D.J. Veale

Items for developing revised classification criteria in systemic sclerosis: Results of a consensus exercise

Classification criteria for systemic sclerosis (SSc; scleroderma) are being updated. Our objective was to select a set of items potentially useful for the classification of SSc using consensus procedures, including the Delphi and nominal group techniques (NGT).

A1.34 Oncostatin M differentially regulates TNFα-induced pro-inflammatory mechanisms in the RA joint

Introduction Oncostatin M (OSM) is a pleiotropic cytokine that has both agonistic and antagonistic effects depending on the inflammatory microenvironment. This study examines the effect of OSM on TNFα-induced pro-inflammatory mechanisms and on Notch-1 signalling in Rheumatoid Arthritis. Methods Primary RA synovial fibroblasts (RASFC) isolated from RA synovial biopsiesobtained at time of knee arthroscopy and human dermal microvascular endothelial cells (HMVEC) were grown to confluence. RASFC and HMVEC were cultured with OSM (10ng/ml) alone or in combination with increasing concentrations of TNFα (0.01 – 1ng/ml). IL-6, IL-8, RANTES, GROα and MCP-1 cytokines/chemokines were quantified in culture supernatants by ELISA. Functionally, angiogenesis and RASFC invasion were assessed by matrigel tube formation and Transwell invasions assays, VEGF in cell lysates by Real-time PCR. Finally, Notch-1, its ligands Delta-like-ligand 4 (DLL4)and Jagged-1 (Jag-1) and downstream transcriptional repressors-Hey-1 and Hey-2 were quantified by Real-time PCR. Results OSM alone significantly induced IL-6 and MCP-1 while inhibiting IL-8 and GROα in RASFC and HMVEC culture supernatants. OSM alone induced RANTES expression in HMVEC with no effect observed for RASFC. OSM potentiated the effect of TNFα on IL-6 and MCP-1 secretion from RASFC and HMVEC. Conversely OSM inhibited TNFα-induced-IL-8 and GROα secretion from RASFC and HMVEC. Interestingly, OSM significantly inhibited TNFa-induced RANTES expression in HMVEC and conversely potentiated this effect in RASFC. At a functional level, OSM induced RASFC and HMVEC invasion and network formation and induced VEGF expression compared to basal control. Finally OSM significantly induced Notch-1 and Hey-2 in RASFC and HMVEC, but interestingly differentially regulated the Notch-1 ligands, with induction of Jag-1 only observed in RASFC, and induction of DLL-4 only observed in HMVEC. Conclusion OSM is a pleiotrophic cytokine that differentiallyregulates pro-inflammatory mechanisms within the inflamed joint, effects that appear to be dependent on cell type and the inflammatory microenvironment. Targeting OSM or downstream signalling pathways may have therapeutic potential.

A1.08 CD141+ CLEC9A+ dendritic cells are enriched in an active state in the inflamedsynovium and contribute to synovial inflammation in rheumatoid arthritis

Introduction Dendritic cells (DC) are a heterogeneous population of professional antigen presenting cells. Currently their classification within blood and skin has been well characterised however their identification within other tissues, in particular in the context of autoimmunity is limited. This work aims to identify DC within the inflamed synovium and elucidate how DC are altered by the microenvironment within the joint. Methods DC immunophenotyping was assessed in whole blood (WB), synovial fluid (SF) and synovial tissue (ST) using 9 colour flow cytometry. CD141+ DC were isolated from SF, cultured overnight +/- TLR3 ligands and stained with a panel of antibodies. Monocyte derived DC were cultured in the presence of explant conditioned media (ECM) or SF. MoDC treated with SF were also cocultured with CD4+ T cells for 5 days, after which T cell proliferation and intracellular cytokines were examined. Results RA patients have a significant decrease (p < 0.05) in mDC in WB compared to HC. CD11c+ HLADR+ myeloid cells are increased in the ST compared to WB, express higher levels of CD80/CD40 and express CD64, CD14 and CD16. These cells may represent a myeloid subset of cells unique to inflammatory tissues. DC in SF are more mature than PB DC (increased CD80/CD40; p < 0.05). SF is enriched in CD141+CLEC9A+DC compared to PB (p < 0.005). These cells are rare in WB and previously unidentified in SF. SF CD141+ DC express TREM1 while blood CD141+ DC do not. SF CD141+ DC express higher levels of CD80/CD86 and CD40 compared to blood CD141+ DC. To examine the effect of the synovial microenvironment on DC, MoDC were cultured in the presence of ECM or SF. Both induced a significant increase in the expression of CD80 (p < 0.05 and p < 0.0001). Finally DC treated with SF and subsequently cocultured with CD4+ T cells have enhanced IFNγ production and T cell proliferation. Conclusion Unique DC subsets are found in both ST and SF within the joint. These cells display a more activated phenotype than DC in circulation and the inflammatory nature of the joint contributes to this activation. These uniquely activated DC induce T cell proliferation and cytokine production.

