Th17 cytokines and arthritis

Rheumatoid arthritis (RA) is characterized by synovial inflammation and destruction of joint cartilage and bone mediated by persistent synthesis of proinflammatory cytokines and matrix metalloproteinases (MMP) [4]. T cell activation and migration occurs as an early consequence of RA, and these cells adopt a proinflammatory phenotype. Classically, autoimmune diseases such as RA were thought to be a Th1- and not a Th2-associated disorder [21]. At present, it is unclear whether RA is a Th1- and/or Th17-mediated disease [52] (Fig. 1). It has been shown that interleukin-17A (IL-17A) is produced by some proinflammatory Th1/Th0 cells isolated from synovial membranes and from synovial fluid of RA patients. In joints of patients with established RA, predominantly, Th1 but not Th17 cells were observed [1]. We detected a relatively high percentage of IL-17A-producing CCR6+ memory T cells in peripheral blood mononuclear cell (PBMC) from treatment-naïve early RA patients [14] (Van Hamburg et al., unpublished observations). Of note, the Th1 interferon (IFN)-gamma and the Th17 IL-17 cytokines are often coexpressed in human memory CD4⁺CD45⁺RO⁺ T cells from treatment-naïve early RA patients [14].

Although the levels of IL-17A in sera of RA patients is hard to detect, elevated levels of this T cell cytokine have been demonstrated in synovial fluid of these patients [10, 41, 100]. In line with these enhance IL-17A levels in the joint, IL-17A-producing CCR6+ memory T cells have been identified in synovial fluid of RA patients [25].

Similar as for the human situation, in the autoimmune collagen-induced arthritis (CIA) model, the mouse model for RA, it is hard to detect IL-17A levels in the serum of arthritic mice. In contrast, elevated levels of IL-17A have been found in inflamed synovium [53]. IL-17A blocking experiments have shown the importance of this T cell cytokine as proinflammatory in the pathogenesis of CIA [8, 53, 54]. Furthermore, as in human RA, IL-17 plays an important role in the additive/synergistic effects induced together with tumor necrosis factor (TNF) and IL-1, two key cytokines in destructive arthritis [55, 62].

In CIA, it is clear that the IL-23/Th17 axis is critical for the development of autoimmune arthritis. Mice that were deficient for IL-23 were completely protected against the development of CIA. Interestingly, these IL-23-deficient mice had no IL-17+ CD4 T cells but normal antigen-specific IFN-gamma-producing Th1 cells [65]. These data show that IL-17-producing CD4+ T cells but not IFN-gamma-producing CD4+ T cells are critical for the initiation of CIA. In line with these results, we found in the T cell-dependent antigen-induced mono-arthritis (AIA) model that lack of IL-23 did not prevent the onset of AIA but protect against progression of joint inflammation into a destructive synovitis [15]. Lower proportion of IL-17-positive T cell receptor gamma delta and IL-17-positive CD4+ T cells was noted in the spleen, lymph nodes, and inflamed synovium in these IL-23-deficient arthritic mice. Interestingly, although mono-arthritis induction in IL-23-deficient mice did not result in a lower proportion of IFN-g-positive CD4+ T cells in the spleen and lymph node, a lower proportion of these cell was detected in the target organ, the joint [15]. Therefore, IL-23 could be required to promote Th1 and Th17 effector responses, especially at the target organ/tissue of inflammation, and further studies are needed to understand this phenomenon.

