti-TNFα of adalimumab, with the binding/blocking capacity comparable to adalimumab -immunoglobulin G (IgG). Tissue-targeting capacity of the BsAb was confirmed on the human arthritic synovium in vitro and in a synovium xenograft Severe combined immune deficient (SCID) mouse model. Peak graft accumulation occurred at 48 h after injection with sustained levels over adalimumab-IgG for 7 days and increased therapeutic effect, efficiently decreasing tissue cellularity, and markers of inflammation with higher potency compared to the standard treatment. This study provides the first description of a BsAb capable of drug delivery, specifically to the disease tissue, and a strong evidence of improved therapeutic effect on the human arthritic synovium, with applications to other existing biologics.

Mast cells (MCs) are immune cells infiltrating the synovial membrane and implicated in the pathogenesis of Rheumatoid Arthritis (RA). Their infiltration in the synovia of early RA patients has been shown to be associated with systemic inflammation, disease activity and autoantibody positivity. Here, we analyzed their presence in matched synovial samples obtained by ultrasound-guided synovial biopsies pre- and post-treatment with conventional synthetic Disease Modifying Anti-Rheumatic Drugs (csDMARDs) (n=20). Upon IHC staining, patients were classified as MC+ve/-ve based on the presence/absence of CD117+ synovial MCs. At baseline, MC+ve patients had significantly higher synovial inflammation, inflammatory markers, disease activity and a higher prevalence of lympho-myeloid aggregates. Synovial biopsies after 6 months of treatment with csDMARDs showed a significant reduction of synovitis scores, but only a partial reduction of MC numbers. Accordingly, 45% of patients (9/20) were MC+ve after treatment, in association with significantly higher degree of synovitis and higher proportion lympho-myeloid aggregates. Finally, significantly lower patients with MC+ve synovitis at 6 months reached Low Disease Activity (LDA), while the association of MCs with disease activity was independent from lymphoid aggregates, after adjustment for BMI and age. Overall, this study confirms the relevance of MCs as part of the inflammatory infiltrate in the synovia of RA patients, warranting further investigations in larger cohorts to clarify their role in disease progression and response to treatment and their relevance as prognostic markers and potential therapeutic targets.

Objectives: To assess whether the histopathological features of the synovium before starting treatment with the TNFi certolizumab-pegol could predict clinical outcome and examine the modulation of histopathology by treatment.Methods: Thirty-seven RA patients fulfilling UK NICE guidelines for biologic therapy were enrolled at Barts Health NHS trust and underwent synovial sampling of an actively inflamed joint using ultrasound-guided needle biopsy before commencing certolizumab-pegol and after 12-weeks. At 12-weeks, patients were categorized as responders if they had a DAS28 fall >1.2. A minimum of 6 samples was collected for histological analysis. Based on H&E and immunohistochemistry (IHC) staining for CD3 (T cells), CD20 (B cells), CD138 (plasma cells), and CD68 (macrophages) patients were categorized into three distinct synovial pathotypes (lympho-myeloid, diffuse-myeloid, and pauci-immune).Results: At baseline, as per inclusion criteria, DAS28 mean was 6.4 ± 0.9. 94.6% of the synovial tissue was retrieved from the wrist or a metacarpophalangeal joint. Histological pathotypes were distributed as follows: 58% lympho-myeloid, 19.4% diffuse-myeloid, and 22.6% pauci-immune. Patients with a pauci-immune pathotype had lower levels of CRP but higher VAS fatigue compared to lympho- and diffuse-myeloid. Based on DAS28 fall >1.2, 67.6% of patients were deemed as responders and 32.4% as non-responders. However, by categorizing patients according to the baseline synovial pathotype, we demonstrated that a significantly higher number of patients with a lympho-myeloid and diffuse-myeloid pathotype in comparison with pauci-immune pathotype [83.3% (15/18), 83.3 % (5/6) vs. 28.6% (2/7), p = 0.022) achieved clinical response to certolizumab-pegol. Furthermore, we observed a significantly higher level of post-treatment tender joint count and VAS scores for pain, fatigue and global health in pauci-immune in comparison with lympho- and diffuse-myeloid patients but no differences in the number of swollen joints, ESR and CRP. Finally, we confirmed a significant fall in the number of CD68+ sublining macrophages post-treatment in responders and a correlation between the reduction in the CD20+ B-cells score and the improvement in the DAS28 at 12-weeks.Conclusions: The analysis of the synovial histopathology may be a helpful tool to identify among clinically indistinguishable patients those with lower probability of response to TNFα-blockade.

