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Background:Liver kinase B1 (LKB1) is known as a tumor suppressor gene and also inhibits reactive oxygen species (ROS) levels. Intracellular ROS are catalyzed by the enzyme complex nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). We previously reported that NOX4 induced the migration and invasion of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA). Although LKB1 is expected to alleviate synovial inflammation through ROS regulation, the role of LKB1 in RA has not been examined.Objectives:To explore whether LKB1 affects RA inflammation, we transfected LKB1 siRNA and analyzed related gene expressions in RA FLS.Methods:Synovial tissues were obtained from RA patients who were undergoing synovectomy or joint replacement. The isolated cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin and maintained in a 5% CO2 incubator at 37 °C. FLS were used for experiments after four to six passages. Cells were transfected with lipofectamine transfection reagent and LKB1 siRNA duplex targeting constructs. After incubation for 24 h, downregulation of target expression was evaluated by real-time PCR and western blot analysis.Results:RA FLS was transfected with LKB1 siRNA and 90% of LKB1 mRNA expression was decreased. LKB1 knock-down also caused the decreased expression of mechanistic target of rapamycin (mTOR; 0.38 fold) and serine/threonine kinase (AKT) 2 (0.40 fold), which are downstream targets of LKB1. NOX4 was significantly increased (4.94 fold) by LKB1 inhibition. On the other side, the down regulated NOX4 induced significantly elevated LKB1 mRNA expression in RA FLS. When the expressions of proinflammatory cytokines were examined, IL-1β, IL-6, TNF-α were highly increased by LKB1 deficiency. FLS migration-related chemokines, IL-8 and MMP-3 were also enhanced compared to control.Conclusion:There was a negative correlation between NOX4 and LKB1 in RA FLS. As LKB1 deficiency induced the expression of proinflammatory cytokines and migration related chemokines, LKB1 could play a critical role in RA pathogenesis.References:[1]Bartok B, Firestein GS. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol Rev. 2010;233(1):233–55.[2]Mateen S, Moin S, Khan AQ, Zafar A, Fatima N. Increased reactive oxygen species formation and oxidative stress in rheumatoid arthritis. PLoS One. 2016;11(4):e0152925.Disclosure of Interests:None declared.

Background:Reactive oxygen species (ROS) and T helper 17 (TH17) cells have been known to play an important role in the pathogenesis of rheumatoid arthritis (RA). However, the interrelationship between ROS and TH17 remains unclear in RAObjectives:To explore whether ROS affect TH17 cells in peripheral blood mononuclear cells (PBMC) of RA patients, we analyzed ROS expressions among T cell subsets following treatment with mitochondrial electron transport chain complex inhibitors.Methods:Blood samples were collected from 40 RA patients and 10 healthy adult volunteers. RA activity was divided according to clinical parameter DAS28. PBMC cells were obtained from the whole blood using lymphocyte separation medium density gradient centrifugation. Following PBMC was stained with Live/Dead stain dye, cells were incubated with antibodies for CD3, CD4, CD8, and CD25. After fixation and permeabilization, samples were stained with antibodies for FoxP3 and IL-17A. MitoSox were used for mitochondrial specific staining.Results:The frequency of TH17 cells was increased by 4.83 folds in moderate disease activity group (5.1>DAS28≥3.2) of RA patients compared to healthy control. Moderate RA activity patients also showed higher ratio of TH17/Treg than healthy control (3.57 folds). All RA patients had elevated expression of mitochondrial specific ROS than healthy control. When PBMC cells were treated with 2.5uM of antimycin A (mitochondrial electron transport chain complex III inhibitor) for 16 h, the frequency of TH17 cells was significantly decreased.Conclusion:The mitochondrial electron transport chain complex III inhibitor markedly downregulated the frequency of TH17 cells in moderate disease activity patients with RA. These findings provide a novel approach to regulate TH17 function in RA through mitochondrial metabolism related ROS production.References:[1]Szekanecz, Z., et al., New insights in synovial angiogenesis. Joint Bone Spine, 2010. 