Although relatively high numbers of Th17 cells have been shown in PBMC from treatment-naïve early RA patients [14] (Van Hamburg JP et al., unpublished observations), the proportion of these cells in PBMC and synovial-fluid mononuclear cells in established RA varies between no Th17 to low Th17 cells (Table 1). Yamada et al. [96] showed that IL-17-positive cells were detected in CD45RO+CD4 T cells in RA, and most IL-17 positive cells produced neither IFN-gamma nor IL-4, but TNF [96]. The frequency of Th17 cells was neither increased in RA nor correlated with the DAS28 disease activity score. Unexpectedly, the frequency of Th17 cells was significantly decreased in the joints compared with PBMC of the same patients with RA, whereas Th1 cells were more abundant in the joints than in PBMC in these established RA patients [96]. Increased levels of IL-17A, IL-6, TGF-beta, and IFN-gamma concentrations in sera and synovial fluid of reactive arthritis (ReA) and undifferentiated spondyloarthropathy (uSpA) compared to RA have been shown, suggesting that Th1 and Th17 cells could be the major agents in inflammation in ReA/uSpA [81] (Table 1). Jandus et al. [34] found increased numbers of circulating Th17 cells in the peripheral blood of patients with seronegative spondylarthritides (psoriatic arthritis and ankylosing spondylitis), but not in patients with RA (Table 1). In addition, Th17 cells from the spondylarthritis patients showed advanced differentiation and were polyfunctional in terms of T cell receptor-driven cytokine production [34].

Subclinical gut inflammation is common in spondylarthritis. A strong and significant upregulation of IL-23p19 transcript was found in the terminal ileum in patients with AS and patients with Crohn's disease (CD). IL-23 was abundantly produced by infiltrating monocyte-like cells in inflamed mucosa from AS and CD patients [12]. Notably, Paneth cells were identified as a major source of IL-23 in patients with AS, patients with CD, and normal controls [12]. It has been shown that Paneth cells can also be a source of IL-17A in a TNF-induced experimental shock model in mice [84]. However, unlike CD, in AS patients, IL-23 was not associated with upregulation of IL-17 and the IL-17-inducing cytokines IL-6 and IL-1beta, indicating that overexpression of IL-23 but not IL-17 is a pivotal feature if subclinical gut inflammation in AS [12].

In patients with systemic sclerosis (SSc) increased IL-17A messenger RNA was expressed in unstimulated PBMC and lymphocytes from the skin and lungs of SSc patients, but not in similar samples from patients with systemic lupus erythematosus (SLE), polymyositis/dermatomyositis, or from healthy donors (Table 1). IL-17 levels were also increased in the serum of SSc patients, but not in that of SLE patients or healthy donors. IL-17 overexpression was significantly related to the early stage of SSc, but not to other clinical features of SSc [45] Furthermore, Radstake et al. showed that CD4, CD45RA, and CD45RO cells from SSc patients highly express the IL-23 receptor, which was associated with high IL-17 expression as well [75]. In contrast, IFN-gamma and TGF-beta were selectively upregulated in subsets of SSc patients. In addition, circulating levels of IL-17-inducing cytokines IL-6, IL-23, and IL-1alpha were increased in all or subsets of SSc patients. The combination of IL-17, IFN-gamma, and TGF-beta levels in CD45RO and CD45RA cells from SSc patients can be useful to distinguish between limited cutaneous SSc, early diffuse cutaneous SSc, or late diffuse cutaneous SSc [75].

Human data implicating IL-17 in lupus has become available [82, 94]. In contrast, some evidence argues against a role for IL-17 in human lupus [45, 61] (Table 1). In the mouse model for lupus-like autoimmunity, the BXD2 mouse model, a dramatic upregulation of serum IL-17 and numbers of Th17 cells have been demonstrated [27]. BXD2 mice form spontaneous germinal centers which was IL-17-dependent, and treatment with IL-17 promoted the secretion of both IgM and IgG autoantibodies which was reduced by crossing BXD2 mice to IL-17R KO mice [27]. A potential role for IL-17-producing T cells has been shown in Ets-1 knockout mice, another mouse model of lupus [63, 90] and in the spontaneous mouse model of lupus, the New Zealand Black (NZB) × SWR F1 cross (SNF1 mice) [36]. These studies reveal that there is increasing evidence in both humans and mouse models that IL-17-producing cells play a role in SLE progression.