Abstract Background Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. Methods Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. Results Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. Conclusions We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.

Abstract Background Plasmablasts and plasma cells play a key role in many autoimmune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). This study was undertaken to evaluate the potential of targeting CD38 as a plasma cell/plasmablast depletion mechanism by daratumumab in the treatment of patients with RA and SLE. Methods RNA-sequencing analysis of synovial biopsies from various stages of RA disease progression, flow cytometry analysis of peripheral blood mononuclear cells (PBMC) from patients with RA or SLE and healthy donors, immunohistochemistry assessment (IHC) of synovial biopsies from patients with early RA, and ex vivo immune cell depletion assays using daratumumab (an anti-CD38 monoclonal antibody) were used to assess CD38 as a therapeutic target. Results We demonstrated that the plasma cell/plasmablast-related genes CD38, XBP1, IRF4, PRDM1, IGJ and TNFSF13B are significantly up-regulated in synovial biopsies from patients with arthralgia, undifferentiated arthritis (UA), early RA and established RA as compared to healthy controls and control patients with osteoarthritis. In addition, the highest CD38 expression was observed on plasma cells and plasmablasts compared to natural killer (NK) cells, classical dendritic cells (DCs), plasmacytoid DCs (pDCs) and T cells, in blood from healthy controls and patients with SLE and RA. Furthermore, IHC showed CD38 staining in the same region as CD3 and CD138 staining in synovial tissue biopsies from patients with early RA. Most importantly, our data show for the first time that daratumumab effectively depletes plasma cells/plasmablasts in PBMC from patients with SLE and RA in a dose-dependent manner ex vivo. Conclusion These results indicate that CD38 may be a potential target for RA disease interception and daratumumab should be evaluated clinically for the treatment of both RA and SLE.

Microvesicles (MVs) are emerging as a novel means to enact cell-to-cell communication in inflammation. Here, we aimed to ascertain the ability of neutrophil-derived MVs to modulate target cell behaviour, the focus being the macrophage.MVs were generated in response to tumour necrosis factor-α, from healthy control neutrophils or those from rheumatoid arthritis patients. MVs were used to stimulate human monocyte-derived macrophages in vitro, or administered intra-articularly in the K/BxN mouse model of arthritis. A macrophage/fibroblast-like synoviocyte co-culture system was used to study the effects of vesicles on the crosstalk between these cells.We demonstrate a direct role for phosphatidylserine and annexin-A1 exposed by the MVs to counteract classical activation of the macrophages, and promote the release of transforming growth factor-β, respectively. Classically-activated macrophages exposed to neutrophil MVs no longer activated fibroblast-like synoviocytes in subsequent co-culture settings. Finally, intra-articular administration of neutrophil MVs from rheumatoid arthritis patients in arthritic mice affected the phenotype of joint macrophages.Altogether these data, with the identification of specific MV determinants, open new opportunities to modulate on-going inflammation in the synovia – mainly by affecting macrophage polarization and potentially also fibroblast-like synoviocytes - through the delivery of autologous or heterologous MVs produced from neutrophils. Keywords: Neutrophils, Macrophages, Vesicles, Rheumatoid arthritis