SAT0007 Distinct macrophage phenotype and bioenergetic profiles in rheumatoid arthritis

Background Diversity of macrophage subsets within the joint remains unknown. The concept of macrophage polarisation into M1 inflammatory macrophages and M2 tissue-resolving macrophages, parelled by changes in the bioenergetic cell profile, has received much attention. Hence, we aimed to examine the phenotype of macrophages within the inflamed RA joint, along with the metabolic and inflammatory capacity of RA monocyte-derived macrophages compared to healthy individuals. Objectives To examine the phenotype, metabolic and inflammatory profile of pro-inflammatory and anti-inflammatory macrophages within the inflamed RA joint. Methods Blood obtained from healthy and RA donors, CD14 +cells sorted and differentiated into macrophages for 8 days. Macrophages were polarised to either M1 (LPS and IFNγ) or M2 (IL-4). Markers of polarisation, metabolism and inflammation were quantified by Real Time-PCR. Seahorse technology measured the major energy-using pathway, oxidative phosphorylation (OCR). Finally, synovial tissue (ST) was digested to yield a single cell suspension, this was then stained using a panel of fluorochrome antibodies (CD45, CD40, CD68, CD64, CD163, CD206, CD253), and subsequently analysed using FlowJo software. Results M1 macrophages were confirmed by increased expression of KLF6 while M2 macrophages expressed high TGM2, PPARG and STAB1). M1 cells had significantly higher expression of pro-glycolytic genes (HIF1α, HK2, LDHA, PFKFB3 and PKM2), which were deficient in M2 macrophages and higher compared to healthy control. This was paralleled by higher pro-inflammatory cytokines levels (IL-8, OSM, MCP-1, RANTES IRAK-1, CCR5 and SOCS3) in M1 vs M2 macrophages, with RA derived macrophages showing higher expressions of pro-inflammatory mediators compared to healthy control. G6PD, PHD3 and PDK1/2 were significantly decreased in M1 yet increased in M2 macrophages, and along with this seahorse technology demonstrated that M2 macrophages have higher baseline OCR. Finally, ST analysis determined approximately 40% of CD45 +cells are positive for the pan-macrophage marker CD68. Interestingly, the classical paradigm of M1 and M2 macrophages is not found. Instead, a spectrum of macrophages were identified with approximately 57% of ST macrophages expressing M2 markers (CD206, CD163) with the M1 activation marker CD40. Conclusions This study demonstrated distinct metabolic profiles in M1/M2 RA macrophages; their opposing roles in perpetuating and resolving inflammation, respectively. We have identified, for the first time, a transitionary subtype of tissuespecific macrophages, suggesting that these cells remain plastic and function according to their microenvironment This study demonstrated distinct metabolic profiles in M1/M2 RA macrophages; their opposing roles in perpetuating and resolving inflammation, respectively. We have identified, for the first time, a dominant transitionary subtype of tissue specific macrophages with at least six other phenotypically distinct subtypes also present, suggesting that these cells remain plastic and function according to their microenvironment. Disclosure of Interest None declared

SAT0021 Human CD4 T cells and synovial fibroblasts cooperate to promote inflammation in the RA synovial joint