77(1): p. 13-9.[2]Prevoo, M.L., et al., Modified disease activity scores that include twenty-eight-joint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum, 1995. 38(1): p. 44-8.Disclosure of Interests:None declared

Background:Rheumatoid arthritis (RA) is a progressive, chronic inflammatory autoimmune disease. Pro-inflammatory molecules, activated lymphocytes, and the migration of inflammatory cells are important in the development of RA. There are many unknown causes of RA. And there are many patients who are refractory to treatment with known disease-modifying anti-rheumatic drugs. So, unknown cause of RA needs to be elucidated.CD70 is a member of the tumor necrosis factor (TNF) superfamily and a ligand for CD27. The interaction of CD70 with its receptor CD27 promotes expansion and differentiation of memory and effector T cells as well as B-cell expansion and plasma cell differentiation. Hypoxia is an important micro-environmental factor in RA synovium. Hypoxia induces activation of hypoxia inducible factor (HIF). The expression of HIF-2α is up-regulated in human RA synovium. Reactive oxygen species (ROS) has been implicated in the pathophysiology of RA.Objectives:In this study, we tried to examine the presence of CD70 in RA synovium and investigate the role of CD70 in the development of RA associated with HIF-2α and ROS.Methods:Fibroblast-like synoviocyte (FLS), peripheral blood (PB) and synovial fluid (SF) were used for experiments. FLS was stimulated with recombinant human (rh)-IL-17 and rh-TNF-α. N-acetyl-L-cysteine (NAC) was used as a ROS scavenger. HIF-2α inhibitor (PT-2385) was used for examine the effect of HIF-2α in RA-FLS. RT-PCR, qPCR, western blotting, flow-cytometry, ELISA, cell migration assay, and scratch wound assay were performed.Results:CD70 mRNA is present and elevated by stimulation with IL-17 and TNF-α in both RA-FLS and osteoarthritis (OA)-FLS (Fig 1). CD70 also expresses on the surface of RA-FLS and OA FLS (Fig 2). CD70 expression on the surface of FLS is elevated by stimulation with IL-17 and TNF-α in both RA and OA. Soluble CD27 is present higher in the supernatant of RA-SF than OA-SF (Fig 3). HIF-2α mRNA, HIF-2α protein, and the amount of ROS were all elevated after treatment with IL-17 and TNF-α in RA-FLS (Fig 4, Fig 5). CD70 expression and the amount of ROS were lowered by treatment with HIF-2α inhibitor in RA-FLS (Fig 6). Decreased amount of ROS results in decreased CD70 expression on the RA-FLS (Fig 7). CD70 influenced on cell migration directly or by HIF-2α (Fig 8).Conclusion:In this study, we found the function of CD70 in RA-FLS associated with HIF-2α and ROS. First, CD70 on RA-FLS interacts with CD27 in the RA-SF and this interaction produces sCD27 (Fig. 9) and CD70 has an influence on the migration of RA-FLS. Second, IL-17 and TNF-α are critical factors to trigger the expression of CD70, HIF-2α and ROS in RA synovium. Third, CD70 is regulated by HIF-2α associated with ROS. From these results, we suggest that CD70 may be a new therapeutic target of RA. And sCD27 also may be an important diagnostic maker of RA.References:[1]Lundy SK, Sarkar S, Tesmer LA, Fox DA. Cells of the synovium in rheumatoid arthritis. T lymphocytes. Arthritis Res Ther. 2007;9(1):202.[2]Nevius E, Gomes AC, Pereira JP. Inflammatory Cell Migration in Rheumatoid Arthritis: A Comprehensive Review. Clin Rev Allergy Immunol. 2016;51(1):59-78.[3]Bowman MR, Crimmins MA, Yetz-Aldape J, Kriz R, Kelleher K, Herrmann S. The cloning of CD70 and its identification as the ligand for CD27. J Immunol. 1994;152(4):1756-61.[4]Kitajima S, Lee KL, Fujioka M, Sun W, You J, Chia GS, et al. Hypoxia-inducible factor-2 alpha up-regulates CD70 under hypoxia and enhances anchorage-independent growth and aggressiveness in cancer cells. Oncotarget. 2018;9(27):19123-35.[5]Gaber T, Dziurla R, Tripmacher R, Burmester GR, Buttgereit F. Hypoxia inducible factor (HIF) in rheumatology: low O2! See what HIF can do! Ann Rheum Dis. 2005;64(7):971-80.Disclosure of Interests:None declared

