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Keywords:

  • interferon regulatory factor 4;
  • IRF-4 binding protein;
  • rheumatoid arthritis;
  • systemic lupus erythematosus

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. STAT proteins
  5. Conclusion
  6. Conflict of interest statement
  7. Acknowledgement
  8. References

Recent work has implicated a novel Th effector cell subset, the Th17 cell subset, in the development of both rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) because of the ability of Th17 cells to produce cytokines like IL-17 and IL-21 that can drive both inflammatory and humoral responses. In this review, we will discuss recent studies that have begun elucidating the factors that regulate the development of Th17 cells and provide a brief overview of the role of Th17 cells in RA and SLE.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. STAT proteins
  5. Conclusion
  6. Conflict of interest statement
  7. Acknowledgement
  8. References

Defects in the appropriate regulation of CD4+ T helper (Th) cell function have been implicated in the pathophysiology of many autoimmune diseases, including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) [1–4]. Recent studies have uncovered the existence of a novel Th effector, the Th17 subset, whose deregulation plays a crucial role in the development of a wide range of autoimmune disorders from multiple sclerosis to inflammatory bowel diseases [5]. There is mounting evidence that inappropriate regulation of Th17 cells also participates in the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus (SLE) [6, 7]. In this review, we will first discuss recent studies, which have started elucidating the pathways and molecular mechanisms that underlie the generation and expansion of Th17 cells and then provide a brief overview of the role of Th17 cells in RA and SLE.

Th17 cells: a novel Th effector subset involved in autoimmunity

Whilst activation of CD4+ T helper cells is critical for an individual’s ability to eliminate a wide array of pathogens, profound pathophysiological consequences can arise if CD4+ T helper cells become erroneously activated in response to a self-antigen. One of the key mechanisms employed by the immune system to prevent autoimmunity is thus to precisely regulate the effector function of CD4+ T helper cells. Extensive work has demonstrated that CD4+ T helper cells can be subdivided into different effector T-cell subsets that secrete mutually distinct profiles of cytokines and thus coordinate different types of immune responses. Until few years ago, CD4+ T helper cells were subdivided into two major T-cell subsets, termed the Th1 and Th2 subsets [8]. Th1 cells were known to secrete IL-2, IFN-γ and TNF-β and to be involved in the generation of delayed type hypersensitivity responses. Th2 cells, on the other hand, produced IL-4, IL-5, IL-6, IL-10 and IL-13 and were known to direct B cells to mount strong humoral responses. Generation of these different Th subsets was found to be regulated by the cytokine environment to which Th cells were exposed upon encountering their cognate antigen. Thus, presence of IL-12 was crucial for driving the differentiation of Th cells towards the Th1 lineage whilst exposure to IL-4 was critical for driving their differentiation towards the Th2 lineage. Whilst excessive polarization of Th cells toward the Th2 phenotype was shown to be associated with the development of allergic responses, excessive polarization of Th cells toward the Th1 phenotype was believed to lead to the development of autoimmunity and, in particular to the development of autoimmune syndromes like RA, multiple sclerosis and Crohn’s disease that were characterized by excessive inflammatory responses.

The paradigm that Th1 cells played an essential role in the development of autoimmunity was, however, challenged when mice deficient in the expression of components of the IL-12 and IFN-γ signalling pathways were found to still develop significant and, at times, even exacerbated autoimmune responses [9, 10]. These findings coupled with the recognition that the expression of IL-17 could be detected in a variety of autoimmune diseases led to the realization that other effector Th subsets might exist. This notion was soon supported by elegant work demonstrating that IL-17 producing T cells represented a subset distinct from Th1 and Th2 cells [11, 12]. Follow-up studies furthermore corroborated this idea by showing that IL-17 producing T cells rather than Th1 cells were pathogenic in classic murine models of autoimmunity like experimental autoimmune encephalomyelitis (EAE), a murine model for multiple sclerosis and collagen induced arthritis (CIA), a murine model for RA [13–15]. These early studies were followed by a rapidly accumulating body of evidence supporting a fundamental role for Th17 cells in mediating a wide range of autoimmune disorders [5].

