Objective The aim of this study was to compare the effects of interleukin (IL)-17A and IL-17F on gene expression and signalling in human rheumatoid arthritis (RA) synoviocytes.
Methods IL-17A- and IL-17F-induced mRNA expression was analysed using Affymetrix microarrays. IL-6 and IL-8 secretion was evaluated by ELISA. Inhibition of two receptors (IL-17RA and IL-17RC) was achieved by small interfering RNA (saran). The effects on mitogen-activated protein kinase (MAPK), activator protein 1 (AP-1) and nuclear factor κB (NF-κB) expression and activation were evaluated by western blotting, qRT-PCR and DNA binding assay.
Results IL-17A and IL-17F induced a molecular pattern characterised by 27 inflammation-related genes for IL-17F and 165 for IL-17A. Virtually all IL-17A and IL-17F inducible genes were dependent on NF-κB activation, whereas a small number were modulated by p38. IL-17A induced activation of all three MAPKs (ERK, p38 and JNK) and downstream transcription factors AP-1 and p65 NF-κB. IL-17F was less potent but induced activation of p50 NF-κB. IL-17A was more potent at inducing IL-6 secretion than IL-17F, which was inactive alone. IL-17A and, to a lesser extent, IL-17F induced TRAF6 but not MyD88. Inhibition of either IL-17RA or IL-17RC expression via siRNA led to near complete abrogation of IL-6 expression mediated by IL-17A and the combination of IL-17F and tumour necrosis factor α.
Conclusion Like IL-17A, IL-17F regulates proinflammatory gene expression by a very similar but not identical signalling pathway involving IL-17RA and IL-17RC.
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Rheumatoid arthritis (RA) is a complex chronic disorder leading to joint destruction and characterised by synovium hyperplasia, neoangiogenesis and local infiltration by immune cells. Among the complex network of inflammatory cells involved in the pathogenesis of RA, Th17 cells have recently been identified as key cells in the promotion of autoimmune processes, joint destruction and angiogenesis.1 The hallmark of the Th17 subset is the production of interleukin (IL)-17A and IL-17F, which share strong homology. Although studies have shown the pathogenic implications of the IL-17 axis in mouse models of RA, our knowledge concerning IL-17F and, to a lesser extent, IL-17A remains limited in human pathology. In particular, the signalling pathways induced by IL-17A and IL-17F are just beginning to be elucidated.2
IL-17A induces activation of at least two major signalling pathways involved in inflammation—the IB kinase/nuclear factor κB (IKK/NF-κB) pathway and the mitogen-activated protein kinase (MAPK) cascade. IL-17A can activate all three classes of MAPK: extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK) and p38.3,–,5 NF-κB exists as dimeric proteins composed of members of the Rel family of proteins which include RelA/p65, c-Rel, RelB, NF-κB1/p50 and NF-κB2/p52.6 p65/p50 constitutes the most abundant form and is increased in the synovium of patients with RA.7 The activator protein 1 (AP-1) family is composed of the Fos (c-Fos, FosB, Fra-1 and Fra-2) and Jun (c-Jun, JunB and JunD) family members.8
We have previously studied the signalling pathways of IL-17A in synoviocytes from patients with RA.9 In this paper we extend the studies to IL-17F. Using oligonucleotide microarray analysis of more than 30 000 genes, the IL-17A and IL-17F expression profile of RA synoviocytes was analysed and the results were validated by extensive statistical analysis. The signal transduction pathways triggered by IL-17A and IL-17F were studied by analysing MAPK (p38, JNK, ERK), AP-1 and NF-κB activation. We show that IL-17A and IL-17F regulate proinflammatory gene expression, and this requires binding to the receptors IL-17RA and IL-17RC and activation of MAPK, NF-κB and AP-1.
