Rheumatoid arthritis (RA) is a chronic inflammatory disease where the contribution of T cells is now supported by clinical results. Among the cytokines produced by T cells, interleukin (IL)-17A (previously known as IL-17) and IL-17F constitute the signature cytokines of the newly described Th17 T helper cell subset. While the effects of IL-17A on RA synoviocytes been well described, those of IL-17F remain less studied. The present review describes the effects of IL-17A and IL-17F on synoviocytes and their contribution to RA pathogenesis. Although IL-17F is less active than IL-17A when used alone, IL-17A and IL-17F induce in synoviocytes a rather similar expression pattern in the presence of tumour necrosis factor α. They enhance their response by stabilising mRNA of cytokines and enhancing receptor expression. They increase the migration, chemokine gene expression and invasiveness of synoviocytes. They contribute to disease chronicity by inhibiting synoviocyte apoptosis. Finally, they enhance metalloprotease secretion leading to cartilage damage. These properties support the combined inhibition of IL-17A and -F to control RA inflammation and joint destruction.
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Rheumatoid arthritis (RA) is a chronic inflammatory disease leading to joint destruction and functional disability. Development of RA is linked to characteristic changes of the synovium,1 with hyperplasia, neoangiogenesis and local infiltration by immune cells, including a significant number of T cells. Among the inflammatory mediators expressed in RA synovium, interleukin (IL)-17 expression has been associated with disease severity.2 This proinflammatory cytokine is produced mainly by Th17 cells. Mouse models have indicated a critical role of IL-17A in RA, since IL-17A deficiency or antagonism has profound anti-arthritic effects.3 4 Results from human in vitro experiments have demonstrated its effects on joint degradation, through the activation of synoviocytes, making IL-17 a key player in RA pathogenesis.5 Indeed recently, two investigational monoclonal antibodies that neutralise IL-17 appeared to be safe and effective in RA in early stage clinical trials.6 7
During the last years, the IL-17 family has been expanding and new results are now available for other members of this family, specifically IL-17F.8 The two cytokines IL-17A and IL-17F exhibit a very similar but not identical expression pattern, and have a 50% homology.9 However, it is still unknown whether IL-17A and IL-17F induce similar, overlapping or divergent gene expression profiles.
The aim of this review is to describe the effect of IL-17A and IL-17F on RA synoviocytes. We briefly describe the role of T cells in RA pathogenesis, and focus on the effects of these cytokines on resident joint cells.
IL-17, Th17 and RA pathogenesis
The RA synovium undergoes striking changes, characterised by inflammatory cell infiltration and synovial hyperplasia. T cells represent a significant proportion of the cells present in the synovial tissue, but their contribution has been controversial for a long time.10 The classical arguments for the role of T cells in RA pathogenesis include the presence of synovial T cell infiltrates, and the evidence for T cell-dependent arthritis in mouse models.11 The best evidence is now illustrated by the development of biological agents that target specifically T cell co-stimulation, such as abatacept.12
A new interest in T cells has come from the discovery of Th17 cells and their contribution to RA.13 The cellular source of IL-17 was found to be different from the Th1 cells and these cells were named Th17.14 15 Following the discovery of Th17 cells, the molecular mechanisms for their differentiation have been extensively studied. It was first established that naïve mouse CD4+ T cells primed with antigen in the presence of transforming growth factor β (TGFβ) and IL-6/IL-1 differentiate into Th17 cells.16 Very similar but not identical results were obtained with human T cells,17 with interactions between TGFβ and others cytokines such as IL-2118 and IL-23.19 Apart from T cells, other cell types have been shown to express IL-17 including neutrophils and mast cells.20 Their respective contribution over that of T cells in RA pathogenesis remains to be clarified.
Concordant results using mouse and human models of RA have shown that IL-17 is involved in joint inflammation.21 The best evidence is the induction of arthritis following a single injection of IL-17 into a normal mouse knee. Furthermore, the continuous administration of IL-17 through gene overexpression induces massive damage with extensive inflammatory cell migration, bone erosion and cartilage degradation.22 23 Conversely, inhibition of IL-17 is protective in collagen-induced arthritis.24 25 For its contribution to human RA, it was first shown that pieces of RA synovium could produce bioactive IL-17.5 This activity was measured with a bioassay where supernatant fractions of RA synovium explants induced a massive production of IL-6 by human RA synoviocytes. Using the first available monoclonal antibody blocking human IL-17, such production was reduced by two-thirds.5 This robust production of IL-6 using supernatant fractions containing IL-17 was in contrast to the limited effect of recombinant IL-17 alone.
