Eunji Kwon

Genetic Studies of Ankylosing Spondylitis in Koreans Confirm Associations with ERAP1 and 2p15 Reported in White Patients

Objective. Investigators from the Australo-Anglo-American Spondyloarthritis Consortium have reported additional genes associated with ankylosing spondylitis (AS) susceptibility including IL1R2, ANTXR2, and gene deserts at 2p15 and 21q22. We evaluated these new candidate genes in a large cohort of Korean patients with AS. Methods. A group of 1164 patients with AS and 752 healthy controls were enrolled for our study. Eight single-nucleotide polymorphisms (SNP) were analyzed to define genetic association with AS by MassARRAY system. Results. Significant positive associations of AS with endoplasmic reticulum aminopeptidase 1 SNP, rs27037 (p = 1.31 × 10−4), and rs27434 (p = 4.59 × 10−6), were observed. The rs10865331 of gene desert at 2p15 also showed a significant association with AS (p = 4.63 × 10−5). Conclusion. This is the first confirmation in a nonwhite population that genetic polymorphisms of rs27037, rs27434, and rs10865331 are associated with AS, implicating common pathogenetic mechanisms in Korean and white patients with AS.

Regulation of TREM-1 expression by 1,25-dihydroxyvitamin D3 in human monocytes/macrophages.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a recently identified cell surface receptor that is expressed mainly on monocytes and neutrophils, and acts as an amplifier of immune responses. In this study, 1,25(OH)2D3 strongly upregulated the expression of TREM-1 in human monocytes and macrophages. 1,25(OH)2D3 stimulated TREM-1 mRNA expression by augmenting transcription, and not by inhibiting mRNA degradation. The upregulated expression of TREM-1 by 1,25(OH)2D3 was dependent on the NF-κB signaling pathway and required new protein synthesis in differentiated U937 macrophages. Our results show that 1,25(OH)2D3 can affect the innate and inflammatory responses by upregulating TREM-1 expression, and suggest that 1,25(OH)2D3 may function as an enhancer of the innate immune response by upregulating TREM-1 expression, in addition to inducing the antimicrobial peptide cathelicidin.

1,25-Dihydroxyvitamin D3 inhibits directly human osteoclastogenesis by down-regulation of the c-Fms and RANK expression.

OBJECTIVE 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is a key molecule to maintain calcium homeostasis and bone metabolism. It was recently reported that 1,25(OH)2D3 directly inhibited osteoclast differentiation in mouse bone marrow cells and human bone marrow-derived colony-forming unit granulocyte macrophage (CFU-GM) cells. However, the direct effects of 1,25(OH)2D3 and its affecting mechanisms on the osteoclast differentiation of human osteoclast precursors remain largely unknown. In this study, we examined the direct effects of 1,25(OH)2D3 on the osteoclastogenesis of human peripheral blood (PB) osteoclast precursors. METHODS In vitro osteoclastogenesis assays were performed using osteoclast precursors from normal PB. The gene expressions were analyzed using real-time PCR. The cell surface proteins, including c-Fms and RANK, were measured by flow cytometry. RESULTS 1,25(OH)2D3 strongly inhibited osteoclast differentiation and it suppressed the expression of RANK in the human PB osteoclast precursors. One mechanism of RANK inhibition by 1,25(OH)2D3 is down-regulation of the M-CSF receptor c-Fms, which is required for the expression of RANK. In contrast to the previous reports on mouse osteoclast precursors, 1,25(OH)2D3 did not affect the expression of c-Fos. Parallel to the inhibition of osteoclastogenesis, 1,25(OH)2D3 increased the expression and phosphorylation of CCAAT enhancer-binding protein β (C/EBPβ), which is a recently discovered inhibitor of osteoclastogenesis. CONCLUSIONS Our results show that 1,25(OH)2D3 inhibits human osteoclastogenesis by decreasing the RANK+ osteoclast precursors, and we suggest that 1,25(OH)2D3 may be a powerful therapeutic agent for treating inflammation-induced bone disease that shows excessive osteoclast activation.