Background Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by synovial tissue proliferation and degradation of articular cartilage. Activated synovial fibroblasts proliferate and express matrix-degrading proteases, adhesion molecules and proinflammatory cytokines, which contribute to cartilage and joint destruction. Moreover, synovial cell activation correlates with infiltration of inflammatory lymphocytes and monocytes which in turn contribute to synoviocyte activation, thus further exacerbating inflammation. Objectives The functional relationship linking fibroblasts and T lymphocytes in this complex microenvironment has yet to be characterised. Therefore, we established an in vitro model to examine the outcomes of co-culturing activated human CD4 T cells with RA synovial fibroblasts. Methods Co-culture assays were carried out using immortalised K4IM RA synovial fibroblasts or synovial fibroblast cells derived from arthroscopy biopsies of RA patients. Human CD4 T cells were stained with a proliferation-tracking dye and co-cultured with pre-seeded synovial fibroblasts for 5 days. The resulting cell cultures and supernatants were examined for proliferation, cytokine production, secretion of matrix metalloproteinases and expression of adhesion molecules. Results We found that CD4 T cells and K4IM cells reciprocally induced an increased expression of adhesion molecules ICAM and VCAM. Furthermore, co-culture of CD4 T cells and synovial fibroblasts resulted in proliferation of CD4 T cells expressing increased levels of the proinflammatory cytokines IFN-γ and IL-17a and RANKL after 5 days. Lastly, co-culture of T cells and synovial fibroblasts resulted in secretion of IL-6, IL-8, IFN-γ and IL-17a and matrix metalloproteinases MMP-1 and MMP-3. Conclusions These results indicate that CD4 T cells work mutually with synoviocytes to create an inflammatory microenvironment likely to promote joint destruction. Future studies will characterise the role of glucose metabolism in these cells and investigate if metabolism is intrinsically coupled to effector functions in these cells. Disclosure of Interest None declared

THU0059 Oncostatin m induces inflammation and differentially regulates tnf alpha-induced pro-inflammatory mechanisms and notch signalling in the ra joint

Background Oncostatin M (OSM) is a pleiotropic cytokine, highly expressed in the RA joint that displays both agonistic and antagonistic effects depending on the inflammatory microenvironment. This study examines the effect of OSM on inflammation, the Notch-1 signalling pathway which plays a critical role in vascular development and angiogenesis and finally on TNFα-induced pro-inflammatory mechanisms in Rheumatoid Arthritis. Objectives To examine the effect of OSM on cytokine/chemokine production, angiogenesis and the Notch-1 signalling pathway in synovial fibroblasts and endothelial cells and whether OSM potentiates the effects of TNFα-induced pro-inflammatory effects. Methods Primary RA synovial fibroblasts (RASFC) isolated from RA synovial biopsies obtained at time of knee arthroscopy and human dermal microvascular endothelial cells (HMVEC) were grown to confluence. RASFC and HMVEC were cultured with OSM (10ngml) alone or in combination with increasing concentrations of TNFα (0.01–1ng/ml). IL-6, IL-8, RANTES, GROα and MCP-1 cytokines/chemokines were quantified in culture supernatants by ELISA. Functionally, angiogenesis and invasion were assessed by matrigel tube formation and Transwell invasions assays respectively, and VEGF in cell lysates quantified by Real-time PCR. Finally, Notch-1, its ligands Delta-like-ligand 4 (DLL-4) and Jagged-1 (Jag-1) and downstream transcriptional repressors – Hey-1 and Hey-2 were quantified by Real-time PCR. Results OSM alone significantly induced IL-6 and MCP-1 while inhibiting IL-8 and GROα in RASFC and HMVEC culture supernatants, compared to basal control. OSM alone induced RANTES expression in HMVEC with little effect observed for RASFC. OSM potentiated the effect of increasing concentrations of TNFα on IL-6 and MCP-1 secretion from RASFC and HMVEC. Conversely OSM significantly inhibited TNFα-induced IL-8 and GROα secretion from both RASFC and HMVEC. Interestingly, OSM significantly inhibited TNFa-induced RANTES expression in HMVEC yet conversely potentiated this effect in RASFC. At a functional level, OSM induced both RASFC and HMVEC invasion and induced network formation and VEGF expression in HMVEC. OSM significantly induced Notch-1 in RASFC and HMVEC in a time dependent manner, but interestingly differentially regulated the Notch-1 ligands, with induction of Jag-1 only observed in RASFC, and induction of DLL-4 only observed in HMVEC. Finally OSM differentially regulated Notch-1 downstream transcriptional repressors in HMVEC, significantly inducing Hey-2 while simultaneously inhibiting Hey-1. Conclusions OSM is a pleiotropic cytokine that displays divergent effects with both pro- and anti-inflammatory mechanisms within the inflamed joint, effects that appear to be dependent on cell type and the inflammatory microenvironment. Targeting OSM or downstream signalling pathways may lead to new potential therapeutic strategies or adjuvant therapies, particularly for those patients who have sub-optimal responses. Disclosure of Interest None declared