More recent results include the demonstration of the expression of IL-17A and IL-17F and their receptors in RA synovial tissue.26 27 Thus we will now discuss the role of IL-17 cytokine family members in RA and focus on its effects on synoviocytes.
The IL-17 family
The IL-17 cytokine family includes six members (A–F). These polypeptides have a dimer structure (figure 1) and consist of 163–202 amino acids with a molecular mass of 23–36 kDa. They have various degrees of similarity with a mean 20–30% amino acid identity and conservation of four cysteine residues that participate in the formation of the intermolecular disulfide bonds.28,–,30
IL-17A (previously known as IL-17) was discovered in 1993 originally as a rodent T cell cDNA transcript, named cytotoxic T lymphocyte-associated antigen 8.15 Human IL-17 was subsequently identified for its effects on fibroblasts and synoviocytes. IL-17 exerts various biological functions in vivo that might be involved in the pathogenesis of a wide range of inflammatory, infectious and autoimmune diseases.31
IL-17F was originally identified in the human genome using the first IL-17A sequence. IL-17F has a 50% homology with IL-17A.8 The gene encoding IL-17F is localised adjacent to that of IL-17A on chromosome 6.32 IL-17F induces proinflammatory cytokines and chemokines in stromal cells. For instance, IL-17F plays a role in angiogenesis through the induction of TGFβ and IL-6 in human umbilical vein endothelial cells.33 Furthermore, induction of chemokine (C-C motif) ligand (CCL) 2, CCL7 and matrix metalloproteinase 1 (MMP-1) by IL-17F in lung fibroblasts cells was reported to be less potent than that of IL-17A.34 The relative potency of IL-17F is of interest because IL-17F and IL-17A can form heterodimeric complexes.35 The contribution of this IL-17 A/F heterodimer remains to be clarified in human pathology. Even if IL-17F alone is commonly less potent than IL-17A, the combination of IL-17F and tumour necrosis factor α (TNFα) is as potent as the combination of IL-17A and TNFα.27
The IL-17 receptor family
Starting with the first IL-17 receptor (now referred to as IL-17RA), the IL-17 receptor (IL-17R) family now includes IL-17RA, IL-17RB, IL-17RC, IL-17RD and IL-17RE.36 Although it is still unclear how the IL-17R subunits interact to form active receptor complexes, it is now established that IL-17RA is a common subunit used by IL-17A and IL-17F.37 38 The affinity of IL-17RA for IL-17A and IL-17-F was lower that estimated from the concentrations required for mediating responses, suggesting that another subunit was involved in binding ligand. This hypothesis was confirmed when IL-17RC was described to be involved in IL-17 signalling.37
IL-17RA is expressed ubiquitously, but the main responses to IL-17A occur in epithelial cells, endothelial cells and fibroblasts, especially RA synoviocytes.33 IL-17RA is a type I transmembrane protein consisting of a 293 amino acid extracellular domain, a 21 amino acid transmembrane domain, and a long 525 amino acid cytoplasmic tail. IL-17RC is another transmembrane receptor able to form a heterodimer receptor complex with IL-17RA (figure 1).39 Using bioinformatics analysis, human IL-17RC was first suspected to be the receptor for IL-17F, but it appears now that it also binds IL-17A. In addition, IL-17RA and IL-17RC are overexpressed in the whole blood and synovium of RA patients, specifically on synoviocytes.40
IL-17A and IL-17F signalling pathway in synoviocytes
Our recent findings have clarified the signalling pathway of IL-17A and IL-17F in RA synoviocytes.41 IL-17A and IL-17F signalling through their receptors remains unusual compared with the typical adaptive T helper cell cytokines. Although they act through signal transducer and activator of transcription 3 (STAT3) as do other cytokines such as IL-6, IL-17A and IL-17F activate proinflammatory pathways more typical of the innate proinflammatory cytokines such as IL-1 and toll-like receptor (TLR) agonists. All these cytokines activate nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK) signalling and the CCAAT/enhancer binding protein (C/EBP) transcription factors.42 Thus, the net effect of IL-17A and IL-17F signalling is to enhance an innate-type inflammatory gene expression programme that mediates potent inflammation response and plays a key role in host defense.43
In RA synoviocytes, IL-17A and to a lesser extent IL-17F induce the binding activity and mRNA expression of p65 and p50, the two major components of the canonical NF-κB pathway. However, there is no evidence that the non-canonical NF-κB pathway is involved in IL-17 signaling.41 44 Indeed IL-17A does not promote the expression of p52 or p100, two components of the non-canonical pathway.41
