Maria Juarez

Expression of FcRL4 defines a pro-inflammatory, RANKL-producing B cell subset in rheumatoid arthritis

Objectives The success of B cell targeting therapies has highlighted the importance of B cells in rheumatoid arthritis pathogenesis. We have previously shown that B cells in the RA synovium are capable of producing pro-inflammatory and bone-destructive cytokines including RANKL. Here we sought to characterise the nature and functional relevance of the RANKL-producing B cell subset in the RA synovium. Methods Synovial fluid and peripheral blood B cells from patients with RA were analysed by flow cytometry for markers of B cell differentiation and activation and for chemokine receptors. FcRL4+ and FcRL4− B cells sorted from synovial fluid were analysed for cytokine expression using Taqman low-density arrays. Synovial tissue biopsies obtained from patients with RA were analysed by immunofluorescence for CD20, RANKL and FcRL4. FCRL4 mRNA expression was determined in synovial tissue of RA patients and non-inflammatory control subjects by real-time PCR. Results RANKL-producing B cells in RA synovial tissue and fluid were identified as belonging to a distinct subset of B cells defined by expression of the transmembrane protein FcRL4. FcRL4+ B cells express a distinct combination of cytokines and surface proteins indicating a function distinct from that of FcRL4− B cells. Notably, FcRL4+ B cells expressed high levels of TNF-α and RANKL mRNA. Conclusions We have identified a novel pro-inflammatory B cell population in the RA synovium which is defined by expression of FcRL4 and responsible for RANKL production. This B cell population expresses high levels of CD20, and its removal by rituximab may contribute to the anti-inflammatory effect of this drug.

Expression of chemokines CXCL4 and CXCL7 by synovial macrophages defines an early stage of rheumatoid arthritis

Background and objectives For our understanding of the pathogenesis of rheumatoid arthritis (RA), it is important to elucidate the mechanisms underlying early stages of synovitis. Here, synovial cytokine production was investigated in patients with very early arthritis. Methods Synovial biopsies were obtained from patients with at least one clinically swollen joint within 12 weeks of symptom onset. At an 18-month follow-up visit, patients who went on to develop RA, or whose arthritis spontaneously resolved, were identified. Biopsies were also obtained from patients with RA with longer symptom duration (>12 weeks) and individuals with no clinically apparent inflammation. Synovial mRNA expression of 117 cytokines was quantified using PCR techniques and analysed using standard and novel methods of data analysis. Synovial tissue sections were stained for CXCL4, CXCL7, CD41, CD68 and von Willebrand factor. Results A machine learning approach identified expression of mRNA for CXCL4 and CXCL7 as potentially important in the classification of early RA versus resolving arthritis. mRNA levels for these chemokines were significantly elevated in patients with early RA compared with uninflamed controls. Significantly increased CXCL4 and CXCL7 protein expression was observed in patients with early RA compared with those with resolving arthritis or longer established disease. CXCL4 and CXCL7 co-localised with blood vessels, platelets and CD68+ macrophages. Extravascular CXCL7 expression was significantly higher in patients with very early RA compared with longer duration RA or resolving arthritis Conclusions Taken together, these observations suggest a transient increase in synovial CXCL4 and CXCL7 levels in early RA.

Inflammatory regulation of glucocorticoid metabolism in mesenchymal stromal cells

Abstract Objective Tissue glucocorticoid (GC) levels are regulated by the GC-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). This enzyme is expressed in cells and tissues arising from mesenchymal stromal cells. Proinflammatory cytokines dramatically increase expression of 11β-HSD1 in stromal cells, an effect that has been implicated in inflammatory arthritis, osteoporosis, obesity, and myopathy. Additionally, GCs act synergistically with proinflammatory cytokines to further increase enzyme expression. The present study was undertaken to investigate the mechanisms underlying this regulation. Methods Gene reporter analysis, rapid amplification of complementary DNA ends (RACE), chemical inhibition experiments, and genetic disruption of intracellular signaling pathways in mouse embryonic fibroblasts (MEFs) were used to define the molecular mechanisms underlying the regulation of 11β-HSD1 expression. Results Gene reporter, RACE, and chemical inhibitor studies demonstrated that the increase in 11β-HSD1 expression with tumor necrosis factor α (TNFα)/interleukin-1β (IL-1β) occurred via the proximal HSD11B1 gene promoter and depended on NF-κB signaling. These findings were confirmed using MEFs with targeted disruption of NF-κB signaling, in which RelA (p65) deletion prevented TNFα/IL-1β induction of 11β-HSD1. GC treatment did not prevent TNFα-induced NF-κB nuclear translocation. The synergistic enhancement of TNFα-induced 11β-HSD1 expression with GCs was reproduced by specific inhibitors of p38 MAPK. Inhibitor and gene deletion studies indicated that the effects of GCs on p38 MAPK activity occurred primarily through induction of dual-specificity phosphatase 1 expression. Conclusion The mechanism by which stromal cell expression of 11β-HSD1 is regulated is novel and distinct from that in other tissues. These findings open new opportunities for development of therapeutic interventions aimed at inhibiting or stimulating local GC levels in cells of mesenchymal stromal lineage during inflammation.