Material and methods
Cell culture and experimental design
RA synoviocytes were obtained from synovial tissue from patients with RA undergoing joint surgery who fulfilled the American College of Rheumatology criteria for RA.10 In brief, synovial tissue was minced into small pieces and incubated for 2 h at 37°C with proteolytic enzymes. Synoviocytes were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen Life Technologies, Carlsbad, California, USA) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin, streptomycin at 37°C and used at passage 3–6 which were >99% negative for CD45, CD1, CD3, CD19, CD14 and HLA-DR and positive for the expression of CD44 (antibodies obtained from PharMingen Europe, Hamburg, Germany). The effect of IL-17A and IL-17F alone or in combination with tumour necrosis factor α (TNFα) was compared at both the protein and mRNA levels. Synoviocytes seeded in 96-well plates (1 × 104 cells /well) were stimulated for 48 h with IL-17A or IL-17F (0.1–100 ng/ml) alone or in combination with TNFα (0.5 ng/ml) to examine protein secretion by ELISA. Synoviocytes seeded in 6-well plates (5 × 105/l) were stimulated for 12 h with IL-17A (50 ng/ml) alone or in combination with TNFα (0.5 ng/ml) to examine target gene mRNA expression by quantitative real-time PCR (RT-PCR). The cells were serum-starved for 12 h and untreated cells were used as negative controls.
RNA extraction and purification
Synoviocytes seeded in 6-well plates (5 × 105 cells/well) were serum-deprived for 2 h and then stimulated for 12 h with IL-17A or IL-17F (50 ng/ml) in DMEM/10% FCS. RNA was extracted from RA synoviocytes using TRIzol reagents (Gibco BRL, Cergy-Pontoise, France), purified using RNeasy kits (Qiagen, Hilden, Germany) and the concentration of RNA was measured by spectrophotometry (SmartSpec 3000, BIO-RAD, Hercules, California, USA).
mRNA Microarray hybridisation and analysis
2 µg RNA from RA synoviocytes were analysed using HG-U133A arrays (Affymetrix, Santa Clara, California, USA). The RNA integrity number (RIN) was assessed using RNA 6000 nano chips and the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Total RNA was used to prepare double-stranded cDNA containing the T7 promoter sequence. cRNA was synthesised and labelled with biotinylated ribonucleotide (GeneChip IVT Labeling Kit, Affymetrix). The fragmented cRNA was hybridised on HG-U133A oligonucleotide arrays (22 283 probe sets). The arrays were washed and stained using the fluidic station FS450 (Affymetrix) (protocol EukGE-WS2v4) and scanned with the Agilent G2500A GeneArray Scanner. Statistical analysis was preformed using the Affymetrix Data Mining Tool Software (MAS 5.0) (see details in online supplement).
DNA binding assays
Nuclear extracts were prepared according to the manufacturer's instructions (Active Motif, Rixensart, Belgium). NF-κB and AP-1 regulation was measured by multiwell colorimetric transcription factor assays using Trans AM AP-1 c-jun and c-fos, NF-κB p50 and p65 transcription factor assay kits (Active Motif). Specific binding was assessed using mutated or wild-type oligonucleotides in accordance with the manufacturer's instructions.
Western blot analysis
RA synoviocytes were serum-starved in DMEM for 24 h, then treated with IL-17A or IL-17F 50 ng/ml for 30 min. MAPK activation in cells was analysed by western blotting in the presence or absence of IL-17A and IL-17F stimulation as previously described.11 In brief, cells were lysed using stress lysis buffer (20 mM Hepes pH 7.7, 2.5 mM MgCl2, 0.1 mM EDTA, 20 mM β-glycerophosphate, 100 mM NaCl, 0.05% Triton X100, 0.5 mM DTT, 0.1 mM sodium ortho-vanadate, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 100 µg/ml phenylmethylsulfonyl fluoride), and the protein concentration was determined by the BCA assay kit (Pierce, Rockford, Illinois, USA). Aliquots containing 80 µg proteins were subjected to electrophoresis on 10% SDS-polyacrylamide gel. After electrophoretic separation, the proteins from the gels were transferred onto nitrocellulose membranes with miniVE blotter (Amersham, Princeton, New Jersey, USA) (see details in online supplement).
