Su-Jin Yoo

Functional Phenotype of Synovial Monocytes Modulating Inflammatory T-Cell Responses in Rheumatoid Arthritis (RA)

Monocytes function as crucial innate effectors in the pathogenesis of chronic inflammatory diseases, including autoimmunity, as well as in the inflammatory response against infectious pathogens. Human monocytes are heterogeneous and can be classified into three distinct subsets based on CD14 and CD16 expression. Although accumulating evidence suggests distinct functions of monocyte subsets in inflammatory conditions, their pathogenic roles in autoimmune diseases remain unclear. Thus, we investigated the phenotypic and functional characteristics of monocytes derived from synovial fluid and peripheral blood in RA patients in order to explore the pathogenic roles of these cells. In RA patients, CD14+CD16+, but not CD14dimCD16+, monocytes are predominantly expanded in synovial fluid and, to a lesser degree, in peripheral blood. Expression of co-signaling molecules of the B7 family, specifically CD80 and CD276, was markedly elevated on synovial monocytes, while peripheral monocytes of RA and healthy controls did not express these molecules without stimulation. To explore how synovial monocytes might gain these unique properties in the inflammatory milieu of the synovial fluid, peripheral monocytes were exposed to various stimuli. CD16 expression on CD14+ monocytes was clearly induced by TGF-β, although co-treatment with IL-1β, TNF-α, or IL-6 did not result in any additive effects. In contrast, TLR stimulation with LPS or zymosan significantly downregulated CD16 expression such that the CD14+CD16+ monocyte subset could not be identified. Furthermore, treatment of monocytes with IFN-γ resulted in the induction of CD80 and HLA-DR expression even in the presence of TGF-β. An in vitro assay clearly showed that synovial monocytes possess the unique capability to promote Th1 as well as Th17 responses of autologous peripheral CD4 memory T cells. Our findings suggest that the cytokine milieu of the synovial fluid shapes the unique features of synovial monocytes as well as their cardinal role in shaping inflammatory T-cell responses in RA.

Prevalence of Pulmonary Arterial Hypertension in Korean Adult Patients with Systemic Sclerosis: Result of a Pilot Echocardiographic Screening Study

Background Pulmonary arterial hypertension (PAH) is a major cause of morbidity and mortality among patients with systemic sclerosis (SSc). Early detection and prompt treatment of PAH associated with SSc (SSc-PAH) result in better prognosis. We conducted echocardiographic study to presume the prevalence of PAH in Korean adult SSc patients and to diagnose SSc-PAH in their early stages with right heart catheterization (RHC). Methods We performed free of charge echocardiographic study including 37 adult SSc patients at the Chungnam National University Hospital. The possibility of PAH is determined by the estimation of pulmonary arterial pressure by peak tricuspid regurgitation velocity of > 3.0 m/s. Patients with possible PAH were recommended to undergo RHC to confirm the diagnosis. Results In 37 patients, 8 patients were suspected with PAH. Among them, 6 patients agreed to be examined with RHC, and 4 were confirmed with PAH. The prevalence of possible PAH was 21.6% (8 of 37 patients), and that of confirmed PAH was 10.8% (4 of 37 patients). Four patients who were confirmed with SSc-PAH through RHC have been treated with specific pulmonary vasodilators and maintained stable. Conclusion Eight patients (21.6%) were possible PAH and 4 (10.8%) were diagnosed as SSc-PAH by RHC after the echocardiographic screening study of 37 adult SSc patients.

TNFα and IL-1β in the synovial fluid facilitate mucosal-associated invariant T (MAIT) cell migration

Abstract Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that primarily affect the joints and inflammatory cell migration into inflamed articular sites contribute to this disease. Among the inflammatory cells, human mucosal‐associated invariant T (MAIT) cells were recently recognized as critical cellular component with a pathological role in RA. However, their migratory characteristics are poorly understood. The aim of this study was to determine whether human MAIT cells preferentially traffick to inflamed synovial sites in rheumatoid arthritis patients and to elucidate the underlying mechanism. First, we found that TNF&agr; and IL‐1&bgr; were elevated in synovial fluid (SF) of RA patients, which resulted in increased expression of E‐selectin, ICAM‐1 and V‐CAM‐1 on blood vessel endothelial cells. To understand whether TNF&agr; and IL‐1&bgr; in the SF facilitated MAIT cell migration, we analyzed CD161+ TCR&agr;7.2+ MAIT and other CD3+ T cells for differences in migratory capacity. Collectively, our results demonstrate that TNF&agr; and IL‐1&bgr; in the SF facilitated MAIT cell migration dependent on expression of selectin ligand, sialyl LewisX (sLeX) and CCR6 on MAIT cells. We also showed that MAIT cells in the SF from RA patients equipped upregulated sLeX compared to the peripheral blood of RA patients and healthy persons, which suggest that TNF&agr; and IL‐1&bgr; mediated expression of E‐selectin preferentially attract sLeX mediated MAIT cell migration into the SF of RA patients.