Synovial DKK1 expression is regulated by local glucocorticoid metabolism in inflammatory arthritis

IntroductionInflammatory arthritis is associated with increased bone resorption and suppressed bone formation. The Wnt antagonist dickkopf-1 (DKK1) is secreted by synovial fibroblasts in response to inflammation and this protein has been proposed to be a master regulator of bone remodelling in inflammatory arthritis. Local glucocorticoid production is also significantly increased during joint inflammation. Therefore, we investigated how locally derived glucocorticoids and inflammatory cytokines regulate DKK1 synthesis in synovial fibroblasts during inflammatory arthritis.MethodsWe examined expression and regulation of DKK1 in primary cultures of human synovial fibroblasts isolated from patients with inflammatory arthritis. The effect of TNFα, IL-1β and glucocorticoids on DKK1 mRNA and protein expression was examined by real-time PCR and ELISA. The ability of inflammatory cytokine-induced expression of the glucocorticoid-activating enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to sensitise fibroblasts to endogenous glucocorticoids was explored. Global expression of Wnt signalling and target genes in response to TNFα and glucocorticoids was assessed using a custom array.ResultsDKK1 expression in human synovial fibroblasts was directly regulated by glucocorticoids but not proinflammatory cytokines. Glucocorticoids, but not TNFα, regulated expression of multiple Wnt agonists and antagonists in favour of inhibition of Wnt signalling. However, TNFα and IL-1β indirectly stimulated DKK1 production through increased expression of 11β-HSD1.ConclusionsThese results demonstrate that in rheumatoid arthritis synovial fibroblasts, DKK1 expression is directly regulated by glucocorticoids rather than TNFα. Consequently, the links between synovial inflammation, altered Wnt signalling and bone remodelling are not direct but are dependent on local activation of endogenous glucocorticoids.

Fibroblasts as therapeutic targets in rheumatoid arthritis and cancer

Rheumatoid arthritis is a common chronic inflammatory disease that causes progressive synovial inflammation resulting in irreversible joint destruction, chronic disability and premature mortality. Although it is recognised that in rheumatoid arthritis, inflammation and its persistence result from complex interactions between haematopoietic and stromal cells, research into the pathogenesis of the disease has traditionally concentrated on cells and cytokines of the immune system, neglecting the role of stromal cells. As a consequence, new biologic treatments have been developed, which have led to a step-change in the management of the disease. Nevertheless, these treatments do not reverse tissue damage or lead to disease cure and are not effective for all patients. Furthermore, at best they induce a significant clinical response (ACR70) in less than 60% of patients, most of whom will relapse on treatment withdrawal, suggesting that additional therapeutic targets, responsible for complete resolution of inflammation, remain to be discovered. An increasing body of evidence implicates rheumatoid arthritis synovial fibroblasts in driving the persistent, destructive characteristics of the disease. In this paper, we discuss the evidence implicating synovial fibroblasts in the pathogenesis of rheumatoid arthritis and explore their role as therapeutic targets.

DKK1 expression by synovial fibroblasts in very early rheumatoid arthritis associates with lymphocyte adhesion in an in vitro flow co-culture system.

BackgroundSynovial fibroblasts play a key role in joint destruction and regulation of the inflammatory infiltrate in established rheumatoid arthritis (RA). The mechanisms by which this occurs in the earliest stages of RA are largely unknown. We investigated the role of Dickkopf-related protein 1 (DKK1) produced by synovial fibroblasts of patients with very early rheumatoid arthritis (VeRA).MethodsFibroblasts were isolated from the disease-modifying anti-rheumatic drug–naive Birmingham early arthritis cohort of patients with new onset of clinically apparent arthritis and inflammatory symptoms of ≤12 weeks’ duration, who at follow-up had either resolving arthritis or RA. Endothelial fibroblast co-cultures were formed using porous filters, and lymphocyte adhesion to co-cultures was assessed using phase-contrast microscopy. DKK1 gene expression and secretion were quantified by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively.ResultsSynovial fibroblasts from patients with VeRA expressed significantly higher levels of DKK1 messenger RNA than those from patients with resolving arthritis. A similar trend was observed for DKK1 protein secretion. In co-culture constructs, more DKK1 tended to be secreted in co-cultures incorporating fibroblasts from VeRA than in co-cultures from non-inflamed joints and resolving arthritis. DKK1 secretion during co-culture positively correlated with lymphocyte adhesion.ConclusionsAlterations in DKK1 could be involved in the pathogenesis and perpetuation of the inflammatory response in the earliest clinically apparent stages of RA.