Background The cold-inducible RNA-binding protein (CIRP) is 18 kDa protein of the glycine-rich RNA binding protein (GRP) family, and these proteins function as RNA chaperones to facilitate translation. Extracellular CIRP is a recently identified endogenous proinflammatory mediator and damage-associated molecular pattern molecules (DAMP) that triggers inflammatory responses in sepsis and inflammatory bowel disease. Objectives This study was planned to investigate the relationship between CIRP and rheumatoid arthritis. Methods Peripheral blood and synovial fluid were collected from 15 patients with rheumatoid arthritis (RA) and 16 patients with osteoarthritis (OA). The concentration of CIRP was measured by sandwich Enzyme-Linked Immunosorbent Assay (ELISA). Results The concentration of serum CIRP was significantly elevated in RA patients group (RA patients=26.39±10.48 pg/ml, OA patients=17.14±7.24 pg/ml, p=0.009). Furthermore, the RA patients group showed significantly higher CIRP concentration than the OA patients group in synovial fluid (153.56±108.93 pg/ml vs. 23.63±16.18 pg/ml, p The mean concentration of synovial fluid CIRP was significantly higher than that of serum in RA patients group (Serum concentration=26.39±10.48 pg/ml, Synovial fluid=153.56±108.93 pg/ml, p DAS28-ESR and DAS28-CRP were positively correlated with synovial fluid concentration of CIRP (DAS28-ESR: r=0.582, p=0.023, DAS28-CRP: r=0.541, p=0.037, by correlation analysis). Conclusions The serum and synovial concentration of CIRP in RA patients was increased compared to OA patients. Also synovial concentration of CIRP in RA patients correlated well with the disease activity, i.e. the DAS28-ESR/CRP. Based on these results, the CIRP mediates the inflammation and is potential marker for synovial inflammation. References Qiang X, Yang WL, Wu R, Zhou M, Jacob A, Dong W, Kuncewitch M, Ji Y, Yang H, Wang H, Fujita J, Nicastro J, Coppa GF, Tracey KJ, Wang P (2013) Cold-inducible RNA-binding protein (CIRP) triggers inflammatory responses in hemorrhagic shock and sepsis. Nat Med 19:1489–95. Nishiyama H, Higashitsuji H, Yokoi H, Itoh K, Danno S, Matsuda T, Fujita J (1997) Cloning and characterization of human CIRP (cold-inducible RNA-binding protein) cDNA and chromosomal assignment of the gene. Gene 204:115–20. Nishiyama H, Danno S, Kaneko Y, Itoh K, Yokoi H, Fukumoto M, Okuno H, Millan JL, Matsuda T, Yoshida O, Fujita J (1998) Decreased expression of cold-inducible RNA-binding protein (CIRP) in male germ cells at elevated temperature. Am. J. Pathol 152:289–96. Nishiyama H, Xue JH, Sato T, Fukuyama H, Mizuno N, Houtani T, Sugimoto T, Fujita J (1998) Diurnal change of the cold-inducible RNA-binding protein (Cirp) expression in mouse brain. Biochem. Biophys. Res. Commun 245:534–8. Disclosure of Interest None declared