Abstract Rheumatoid arthritis (RA) has been associated with endothelial dysfunction, a pathophysiological feature of atherosclerosis. Our aim was to determine whether TNF-α blockade has a beneficial effect on endothelial function in RA. We performed a systematic review with meta-analysis to evaluate the effect of anti-TNF-α agents on endothelial function in RA patients. MedLine, Cochrane CENTRAL and SCOPUS were searched up to March 2016. Inclusion criteria were: 1) randomised controlled trial (RCT), quasi-RCT, before-after cohort study; 2) including RA patients; 3) treatment with anti-TNF-α medications; 4) evaluating the change from baseline in endothelial function. The search strategy retrieved 180 records, of which 20 studies were included in the systematic review. Pooled analysis using a random-effects model demonstrated a significant improvement in endothelial function following anti-TNF-α treatment (SDM 0.987, 95%CI [0.64–1.33], p

Summary: There is a current imperative to unravel the hierarchy of molecular pathways that drive the transition of early to established disease in rheumatoid arthritis (RA). Herein, we report a comprehensive RNA sequencing analysis of the molecular pathways that drive early RA progression in the disease tissue (synovium), comparing matched peripheral blood RNA-seq in a large cohort of early treatment-naive patients, namely, the Pathobiology of Early Arthritis Cohort (PEAC). We developed a data exploration website (https://peac.hpc.qmul.ac.uk/) to dissect gene signatures across synovial and blood compartments, integrated with deep phenotypic profiling. We identified transcriptional subgroups in synovium linked to three distinct pathotypes: fibroblastic pauci-immune pathotype, macrophage-rich diffuse-myeloid pathotype, and a lympho-myeloid pathotype characterized by infiltration of lymphocytes and myeloid cells. This is suggestive of divergent pathogenic pathways or activation disease states. Pro-myeloid inflammatory synovial gene signatures correlated with clinical response to initial drug therapy, whereas plasma cell genes identified a poor prognosis subgroup with progressive structural damage. : Lewis et al. use histology and RNA-seq of synovial biopsies from a cohort of early rheumatoid arthritis individuals to identify three histological pathotypes and reveal gene modules associated with disease severity and clinical response. Keywords: rheumatoid arthritis, RNA sequencing, personalized medicine, synovial biopsy, ectopic lymphoid structures, lymphoid neogenesis, transcriptomics, Pathobiology of Early Arthritis Cohort study, PEAC

Objectives To report on fatigue in patients from the United Kingdom primary Sjögren’s syndrome (pSS) registry identifying factors associated with fatigue and robust to assignable causes such as comorbidities and medications associated with drowsiness.Methods From our cohort (n = 608), we identified those with comorbidities associated with fatigue, and those taking medications associated with drowsiness. We constructed dummy variables, permitting the contribution of these potentially assignable causes of fatigue to be assessed. Using multiple regression analysis, we modelled the relationship between Profile of Fatigue and Discomfort physical and mental fatigue scores and potentially related variables.Results Pain, depression and daytime sleepiness scores were closely associated with both physical and mental fatigue (all p ≤ 0.0001). In addition, dryness was strongly associated with physical fatigue (p ≤ 0.0001). These effects were observed even after adjustment for comorbidities associated with fatigue or medications associated with drowsiness.Conclusions These findings support further research and clinical interventions targeting pain, dryness, depression and sleep to improve fatigue in patients with pSS.This finding is robust to both the effect of other comorbidities associated with fatigue and medications associated with drowsiness.

Prof Josef Smolen opened the symposium and briefly described the aims of the meeting. Co-host Prof Constantino Pitzalis first discussed the pathophysiology of rheumatoid arthritis (RA), identifying the pro-inflammatory cytokines involved and explaining why specific drugs only work in certain conditions. Prof Simon Jones followed with a discussion on comorbidities and adverse events associated with interleukin (IL)-6 intervention in rheumatic disease. Dr Frank McKenna presented on the psychological impact of RA, including mood changes and development of depressive disorders, and Prof Smolen described the upcoming therapeutic approaches for the condition while also comparing and contrasting existing treatment options. The symposium concluded with a question and answer session.