One-Pot Template-Free Synthesis of Monodisperse Hollow Hydrogel Microspheres and their Resulting Properties

Monodisperse poly(methacrylic acid/ethyleneglycoldimethacrylate) (MAA/EGDMA) hollow microcapsules, which exhibit pH-responsive behavior, are prepared by diffusion of cationic surfactants and hydrophobic interaction. During the association of the negatively charged hydrogel microspheres and an oppositely charged surfactant (cetyltrimethylammonium bromide, CTA(+)B), the hydrophobic polymer-surfactant complexes that form are separated from the internal water; consequently, a hollow structure can be formed. Confocal laser scanning microscopy, UV spectro-scopy and zeta potential are employed to study the formation of the hollow structure during the diffusion of the cationic surfactant. The controlled release behavior of methylene blue as a model drug from the as-prepared poly(MAA/EGDMA) microcapsules with a hollow structure is investigated under different pH conditions. The hollow structure can be retained, even during repetitive pH changes.

1α,25-Dihydroxyvitamin D3 upregulates HIF-1 and TREM-1 via mTOR signaling

Triggering receptor expressed on myeloid cells-1 (TREM-1) is induced by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in human monocytes/macrophages and epithelial cells. However, little information is available regarding the mechanism of 1,25(OH)2D3-induced TREM-1 expression in human monocytes/macrophages. In this study, 1,25(OH)2D3 was shown to strongly upregulate hypoxia-inducible transcription factor (HIF) in PMA-differentiated U937 cells. However, HIF was not mainly involved in 1,25(OH)2D3-induced TREM-1 expression. Instead, 1,25(OH)2D3-induced expression of TREM-1 was inhibited by rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway, indicating the involvement of mTOR. Induction of HIF proteins by 1,25(OH)2D3 was also inhibited by rapamycin. In addition, 1,25(OH)2D3 induced the phosphorylation of p70S6 kinase, a target of mTOR complex 1 (mTORC1). Our results suggest that 1,25(OH)2D3 induces the expression of TREM-1 through the mTOR signaling pathway in human macrophages.

A novel role for bone-derived cells in ankylosing spondylitis: Focus on IL-23

The main aim of this study are to explore the role of bone-derived cells (BdCs) in ankylosing spondylitis (AS) and determine the underlying molecular mechanisms of IL-23 production. Primary BdCs were isolated from diced bone of facet joints obtained during surgery from seven AS patients and seven disease control (Ct) patients. Osteoblastic activity of BdCs was assessed by measuring their alkaline phosphatase activity and by alizarin red staining. Osteoblast and endoplasmic reticulum (ER) stress-related genes were assessed by quantitative PCR, immunoblotting, immunofluorescence, and immunohistochemistry. In addition, expression of IL-23 in response to BIX (selective BIP inducer X)-induced ER stress was evaluated by qPCR and ELISA. Protein interaction and binding to IL-23 promoter were confirmed by Immunoprecipitation and Chromatin immunoprecipitation, respectively. Transcript levels of genes involved in osteoblast function, as well as of the ER stress marker were higher in the AS group than the Ct group, and elevated RUNX2, BiP and IL-23 expression were observed in the BdCs, serum, and bone biopsies from the AS group. BIX-induced ER stress stimulated osteoblastic activity and IL-23 secretion by upregulating RUNX2 expression. Furthermore, in AS BdCs, RUNX2 interacted with C/EBPβ to bind to IL-23 promoter and RUNX2 knockdown suppressed IL-23 secretion. These finding may provide a molecular mechanism involved in sustained ER stress in AS BdCs stimulates the activation of RUNX2 and C/EBPβ genes, leading to IL-23 production.

