synovial inflammation and/or hyperplasia may contribute to disease chronicity [3]. Recently, synoviolin a novel E3 ubiquitin ligase present in the endoplasmic reticulum, was identified in RA FLS and synovial lining tissue, and may be implicated in dysregulated proliferation and apoptosis in RA [4]. The overexpression of synoviolin leads to destructive arthritis in mice [4]. In murine arthritis, synoviolin heterozygous knockout mice are protected against arthritis development associated with reduced synovial hypercellularity. In addition, synoviolin is increased during the course of murine collagen induced arthritis (CIA) [5]. In contrast to murine studies, the role of synoviolin in human RA is less well understood. In vitro, synoviolin has been reported to protect against both ER-stress-induced apoptosis in RA FLS [4] and p53-mediated apoptosis in 293 cells [6]. In RA, the proinflammatory cytokines IL-1 and TNF derived predomi- nantly from synovial macrophages lead to sustained synoviolin expression [3,5,7]. In vivo, synoviolin has been reported to be elevated in a subset of circulating immune cell subsets which may be associated with anti-TNF nonresponse [3]. IL-17 is a pleiotropic proinflammatory cytokine produced from Th17 cells [8]. IL-17A is the prototypic member belonging to a family of 6 ranging from IL-17A to IL-17F [9]. IL-17A mediates its biological effects through binding to a receptor complex consisting of IL-17RA and IL-17RC subunits which are expressed in RA FLS [10]. The IL-17 receptor complex interacts with a STIR (SEFIR, similar expression to fibroblast growth factor genes) subdomain leading to activation of act1, TRAF6, nuclear factor kappa B (NFkB), activator protein 1 (AP-1) and mitogen protein kinases (MAPK) [10,11,12,13,14]. IL-17 has a critical role in RA joint inflammation and destruction [15]. IL-17 has been described to be implicated in synovial hypercellularity in murine arthritis models [16]. In addition, IL-17RC has been reported to regulate apoptosis [17]. The role of T-cell derived cytokines such as IL-17 in regulation of dysregulated apoptosis and proliferation in RA FLS is not known. In this study, we demonstrate that IL-17R- mediated induction of synoviolin contributes to chronic synovial hyperplasia and inflammation by prolonging the survival of RA FLS and germinal centre reactions. Results Synoviolin induction by IL-17 in RA FLS We investigated the expression of synoviolin in cultured RA FLS treated with IL-17A or IL-17F compared to IL-1, TNF and LPS by real time RT-PCR and Western blot. Synoviolin was constitutively expressed in RA FLS, and induced by IL-17A, IL- 17F, IL-1 or TNF at 24 h (Figure 1A, upper panel). The induction of synoviolin expression by IL-17A at 12 h was weaker than induction by IL-1b or LPS but stronger than IL-17F or TNF-a. IL-17-induced synoviolin expression was confirmed at the protein level. IL-17A was a more potent inducer of synoviolin compared to IL-17F (Figure 1A, lower panel). Given that IL-17A induced more robust synoviolin induction than IL-17F, subsequent experiments were carried out with IL-17A alone and hereby referred to as IL-17. We next determined the dose range of synoviolin induction by IL-17. A dose dependent increase in synoviolin mRNA by IL-17A was observed (Figure 1B, left panel). The timecourse of IL-17 50 ng/ml induction of synoviolin was examined over 24 hours. IL-17A induced significant synoviolin mRNA expression at 6 h which increased and remained elevated over 24 h (Figure 1B, right panel). IL-17A increased synoviolin mRNA 4.9 fold at 24 h (P,0.05). In comparison with OA FLS or OA dermal fibroblasts, IL-17-induced synoviolin expression was more marked in RA FLS (Figure 1C). IL-17 and Synoviocyte Survival ERK and JNK activation is required for IL-17 induced expression of synoviolin ERK MAPK and ETS1 transcription factor activation have been described to modulate synoviolin expression in murine cells[5]. To investigate the involvement of MAPK activation in IL- 17 induced synoviolin expression, RA FLS were preincubated with MAPK inhibitors for 1 hour [18,19] and then treated with IL-17 50 ng/ml over 12 h. Inhibition of ERK (PD98059) or JNK (SP600125) was associated with a marked reduction in synoviolin mNA expression (Figure 1D). Although there was a trend, we did not observe a statistically significant reduction in IL-17 induced synoviolin expression in the presence of the p38 pathway inhibitor (SB203580). IL-17 protects RA FLS from SNP-induced apoptosis To determine the functional significance of IL-17-induced synoviolin expression in RA FLS, we examined the effects of IL-17 treatment on SNP-induced apoptosis. SNP induces apoptosis by NO donation and leads to cell death by both ER-stress and p53- mediated apoptosis [20,21,22]. RA FLS demonstrated low rates of spontaneous apoptosis. SNP (0.1–1 mM) induced a significant increase in apoptosis up to 70–80% of annexin V positive cells by FACs analysis (data not shown) or by SS DNA fragmentation, associated with decreased synoviolin expression at 6 and 24 h (Figure 2A, B, P,0.05). Treatment of RA FLS by IL-17 over 24 h significantly impaired SNP-induced apoptosis (Figure 2C). This effect of IL-17 was confirmed by a reduction in SNP-induced annexin V positive cells (Figure 2D, P,0.05). Effect of IL-17RA or IL-17RC RNA interference on RA FLS apoptosis To confirm a protective role for IL-17R signaling in SNP- induced apoptosis we next examined the effect of IL-17RA or IL- 17RC RNA interference in RA FLS. IL-17RA siRNA lead to a 72% inhibition of IL-17RA mRNA and protein expression, whereas IL-17RC siRNA inhibited IL-17RC mRNA and protein expression by 70% (Figure 3A, B). To determine the specificity of RNA interference we examined the effects of IL-17RA or IL- 17RC interference on IL-17RA or IL-17RC expression. IL-17RA RNA interference had either no effect or slightly increased IL- 17RC mRNA expression (Figure 3B). IL-17RC RNA interference had no effect on IL-17RA mRNA expression (Figure 3B). In the presence of IL-17, IL-17RA or IL-17RC RNA interference significantly enhanced SNP 0.1 mM induced apoptosis in RA FLS, with a 3 or 2.7 fold increase in apoptosis respectively compared to sicontrol (Figure 3C, P,0.05), confirming a protective effect of IL-17 on RA FLS apoptosis. Effect of IL-17RA or IL-17RC RNA interference on synoviolin expression We next determined the effects of IL-17RA or IL-17RC RNA interference on synoviolin expression. Both IL-17RA or IL-17RC siRNA lead to near complete abrogation of IL-17-induced synoviolin expression in RA FLS (Figure 3D). As expected IL- 17RA or IL-17RC RNA interference antagonism also reduced IL- 17-mediated p65 induction, whereas IL-17RA RNA interference more potently suppressed IL-17-induced c-fos expression com- pared to IL-17RC (data not shown). We confirmed IL-17R- mediated modulation of synoviolin by the use of neutralising Abs against IL-17RA or IL-17RC [23]. Anti-IL-17RA or anti-IL- 17RC Abs significantly antagonised IL-17-induced synoviolin expression (Figure 3E, P,0.05). PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13416