Chemokine synergy-inducing molecules are emerging as regulating factors in cell migration. The alarmin HMGB1, in its reduced form, can complex with CXCL12 enhancing its activity on monocytes via the chemokine receptor CXCR4, while the form containing a disulfide bond, by binding to TLR2 or TLR4, initiates a cascade of events leading to production of cytokines and chemokines. So far, the possibility that the CXCL12/HMGB1 heterocomplex could be maintained in chronic inflammation was debated, due to the release of reactive oxygen species. Therefore, we have assessed if the heterocomplex could remain active in Rheumatoid Arthritis (RA) and its relevance in the disease assessment. Monocytes from RA patients with active disease require a low concentration of HMGB1 to enhance CXCL12-induced migration, in comparison to monocytes from patients in clinical remission or healthy donors. The activity of the heterocomplex depends on disease activity, on the COX2 and JAK/STAT pathways, and is determined by the redox potential of the microenvironment. In RA, the presence of an active thioredoxin system correlates with the enhanced cell migration, and with the presence of the heterocomplex in the synovial fluid. The present study highlights how, in an unbalanced microenvironment, the activity of the thioredoxin system plays a crucial role in sustaining inflammation. Prostaglandin E2 stimulation of monocytes from healthy donors is sufficient to recapitulate the response observed in patients with active RA. The activation of mechanisms counteracting the oxidative stress in the extracellular compartment preserves HMGB1 in its reduced form, and contributes to fuel the influx of inflammatory cells. Targeting the heterocomplex formation and its activity could thus be an additional tool for dampening the inflammation sustained by cell recruitment, for those patients with chronic inflammatory conditions who poorly respond to current therapies.

OBJECTIVES To assess the use of the Clinical EULAR Sjögren’s Syndrome Disease Activity Index (ClinESSDAI), a version of the ESSDAI without the biological domain, for assessing potential eligibility and outcomes for clinical trials in patients with primary Sjögren’s syndrome (pSS), according to the new ACR-EULAR classification criteria, from the UK Primary Sjögren’s Syndrome Registry (UKPSSR). METHODS A total of 665 patients from the UKPSSR cohort were analysed at their time of inclusion in the registry. ESSDAI and ClinESSDAI were calculated for each patient. RESULTS For different disease activity index cut-off values, more potentially eligible participants were found when ClinESSDAI was used than with ESSDAI. The distribution of patients according to defined disease activity levels did not differ statistically (chi2 p = 0.57) between ESSDAI and ClinESSDAI for moderate disease activity (score ≥5 and

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.

Porphyromonas gingivalis peptidylarginine deiminase (PPAD) is the focus of several studies due to its ability to citrullinate in vitro human proteins, which have been linked to the aetiopathogenesis of rheumatoid arthritis (RA). The aim of this work was the generation by homologous recombination and characterisation of a P. gingivalis W50 mutant lacking pad gene (PG1424) to study the role of PPAD in RA. To confirm the absence of PPAD activity in P. gingivalis PG1424, cells were incubated with arginine-containing substrates and citrullination of L-arginine measured using a colorimetric assay and thin-layer chromatography. Furthermore, arginine and lysine protease (gingipain) activities were assessed and immunoblotting was performed using monoclonal antibody 1B5 (mAb1B5) and a commercial anti-modified citrulline antibody (AMC) to detect differences in virulence factor expression. The deletion of pad gene in P. gingivalis PG1424 completely abolished the ability to autocitrullinate P. gingivalis proteins in the mutant strain and also the citrullination of used substrates but not free L-arginine. Moreover, the wild-type and mutant strains had similar total gingipain activities and reactivity with mAb1B5. In conclusion, this work has produced a well-characterised PPAD-deleted P. gingivalis strain, which can be used to help determine the role of citrullination by this microorganism in RA.