Human Lyme arthritis is caused by Borrelia burgdorferi and is characterized by an inflammatory infiltrate that consists mainly of neutrophils and T cells. B. burgdorferi, Mycobacterium tuberculosis, and synthetic lipopeptides derived from B. burgdorferi outer surface lipoproteins induced IL-17 expression in both murine and human Th cells [30]. The IL-17-producing Th population is characterized by the coexpression of the proinflammatory cytokines IL-17, TNF, and granulocyte-macrophage colony-stimulating factor [30]. Anti-NapA (neutrophil-activating protein A of B. burgdorferi) antibodies were found in 48% of the patients with Lyme arthritis but were undetectable in the healthy controls [13]. T cells from the synovial fluid of patients with Lyme arthritis produced IL-17 in response to NapA. Moreover, NapA was able to induce the expression of IL-23 in neutrophils and monocytes, as well as the expression of IL-6, IL-1beta, and TGF-beta in monocytes via Toll-like receptor 2 [13]. Therefore, NapA of B. burgdorferi is able to elicit a synovial fluid Th17 cell response that might play a crucial role in the pathogenesis of Lyme arthritis [13] (Table 1). In addition, IL-23 is required for the development of arthritis in mice vaccinated and challenged with Borrelia species [42].

In patients with Wegener's granulomatosis (WG) in remission, the percentage of Th17 cells and Th2 cells within the activated CD69+, CD4+ T cell population were significantly increased, while no difference was found in Th1 cells compared with the percentage in healthy controls (Table 1). Increased percentages of Th17 cells in response to tetanus toxoid and staphylococcal enterotoxin B were found both in antineutrophil cytoplasmic antibody (ANCA)-positive and in ANCA-negative patients, while an increased frequency of PR3-specific Th17 cells was restricted to ANCA-positive patients. Therefore, a skewed Th17 response found in ANCA-positive WG patients following stimulation with the autoantigen PR3 suggests that IL-17 is involved in disease pathogenesis and could constitute a new therapeutic target for WG [3].

Also, in Sjogren's syndrome (SS), an upregulation has been shown of the Th17/IL-23 system at the time of disease [68] (Table 1). Salivary gland biopsy specimens from SS patients revealed strong positive staining for both IL-17 and IL-23 within lymphocytic foci and diffuse staining on epithelial tissues. In sera and saliva from SS patients, IL-17 and IL-6 were present at varying levels [68].

In addition, IL-18 and Th17 cells detected in the salivary glands in SS patients are associated with the pathogenesis of SS in the salivary glands [79].

IL-17+ T cells were also detected in the joints of children with juvenile idiopathic arthritis (JIA), and these cells were enriched in the joint compared to the blood of JIA patients [69] (Table 1). Of note, IL-17+ T cell numbers were higher in patients with extended oligoarthritis, the more severe subtype of JIA, as compared with patients with persistent oligoarthritis, the milder subtype [69]. Within the joint, there was an inverse relationship between IL-17+ T cells and FoxP3+ Treg cells [69].

These studies suggest that IL-17+ T cells contribute to the pathogenesis of different arthropathies and that the stage of the disease and the site of expression might be important for the role of these IL-17+ T cells in the pathogenesis of the disease. Furthermore, the balance between, on one hand, Th17 and Th1 and, the other hand, Th17 and Treg may be critical in disease outcome.

Th17 cells have been detected in different arthropathies, but their functional role in the human disease has not been established. Both Th1 and Th17 cells have been recognized in RA; however, it remains unclear whether Th1 and/or Th17 cells drive disease chronicity [52]. In experimental arthritis models, Th17 cells have been recognized as important contributors to the inflammatory processes [52, 55], and preferential recruitment of CCR6-expressing Th17 cells to inflamed joints via CCL20 in RA and its animal model has been shown [25]. However, in experimental autoimmune encephalitis (EAE) and experimental autoimmune uveoretinitis models, evidence also suggest the potential of Th1 cells as pathogenic mediators [44, 57]. In arthritis, both Th17- and Th1-dependent experimental models have been described [19, 25, 65, 67, 88]. Th17-dependent models can be induced by a specific antigen with complete Freund's adjuvant, but also Th17-dependent spontaneous arthritis models were reported [25, 65, 67, 88]. On the other hand, the development of proteoglycan-induced arthritis has been shown to be IL-17-independent, and the severity of arthritis is dependent on the production of IFN-gamma [19].