OP0027 Characteristics of cytokines and changes in interleukin-17 levels in the synovial fluid of patients with ankylosing spondylitis on treatment with biologics

Background Biologic drugs targeting the inflammatory cytokines have been recommended in rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Yet, some patients require a change in treatment because an adequate response is not achieved. Objectives The current study aimed to evaluate the levels of tumour necrosis factor alpha (TNF-α), interleukin (IL)−17, IL-23, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the joint fluid in patients with AS and RA and identify the important cytokines related with treatment-response. Methods Synovial fluid was obtained from 18 patients with AS and 19 with RA who suffered from arthritis of the knee; and the levels of the cytokines were measured. The differences in their levels between patients with AS and RA, and between patients treated with and without biologics (biologics group and non-biologics group) were analysed. Results TNF-α and GM-CSF levels in patients with AS were significantly lower than those in patients with RA (figure 1A, both p<0.01); however, IL-17 and IL-23 levels were not significantly different between the two groups. Furthermore, levels of IL-17 were markedly elevated in the biologics group compared with the non-biologics group in AS (figure 1B, p=0.04). However, in RA, there were no significant differences between the non-biologics and biologics group (figure 1C). Conclusions In AS, IL-17 in synovial fluid is a good marker of non-response to biologics and may be a good target for non-responders to TNF inhibitor. Disclosure of Interest None declared

Expression of PRDM10 in arthritic synovial derived tissues

We previously reported the expression of PRDM10 in skeletal cartilage during mouse embryonic development. In this study, we examined PRDM10 expression in blood, synovial fluid and synovium from patients with rheumatoid arthritis (RA) or osteoarthritis (OA). PRDM10 mRNA was detected in all peripheral blood and RA synovial fluid, as well as in normal blood. We also performed immunohistochemistry with human synovium tissues. Compared to other PR/SET proteins including PRDM14, PRDM12, and SUV39H1, PRDM10 expression was conspicuous in cells of the synovial lining layer, subintimal layer, and perivascular layer in RA and OA synovium. Immunostaining using cultured fibroblast like synoviocytes detected PRDM10 mainly in the nucleus, with a small amount detected in the cytoplasm. Transforming growth factor-beta (TGF-β) treatment decreased PRDM10 expression in OA-derived FLS cells, whereas polyinosinic:polycytidylic acid or lipopolysaccharide treatment did not produce a distinct change of PRDM10 expression, PRDM10 expression was reversed by the smad inhibitor SB431542. Given that PRDM10 is highly expressed in human synovial tissues and is transcriptionally regulated by TGF-β, we suggest that PRDM10 might be involved in the pathogenesis of arthritis.

THU0027 TREM-1 stimulation inhibits human osteoclastogenesis via down-regulation of M-CSF receptor expression

Background Triggering receptor expressed on myeloid cells (TREMs) are a family of cell surface receptors that play important roles in innate and adaptive immunity. Among then, TREM-2 has been extensively studied in the osteoclast differentiation and the essential role of TREM-2 in human osteoclastogenesis has been well established. However, much less has been known about the role of TREM-1 in human osteoclast differentiation. Objectives In this study, we investigated the role of TREM-1 in human osteoclast differentiation. Methods In vitro osteoclastogenesis assays were performed using osteoclast precursors from normal peripheral blood. Gene expressions were analyzed using real-time PCR. Results Consistent with previous reports, TREM-2 expression was strongly increased during generation of human osteoclast precursors (pOCs). In contrast, TREM-1 expression was significantly decreased during generation of human pOCs. Stimulation of TREM-1 using agonistic TREM-1 antibody resulted in a significant suppression of RANKL-induced osteoclastogenesis, as evidenced by diminished formation of TRAP+ multinucleated cells. In addition, TREM-1 stimulation strongly suppressed RANKL-induced expression of osteoclast-related genes such as cathepsin K, integrin β3 and NFATc1. TREM-1 stimulation also down-regulated gene expression and cell surface expression of M-CSF receptor that is essential for osteoclast differentiation and survival. Conclusions In this study, we demonstrated that TREM-1 play a negative regulator in human osteoclast differentiation and our findings identify a new mechanism of negative regulation of osteoclastogenesis that play a role during inflammation. Disclosure of Interest None Declared