Although great progress has been made in the past decade toward understanding the pathogenesis of rheumatoid arthritis (RA), clinicians remain some distance from a goal of personalized health care. The capacity to diagnose RA early, predict prognosis, and moreover predict response to biologic therapies has been a research focus for many years. How currently available clinical prediction models can facilitate such goals is reviewed in this article. In addition, the role of current imaging techniques in this regard is also discussed. Finally, the authors review the current literature regarding synovial biomarkers and consider whether integration of synovial pathobiology into clinical prediction algorithms may enhance their predictive value.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by inflammation of the synovial membrane, with thickening of the synovial layer, cellular hyperplasia, and infiltration of immune cells. Mast cells (MCs) are cells of the innate immunity present in healthy synovia and part of the cellular hyperplasia characterizing RA synovitis. Although their presence in synovia has been well described, the exact functions and the correlation of MCs with disease development and progression have been debated, particularly because of contradictory data obtained in animal models and from patients with longstanding disease. Here, we present a revision of the literature on MCs in RA, including the most recent observations obtained from patients with early RA, indicating MCs as relevant markers of disease severity in early RA.

The interleukin (IL)-1 family of cytokines is composed of 11 members, including the most recently discovered IL-36α, β, γ, IL-37, and IL-38. Similar to IL-1, IL-36 cytokines are initiators and amplifiers of inflammation, whereas both IL-37 and IL-38 display anti-inflammatory activities. A few studies have outlined the role played by these cytokines in several inflammatory diseases. For instance, IL-36 agonists seem to be relevant for the pathogenesis of skin psoriasis whereas, despite being expressed within the synovial tissue, their silencing or overexpression do not critically influence the course of arthritis in mice. In this review, we will focus on the state of the art of the molecular features and biological roles of IL-36, IL-37, and IL-38 in representative skin- and joint-related inflammatory diseases, namely psoriasis, rheumatoid arthritis, and psoriatic arthritis. We will then offer an overview of the therapeutic potential of targeting the IL-36 axis in these diseases, either by blocking the proinflammatory agonists or enhancing the physiologic inhibitory feedback on the inflammation mediated by the antagonists IL-37 and IL-38.

Ectopic lymphoid structures (ELS) often develop at sites of inflammation in target tissues of autoimmune diseases such as rheumatoid arthritis, Sjögren’s syndrome, multiple sclerosis, myasthenia gravis and systemic lupus erythematosus. ELS are characterised by the formation of organized T/B cells aggregates which can acquire follicular dendritic cells network supporting an ectopic germinal center (GC) response. In this review, we shall summarise the mechanisms which regulate the formation of ELS in tertiary lymphoid organs, with particular emphasis on the role of lymphoid chemokines in both formation and maintenance of ELS, the role of emerging positive and negative regulators of ELS development and function, including T follicular helper cells and IL-27, respectively. Finally, we shall discuss the main functions of ELS in supporting the affinity maturation, clonal selection and differentiation of autoreactive B cells contributing to the maintenance and perpetuation of humoral autoimmunity.

Psoriasis is a chronic systemic inflammatory disease causing erythematosus and scaly skin plaques; up to 30% of patients with psoriasis develop Psoriatic Arthritis (PsA), which is characterised by inflammation and progressive damage of the peripheral joints and/or the spine and/or the entheses. The pathogenic mechanisms driving the skin disorder in psoriasis and the joint disease in PsA are sustained by the activation of inflammatory pathways that can be overlapping, but also, at least partially, distinct. Cytokines members of the IL-23/IL-17 family, critical in the development of autoimmunity, are abundantly expressed within the cutaneous lesions but also seem to be involved in chronic inflammation and damage of the synovium though, as it will be here discussed, not in all patients. In this review, we will focus on the state of the art of the molecular features of psoriatic skin and joints, focusing on the specific role of the IL-23/IL-17 pathway in each of these anatomical districts. We will then offer an overview of the approved and in-development biologics targeting this axis, emphasising how the availability of the “target” in the diseased tissues could provide a plausible explanation for the heterogeneous clinical efficacy of these drugs, thus opening future perspective of personalised therapies.