Quantitative RT-PCR analysis
RA synoviocytes were serum-starved for 12 h and treated with IL-17A or IL-17F 50 ng/ml for 30 min. RNA was extracted from RA synoviocyte cells using TRIzol reagent (Invitrogen) and 1 µg of RNA was reverse transcribed using the Superscript reverse transcription system (Invitrogen). PCR amplification was performed on a LightCycler (Roche Diagnostics, Switzerland) using Fast-Start DNA Master SYBR Green I real-time PCR kit (Roche Molecular Biochemicals, Switzerland) as previously described (see details in online supplement).12
Gene-specific siRNA corresponding to a 19-nucleotide sequence targeting 1623–1641 of human IL-17RA (NM_014339) or 985–1003 of human IL-17RC (NM_153460) were purchased from Dharmacon (Lafayette, Colorado, USA). A non-targeting siRNA (siCONTROL) was used as a control for non-sequence-specific effects. RA synoviocytes (1 × 105) were transfected with 0.5 μg IL-17RA, IL-17RC or siCONTROL siRNA duplexes using the Nucleofector Kit (Amaxa GmbH, Cologne, Germany) according to the recommended protocol from the manufacturer. After 24 h of transfection, the cells were serum-starved for 12 h and then treated with 50 ng/ml IL-17A or IL-17F. IL-8 and IL-6 mRNA and protein levels were assessed by qRT-PCR and ELISA.
Cytokines, antibodies and ELISA
Human recombinant TNFα was purchased from Sigma-Aldrich (St Louis, Missouri, USA). Human recombinants IL-17A and IL-17F were purchased from R&D Systems (London, UK). RA synoviocytes seeded in 6-well plates (5 × 105 cells/well) or 10 mm Petri dishes were stimulated with IL-17A or IL-17F (50 and 200 ng/ml) for 12 h or 1 h for mRNA or functional studies, respectively. RA synoviocytes were stimulated with recombinant human IL-17A or IL-17F (R&D Systems, Minneapolis, Minnesota, USA) 50 ng/ml for 12 or 48 h.
mRNA expression of target genes was normalised with GAPDH mRNA expression and data expressed as the fold induction compared with untreated controls. Data are expressed as mean±SEM. The statistical significance of changes was determined by the Student t test. Differences with p values <0.05 were considered statistically significant. All data are the result of at least three separate experiments (see details in online supplement).
IL-17A and IL-17F induce different expression patterns in RA synoviocytes
Microarray analysis of IL-17A- and IL-17F-induced mRNA expression in RA synoviocytes showed the activation of several pathways. The microarray-derived dataset has previously been validated by computer simulation and by RT-PCR focusing on chemokines.13 Restricting the inclusion criteria to at least a 2.5-fold increase in gene expression or a clear on/off switch (for details see Materials and methods section), 27 genes were induced by exposure to IL-17F compared with 165 genes with IL-17A. Furthermore, genes increased by IL-17F and decreased by IL-17A or vice versa were not identified. Most genes could be assigned to functional groups such as chemokines/cytokines, cell surface receptors, components of the coagulation system, inflammatory response, signal and transcription factors, apoptosis or cell proliferation, metabolism components or cell transporters/channels (table 1). All genes induced by IL-17F were also induced by IL-17A, usually in a more potent manner. The greatest effect was observed for chemokine mRNA induction which reached 755–4571-fold induction with IL-17A compared with 20-fold induction with IL-17F (table 1).
Focusing on a few new genes of potential interest in RA, the effect of IL-17A and IL-17F was compared at the mRNA level. For example, IL-17A and IL-17F significantly upregulated the expression of two key antioxidant enzymes (SOD2 and catalase), suggesting a direct effect of IL-17A and IL-17F in preventing intracellular disequilibrium of redox status. This is in accordance with the effect of IL-17A and IL-17F on the expression of genes implicated in cell survival. Indeed, both cytokines upregulated the expression of Bcl2A1 and mcl-1, two bcl2-related members, and NR4 family members (NR4A1, NR4A2 and NR4A3) which promote cell survival.
The expression profiles of synoviocytes treated with IL-17A or IL-17F also included the upregulation of genes implicated in joint lubrication. For example, IL-17A induced a 21-fold induction of hyaluronan synthase 1 (HAS 1), an enzyme implicated in the production of hyaluronic acid. IL-17A and IL-17F had effects on the expression of several growth factors such as epiregulin (EREG), a member of the epithelial growth factor family. The regulatory effect of IL-17A and IL-17F on EREG was validated by RT-PCR, and showed that IL-17A and IL-17F had an additive effect with TNFα (figure 1). To assess the role of IL-17F in joint destruction, the effect of IL-17F on matrix metalloproteinase 3 (MMP-3) mRNA expression was analysed. IL-17F alone had no effect but, as for IL-17A, a combination of IL-17F and TNFα had a synergistic effect on MMP-3 expression. Although the combination of IL-17A or IL-17F with TNFα usually increased expression, the combination did inhibit the expression of a few genes. For instance, the combination of IL-17A or IL-17F with TNFα inhibited the expression of COLA1A, a gene involved in collagen synthesis, in line with an inhibitory effect on repair.