However, whether this is also the case in human arthritis needs further clarification including the functional role of these T cell subsets with focus on different stages of the disease.

T cells and the cytokines IL-17A and TNF-alpha have been shown to activate RA synovial fibroblast (RASF), resulting in the expression of proinflammatory cytokines such as IL-6 and IL-8, which are mediators of joint inflammation [6, 7, 10, 60, 62, 73, 85]. In addition, an IL-17A-triggered positive-feedback loop of IL-6 signaling in fibroblast has been described in experimental arthritis and discussed for human Th17-mediated autoimmune disorders [60, 72]. However, characterization of the T cell population that activates RASF and responsible for IL-6 and IL-8 cytokine induction has not been established. We identified IL-17+TNF+Th17 cells in PBMC from treatment-naïve early RA patients. These cells are identified as Th17 cells since they express CCR6, IL-17F, IL-22, IL-26, RORc, CCL20, low T-bet, low FoxP3, and low IFN-gamma. Their functional potential was examined using cocultures with early RASF. CCR6+ (Th17) but not CCR6− (Th1) memory T cells were potent inducers of IL-6, IL-8, and MMPs by the synovial fibroblast in these cocultures. Of interest, also IL-17A expression was increased in these cocultures with synovial fibroblast indicating the induction of a proinflammatory feedback loop. This Th17-RASF proinflammatory feedback loop might be an important mechanism driving persistent arthritis (Van Hamburg JP et al., unpublished observations; Fig. 1). Blocking experiments revealed that both TNF and IL-17A needed to be blocked for optimal suppressive effect, indicating additional value of blocking Th17 activity to anti-TNF neutralization in early RA (Van Hamburg JP et al., unpublished observations).

These are the first data indicating potential pathogenicity of Th17 cells in the human situation. Interestingly, data from first clinical trials with anti-IL-17A antibody treatment in psoriatic arthritis patients and RA patients looks promising. Since additional neutralization of the Th17 cytokine IL-17A to TNF-alpha was needed to suppress the mechanism that drives the proinflammatory feedback loop in cocultures of cells from treatment-naïve early RA patients, blocking Th17 activity in addition to anti-TNF in early RA and potentially other Th17-mediated disorders may be essential to reach the ultimate goals and that is (1) to prevent the development of persistent and destructive arthritis at the very early stage of the disease and (2) reach permanent remission of the disease.

Th17 cells appear to be associated with Treg cells, which are indicated by the use of a common inducer, TGF-beta, an overlapping chemokine receptor profile, and the expression of the Th17-associated transcription factor RORgammat [32, 87]. In mice, it has been shown that Treg cells can be converted to IL-17-producing T cells [74, 95, 98]. Additionally, also human Treg cells, defined as CD4+CD25highFoxP3+CD127-CD27+, were reported to differentiate to IL-17-producing T cells. This was accompanied by an upregulation of RORgammat and CCR6 expression [38].

Next to plasticity between Treg and Th17 cells new data emerged that Th17 cells are closely related to Th1 cells. In the mouse, differentiated Th17 cells responded rapidly in vitro to IL-12, by upregulating the expression of IFN-gamma and downregulating IL-17 expression. The development of IFN-gamma-producing effector T cells from IL-17-producing progenitor cells is inhibited in the presence of TGF-beta, which is important for the maintenance of IL-17 expression by Th17 polarized cells [49, 50, 58].