Lactate has long been considered a "waste" by-product of cell metabolism, and it accumulates at sites of inflammation. Recent findings have identified lactate as an active metabolite in cell signalling, although its effects on immune cells during inflammation are largely unexplored. Here we ask whether lactate is responsible for T cells remaining entrapped in inflammatory sites, where they perpetuate the chronic inflammatory process. We show that lactate accumulates in the synovia of rheumatoid arthritis patients. Extracellular sodium lactate and lactic acid inhibit the motility of CD4+ and CD8+ T cells, respectively. This selective control of T cell motility is mediated via subtype-specific transporters (Slc5a12 and Slc16a1) that we find selectively expressed by CD4+ and CD8+ subsets, respectively. We further show both in vitro and in vivo that the sodium lactate-mediated inhibition of CD4+ T cell motility is due to an interference with glycolysis activated upon engagement of the chemokine receptor CXCR3 with the chemokine CXCL10. In contrast, we find the lactic acid effect on CD8+ T cell motility to be independent of glycolysis control. In CD4+ T helper cells, sodium lactate also induces a switch towards the Th17 subset that produces large amounts of the proinflammatory cytokine IL-17, whereas in CD8+ T cells, lactic acid causes the loss of their cytolytic function. We further show that the expression of lactate transporters correlates with the clinical T cell score in the synovia of rheumatoid arthritis patients. Finally, pharmacological or antibody-mediated blockade of subtype-specific lactate transporters on T cells results in their release from the inflammatory site in an in vivo model of peritonitis. By establishing a novel role of lactate in control of proinflammatory T cell motility and effector functions, our findings provide a potential molecular mechanism for T cell entrapment and functional changes in inflammatory sites that drive chronic inflammation and offer targeted therapeutic interventions for the treatment of chronic inflammatory disorders.

BackgroundFatigue is a debilitating condition with a significant impact on patients' quality of life. Fatigue is frequently reported by patients suffering from primary Sjögren's Syndrome (pSS), a chronic autoimmune condition characterised by dryness of the eyes and the mouth. However, although fatigue is common in pSS, it does not manifest in all sufferers, providing an excellent model with which to explore the potential underpinning biological mechanisms.MethodsWhole blood samples from 133 fully-phenotyped pSS patients stratified for the presence of fatigue, collected by the UK primary Sjögren's Syndrome Registry, were used for whole genome microarray. The resulting data were analysed both on a gene by gene basis and using pre-defined groups of genes. Finally, gene set enrichment analysis (GSEA) was used as a feature selection technique for input into a support vector machine (SVM) classifier. Classification was assessed using area under curve (AUC) of receiver operator characteristic and standard error of Wilcoxon statistic, SE(W).ResultsAlthough no genes were individually found to be associated with fatigue, 19 metabolic pathways were enriched in the high fatigue patient group using GSEA. Analysis revealed that these enrichments arose from the presence of a subset of 55 genes. A radial kernel SVM classifier with this subset of genes as input displayed significantly improved performance over classifiers using all pathway genes as input. The classifiers had AUCs of 0.866 (SE(W) 0.002) and 0.525 (SE(W) 0.006), respectively.ConclusionsSystematic analysis of gene expression data from pSS patients discordant for fatigue identified 55 genes which are predictive of fatigue level using SVM classification. This list represents the first step in understanding the underlying pathophysiological mechanisms of fatigue in patients with pSS.

Sjögren's syndrome (SS) is an autoimmune disease characterised by breach of self-tolerance towards nuclear antigens resulting in high affinity circulating autoantibodies. Although peripheral B cell disturbances have been described in SS, with predominance of naïve 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 naïve and memory compartment of SS patients. Single CD27-IgD+ naïve, CD27+IgD+ memory unswitched and CD27+IgD- memory switched B cells were sorted by FACS from the peripheral blood of 7 SS patients. To detect the frequency of polyreactive and autoreactive clones, paired Ig VH and VL genes were amplified, cloned and expressed as recombinant monoclonal antibodies (rmAbs) displaying identical specificity of the original B cells. IgVH and VL gene usage and immunoreactivity of SS rmAbs were compared with those obtained from healthy donors (HD). From a total of 353 VH and 293 VL individual sequences, we obtained 114 rmAbs from circulating naïve (n = 66) and memory (n = 48) B cells of SS patients. Analysis of the Ig V gene repertoire did not show significant differences in SS vs. HD B cells. In SS patients, circulating naïve B cells (with germline VH and VL genes) displayed a significant accumulation of clones autoreactive against Hep-2 cells compared to HD (43.1% vs. 25%). Moreover, we demonstrated a progressive increase in the frequency of circulating anti-nuclear naïve (9.3%), memory unswitched (22.2%) and memory switched (27.3%) B cells in SS patients. Overall, these data provide novel evidence supporting the existence of both early and late defects in B cell tolerance checkpoints in patients with SS resulting in the accumulation of autoreactive naïve and memory B cells.