Induction of IL-6 and IL-8 secretion by IL-17F and IL-17A in RA synoviocytes
To compare IL-17A with IL-17F, the expression and production of IL-6 and IL-8 evaluated by qRT-PCR and ELISA, respectively, were used as read-out assays. Suboptimal concentrations of IL-17F alone had a limited or no effect but, in combination with a suboptimal concentration of TNFα, it induced a rapid increase in the accumulation of IL-6 and IL-8 mRNA and protein with a clear synergistic effect (figure 2). IL-17F 50 ng/ml induced an approximately 3.2-fold increase in IL-8 mRNA compared with a 47.1-fold increase with IL-17A, confirming the weaker effects of IL-17F compared with IL-17A.
IL-17A and IL-17F regulate inflammatory gene expression via IL-17RA and IL-17RC
In order to study the signalling cascade following activation with IL-17A or IL-17F, the first step was to clarify the contribution of IL-17 receptors to the effect of IL-17F compared with IL-17A. Two receptors, IL-17RA and IL-17RC, have been identified for the signalling of IL-17A.14 However, the respective contributions of IL-17RA and IL-17RC to the signalling of IL-17F remain to be clarified. Specific downregulation of IL-17RA and IL-17RC was obtained using siRNA technology. IL-17RA or IL-17RC expression was downregulated via transfection with specific siRNA. Compared with siCONTROL, IL-17RA and IL-17RC siRNA significantly decreased the mRNA level of target genes (here IL-6) (figure 3A). Similar experiments with IL-17F alone were difficult to interpret because of its limited effects when used alone. Using the combination with TNFα (figure 2), a threefold decrease in IL-6 mRNA expression after knockdown of IL-17RA and IL-17RC was observed (figure 3B), indicating the role of both IL-17RA and IL-17RC in the signalling of IL-17F.
Modulation of transcription factor activation by IL-17A versus IL-17F
To further compare the effect of IL-17A and IL-17F, the activation of several signalling pathways was studied (figure 4). RA synoviocytes were stimulated with IL-17A or IL-17F for 1 h and p65, p50, p52, RelB NF-κB DNA binding activity and mRNA expression were analysed. IL-17A but not IL-17F induced p65 binding activity (figure 4A left panel) and mRNA expression (figure 4B left panel, p<0.05). Furthermore, IL-17A and IL-17F induced p50 DNA binding activity (figure 4A right panel, p<0.05). However, IL-17A and IL-17F did not induce p52 mRNA expression (data not shown). To confirm the role of NF-κB activation, the effect of a chemical NF-κB inhibitor (BAY) on IL-6 mRNA expression was assessed. RA synoviocytes were preincubated for 1 h with the inhibitor and then treated with IL-17F (50–200 ng/ml) for 12 h. IL-6 mRNA expression decreased dose-dependently in the presence of the inhibitor. The experiments were performed for IL-17F at 200 ng/ml, a concentration high enough to induce IL-6 mRNA expression; a fourfold decrease in IL-6 mRNA expression was observed with BAY in a dose-related fashion (p<0.05; figure 4B, right panel). The specificity of this reaction was confirmed by the addition of cold oligo-DNA (data not shown).
Similar experiments were performed for the activity and expression of c-fos and c-jun, which are two components of the AP-1 complex. After 1 h of stimulation, IL-17A and IL-17F significantly increased c-jun or c-fos DNA binding (figure 4D) and mRNA expression (figure 4C, p<0.05).
Effect of IL-17A versus IL-17F on MAPK activation
The MAPK family has been shown to play an important role in regulating the gene expression response to inflammatory mediators.15 To compare IL-17A and IL-17F, RA synoviocytes were stimulated for 1 h to measure the activation of MAPK family members (p38, JNK and ERK). As shown in figure 5A, IL-17A and IL-17F increased the phosphorylation of all three MAPKs with a maximal effect at 30 min. At an optimal dose of 50 ng/ml, IL-17A was not statistically more potent than IL-17F in inducing the three MAPKs (figure 5B). However, IL-17A was more potent than IL-17F in inducing the upstream signalling molecules TRAF6 and MEKK3 (figure 5C left panel). IL-17A and IL-17F had no effect on MyD88 activation at the mRNA or protein level (figure 5C, right panel).