Detection of antibodies to citrullinated tenascin-C in patients with early synovitis is associated with the development of rheumatoid arthritis.

Early treatment of rheumatoid arthritis (RA) results in more effective disease suppression and can be key to a successful patient response. However, not all people who exhibit early synovitis develop RA; for example, in some, synovial inflammation resolves spontaneously.1 The factors that drive RA development remain unclear and clinical tools to predict RA development are imperfect. Tenascin-C is a proinflammatory matrix molecule that is absent from healthy joints but highly expressed in the joints of patients with RA.2 ,3 We identified an immunodominant peptide in citrullinated tenascin-C, cTNC5, antibodies against which are detected in around half of the patients with RA, and can be found years before disease onset in some individuals.4 Here, we sought to determine if anti-cTNC5 antibodies can discriminate among people with early synovial inflammation those who develop RA and those with other outcomes. Sera from 263 patients in the Birmingham early arthritis cohort were analysed. Patients were disease-modifying antirheumatic drug (DMARD) naive with clinically apparent synovitis of ≥1 joint and with inflammatory joint symptoms of ≤3 months’ duration. Patients were followed for 18 months to ensure development of full disease phenotype and to allow any resolving arthritis time to resolve. At 18 months, patients were assigned to the following outcome categories: persistent RA according to the American College of Rheumatology (ACR) 2010 criteria5 (RA, n=101), persistent non-RA arthritis (PNRA, n=66) and resolving arthritis (no clinically apparent joint swelling, no DMARD/steroid use in the previous 3 months, n=96). …

1.66 CXCL4 and CXCL7 expression on macrophages: a potential predictor of disease outcome in patients presenting with early synovitis?

Background and Objectives Within the context of a comprehensive study of the pathology of the early stages of rheumatoid arthritis (RA) we compared cytokine mRNA expression in the synovium of patients with early inflammatory arthritis who later progressed to rheumatoid arthritis with that of patients with a resolving disease course. Interestingly, we found a trend towards higher expression of platelet related chemokines CXCL4 and CXCL7 mRNA in early RA synovium. We therefore investigated CXCL4 and CXCL7 expression at the protein level and its co-localisation with platelets, macrophages and blood vessels. Methods Synovial tissue biopsies were obtained from treatment naïve patients presenting with at least one clinically swollen joint within the first 12 weeks of symptom onset. Patients who went on to develop RA (according to the 1987 ACR criteria) at an 18 month follow-up (n = 8), as well as patients whose arthritis spontaneously resolved (n = 9) were included. In addition, biopsies collected from longer duration (> 12 weeks) treatment naïve RA patients (n = 10) and patients with mechanical symptoms undergoing knee arthroscopy without obvious signs of inflammation were included as controls (n = 7). Synovial tissue sections were stained with antibodies specific to CXCL4 or CXCL7, CD41, CD68 and vWF using immunofluorescence and staining was quantified using Zeiss imaging software. Specificity of CXCL7 staining was confirmed by blocking with recombinant cytokine. Results We observed a statistically significant increase of CXCL4 and CXCL7 protein expression in patients with early RA when compared to early resolvers (CXCL4 p = 0.036, CXCL7 p = 0.011). This increase reflected a transient stage of early disease, as in treatment-naïve patients with more than 12 weeks disease duration the expression level of these chemokines was found at levels comparable to non-inflamed synovium. Both CXCL4 and CXCL7 co-localised with blood vessels, platelets and CD68+ macrophages within the synovial tissue. However, only staining found outside blood vessels, which co-localised largely with CD68 as a marker for macrophages, differed between synovium from patients with resolving, early and established arthritis (CXCL4 p = 0.063, CXCL7 p = 0.028). Conclusions We have identified two chemokines, CXCL4 and CXCL7, that are expressed at higher levels on macrophages during a transient phase in early RA. Future work will investigate whether these chemokines play a role in disease progression and/or may be used as biomarkers for prediction of disease outcome.