SAT0013 The pathogenic role of myeloid CD141+ dendritic cells in inflammatory arthritis

Background Dendritic cell (DC) are a heterogeneous group of antigen presenting cells that can be subdivided into CD1c+ & CD141+ DC. CD141+ DC are a rare population of DC that were first discovered in 2010 in human peripheral blood. Due to their rarity very little is known about the function of these cells in other tissue in or indeed disease. These newly described DC subset have thus never been described in Inflammatory Arthritis (IA) or any of the rheumatic diseases. Objectives To identify CD141 DC in IA synovium and functionally assess if these cells play a pathogenic role in IA. Methods CD141+DC were magnetically purified from synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) stimulated and stained with a panel of fluorochrome conjugated antibodies for multicolour flow cytometry. CD141+ DC isolated and purified from IA synovial fluid were subsequently cocultured with allogenic CD3+ T cells for 6d after which intracellular cytokine production was assessed by flow cytometry. Supernatants from this DC-T cell cocultures were used to treat synovial fibroblasts & the expression of adhesion molecules, cytokines & MMPs was measured. Finally using sorted populations of CD141+ DC from SFMC and PBMC, RNA sequencing was performed and differentially expressed genes and interaction network analysis were identified using the DeSeq2 R package, Ingenuity® Pathway Analysis (IPA) and InnateDB and Cytoscape. Results Within IA synovial fluid (SF), CD141+ DC are significantly enriched compared to WB & express higher levels of the costimulatory activation markers CD80 CD86 and CD40. Following coculture of these SF CD141+ DC with CD3+ T cells, CD141+ DC induce both CD8+ & CD4+ T cell proliferation. SF CD141+ DC induce Granzyme B production from CD8+ T cells & TNFα, IFNγ & GMCSF from CD4+ T cells. The IA synovium consists of a complex interplay of multiple cell types. Therefore next we examined the effect of this CD141+ DC-T cell interaction on the key invasive cells in the synovium – synovial fibroblasts. Supernatants from CD141 activated T cells were cultured with fibroblasts & induced expression of ICAM-1, IL-6, IL-8, MMP1 & MMP3. SF CD141 expressed significantly higher levels of the the hypoxia marker TREM1, activation of which induces further expression of CD80, CD86 and CD40. Coculture of these TREM1 activated CD141 with CD3+T cells increases IFNγ and IL-17a production. Finally RNASeq analysis revealed that there are 2089 differentially expressed genes between SF CD141+DC & WB CD141+DC. These genes are involved in a number of key pathways such as energy metabolism, chemokine & cytokine signalling. Principal Component Analysis (PCA) revealed that CD141+ DC with the synovium are distinctly different from blood CD141+ DC Conclusions CD141+ DC are enriched in the IA joint in an active state. RNASeq analysis revealed they are distinct from blood CD141+DC and our in vitro data would support the hypothesis that these CD141+DC contribute to synovial inflammation and joint destruction. Disclosure of Interest None declared

