Ronan Mullan

Human rheumatoid arthritis tissue production of IL-17A drives matrix and cartilage degradation: synergy with tumour necrosis factor-α, Oncostatin M and response to biologic therapies

IntroductionThe aim of this study was to examine IL-17A in patients, following anti-TNF-α therapy and the effect of IL-17A on matrix turnover and cartilage degradation.MethodsIL-17A expression was examined by ELISA and immunohistology in the rheumatoid arthritis (RA) joints. RA whole synovial tissue explant (RA ST), primary synovial fibroblasts (RASFC), human cartilage and chondrocyte cultures were stimulated with IL-17A +/- TNF-α and Oncostatin M (OSM). Matrix metalloproteinase (MMP) and tissue inhibitor (TIMP-1) were assessed by ELISA and zymography. Cartilage proteoglycan release was assessed histologically by Safranin-O staining. Clinical parameters, IL-17A, MMP/TIMP were assessed in patients pre/post biologic therapy.ResultsIL-17A levels were higher in RA vs osteoarthritis (OA)/normal joints (P < 0.05). IL-17A up-regulated MMP-1, -2, -9, and -13 in RA ST, RASFC, cartilage and chondrocyte cultures (P < 0.05). In combination with TNF-α and OSM, IL-17A shifted the MMP:TIMP-1 ratio in favor of matrix degradation (all P < 0.05). Cartilage proteoglycan depletion in response to IL-17A was mild; however, in combination with TNF-α or OSM showed almost complete proteoglycan depletion. Serum IL-17A was detected in 28% of patients commencing biologic therapy. IL-17A negative patients demonstrated reductions post therapy in serum MMP1/TIMP4, MMP3/TIMP1 and MMP3/TIMP4 ratios and an increase in CS846 (all P < 0.05). No significant changes were observed in IL-17A positive patients.ConclusionsIL-17A is produced locally in the inflamed RA joint. IL-17A promotes matrix turnover and cartilage destruction, especially in the presence of other cytokines, mimicking the joint environment. IL-17A levels are modulated in vivo, following anti-TNF therapy, and may reflect changes in matrix turnover.

Acute Serum Amyloid A Induces Migration, Angiogenesis, and Inflammation in Synovial Cells In Vitro and in a Human Rheumatoid Arthritis/SCID Mouse Chimera Model

Serum amyloid A (A-SAA), an acute-phase protein with cytokine-like properties, is expressed at sites of inflammation. This study investigated the effects of A-SAA on chemokine-regulated migration and angiogenesis using rheumatoid arthritis (RA) cells and whole-tissue explants in vitro, ex vivo, and in vivo. A-SAA levels were measured by real-time PCR and ELISA. IL-8 and MCP-1 expression was examined in RA synovial fibroblasts, human microvascular endothelial cells, and RA synovial explants by ELISA. Neutrophil transendothelial cell migration, cell adhesion, invasion, and migration were examined using transwell leukocyte/monocyte migration assays, invasion assays, and adhesion assays with or without anti–MCP-1/anti–IL-8. NF-κB was examined using a specific inhibitor and Western blotting. An RA synovial/SCID mouse chimera model was used to examine the effects of A-SAA on cell migration, proliferation, and angiogenesis in vivo. High expression of A-SAA was demonstrated in RA patients (p < 0.05). A-SAA induced chemokine expression in a time- and dose-dependent manner (p < 0.05). Blockade with anti-scavenger receptor class B member 1 and lipoxin A4 (A-SAA receptors) significantly reduced chemokine expression in RA synovial tissue explants (p < 0.05). A-SAA induced cell invasion, neutrophil–transendothelial cell migration, monocyte migration, and adhesion (all p < 0.05), effects that were blocked by anti–IL-8 or anti–MCP-1. A-SAA–induced chemokine expression was mediated through NF-κB in RA explants (p < 0.05). Finally, in the RA synovial/SCID mouse chimera model, we demonstrated for the first time in vivo that A-SAA directly induces monocyte migration from the murine circulation into RA synovial grafts, synovial cell proliferation, and angiogenesis (p < 0.05). A-SAA promotes cell migrational mechanisms and angiogenesis critical to RA pathogenesis.