Background Behcet9s disease (BD) is a multisystemic vasculitis of unknown etiology. Cardiovascular (CV) involvement has been reported in 1–6% of BD patients. In case of CV involvement, prognosis is poor. The incidence of the CV involvement in Korean patients with BD is not clearly documented. Objectives The aim of this study is to evaluate the CV findings in Korean patients with BD and to prevent or lower mortality and morbidity through early diagnosis and early treatment. Methods We performed Ankle Brachial Index (ABI), Pulse wave velocity (PWV) and Echocardiographic screening in 81 patients with BD who fulfilled the International Study Group criteria. PWV was calculated as the brachial-ankle path length divided by the brachial-ankle transit time (baPWV). We compared the result between two groups of patients with BD. Two groups are patients who have been treated with immunosuppressant agent and without immunosuppressant agent. The patients of former group are patients who have been suffered from severe complication or refractory disease. Results Fifty-nine (72.8%) patients were female. Mean age was 50.6 ± 2.5 years old. Mean age at diagnosis was 40.5 ± 2.3 years old and disease duration was 10.2 ± 1.5 years. Mean ABI was 1.10 ± 0.03. There was no significant difference between with and without immunosuppressant agent treated group. (1.11 ± 0.03 vs. 1.09 ± 0.03, P=0.167). The number of patients with abnormal ABI was 7 (17.5%) vs. 6 (14.6%), P=0.781. Mean PWV (m/sec) was 14.0 ± 0.67. There was no significant difference between two groups (13.8 ± 1.09 vs. 14.2 ± 0.83, P=0.144). But the immunosuppressant treated group had more patients with elevated levels of PWV (>12.0 m/sec) compared to non-immunosuppressant treated group [n =34 (82.9%) vs. n=25 (62.5%), P=0.024]. Echocardiography was done. Left ventricular ejection fraction (%) (64.9 ± 1.5 vs. 63.7 ± 1.7, P=0.254), Left ventricular mass index (g/m 2 ) (74.9 ± 5.0 vs 74.1 ± 5.7, P=0.970), E/E9 ratio (8.35 ± 0.9 vs 9.17 ± 1.0, P=0.228), Tricuspid regurgitating velocity (TRV) max (m/sec) (2.35 ± 0.09 vs 2.39 ± 0.11, P=0.572) were similar in both groups. We detected 39 minimal mitral, aortic and tricuspid valve insufficiencies. One patient showed suspiciously resting pulmonary hypertension (TRVmax – 3.3m/sec). In two patients, dilatation of ascending aorta was showed. There was aortic arch mass in one patient. Conclusions The CV involvement in BD can be a cause of death. In this screening study, asymptomatic patients showed many CV manifestations. And in 59 (72.8%) patients, PWV was elevated. Arterial stiffness can be a surrogate marker of CV disease. Larger study about CV manifestations in BD must be planned. And during treatment of patients with BD, CV screening study needs to be considered. References H L Chang, S K Kim, S S Lee, M Y Rhee. Arterial stiffness in Behcet9s disease: increased regional pulse wave velocity values. Ann Rheum Dis. 2006 Mar; 65(3): 415–416. Geri G, Wechsler B, Huong DLT, et al. Spectrum of cardiac lesions in Behcet disease: a series of 52 patients and review of literature. Medicine (Baltimore). 2012 Jan;91(1):25–34. C.A. Caldas, E.F. Borba, L.A. Bortolotto, D.M. Medeiros, E. Bonfa and C.R. Goncalves. Increased arterial stiffness assessed by pulse wave velocity in Behcet9s disease and its association with the lipid profile. J Eur Acad Dermatol Venereol. 2013 Apr;27(4):454–9 Disclosure of Interest None declared

Background Myositis-specific antibodies in patients with inflammatory myopathies are known to be associated with various clinical manifestations, classifications and diagnosis. Among them, recently found anti-transcriptional intermediary factor 1 (TIF1) α, β, or γ antibodies, has been reported to be associated with dermatomyositis (DM) accompanied by cancer. Objectives Although previous studies have evaluated the association of the antibodies in serum and clinical subtypes, the information about the target antigen is insufficient. The purpose of this study was to confirm the overexpression of TIF1s in the muscle and skin tissues of patients with inflammatory myopathies. Methods From February 2004 to November 2014, skin and muscle biopsies were performed on 45 patients diagnosed with dermatomyositis and polymyositis. We stained skin and muscle tissue by immunohistochemistry using anti-TIF1α, β, or γ and compared with the results of healthy control. We analyzed the association between the clinical manifestations and protein