Both IL-17A and IL-17F induce activator protein 1 (AP-1) expression and its DNA binding activity. After stimulation of synoviocytes, IL-17A and IL-17F significantly increase DNA binding and mRNA expression of c-jun and c-fos, two components of the AP-1 complex.41 IL-17F signals independently of myeloid differentiation primary response gene 88 (MyD88), as has already been shown for IL-17A using MyD88-deficient mice fibroblasts and confirmed recently for RA synoviocytes.45 41
Effect of IL-17A and IL-17F on MAPK activation
The MAPK family plays an important role in regulating gene expression response to inflammatory mediators, by inducing the activation of AP1.46 47 The most important consequence of MAPK pathway activation by IL-17 is the enhancing effect on the stability of mRNA transcripts.48 This effect is crucial because many IL-17A and IL-17F target genes in human RA synoviocytes are chemokine or cytokine genes, the transcripts of which are frequently destabilised by adenylate uridylate (AU)-rich elements (AREs) located in the 3' non-translated region.49
Effects of IL-17A and IL-17F on human RA synoviocytes
Having defined the interaction between IL-17A and IL-17F and their receptors, we will now focus on the functional consequences of these interactions.
Role of IL-17A and IL-17F in synoviocyte activation
Effects on cytokine production
IL-17A was identified for its ability to activate human RA synoviocytes promoting IL-6 and IL-8 mRNA expression and production.31 This production was inhibited with an anti-IL-17 antibody added to supernatant fractions of RA synovial tissue cultures.51 To assess the contribution of soluble factors in the interaction between T cells and synoviocytes in RA, the effect of IL-17A on these cells was evaluated in the presence of cytokines classified as pro- (IL-1) and anti- (IL-4, IL-13, IL-10) inflammatory. Both human IL-1 and IL-17A induced IL-6 and leukaemia inhibitory factor (LIF) production by RA synoviocytes.52 Both IL-4 and IL-13 had a modest stimulatory effect on IL-1- and IL-17A-induced production of IL-6, but inhibited that of LIF.
Comparison between IL-17A and IL-17F effect
When comparing IL-17A with IL-17F used alone for the expression and production of IL-6 and IL-8, IL-17F alone has a limited effect or no effect.27 Using microarray studies, IL-17A and IL-17F were able to regulate the expression of genes involved in inflammation, bone destruction, cartilage damage and angiogenesis. Based on this analysis, 124 genes were induced by IL-17A and IL-17F used alone but IL-17A induced more genes than IL-17F (350 versus 150, respectively).41 There was no gene induced only by IL-17F and not by IL-17A. The most important genes induced by IL-17F alone are summarised in table 1.
Synergistic effect of IL-17A and IL-17F with other cytokines
Using low concentrations of IL-17 and IL-1 in combination, a synergistic effect was observed for the production of IL-6, with an additive effect for LIF. These early findings indicate that low levels of cytokines produced by monocytes (IL-1 and TNFa) and T cells (IL-17) can act together and often synergistically in RA synoviocytes.52 Extending this observation to microarrays and using a linear mixed model, the cooperation between IL-17A and IL-17F with TNFα was found to be largely positive. Among the 1968 genes modified by the IL-17A and TNFα combination, 80 genes showed a pattern of inhibition, 1758 of additivity and 130 of synergy. Among the major genes synergistically induced by IL-17A or IL-17F in combination with TNFα, IL-6, IL-8, chemokine (C-X-C motif) ligand (CXCL) 1, CCL20, CXCL5, IL-23, p19, E-selectin, early growth response 1 (EGR-1) and granulocyte colony-stimulating factor (GCSF) are of importance in RA. The synergistic effect of IL-17A and IL-17F with TNFα is probably essential to maintain a high level of bioactivity, leading to chronicity.27 The molecular mechanisms of such synergy remain to be fully defined. One explanation is the ability of IL-17 to stabilise mRNA of cytokines. Another explanation is the enhancing effect on the expression of the TNF type II receptor with no effect on the type I or IL-17RA and IL-17RC receptors.53 The kinetic sequence includes IL-17A or IL-17F acting first in order for cells to respond differently (often better) to TNFα. Table 2 summarises the synergistic target genes induced by IL-17 in combination with other cytokines in RA synoviocytes.