Acute‐phase serum amyloid A regulates tumor necrosis factor α and matrix turnover and predicts disease progression in patients with inflammatory arthritis before and after biologic therapy

OBJECTIVE To investigate the relationship between acute-phase serum amyloid A (A-SAA) and joint destruction in inflammatory arthritis. METHODS Serum A-SAA and C-reactive protein (CRP) levels, the erythrocyte sedimentation rate (ESR), and levels of matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, MMP-9, MMP-13, tissue inhibitor of metalloproteinases 1 (TIMP-1), vascular endothelial growth factor (VEGF), and type I and type II collagen-generated biomarkers C2C and C1,2C were measured at 0-3 months in patients with inflammatory arthritis commencing anti-tumor necrosis factor α (anti-TNFα) therapy and were correlated with 1-year radiographic progression. The effects of A-SAA on MMP/TIMP expression on RA fibroblast-like synoviocytes (FLS), primary human chondrocytes, and RA/psoriatic arthritis synovial explant cultures were assessed using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, antibody protein arrays, and gelatin zymography. RESULTS Serum A-SAA levels were significantly (P < 0.05) correlated with MMP-3, the MMP-3:TIMP-1 ratio, C1,2C, C2C, and VEGF. The baseline A-SAA level but not the ESR or the CRP level correlated with the 28-joint swollen joint count and was independently associated with 1-year radiographic progression (P = 0.038). A-SAA increased MMP-1, MMP-3, MMP-13, and MMP/TIMP expression in RA FLS and synovial explants (P < 0.05). In chondrocytes, A-SAA induced MMP-1, MMP-3, and MMP-13 messenger RNA and protein expression (all P < 0.01), resulting in a significant shift in MMP:TIMP ratios (P < 0.05). Gelatin zymography revealed that A-SAA induced MMP-2 and MMP-9 activity. Blockade of the A-SAA receptor SR-B1 (A-SAA receptor scavenger receptor-class B type 1) inhibited MMP-3, MMP-2, and MMP-9 expression in synovial explant cultures ex vivo. Importantly, we demonstrated that A-SAA has the ability to induce TNFα expression in RA synovial explant cultures (P < 0.05). CONCLUSION A-SAA may be involved in joint destruction though MMP induction and collagen cleavage in vivo. The ability of A-SAA to regulate TNFα suggests that A-SAA signaling pathways may provide new therapeutic strategies for the treatment of inflammatory arthritis.

Downregulation of the inhibitor of apoptosis protein survivin in keratinocytes and endothelial cells in psoriasis skin following infliximab therapy

Background  Survivin, an inhibitor of apoptosis protein (IAP), has been implicated in endothelial cell stability, through inhibition of apoptosis and in cell proliferation.

A role for the high-density lipoprotein receptor SR-B1 in synovial inflammation via serum amyloid-A.

Acute phase apoprotein Serum Amyloid A (A-SAA), which is strongly expressed in rheumatoid arthritis synovial membrane (RA SM), induces angiogenesis, adhesion molecule expression, and matrix metalloproteinase production through the G-coupled receptor FPRL-1. Here we report alternative signaling through the high-density lipoprotein receptor scavenger receptor-class B type 1 (SR-B1). Quantitative expression/localization of SR-B1 in RA SM, RA fibroblast-like cells (FLCs), and microvascular endothelial cells (ECs) was assessed by Western blotting and immunohistology/fluorescence. A-SAA-mediated effects were examined using a specific antibody against SR-B1 or amphipathic alpha-Helical Peptides (the SR-B1 antagonists L-37pA and D-37pA), in RA FLCs and ECs. Adhesion molecule expression and cytokine production were quantified using flow cytometry and ELISA. SR-B1 was strongly expressed in the RA SM lining layer and endothelial/perivascular regions compared with osteoarthritis SM or normal control synovium. Differential SR-B1 expression in RA FLC lines (n = 5) and ECs correlated closely with A-SAA, but not tumor necrosis factor alpha-induced intercellular adhesion molecule-1 upregulation. A-SAA-induced interleukin-6 and -8 production was inhibited in the presence of anti-SR-B1 in human microvascular endothelial cells and RA FLCs. Moreover, D-37pA and L-37pA inhibited A-SAA-induced vascular cell adhesion molecule-1 and intercellular adhesion molecule expression from ECs in a dose-dependent manner. As SR-B1 is expressed in RA synovial tissue and mediates A-SAA-induced pro-inflammatory pathways, a better understanding of A-SAA-mediated inflammatory pathways may lead to novel treatment strategies for RA.