Although IL-17A has been relatively well studied, the effects of IL-17F remain poorly understood. The aim of this study was to describe the effects and the signalling of IL-17F compared with IL-17A in RA synoviocytes. We have previously observed that IL-17F stimulates many genes through Affymetrix microarray analysis focusing on chemokines.13 These first results showed that IL-17F had a very similar regulation to IL-17A.13 The current study provides additional findings on IL-17F signalling. IL-17F alone induced the expression of only 27 genes compared with 165 genes for IL-17A. No gene was increased by IL-17F and decreased by IL-17A or vice versa. Of the 27 genes induced by IL-17F, no gene was identified as being specific to IL-17F. However, their effects can still be different in vivo. For instance, IL-17F is not needed to initiate experimental allergic encephalitis in mouse models but it acts as a proinflammatory cytokine in acute experimental colitis. Indeed, IL-17F knock-out mice were protected whereas IL-17A deficiency increased colon damage.16
Using siRNA, downregulation of either IL-17RA or IL-17RC led to near complete abrogation of IL-6 activation, in line with results in the mouse.17 At the time IL-17A was identified, the first receptor was also described and designated IL-17R,2 now referred to as IL-17RA. Bioinformatic analysis led to the identification of four additional IL-17 receptor-related molecules and these are now designated IL-17RB, IL-17RC, IL-17RD and IL-17RE.18 It has been clearly shown that IL-17A binds to IL-17RA and IL-17E binds to IL-17RB.19 In addition, it has been reported that IL-17RA and IL-17RC function as a heterodimer using mouse IL-17RA-deficient fibroblasts.20 Using bioinformatic analysis, human IL-17RC was suspected to be the receptor for human IL-17F, but it appears now that it also binds human IL-17A.20 In addition, we have already shown that IL-17RA and IL-17RC are overexpressed in whole blood of patients with RA.21
The basis for the functional differences between IL-17A and IL-17F could be the markedly decreased strength of signalling triggered by IL-17F compared with IL-17A, as IL-17F-induced responses are 10–30-fold weaker in terms of downstream gene activation than those of IL-17A, with IL-17A–IL-17F heterodimers acting at an intermediate level.22 23 As IL-17RA is not homologous to any known receptors,24 it was not possible to investigate signal transduction simply with reference to other cytokine receptor systems such as the IL-1 and TNFα receptors. However, studies defining IL-17A-induced genes showed that IL-17A activates a highly proinflammatory programme of genes which is very similar to that induced by the IL-1 and TNF receptors.25 26
Both IL-17A and IL-17F significantly induced activation of p50 NF-κB, although IL-17A was again more potent. Both IL-17A and IL-17F induced AP-1 expression and its DNA binding activity. IL-17A induced phosphorylation of all three MAPKs in a dose-dependent manner; IL-17F showed less activity on MAPKs. Regarding the induction of other pathways known to be activated by IL-17A, IL-17F activated TRAF6 in a dose-dependent manner. We also confirmed that IL-17F signals independently of MyD88, as already shown for IL-17A using MyD88-deficient mice fibroblasts.27
Understanding the differential contribution of IL-17A versus IL-17F and IL-17R-mediated intracellular signalling pathways in RA synoviocytes may lead to the development of novel therapies. Positive results have been observed with IL-17A inhibition in RA and psoriasis.28 Our results indicate that IL-17F could also be targeted based on the synergistic interaction between IL-17F and TNFα. The same concept applies to the targeting of IL-17 receptors and their signalling pathways.29
Funding This work was supported in part by grants from the Hospices Civils de Lyon and the Region Rhône-Alpes. AH is supported by the Société Nationale de Médecine Interne. SZ was supported by a scholarship from the Region Rhône-Alpes. M-LT was supported by a fellowship from the Region Rhône-Alpes.
Competing interests None.
Patient consent Obtained.
Ethics approval This study was conducted with the approval of the ethics committee of the Hospitals of Lyon.
Provenance and peer review Not commissioned; externally peer reviewed.