A8.13 TLR regulated MIR-23A down-regulated in psoriatic arthritis

Background and purpose Psoriatic Arthritis (PsA) is amultifaceted disease associated with Psoriasis. Key pathogeneic mechanisms seen at all disease sites include angiogenesis, invasion, migration, proliferation and more specifically to joint involvement, osteoclastogenesis. These aberrant processes have been previously associated with dysregulated miRNA expression, a class of small non-coding RNAs which exert their function through suppression of specific target genes. More specifically, the miR-23a Cluster (miR-23a, miR-27a and miR-24–2) as in other chronic conditions, such as cancer. Methods Synovial tissue biopsies and/or peripheral blood mononuclear cells (PBMC) were obtained from PsA (n = 8), osteoarthritis (OA) (n = 7), and healthy controls (n = 8). To determine factors involved in regulating miRNA expression, primary PsA synovial fibroblasts (SFC) were isolated and cultured with candidate pro-inflammatory stimuli including TLR ligands: Pam3CSK4 (1 µg/ml), LPS (1 µg/ml), polyIC (10 µg/ml) and pro-inflammatory cytokines: IL-1β (1 ng/ml), TNFα (10 ng/ml) and IL-17 (20ng/ml). MiR-23a, miR-27a and miR-24–2 expression was quantified by RT-PCR using RNU48 as an endogenous control. Clinical demographics such as synovitis, vascularity, DAS-28 CRP, TJC, SJC and patient global were also assessed. Results All members of the miR-23a cluster, miR-23a, miR-27a and miR-24–2, were significantly decreased PsA synovial tissue versus OA (p = 0.0172). Synovial miR-23a expression negatively correlated with matched PBMC miR-23a (r=-1.00, p = 0.0167), demonstrating a dissociation in miR-23a expression between systemic and local inflammation. In addition, miR-23a expression in PsA synovial biopsies inversely correlated with DAS28-CRP (r= -0.5294, p = 0.035) and distinguished patient synovial vascularity and synovitis into high versus low groupings (all < 0.05). TLR activation via PolyIC (TLR3) and LPS (TLR4), but not Pam3CSK4 (TLR2), significantly decreased miR-23a expression in PsA SFC (all p < 0.05), with no effect observed for pro-inflammatory cytokines. Finally, in silico analysis identified putative targets for miR-23a, including PDE4B and PTK2B, which are known mediators in immune pathways, osteoclast function and angiogenic mechanisms. Conclusion Our data provides evidence that the miR-23a cluster is significantly decreased in PsAsynovium. Synovial miR-23a levels, regulated by TLRs, correlate and distinguish clinical markers. Both previously confirmed and new potential targets identified here may have putative pathogenic implications for PsA pathogenesis. Thereby, the miR-23a cluster may represent a potential novel biomarker for disease activity or elucidate new therapeutic strategies.

A8.12 Decreased expression of MIR-125A in psoriatic arthritis. Implications for joint pathogenesis

Background and aims Psoriatic Arthritis (PsA) is a chronic immune-mediated inflammatory disease, characterised by proliferation of synovial tissue and destruction of articular cartilage/bone with associated psoriasis. Dysregulated angiogenesis is one of the key early pathogenic processes in PsA. These processes may be governed by microRNA (miRNA), a class of evolutionary conserved short non-coding RNAs which function as post-transcriptional repressors of gene expression. On such miRNA is miR-125a-5p which, in cancer, has been previously associated with altered angiogenesis, invasion and migration. Objectives To examine the expression and angiogenic associations of miR-125a-5p in PsA. Methods Synovial tissue biopsies and peripheral blood mononuclear cells were obtained from patients with PsA, osteoarthritis (OA) and/or healthy controls (HC). MiR-125a-5p levels were analysed by real-time PCR using RNU48 as an endogenous control. To examine possible factors involved in regulating miR125a-5p expression, primary synovial fibroblasts (SFC) and microvascular endothelial cells (HMVEC) microvascular endothelial cells (HMVEC) were cultured with candidate pro-inflammatory stimuli including; TLR ligands (PAM, PolyIC, LPS), pro-inflammatory cytokines (TNFa, IL-1b, IL-17) and growth factors (VEGF, Ang2). Immunoistochemical analysis was performed using factor VIII (DAKO) and appropriate IgG controls, on cryostat synovial tissue sections. Clinical markers, including synovitis, vascularity, ESR, CRP, DAS28, ascertained at the time of arthroscopy were correlated with synovial miRNA expression. Results Expression of miR-125a-5p was significantly decreased in PsA synovial biopsies (n = 8) compared to OA (n = 4) (p < 0.05). No significant difference was observed at the PBMC level across PsA (n = 6), RA (n = 5) and Healthy controls (n = 5). Angiogenic growth factor, Ang2, induced miR-125a-5p in HMVEC (p < 0.05). Lower synovial expression of miR-125a-5p displayed a higher degree of factor VIII staining compared to patients with higher miR-125a-5p expression. This was further demonstrated in representative arthroscopic images. Conclusion Decreased expression of miR-125a-5p in the joint and it’s association with vascularity, which is a hallmark of PsA, suggests it may be important in mediating key pro-angiogenic/pro-inflammatory mechanisms in the synovium. Correcting these microRNA deficiencies, either by conventional pharmacological agents or as novel targets, may provide a therapeutic benefit, especially in early disease stages.