Preferential Induction of the T Cell Auxiliary Signaling Molecule B7-H3 on Synovial Monocytes in Rheumatoid Arthritis

B7-H3, a newly identified B7 family member, has functional duality as a co-stimulator and co-inhibitor that fine-tunes T cell-mediated immune responses. Given that B7-H3 expression on human monocytes and dendritic cells is enhanced by inflammatory cytokines, its potential inmmunoregulatory role at sites of inflammation has been suggested. Further, monocytes play crucial roles 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. Thus, we aimed to investigate the possible roles of monocyte B7-H3 in the pathogenesis of RA. Synovial monocytes, but not peripheral monocytes, in RA patients predominantly express surface B7-H3. The 4Ig isoform of B7-H3 is exclusively induced on the cell surface, whereas the 2Ig B7-H3 isoform is constitutively expressed in the intracytoplasmic region of both peripheral and synovial monocytes. B7-H3 knockdown experiments reveal that surface B7-H3 has an inhibitory effect on IFN-γ production in CD4 memory cells. Moreover, surface B7-H3 expression on synovial monocytes inversely correlates with RA clinical parameters. Our findings demonstrate that activation-induced B7-H3 expression on synovial monocytes has the potential to inhibit Th1-mediated immune responses and immunomodulatory roles affecting RA pathogenesis.

THU0548 Interleukin-7 Accelerates Osteoclast Formation from Monocytes in Synovial Fluid from Patients from Rheumatoid Arthritis

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

Intrinsic changes of left ventricular function in patients with Behçet disease and comparison according to systemic disease activity

Although cardiac manifestation of Behçet disease (BD) has been described in sporadic reports, its timely diagnosis remains difficult. The objective of this study was to describe early cardiac manifestations of BD. We also performed a comprehensive classification of systemic BD activity and compared their cardiac manifestations.

THU0093 Nadph oxidases associated production of reactive oxygen species in rheumatoid arthritis

Background Reactive oxygen species (ROS) is produced during metabolism of Oxygen. ROS is important in cell signalling and homeostasis. Production of ROS can be elevated in stressful condition. Oxidative stress has been known to be related with the disease like infection and malignancy. NADPH oxidases (Nox) are membrane proteins which produce ROS. Objectives In this study we aimed to investigate the role of Nox in rheumatoid arthritis (RA) associated with production of ROS. Methods Nox and Granulocyte macrophage colony-stimulating factor (GMCSF) messenger ribonucleic acid (mRNA) were analysed in fibroblast like synoviocyte (FLS) of patients with RA and osteoarthritis (OA) by reverse transcription polymerase chain reaction. Amount of ROS which is produced in FLS of patients with RA and OA is determined using the cell permeant fluoroprobe 5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) by flowcytometry. Same experiments were performed after treatment with cytokine, interleukin (IL)−17 and tumour necrosis factor-α (TNF-α). Results Among Nox subunits (Nox1, Nox2, Nox4, Nox5, DUOX1, DUOX2, NOXA1, NOXO1), NOXA1 and NOXO1 mRNA were expressed higher in RA FLS than in OA FLS. After treatment with IL-17 and TNF-α for 24 hours GMCSF, Nox1 and NOXO1 mRNA in RA FLS were elevated. Amount of ROS production was also elevated after treatment with IL-17 and TNF-α. When RA FLS were treated with bromopyruvic acid (BrPa), glucolysis inhibitor by inhibition of hexokinase II, GMCSF mRNA and ROS were decreased and Nox1 and Nox4 mRNA showed no diffrence. Conclusions Several factors may be involved between ROS and Nox in RA FLS. Both ROS and Nox were elevated in inflammatory condition in RA FLS. From this result we expect that Nox-targeted therapy may be effective for treatment with RA. References [1] Drummond GR, Selemidis S, Griendling KK, Sobey CG. Combating oxidative stress in vascular disease: NADPH oxidases as therapeutic targets. Nat Rev Drug Discov2011;10:453–71. [2] Neumann E, Lefèvre S, Zimmermann B, Gay S, Müller-Ladner U. Rheumatoid arthritis progression mediated by activated synovial fibroblasts. Trends Mol Med2010;16:458–68. [3] Rodiño-Janeiro BK, Paradela-Dobarro B, Castiñeiras-Landeira MI, Raposeiras-Roubín S, González-Juanatey JR, Alvarez E. Current status of NADPH oxidase research in cardiovascular pharmacology. Vasc Health Risk Manag2013;9:401–28. [4] Xiao C, Li J, Dong X, He X, Niu X, Liu C, Zhong G, Bauer R, Yang D, Lu A. Anti-oxidativeand TNF-α suppressive activities of puerarin derivative (4AC) in RAW264.7 cells and collagen-induced arthritic rats. Eur J Pharmacol2011;666:242–50. [5] Yun JM, Chien A, Jialal I, Devaraj S. Resveratrol up-regulates SIRT1 and inhibits cellular oxidative stress in the diabetic milieu: mechanistic insights. J Nutr Biochem2012;23:699–705. Disclosure of Interest None declared

AB0113 The Serum and Synovial Fluid Levels of Cold-Inducible Rna-Binding Protein (Cirp) in Patients with Rheumatoid Arthritis

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<0.001) (Fig.1). 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<0.001). Also, we found the tendency that the CIRP concentration of synovial fluid was significantly higher than that of serum in same patient (P=0.025) (Fig.2). 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, Millán 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

SAT0190 Histological Study on The Expression of Transcriptional Intermediary Factor 1 (TIF 1) in The Patients with Inflammatory Myopathies

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<0.001; TIF1γ, muscle, 10/35 [29%] vs. skin, 13/15 [87%], p<0.001). When analyzing DM patients only, the result was similar (TIF1α, muscle, 1/19 [5%] vs. skin, 12/15 [80%], p<0.001; TIF1γ, muscle, 8/19 [42%] vs. skin, 13/15 [87%], p=0.013). TIF1β showed strong positivity in all tissues of myositis or healthy control. 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

AB0039 Preferential Induction of 4IG B7-H3 on Synovial Monocytes in Rheumatoid Arthritis (RA): Possible Role of 4IG B7-H3 as a Co-Inhibitory Ligand

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