Recently, a role for epigenetic regulation of effector T cell plasticity was found. For example, the gene encoding for T-bet, the master regulator of Th1 differentiation, was found to be in an active state, according to histone methylation marks, in both Th17 and Treg cells. This implies that Th17 and Treg cells remain to have the potential to upregulate the expression of T-bet and to differentiate towards Th1 cells [92].

Associations between T cell subsets such as Th17, Th1, and Treg cells have been found in inflammation and progression of RA. In joint inflammation of RA, high levels of IL-17 can be found, and hardly any IFN-gamma expression can be detected in the joint synovium [10, 76]. This was in line with studies in mice, which indicate the importance for IL-17 in the induction of arthritis [55]. In contrast, IFN-gamma cytokine levels were detected in the joint synovium in later stages of RA, and IFN-gamma expression was detected in joint infiltrating CD4 T cells and in CD4 T cell clones, obtained from inflamed synovium [1, 18, 64]. In line with these findings, increased levels of IFN-gamma were detected in lymph nodes and synovium at later time points when adjuvant arthritis was induced in rats. In contrast, IL-17 was induced shortly after initiation and declined in time [9]. In a 2-year predictive study, it became even evident that IFN-gamma was playing a role in reducing joint progression. Inhibition of IFN-gamma in later disease was even exacerbating the disease [37].

Next to Th1 cells, an inverse correlation between IL-17-producing and FoxP3+ Treg cells has been identified. In children with JIA, the balance between IL-17+ cells and Treg cells may even be critical for the disease outcome [69]. In addition, Treg cells are present in higher numbers in inflamed joints in patients with a mild RA phenotype, compared to patients with a more severe disease [17, 78].

These observations indicated that plasticity between Treg, Th1, and Th17 subsets exist and that differentiation of these subsets was not completely restricted to separate lineages. All together, these recent advances provide a complete new concept of T cell plasticity that may be relevant to the arthritis process (Fig. 1).

The role of IL-1, IL-6, IL-23, TGF-beta, and TNF-alpha in the differentiation of mouse and human Th17 cells has been reported [40]. However, the role of IL-23 in the effector function of Th17 cells is not clearly understood. The essential role for IL-23 in the pathogenic function of Th17 cells was shown in EAE [59]. In this experiment, Th17 cells, which were polarized under IL-23 polarizing conditions, were potent inducers of EAE, while the pathogenic function in TGF-beta/IL-6-polarized Th17 cells was completely abrogated. A possible explanation for this discrepancy could be the inhibited induction of IL-10 in the IL-23 polarized cells. Interestingly, TGF-beta/IL-6-polarized Th17 cells modulated the induction of EAE by IL-23-polarized Th17 cells [59]. These data indicate that IL-23 is essential for the pathogenic function of Th17 cells. In line with this, we have shown that IL-23 and TGF-beta/IL-6 differentially regulate T helper specific transcription factors during Th17 development in autoimmune experimental arthritis, which may explain the pathogenic potential of IL-23 [66]. In CD4+ T cells from naïve and collagen-type II immunized DBA/1 mice, IL-23 increased the expression of IL-17A, IL-17F, and RORgammat. In contrast to TGF-beta/IL-6, IL-23 inhibited the Th1 and regulatory T cell-specific transcription factors T-bet and FoxP3 respectively [66].

In addition to IL-23, IL-1beta signaling in T cells has been shown to induce a robust and durable primary and secondary CD4 responses [5]. Mice defective in IL-1R1 signaling were resistant to EAE and exhibit a severe defect in the generation of IL-17-producing T cells, suggesting that IL-1 is important for mouse Th17 cell regulation in vivo [83]. On the other hand, IL-1Ra-deficient mice spontaneously developed chronic inflammatory arthropathy which could be completely suppressed when crossing these mice with IL-17A-deficient mice or TNF-alpha-deficient mice [26, 67]. Moreover, IL-1 signaling regulated early Th17 cell differentiation [11]. IL-1 receptor 1 (IL-1R1) expression in T cells, which was induced by IL-6, was necessary for the induction of experimental autoimmune encephalomyelitis and for early Th17 cell differentiation in vivo [11]. Moreover, IL-1 signaling in T cells was required in dendritic cell-mediated Th17 cell differentiation from naïve or regulatory precursors and IL-1 synergized with IL-6 and IL-23 to regulate Th17 cell differentiation and maintain cytokine expression in effector Th17 cells [11]. IL-1 regulated the expression of the transcription factors IRF4 and RORgammat during Th17 cell differentiation [11].