Effect of IL-17A and IL-17F on chemokine production
IL-17A and IL-17F contribute to the inflammatory cell accumulation, specifically of neutrophils by the induction of ELR chemokines defined by the Glu–Leu–Arg + CXC sequence. IL-17A induces IL-8 mRNA expression more than IL-17F alone, and the combination with TNFα is synergistic.54 Regarding chemokines for monocytes, such as CCL5, IL-17A and IL-17F have no effect on CCL5 mRNA expression, whereas TNFα is effective. IL-17A and IL-17F induce a massive inhibition of TNFα-induced CCL5 expression.27 Regarding the expression of other CXC chemokines, IL-17A and IL-17F increase the secretion of CXCL12 (also known as SDF1) and the expression of its receptor CXCR4. IL-17A and IL-17F further induce CXCR4 expression in the presence of TNFα, with no effect on other chemokine receptors. This suggests a role for IL-17A and IL-17F in synoviocyte migration through a CXCL12 gradient where there is a reciprocal action between IL-17 from T cells and CXCL12 from synoviocytes. T cells migrate into the inflamed synovium under the attraction of CXCL12 produced by synoviocytes. Local Th17 cells produce IL-17, resulting in the production of CXCL12 from synoviocytes. IL-17 regulates the expression of CXCL12 in RA synoviocytes via pathways mediated by phosphoinositide (PI) 3-kinase, NF-κB and AP-1.55
Regarding the specific migration of Th17 cells, IL-17A has a synergistic effect with TNFα to promote CCL20 (also known as macrophage inflammatory protein 2 alpha (MIP3α)) expression leading to the local recruitment of chemokine (C-C motif) receptor 6 (CCR6)-positive cells, which include immature dendritic cells, naive B cells and Th17 cells. IL-17 and TNFα augment CCL20 production, whereas interferon γ (IFNγ) and IL-4 inhibit its production in synoviocytes.56 These results indicate how activated synoviocytes recruit CCR6-expressing Th17 cells to the site of Th17 cell-mediated joint inflammation.57
Among the other CC chemokines, IL-17A has a positive effect on CCL7, whereas it inhibits CCL2 and CCL5 mRNA levels. This specific regulatory effect of IL-17A highlights its role in neutrophil recruitment into the joint, but also supports its specific promoting effect on angiogenesis. This effect is linked to an increase of the production of vascular endothelial growth factor (VEGF) in RA synoviocytes,58 and to local angiogenesis.59
Effect of IL-17A and IL-17F on matrix destruction
IL-17 stimulates the release of degradative enzymes by RA synoviocytes, promoting synovium, ligament and cartilage matrix destruction. IL-17 increases the production of enzymes and the destruction of collagen, but its effect is less potent than that of IL-1.5 IL-17A upregulates the expression of the collagenases MMP-1, -2, -9 and -13 in synovial tissue explants, enhancing matrix turnover and cartilage destruction.60 IL-4 and IL-13 inhibit the spontaneous and stimulated production of MMP-1 by synoviocytes.61 IL-10 has no effect, probably because of the absence of IL-10 receptors on synoviocytes. Moreover, IL-17 is able to promote the expression of receptor activator of NF-κB ligand in synoviocytes and osteoblasts and then contributes to bone destruction.62 This effect is further increased with IL-1 and TNFa.