OP0083 Mir-125a Decreased in Psa Synovium and Peripheral Blood CD14+ Monocytes and Correlates To Joint Angiogenesis

Background Psoriatic Arthritis (PsA) is a chronic immune-mediated inflammatory disease, characterised by proliferation of synovial tissue and destruction of articular cartilage/bone with associated psoriasis. Dysregulated angiogenesis is one of the key early pathogenic processes in PsA. Monocyte subpopulations are increasingly recognized contributors to angiogenesis. Circulating monocytes can differentiate into tissue resident macrophages, monocyte derived dendritic cells and osteoclasts, which orchestrate both pro-inflammatory and pro-angiogenic signalling. These processes may be governed by microRNA (miRNA), a class of evolutionary conserved short non-coding RNAs which function as post-transcriptional repressors of gene expression. On such miRNA is miR-125a-5p, which in cancer, has been previously associated with altered angiogenesis, invasion and migration. Objectives To examine the expression and angiogenic associations of miR-125a-5p in PsA. Methods Synovial tissue biopsies were obtained from patients with PsA and osteoarthritis (OA). In parallel, CD14+ monocytes and CD3+ T cells were isolated from peripheral blood mononuclear cells. MiR-125a-5p levels were analysed by real-time PCR. To examine possible factors involved in regulating miR-125a-5p expression in endothelial cells,microvascular endothelial cells (HMVEC) were cultured with candidate pro-inflammatory stimuli including; TLR ligands (PAM, PolyIC, LPS), pro-inflammatory cytokines (TNFa, IL-1b, IL-17) and growth factors (VEGF, Ang2). Matrigel tube formation assays were performed with pre- and anti- 125a treated HMVEC to elucidate angiogenic function. Immunoistochemical analysis of synovial vasculature was performed using factor VIII (DAKO).Cliical markers assertained at the time of arthroscopy, includding synovitis and vascularity were correlated with synovial miRNA expression. Results Expression of miR-125a-5p was significantly decreased in PsA synovial biopsies (n=8) compared to OA (n=4) (p<0.05). CD14+ peripheral blood monocytes showed a significant decrease in miR-125a-5p compared to whole PBMC (p<0.05). No difference was observed for CD3+ T cells. Anti-125a-5p treated HMVEC displayed increased tube formations. Angiogenic growth factor, Ang2, induced miR-125a-5p in HMVEC (p<0.05). Lower synovial expression of miR-125a-5p displayed a higher degree of factor VIII staining compared to patients with higher miR-125a-5p expression. This was further demonstrated in representative arthroscopic images. Conclusions Our data demonstrates decreased expression of miR-125a in the joint and peripheral CD14+ monocytes in PsA patients. MiR-125a expression was associated with joint vascularity, a hallmark of PsA, suggesting its important role in mediating key pro-angiogenic and thereby pro-inflammatory mechanisms in the synovium. Correcting these microRNA deficiencies, either by conventional pharmacological agents or as novel targets, may provide a therapeutic benefit, especially in early disease stages. Disclosure of Interest None declared