A1.53 A novel pro-inflammatory B cell population in the rheumatoid synovium can be identified by expression of FCRL4

Background and Objectives The importance of B cells in the pathogenesis of rheumatoid arthritis (RA) is highlighted by the clinical effectiveness of rituximab. Potential mechanisms for B cells in driving disease processes include autoantibody production, antigen presentation and cytokine secretion. Our recent findings suggest a pro-inflammatory and destructive role for B cells via production of RANKL, TNF-α and IL-6. A memory B cell subset in tonsils which expresses Fc receptor-like 4 (FcRL4) has previously been found to produce RANKL. We sought to determine whether the RANKL-expressing B cells present in the RA synovium belong to this subset. Methods: Synovial fluid and peripheral blood cells from RA patients were analysed for B cell differentiation markers by flow cytometry. Synovial tissue samples from RA patients were assessed using immunofluorescence and immunohistochemistry. FcRL4 mRNA expression was determined in synovial samples from RA patients and uninflamed control subjects by real-time PCR. FcRL4+ and FcRL4- CD19+ B cells were sorted from synovial fluid using a MoFlo cell sorter. mRNA expression of 48 cytokines was determined using Taqman microfluidic cards. Results FcRL4 expressing B cells were detected in the synovial fluid (median 14% of B cells) at a significantly higher frequency compared to peripheral blood (median 0.04%; p = 0.004). RANKL was significantly enriched in the FcRL4+ B cell population compared to the FcRL4- population. FcRL4+ B cells were predominantly switched memory B cells, expressing significantly higher levels of CD80, CD86, CD95, CD20 and CD11c than FcRL4- cells. Sorted FcRL4+ B cells expressed significantly higher mRNA levels of RANKL and TNF-α than FcRL4- B cells. FcRL4+ B cells expressing RANKL were present in synovial tissue, and were distributed under the synovial lining layer and in perivascular cuffs. FcRL4 mRNA was detected in synovium from RA patients but not uninflamed controls, and FcRL4 expression in RA synovium correlated positively with the degree of leukocyte infiltration. Conclusions We have identified a novel pro-inflammatory subset of B cells in the RA synovium which expresses RANKL and TNF-α and shows considerable expression of co-stimulatory molecules indicating a potential pathogenic role in RA. As these cells express high levels of CD20 it is conceivable that their removal contributes to the anti-inflammatory clinical effect of rituximab.

FRI0355 Microarray Analysis of Early and Late RA Human Synovial Fibroblasts Reveals A Unique Gene Expression Pattern in Early Disease

Background Human synovial fibroblasts are implicated in rheumatoid arthritis (RA) pathogenesis at two levels: they promote cartilage and bone destruction and contribute to persistent joint inflammation. Significant damage to RA joints occurs within the first year of disease and thus early aggressive treatment is advocated. Special attention has been given to the “early window of opportunity” in the first 3 months of disease. Despite this, whereas much is known about synovial fibroblasts taken from very late disease at joint replacement, little is known about fibroblast biology in early RA. We hypothesised that by studying synovial fibroblasts from patients in this early window we would identify novel stromal biomarkers and therapeutic targets in very early disease. Objectives To describe the gene expression pattern of human synovial fibroblasts from patients with early RA of up to three months symptom duration and compare it to that of synovial fibroblasts from patients with longstanding RA. Methods Synovial tissue was obtained from patients with longstanding RA undergoing joint replacement (n=8, mean disease duration 20 years) or patients with synovitis of at least one joint and symptom duration of up to 3 months fulfilling 1987 criteria for RA by 18 months follow-up (n=14) via ultrasound guided synovial biopsy. Normal (n=8) and resolving arthritis (n=16) fibroblasts were used as controls. RNA was extracted and treated with the RNA clean and concentrator kit. Samples were hybridised onto Agilent Sure Print G3 8x60k v2 arrays. Scanned images were analysed with Agilent Feature Extraction Software. Partek Genomics Suite was used to analyse gene expression using a 1-way fixed model ANOVA to create pairwise contrasts with a corrected step-up p-value (False Discovery Rate by Benjamini) and a fold change for differences in gene expression. Age, gender and joint of origin were found to contribute to variance and thus included in the ANOVA model. Antibody status did not contribute to variance of the datasets and was thus excluded. Results 73 out of a total of 50,599 probes, detected differentially expressed transcripts between early and late RA (FDR<0.05, fold change ±1.5). Some of the probes denoted protein coding genes whilst others denoted pseudogenes, small nuclear and long non-coding RNAs. To ascertain those genes that were uniquely differentially expressed between early and late RA, normal and resolving samples were compared to late RA samples. In total 13 probes were shared between this analysis and that of early and late RA, leaving 60 probes exclusively differentiating early RA. Of these, 27 were upregulated and 33 downregulated in early RA compared to late RA. Gene ontology revealed that 38.2% of the differentially expressed genes were involved in transcription regulation and 23.5% in RNA metabolic processes. Detailed analysis and validation of differentially expressed targets is ongoing. Conclusions We have identified a distinct gene expression pattern in synovial fibroblasts of patients with early RA. By analysing normal and resolving arthritis samples, we have been able to ensure that this pattern is unique to early RA fibroblasts and not due to non-specific joint inflammation. Ongoing in depth analysis and validation of targets will allow unravelling of modifiable disease mechanisms in early RA. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3703