IL-17 and Synoviocyte Survival pretreated with IL-17 100 ng/ml for 2 h then cotreated with SNP 0.1-1 mM for 24 h and apoptosis measured by SS DNA apoptosis kit (C). The results are presented as the fold induction in apoptosis relative to control samples, n = 3, mean 6 SEM of duplicate experiments from 3 different RA donors. * P,0.05 compared to no treatment control. RA FLS were treated as above and annexin V analysed. Representative serial fluorescent microscopic pictures of Annexin V positive cells (green), double labelled with Propidium iodide (PI, red) at magnification x 200 (D). doi:10.1371/journal.pone.0013416.g002 for IL-17RC in protection against apoptosis has been reported in a prostate cancer line whereby IL-17RC inhibits caspase 2, 8 and subsequently caspase 3 [17]. In contrast an anti-apoptotic effect of IL-17RA has not previously been described. Similarly both IL- 17R components were involved in IL-17 induced p65 NFkB and c-fos AP-1 activation in RA FLS. This is consistent with human and murine data demonstrating the requirement for both IL- 17RA and IL-17RC subcomponents in the biological effects of IL- 17 [23,33]. In contrast to the clear anti-apoptotic effects of IL-17, IL-17 (1-100 ng/ml) over 6 h, 24 h, 48 h or 7 days did not significantly stimulate RA FLS proliferation (data not shown). We observed that IL-17R mediated ERK and JNK activation were required in RA FLS synoviolin expression. Cytokine induced ERK MAPK and Ets 1 transcription factor have been reported to control synoviolin expression in mouse cells [5]. In contrast to IL- 17-dependent synoviolin in human RA FLS, in mouse cells Gao et al [5] demonstrated that JNK activation is dispensable for IL- 1b-dependent synoviolin expression. The differences observed may be cytokine dependent and/or associated with mouse/human cell differences. NFkB binding sites are not present in the synoviolin promoter. These data suggest that IL-17R-induced synoviolin expression via ERK and JNK MAPK activation contributes to protection against apoptosis in RA FLS. MAPK and AP-1 activation have an established role in cell survival. The underlying mechanism for IL-17-mediated protection against RA FLS apoptosis may be associated at least in part to IL-17 induced synoviolin expression. Synoviolin RNA interference enhances ER stress induced apoptosis in RA FLS[34], and in this study synoviolin siRNA augments SNP-induced RA FLS apopto- sis. Herein, IL-17 treatment subsequently reduced synoviolin siRNA-induced RA FLS apoptosis, rescued RA FLS from SNP- induced apoptosis and dose dependently induced synoviolin expression. Furthermore, either IL-17 or synoviolin inhibition enhanced SNP-induced apoptosis. Complete abrogation of synoviolin knockdown-induced apoptosis at high doses of SNP required the combination of both IL-17 and TNF consistent with the additive effects of this cytokine combination on synoviolin induction. IL-17 had more potent effects than TNF on synoviolin induction and protection against RA FLS apoptosis. Thus, in addition to additive effects of IL-17 and TNF on inflammation and bone destruction this cytokine combination enhanced anti- apoptotic effects of IL-17. In the mixed cytokine milieu of an inflammed arthritic joint, other proinflammatory cytokines may enhance the protective effects of IL-17 against FLS apoptosis implicated in synovial hyperplasia contributing to pannus persistence and chronicity. It is possible that IL-17 may also exert its anti-apoptotic effects in a synoviolin independent manner such as through induction of NFkB, MAPK and AP-1. Both endogenous and cytokine induced synoviolin expression in RA FLS promote RA FLS survival. It is unclear how IL-17-induced or endogenous synoviolin expression may be anti-apoptotic in RA FLS. Recent data suggests that the tumour suppressor gene, p53 may be regulated by synoviolin [6]. Synoviolin binds to, ubiquitinates and degrades cytoplasmic p53. Furthermore, IL-17 rescued RA FLS from SNP-induced apoptosis. SNP is a nitric oxide (NO) donor, and elevated NO levels play a key role in apoptosis of cells involved in the inflammatory response. NO mediates apoptosis by both p53-mediated apoptosis [35] and ER-stress induced apoptosis [36,37]. Although it is tempting to speculate that IL-17 may indirectly modulate p53 expression through synoviolin expression we were unable to confirm this hypothesis at least at timepoints up to 24 h of IL-17 treatment (data not shown). IL-17 did not suppress p53 protein expression in timepoints up to 24 h despite rescue of RA FLS from SNP- induced apoptosis by IL-17, associated with increased synoviolin expression. IL-17 increased p53 expression prior to synoviolin expression at 4 h and 6 h respectively following IL-17A treatment which was sustained out to 24 h (data not shown). However, the time course needs to be extended to examine whether p53 degradation occurs following synoviolin induction beyond 24 h. Moreover, synoviolin sequesters p53 in the ER. It would be interesting to examine p53 sequestration following IL- 17-induced synoviolin upregulation, and the ubiquitination status of p53 at later timepoints. Although, IL-17 was capable of inducing both synoviolin and p53 the net effect was anti- apoptotic. Other proinflammatory cytokines such as MIF have clear protective effects against SNP-induced apoptosis in RA FLS through marked downregulation of p53 expression [32]. Interestingly we identified synoviolin and IL-17 coexpressing cells in synovial germinal centre and follicle-like structures in WT mice with SCW-induced arthritis. Th17 cells have been recently described to play a key role in germinal centre formation [38]. We previously described synoviolin expression in peripheral blood CD3 + T cells from RA patients [3]. In addition, such synoviolin expressing Th17 cells were found in close contact with synoviolin expressing CD19 + B cells in follicle-like structures of these mice. Subsequent to this finding we cell sorted human CD19 + B cells from the peripheral blood of RA patients and found that synoviolin was also highly expressed in the blood (data not shown). Germinal centre-like structures including synoviolin expression were completely absent in IL-17R deficient mice. IL- 17R signaling has been demonstrated to be critical for the formation of such structures in an autoimmune model of arthritis as in autoimmune BXD2 mice [38]. In this study the coexistence of Th17 cells and B cells expressing synoviolin suggests that it may be a prosurvival factor for such cells in addition to RA FLS. B cells have a clear pathogenic role in RA as demonstrated by the efficacy of B cell depletion with anti-CD20 mAbs in the clinic [39]. It remains to be determined if IL-17 antagonists may directly reduce circulating and synovial B-cells in a similar way to current anti- TNF biologics or anti-B cell therapies [39,40]. We describe a novel role for IL-17 in RA FLS survival via downregulation of FLS apoptosis. IL-17RA and IL-17RC mediated signaling and synoviolin expression may contribute to dysregulated RA FLS growth. Anti-apoptotic effects of IL-17- induced synoviolin are enhanced by TNF. Persistence of synovial Th17 synoviolin-expressing cells in close contact with B cells in germinal centre structures may further contribute to chronicity. These observations have important implications in the interaction between T cells and other stromal cells in the progression from initial synovial inflammation to hyperplastic pannus formation. Future IL-17 antagonists may limit both synovial inflammation and hyperplasia. Targeting IL-17 may improve efforts to control the chronicity of the disease, possibly in addition or following the control of inflammation with current cytokine inhibitors. PLoS ONE | www.plosone.org 6 October 2010 | Volume 5 | Issue 10 | e13416