IL-17 and Synoviocyte Survival SEM. * P,0.05 compared to no treatment control. RA FLS were treated with IL-17A or IL-17F (50 ng/ml) for 24 h and synoviolin expression measured by Western Blotting (A, lower panel). The membrane was stripped and reprobed for b-actin and the result is representative of n = 3. RA FLS were treated with IL-17 5-200 ng/ml for 24 h (B, left panel) or IL-17 50 ng/ml over 24 h (B, right panel) and synoviolin expression measured by real-time RT-PCR, n = 3, mean 6 SEM, * P,0.05. Synoviolin mRNA expression in RA FLS, OA FLS and OA dermal fibroblasts that were untreated (–) or were treated with 50 ng/ml or 10 ng/ml of IL-1 for 24 h (C). * P,0.05 versus untreated FLS. RA FLS were preincubated with PD98059 (50mM), SB203580 (5mM) or SP600125 (50mM) for 1 hour, then treated with IL-17 50 ng/ml for 1 hour. Synoviolin expression was measured by real-time PCR, mean 6 SEM, n = 3 (D). * p,0.05 compared to IL-17 treatment. doi:10.1371/journal.pone.0013416.g001 Effect of synoviolin RNA interference on RA FLS apoptosis To determine if synoviolin may inhibit SNP-induced apoptosis in RA FLS, we determined effects of synoviolin RNA interference in RA FLS. We confirmed 80% knockdown of synoviolin mRNA and protein by RNA interference in RA FLS (Figure 4A). Duplex 2 (S-02) was the most efficient and was chosen for subsequent experiments. Synoviolin RNA interference increased SNP-induced apoptosis by 4 fold in RA FLS (Figure 4B, P,0.05) demonstrating the specificity of its contribution. To confirm that the anti- apoptotic effects of IL-17 were mediated by synoviolin, we demonstrated that the protective effects of IL-17 were completely abrogated in the presence of synoviolin knockdown at the lowest dose of SNP (Figure 4C, P,0.05). However, at higher concentra- tions of SNP the anti-apoptotic effects of IL-17 could be reversed. Effect of IL-17 and TNF on synoviolin expression and apoptosis in RA FLS IL-17 and TNF have synergistic or additive effects on many of the biological activities of IL-17 [24]. As noted previously TNF had a weaker effect on induction of synoviolin than IL-17A. IL-17 100 ng/ml in combination with TNF (1 or 10 ng/ml) had additive effects on synoviolin induction in comparison with TNF alone (P,0.05), but only trended to significance compared to IL-17 alone (Figure 5A). Similarly, at the higher concentrations of SNP 0.5 mM (or 1 mM data not shown) only the combination of IL-17 and TNF was capable of completely abrogating RA FLS apoptosis in the presence of synoviolin knockdown compared to either cytokine alone (Figure 5B). At the lowest concentration of SNP 0.1 mM, IL- 17 but not TNF was capable of completely protecting RA FLS from apoptosis induced by synoviolin knockdown. Some variabil- ity in the potency of cytokine-induced synoviolin induction and apoptosis was noted between different cell lines. Reduced synoviolin expression in IL-17R deficient mice during chronic SCW-induced arthritis. To confirm the requirement of IL-17R-dependent signaling in IL-17-induced synoviolin expression we examined the effects of IL-17R deficiency on synoviolin expression. We first assessed synoviolin expression histologically in injected knee joints of WT mice during chronic SCW-induced arthritis. The synoviolin positive cells in murine synovium were observed either diffusely or in some sections almost entirely limited to follicle-like structures. Serial sections of follicle-like structures, demonstrated that synoviolin positive cells colocalised with IL-17 and CD19 positive staining suggesting that synoviolin was expressed by both Th17 cells and CD19+ B cells (Figure 6A-E, isotype control 6F). Synoviolin expression was also observed in RA FLS invading cartilage at the pannus-cartilage junction in WT mice (Figure 6G). As previously described [25], compared to WT mice, IL-17R 2/2 mice had reduced synovial hypercellularity leading to significantly decreased macroscopic arthritis severity scores (Figure 7A, mean 6 SEM score 1.560.17 in WT versus 0.3160.10 in IL-17R 2/2 mice; P,0.05). Similarly, in comparison to WT animals, IL-17R 2/2 mice histologically displayed markedly reduced inflammatory infiltrate scores (mean 6 SEM score 2.560.43 in WT versus 0.8760.27 in IL-17R 2/2 mice ; P,0.05). Synoviolin expression was reduced in the synovial knee sections of IL-17R 2/2 compared to WT mice (Figure 7B). In IL-17R 2/2 mice, synoviolin expression was either weak or limited throughout the inflammatory infiltrate. We next examined synovial apoptosis by TUNEL staining of synovial knee joints from mice with chronic SCW-induced arthritis. TUNEL staining was significantly increased in synovial knee joints from IL-17R 2/2 mice than from WT mice (mean 6 SEM TUNEL-positive cells per high-power field 203621 versus 63618; P,0.05, Figure 7C). In situ proliferation was detected by PCNA staining. In WT mice with chronic SCW-induced arthritis, the number of proliferating cells detected was 219652 PCNA-positive cells 6 SEM per high- power field which was further reduced in IL-17R 2/2 mice compared to WT mice (110615, Figure 7D). Discussion The chronicity of RA disease is not well understood. Persistence of biological abnormalities in RA FLS or synovial tissue despite disease-modifying treatment is supported by a substantial proportion of nonresponders and the transient effects of current gold standard treatment such as anti-TNF biologics. New treatment regimens will need to go beyond the simple control of inflammation if more prolonged remissions are to be achieved. Synoviolin is an E3 ubiquitin ligase implicated in murine arthritis pathogenesis. Overexpression of synoviolin leads to spontaneous destructive arthritis, and synoviolin 2/+ mice are resistant to CIA [4]. The protective role of synoviolin in ER stress-induced apoptosis and cell survival has been demonstrated in RA FLS and other cell types [26,27,28,29]. In RA FLS, we and others have demonstrated that synoviolin plays an important role in RA FLS growth [4]. Synoviolin knockdown augments ER- stress induced apoptosis and diminishes intrinsic and cytokine- induced FLS proliferation. We recently observed expression of synoviolin in T cells, macrophages and RA FLS, which is increased by the predominantly macrophage-derived cytokines IL-1 and TNF known to regulate RA FLS cell growth and survival [3]. In contrast, IL-17 derived from Th17 cells has not been reported to modulate RA FLS cell growth. Indeed, reports in the literature suggest that IL-17 regulates cell growth in only certain cell types such as lymphocytes and data in cancer is controversial. In this study, IL-17 exerts an anti-apoptotic effect, mediated by IL-17RA and IL-17RC, associated with increased synoviolin expression. These data suggest that IL-17 contributes to RA chronicity through both synovial inflammation and hyperplasia. An anti-apoptotic role for IL-17 is supported by data in IL-17R knockout mice associated with markedly reduced synovial hypercellularity [30]. Other proflammatory cytokines such as TNF [31] and MIF [32] have been reported to contribute to synovial hyperplasia and the increased survival of RA FLS. IL-17 rescued RA FLS from SNP-induced apoptosis and IL-17RA or RC siRNA augmented SNP-induced apoptosis. An important role PLoS ONE | www.plosone.org 4 October 2010 | Volume 5 | Issue 10 | e13416