Consistency in assessing the Disease Activity Score-28 in routine clinical practice

With the increasing availability of biological therapies for patients with rheumatoid arthritis (RA), the need to include reliable and reproducible clinical measures of disease activity in routine practice has been emphasised.1 Disease Activity Score-28 (DAS28) is validated for use in clinical trials;2–5 however, it is not known how it performs in a clinical setting. The aim of this study was to evaluate consistency in the measurement of DAS28, and its individual components, by clinic staff in one centre with different levels of experience. Twelve patients with RA (six per exercise) were assessed for the inter-observer exercises. Five patients were assessed for the intra-observer exercise. Six members of the rheumatology team (two staff rheumatologists, two trainees in rheumatology, …

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

01.04 Enrichment of activated cd141+ dendritic cells in inflammatory arthritis

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

FRI0211 High Body Mass Index in Ankylosing Spondylitis is Associated with Greater Disease Activity and More Functional Impairmairment

Background In 2013, the first patients were entered in to ASRI – the Ankylosing Spondylitis Registry of Ireland. The primary objectives of ASRI are to provide basic descriptive epidemiological data on the Ankylosing Spondylitis (AS) population in Ireland, and to establish a registry for potential future studies of genetics, aetiology and therapeutics. Objectives In this current study we were interested in exploring the issue of obesity in an AS cohort, and the effect this has on disease activity and functional impairment. Methods A standardised detailed clinical assessment of patients is performed on each patient and entered in a web-based database. Specific measures of disease activity (BASDAI), function (BASFI and HAQ), and quality of life (ASQoL) were obtained. Body Mass Index (BMI) was calculated. The cohort was divided in to those of normal weight (BMI <25), and those that were overweight or obese (BMI >25). The 2 cohorts were compared using a number of different clinical variable using standard t-test. Results As of Dec 2014, 267 patients have been entered in the database (212 males, 55 females). The average age of the cohort is 47.8 years. The average disease duration is 21.7 years. The average delay in diagnosis is 8.2 years. As regards extra-spinal manifestations, 15.8% have Psoriasis, 40.4% have uveitis, 13% have Crohns/IBD. Of the 267 patients, 183 were overweight or obese (68.5%). There is a significant difference in BASFI scores between normal weight AS patients versus those that are overweight/obese. There is also a trend towards higher BASDAI, HAQ and ASQoL in those that are overweight/obese also. See Table below. There were a higher percentage of patients with Hypertension (30% versus 13%), Hyperlipidaemia (15% versus 7%) and Diabetes (7% versus 4%) in those that were overweight/obese. The rate of current or past smokers was very similar between the 2 cohorts, 57% (normal weight cohort) and 60% (overweight/obese cohort). Total cohort Normal weight Overweight or obese p (BMI <25) (BMI >25) No. of patients 267 84 183 BASDAI (mean ± SD) 3.9±2.4 3.5 ±2.3 4.0±2.4 p=0.08 BASFI (mean ± SD) 3.7 ±2.7 3.1 ±2.7 4.0 ±2.6 p=0.02 HAQ (mean ± SD) 0.53±0.5 0.48±0.48 0.56±0.51 Not sig ASQoL (mean ± SD) 5.9±5.2 5.3±5.0 6.1±5.3 Not sig Conclusions The majority of AS patients in our cohort were overweight or obese. These patients have a greater burden of symptoms and more functional impairment. They also have a higher percentage of other cardiovascular risk factors. As our awareness of the increased risk of cardiovascular disease in AS patients improves this is important information in on going management. Acknowledgements I would like to acknowledge Abbvie and Pfizer for the unrestricted support of ASRI. I would also like to acknowledge all the effort from the rheumatology community in Ireland for their hard work, enthusiasm and support of ASRI. Disclosure of Interest None declared