Role of IL-17 A and IL-17F in synoviocyte proliferation and survival
The role of IL-17 in the proliferation of synoviocytes remains controversial. It is still unclear whether IL-17 induces cell proliferation or cell survival by inhibiting apoptosis. IL-17 could stimulate synoviocyte proliferation through the induction of cysteine-rich angiogenic inducer 61 (CYR61), which is known to regulate cell proliferation. This protein reduces synoviocyte apoptosis. IL-17 has a complex effect on synoviocyte apoptosis, as it enhances the expression of anti-apoptotic genes and downregulates pro-apoptotic genes.63 We have recently demonstrated that IL-17 has an anti-apoptotic effect on RA synoviocytes by inducing synoviolin, an E3 ubiquitine ligase with anti-apoptotic effects. Inhibition of IL-17 response in a mouse model of arthritis reduced joint inflammation by increasing apoptosis in the synovium.64
Role of IL-17A and IL-17F in RA synoviocyte migration
IL-17A is able to promote cytoskeleton rearrangement genes such as aquaporin 1, gelsolin and cystatin B. Moreover, IL-17 enhances synoviocyte migration and invasion in in vitro assays of wound repair and invasion. This effect is inhibited by CXCL1 and CCL2, suggesting that IL-17 induces chemokine-dependent cell migration and invasion.65
IL-17A and production of auto-antigen by synoviocytes
Synoviocytes can function as antigen-presenting cells and present peptides. The human cartilage glycoprotein of 39 kDa molecular mass (HC-gp39) is a protein found in the synovium and is able to activate T cells in vitro. Synoviocytes can extract and present HC-gp39 from synovial fluid.66 With IL-17, higher concentrations of HC-gp39 protein were found in cell supernatant fractions of synoviocytes with an additive effect with TNFα. This effect was only seen with IL-17A and not with IL-1 or TNFα alone. These findings suggest a positive feedback loop, involving T cell activation by an auto-antigen.67
Therapeutic implications for anti-cytokine therapy
Blocking TNFα has become the gold standard for treating RA patients, but about 40% of patients do not respond.68 It is possible that anti-TNF therapy directly inhibits the generation of Th17 cells and hence the production of IL-17A and IL-17F. The levels of IL-17A and IL-22 in psoriatic lesions were decreased in patients treated with etanercept, a soluble receptor of TNFα.69 More recently, blocking the IL-6 receptor with tocilizumab has been effective in RA, and IL-6 is both a regulator of the development of Th17 cells and one of the major gene targets of IL-17A and IL-17F.70
Blocking IL-17A is very efficacious in various mouse models of arthritis.21 24 At the same time, blocking IL-17A may enhance the susceptibility to common infections. Indeed, studies using IL-17A- or IL-17RA-deficient mice have shown an important role for this cytokine in preventing infections with a long list of microbial pathogens, particularly extracellular bacteria and Candida albicans.71
Inhibition of IL-17A has been tested in patients with psoriasis, rheumatoid arthritis, uveitis and spondylitis. Promising results were obtained with LY2439821, a humanised monoclonal antibody, and AIN457, a human monoclonal antibody.6 The initial safety and efficacy profile of this approach will need to be further explored in larger-scale clinical trials. These and other anti-IL-17 inhibitors are presently in Phase I or II clinical trials for a variety of other autoimmune diseases, including multiple sclerosis, psoriatic arthritis and Crohn's disease. Further studies are needed to assess the effect of IL-17 inhibition on RA bone and joint damage.72 As these inhibitors target only IL-17A, the contribution of IL-17F is left untouched. The soluble receptor approach that blocks both cytokines could be more effective. Finally, preclinical results have indicated the potency of targeting IL-17 and TNFα at the same time,60 but this has not been tested in the clinic.
Although numerous cytokines participate in RA pathogenesis, it becomes increasingly clear that IL-17 family members are essential players in joint damage. IL-17A and IL-17F induce a rather similar expression pattern in the presence of TNFα in synoviocytes. They enhance the response of synoviocytes by stabilising mRNA of cytokines and enhancing receptor expression. They increase the migration, chemokine gene expression and invasiveness of synoviocytes. They contribute to disease chronicity by inhibiting synoviocyte apoptosis. Finally, they enhance metalloprotease secretion leading to joint damage. These properties support the combined inhibition of IL-17 A and IL-17F to control RA inflammation and joint destruction.
Funding This work has been 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.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.
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