expression in each tissue. Results When compared with the control group, any antigens showed no significant overexpression in the muscle. However, TIF1α showed higher positive rate in the skin of DM (12/15 [80%]) than in the skin of healthy control (0/7 [0%]) (p=0.001). TIF1γ expression was higher in the muscle of patients with DM while there was no expression in the muscle of healthy controls (DM, 8/19 [80%] vs. healthy control 0/7 [0%], p=0.039). In the tissues of inflammatory myopathies, TIF1α and TIF1γ demonstrated higher positive rates in the skin than in the muscle (TIF1α, muscle, 4/35 [11%] vs. skin, 12/15 [80%], p Analyzing the association with TIF1s expression and cancer, there was no significant difference in the positive rate of TIF1α or γ in the muscle or skin between the myositis patient with or without cancer. Conclusions TIF1α was expressed more in the skin of DM patients than that in that of control group and TIF1γ in the muscle of DM patients than in that of control. The expression of TIF1α in the skin and TIF1γ in the muscle of cancer associated DM was not higher than those of DM without cancer. Thus the expression levels of TIF1α in the skin and TIF1γ in the muscle may be associated with myositis rather than with cancer. References Hoshino K, et al. Anti-MDA5 and anti-TIF1-gamma antibodies have clinical significance for patients with dermatomyositis. Rheumatology (Oxford) 2010;49(9):1726–1733. Fiorentino D. Casciola-Rosen L, Autoantibodies to transcription intermediary factor 1 in dermatomyositis shed insight into the cancer-myositis connection. Arthritis Rheum 2012;64(2):346–349. Disclosure of Interest None declared

Background B7-H3 (B7-H3), newly identified B7 family member, has functional duality as a T-cell costimulator or coinhibitor for fine-tuning T-cell mediated immune responses. Given that B7-H3 expression is enhanced in human monocyte and dendritic cells by inflammatory cytokines, it has been suggested their potential inmmunoregulatory function at sites of inflammation. Monocytes play a crucial role in the pathophysiology of various inflammatory disorders including autoimmune diseases. However, the immunological role of B7-H3 in rheumatoid arthritis (RA) has not been defined. Objectives We aimed to investigate the possible roles of B7-H3 on monocyte in pathogenesis of RA. Methods Synovial fluid and peripheral blood from 33 RA patients and peripheral blood from 19 healthy controls (HCs) were obtained. The expression of B7-H3 on monocytes in RA patients and HCs were analyzed using flow cytometic analysis and quantitative RT-PCR. Purified synovial and peripheral monocytes were used for immunoblotting and intracellular flow cytometic analysis to investigate the distinct expression and localization of two isoforms of B7-H3. B7-H3-expressing THP-1 cells were transfected with B7-H3-specific siRNA and subjected to coculture assay with CD4 T cells. Proliferation and cytokine production of the cocultured T cells were assessed by CFSE dilution and ELISA, respectively. Clinical relevance of enhanced B7-H3 expression in RA was tested by comparison with clinical parameters and disease severity in the patients and with synovial cytokine levels measured by multiplex cytokine assay. Results Synovial monocytes but not peripheral monocytes in RA patients predominantly induce surface B7-H3 expression. The induced B7-H3 is exclusively 4Ig isoform, whereas 2Ig B7-H3 isoform constitutively express the intracytoplasmic region both peripheral and synovial monocytes. The B7-H3 knockdown experiments provided the evidence that 4Ig B7-H3 isoform has an inhibitory effect on interferon-gamma (IFN-γ) production of CD4 memory cells. Moreover, the expression level of 4Ig B7-H3 on synovial monocytes was inversely correlated with clinical parameters such as C-reactive protein (CRP) level, erythrocyte sedimentation rate (ESR), and Disease Activity Score in 28 joints (DAS28). Conclusions Our findings demonstrate that activation-induced B7-H3 expression on synovial monocytes has the potential to inhibit Th1-mediated immune response and immunomodulatory role affecting pathogenesis of RA. Disclosure of Interest None declared