THU0150 High Acute Phase Reactants Predict Cardiovascular Events and Rapid Radiological Progression in Inflammatory Arthritis

Background Inflammatory arthritis (IA) is associated with an increased risk of cardiovascular events1,2, similarly known that high baseline serum amyloid A (SAA) levels and erosive disease are predictors of aggressive arthritis3. Objectives To assess the influence of markers of early disease activity on long term cardiovascular outcome and radiological progression in IA. Methods Patients were recruited and prospectively followed for 4 years after commencing biological therapy (n=62). Radiological progression was estimated using Modified Sharp score (mSS) method at baseline, 3 months and 4 years posttherapy on hands and feet radiographs. Radiological progression was defined as ΔmSS>1.5 with a cutoff of rapid radiological progression at ΔmSS>30. Cardiovascular (CV) disease was defined as any new CV event during follow up. Markers of disease activity were collected at each timepoint including erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), tender joint count out of 28 joints (TJC28), swollen joint count out of 28 joints (SJC28) and patient global health assessment (PGH). Disease activity score 28 (DAS28) remission was defined by DAS28<2.6. Results Median (range) patient age was 50,5 (18-77) yrs with disease duration of 9.75 (0.3-48) yrs. Eight patients (13%) sustained CV events and they were older, had higher baseline CRP and SAA levels compared to those who did not (p=0.029, p=0.0001). Baseline CRP and SAA levels showed significant correlation with each other (all p<0.0001). Radiological progressor status deteriorated from 66.7 % (1st year) to 77% after 4 yrs. Patients with rapid radiological progression (RRP) at 4 yrs sustained more CV events (p=0.003), moreover they had higher baseline SAA (p=0.004) and CRP levels (NS) compared to nonprogressors. In addition, patients suffering CV events or RRP had higher 3 months DAS28 scores than non-progressors (p=0.013, p=0.001), probably due to higher ESR (p=0.005, p<0.0001) and SJC28 (p=0.009, p=0.012) at 3 months. RA patients in DAS28 remission at 3 months had no evidence of CV events or RRP in long term. Conclusions In this extended inflammatory arthritis cohort, CV events and RRP appear to be predicted by high baseline SAA and CRP, suggesting a direct link to the initial level of inflammation. RA remission is not associated with CV events or RRP. References Inflammatory arthritis as a novel risk factor for cardiovascular disease. John H, Kitas G. Eur J Intern Med. 2012 Oct;23(7):575-9. Traditional cardiovascular risk factors, inflammation and cardiovascular risk in rheumatoid arthritis. Liao KP, Solomon DH. Rheumatology (Oxford).2013 Jan;52(1):45-52. Serum amyloid A in the assessment of early inflammatory arthritis. Cunnane G, Grehan S, Geoghegan S, McCormack C, Shields D, Whitehead AS, Bresnihan B, Fitzgerald O. J Rheumatol. 2000 Jan;27(1):58-63. Acknowledgements Translational Research Group, DAMC, SVUH Disclosure of Interest E. Balogh Grant/research support from: MSD, C. Ng: None Declared, T. Saber: None Declared, J. Dias: None Declared, R. Mullan: None Declared, U. Fearon: None Declared, D. Veale Grant/research support from: Abbott, Opsona, Pfizer, Roche, Consultant for: MSD, Pfizer, Roche, Speakers bureau: Janssen, MSD, Pfizer, UCB