The resolution of inflammation is now known to be an active process, armed with a multitude of mediators both lipid and protein in nature. Melanocortins are peptides endowed with considerable promise with their proresolution and anti-inflammatory effects in preclinical models of inflammatory disease, with tissue protective effects. These peptides and their targets are appealing because they can be seen as a natural way of inducing these effects as they harness endogenous pathways of control. Whereas most of the information generated about these mediators derives from several acute models of inflammation (such as zymosan induced peritonitis), there is some indication that these mediators may inhibit chronic inflammation by modulating cytokines, chemokines, and leukocyte apoptosis. In addition, proresolving mediators and their mimics have often been tested alongside therapeutic protocols, hence have been tested in settings more relevant to real life clinical scenarios. We provide here an overview on some of these mediators with a focus on melanocortin peptides and receptors, proposing that they may unveil new opportunities for innovative treatments of inflammatory arthritis.

Follicular structures resembling germinal centres (GCs) that are characterized by follicular dendritic cell (FDC) networks have long been recognized in chronically inflamed tissues in autoimmune diseases, including the synovium of rheumatoid arthritis (RA). However, it is debated whether these ectopic structures promote autoimmunity and chronic inflammation driving the production of pathogenic autoantibodies. Anti-citrullinated protein/peptide antibodies (ACPA) are highly specific markers of RA, predict a poor prognosis, and have been suggested to be pathogenic. Therefore, the main study objectives were to determine whether ectopic lymphoid structures in RA synovium: (i) express activation-induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class-switch recombination (CSR) of Ig genes; (ii) support ongoing CSR and ACPA production; and (iii) remain functional in a RA/severe combined immunodeficiency (SCID) chimera model devoid of new immune cell influx into the synovium.Using immunohistochemistry (IHC) and quantitative Taqman real-time PCR (QT-PCR) in synovial tissue from 55 patients with RA, we demonstrated that FDC+ structures invariably expressed AID with a distribution resembling secondary lymphoid organs. Further, AID+/CD21+ follicular structures were surrounded by ACPA+/CD138+ plasma cells, as demonstrated by immune reactivity to citrullinated fibrinogen. Moreover, we identified a novel subset of synovial AID+/CD20+ B cells outside GCs resembling interfollicular large B cells. In order to gain direct functional evidence that AID+ structures support CSR and in situ manufacturing of class-switched ACPA, 34 SCID mice were transplanted with RA synovium and humanely killed at 4 wk for harvesting of transplants and sera. Persistent expression of AID and Igamma-Cmu circular transcripts (identifying ongoing IgM-IgG class-switching) was observed in synovial grafts expressing FDCs/CD21L. Furthermore, synovial mRNA levels of AID were closely associated with circulating human IgG ACPA in mouse sera. Finally, the survival and proliferation of functional B cell niches was associated with persistent overexpression of genes regulating ectopic lymphoneogenesis.Our demonstration that FDC+ follicular units invariably express AID and are surrounded by ACPA-producing plasma cells provides strong evidence that ectopic lymphoid structures in the RA synovium are functional and support autoantibody production. This concept is further confirmed by evidence of sustained AID expression, B cell proliferation, ongoing CSR, and production of human IgG ACPA from GC+ synovial tissue transplanted into SCID mice, independently of new B cell influx from the systemic circulation. These data identify AID as a potential therapeutic target in RA and suggest that survival of functional synovial B cell niches may profoundly influence chronic inflammation, autoimmunity, and response to B cell-depleting therapies.