Background In osteoarthritis (OA), synovial inflammation may contribute disease progression. Moreover, monocytes and Toll-like receptors 2 (TLR2) or 4 (TLR4) may play a role in promoting inflammation of OA. However, inflammatory cells in osteoarthritis synovial fluid (OASF) have been rarely studied. Objectives In order to reveal the inflammatory cells in OASF, we investigated cell markers of mononuclear cells in OA synovial fluid and TLR2 or TLR4 expression in the subtypes of monocyte. Methods Mononuclear cells were purified by Ficoll density gradient methods from OASFs. Mononuclear cells were stained using anti-CD3, anti-CD4, anti-CD14, anti-CD16, anti-CD64, anti-CD90, anti-TLR4, or anti-TLR2 and analyzed using FACS Canto II. Results In OASF, there are many CD14+CD16- cells (10.9±10.0%) or CD14+CD16+ cells (21.2±21.8%), CD3+CD4+ cells (16.7±12.9%), CD3+CD4- cells (20.7±12.8%) but rare CD90+ cells (1.7±1.3%). In the majority of CD14+ cells, CD64 and low level CD4 was expressed (CD64, 84.6±1.1%; CD4, 71.0±20.5%). TLR4 expression was higher in CD14+CD16+ cells than in CD14+CD16- cells or CD14-CD16+ cells (mean fluorescence intensity [MFI], CD14+CD16-: 159±128, CD14+CD16+: 307±272, CD14-CD16+: 57±92, p Conclusions In OASF, CD3+ T cells and CD14+ monocytes were abundant and majority of CD14+ cells showed CD64 and CD4. Monocyte subtypes are mainly CD14+CD16+ or CD14+CD16- and those subtypes expressed TLR4 and TLR2. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3425

Background Interleukin-7 (IL-7), a member of the common gamma-chain cytokine family, plays a crucial role in T cell development, which induces T cell survival, proliferation, differentiation in mature naive and memory T cells. IL-7 is expressed by stromal cells, epithelial cells, and fibroblasts. IL-7 is highly expressed in synovial tissue (ST) of patients with rheumatoid arthritis (RA) and its expression level correlates with the disease activity. Although IL-7Rα is mainly expressed in T cells, IL-7 receptor α (IL-7Rα) is able to be expressed in CD19-positive B cells and monocytes of synovial fluid mononuclear cells (SFMC) in RA patients. IL-7 promotes the secretion of osteoclastogenic factors, such as M-CSF and RANKL, in T cells, and eventually induces osteoclastogenesis. In contrast, It has been shown that IL-7α-deficient mice promote osteoclastogenesis and IL-7 treatment inhibits the osteoclastogenesis in vitro. Thus, the role of IL-7 involved in osteoclastogenesis is still unclear. Objectives In order to resolve this discrepancy, we investigated the effect of IL-7 on the differentiation of monocytes into osteoclasts. Methods First, we examined the expression level of IL-7Rα in several subsets from PBMC and SFMC of RA patients as well as healthy subjects. Second, we determined the direct role of IL-7 in osteoclast differentiation of monocytes expressing IL-7Rα in vitro. Results As results, we found that IL-7Rα was highly expressed in monocytes from RA SFMC compared to health PBMC, and its expression was confined to specific monocyte-subsets, intermediate monocyte (CD14+CD16+) and inflammatory monocyte (CD14-CD16++). Furthermore, IL-7 induced osteoclastogenesis from RA monocytes in the presence or absence of M-CSF and RANKL, and also promoted the osteoclastogenesis in earlier than done by M-CSF and RANKL. Conclusions These finding suggest that IL-7 may directly affect monocyte differentiation in osteoclast cells in inflammatory condition, such as synovial fluid of RA, and blocking IL-7 or IL-7Rα as therapy may give a new pathway to treatment of bone erosion in patient with RA. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.1500