Thus, similar to the human system, IL-1 plays a unique, nonredundant role during murine Th17 cell polarization [11], and its role in Th17 cell/Th17 cytokine regulation in arthritis may be important [2, 56].

Since Th17 cells and Th17 cytokines can have a proinflammatory effect in arthritis, it will be of interest to understand how to regulate the activity of these cells/cytokines. It has been shown that overexpression of the Th2-specific transcription factor, GATA3, in T cells can modulate Th17 cell differentiation and protects against severe joint inflammation and bone erosion in experimental arthritis [88]. Moreover, GATA3 overexpression resulted in reduced gene expression of the Th17-associated transcription factor RORgammat [88].

In addition, treatment of mice with CIA with anti-IL-6R mAb on day 0 markedly suppressed the induction of Th17 cells and arthritis development, but treatment with this antibody on day 14 failed to suppress both Th17 differentiation and arthritis [22]. Of note, treatment of mice with TNFR-Fc from day 0 to day 14 suppressed neither Th17 differentiation nor arthritis, but treatment from day 21 to day 35 successfully ameliorated arthritis without inhibiting Th17 induction [22]. This study indicate that the protective effect of IL-6 blockade, but not TNF blockade in CIA, correlated with the inhibition of Th17 differentiation and suggest that IL-6 blockade in RA in human likely involve a therapeutic mechanism distinct from that of TNF blockade [22]. Moreover, Notley et al. [70] showed that TNF blockade using TNFR-Fc fusion protein or anti-TNF monoclonal antibody reduced collagen arthritis severity but, unexpectedly, expanded populations of Th1 and Th17 cells, which were shown by adoptive transfer to be pathogenic [70]). Th1 and Th17 cell populations were also expanded in CII-immunized TNFR p55KO but not p75KO. The expansion of Th1/Th17 cells was abrogated by blockade of p40 [70]. However, although TNF blockade increased numbers of Th1 an Th17 cells in lymph nodes, it inhibited their accumulation in the joint, thereby providing an explanation for the paradox that anti-TNF therapy ameliorates arthritis despite increasing numbers of pathogenic T cells [70].

Lai Kwan Lam et al. showed that local B cell-activating factor (BAFF) gene silencing suppresses Th7 cell generation and ameliorates autoimmune arthritis [46]. TNF superfamily member BAFF plays an important role in humoral immunity and in autoimmune diseases, including RA. Local BAFF gene targeting inhibited proinflammatory cytokine expression, suppressed generation of plasma cells and Th17 cells, and markedly ameliorated joint pathology [46]. This study revealed a previously unrecognized role for BAFF in promoting the expansion of Th17 cells and demonstrated IL-17 as a crucial effector cytokine for BAFF-mediated proinflammatory effects during CIA development [46].

Apart from regulation of Th17 cells in experimental mouse models for arthritis, Colin et al. showed modulatory effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)₂D₃) on the memory Th17 activity in untreated early RA patients [14]. 1,25-(OH)₂D₃ reduced the levels of IL-17A and IFN-gamma and increased IL-4 in stimulated PBMC from treatment-naïve early RA patients [14]. Interestingly, 1,25-(OH)₂D₃, in contrast to dexamethasone, directly modulated human Th17 polarization accompanied with suppression of IL-17A, IL-17F, TNF-alpha, and IL-22 production by FACS sorted memory T cells from these early RA patients [14].