Pathways regulating the differentiation of Th17 cells

Th17 cells have been shown to develop via a pathway distinct from Th1 and Th2 cells [16, 17]. Similarly, to the differentiation of other Th subsets, the generation of Th17 cells is controlled by exposure to selected cytokines (Fig. 1). In the murine system, the critical cytokines controlling the initial commitment of a CD4+ T helper cell to become a Th17 cell are TGFβ and IL-6. As presence of TGFβ by itself regulates the development of Foxp3+ regulatory T cells (Tregs), there appears to be a reciprocal relationship between Th17 cells and Tregs whereby, in the setting of a proinflammatory environment (i.e. IL-6), TGFβ will drive the development of an inflammatory Th cell subset (Th17 cells) rather than promote the generation of an immunosuppressive subset (Tregs). Interestingly, the dose of TGFβ appears to be important in this lineage decision as lower doses of TGFβ favour commitment of a Th cell towards becoming a Th17 cell whilst higher doses of TGFβ skew differentiation of a Th cell towards the regulatory fate [18]. The cytokine milieu favouring the generation of human Th17 cells has been the subject of some controversy possibly because of confounding effects of cell culture conditions and the challenges of purifying truly naïve T-cell subsets from human samples. It does, however, appear that TGFβ, together with inflammatory mediators, is also required for the generation of human Th17 cells [19–21].

image

Figure 1.  Diagram of CD4+ T helper (TH) cell differentiation. Naïve CD4+ T cells can differentiate into TH1, TH2 or TH17 effector cells or into Tregs depending on the cytokine milieu that they encounter upon exposure to the antigen. The different TH effector subsets will produce distinct cytokine profiles and thus exert different effector functions.

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Although exposure to TGFβ and IL-6 initiates the commitment a naïve Th cell to the Th17 lineage, additional stimuli are required for the full acquisition of a Th17 cell programme (Fig. 2). An important step in this process is the ability of a developing Th17 cell to attain the capacity of producing IL-21, a member of the γc chain family of cytokines [22–24]. The acquisition of IL-21 production is believed to play a key amplification role in the generation of Th17 cells as IL-21 functions in an autocrine manner to reinforce the commitment of the newly differentiating Th17 cell towards the Th17 lineage. IL-21 can also substitute for IL-6 in promoting the initial differentiation of murine Th17 cells in certain experimental settings as well as drive the differentiation of human Th17 cells [21]. Another cytokine, IL-23, a member of the IL-12 family of cytokines, subsequently plays a crucial role in the expansion and maintenance of committed Th17 cells [24, 25]. Although IL-23 was initially thought to participate in the initial differentiation of a Th cell towards the Th17 lineage [12], it was later found that naïve T cells are unresponsive to IL-23 and upregulate the expression of the IL-23R only after committing to the Th17 fate, thus positioning IL-23 at a later stage of the Th17 developmental process.

image

Figure 2.  Regulation of TH17 cell differentiation. The differentiation of TH17 cells proceeds in a stepwise manner. The initial commitment to the TH17 fate is regulated by exposure to TGF-β and IL-6. These stimuli lead to the production of IL-21, which acts in an autocrine manner to amplify the commitment toward the TH17 cell fate. Exposure to IL-23 produced by myeloid cells will then help maintain the TH17 cells.

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The generation of Th17 cells can be inhibited by various cytokines [16, 17]. Importantly, cytokines produced by Th1 and Th2 cells, IFN-γ and IL-4 respectively, are known to interfere with the development of Th17 cells consistent with the finding that once effector Th cells differentiate towards a specific lineage, the cytokines that they produce inhibit commitment to alternative Th effector fates. Presence of IL-2 has also been shown to inhibit differentiation towards the Th17 lineage and instead favours the generation of a regulatory T-cell phenotype [26]. Development of Th17 cells can also be suppressed by the presence of IL-27, another member of the IL-12 family of cytokines [27, 28]. Consistent with this effect, mice that lack the IL-27 receptor are highly susceptible to EAE because of increased IL-17 production.

Although most of the attention has been focused on the pathways that control the differentiation of CD4+ T cells into Th17 cells, it is important to note that the production of IL-17 is not confined to CD4+ T cells. Indeed, CD8 T cells lacking both T-bet and eomesodermin can differentiate into an interleukin-17-secreting lineage [29]. Subpopulations of NKT cells have been shown to be able to produce high levels of IL-17 rapidly upon activation [30, 31] and rapid production of IL-17 has also been detected upon stimulation of lymphoid tissue inducer-like cells by zymosan [32]. Interestingly, a recent study also reported IL-17 production by CD3+CD4CD8 double negative (DN) T cells, a population that is expanded in SLE patients [33]. The factors and mechanisms that control IL-17 production by these other cell-lineages have, however, not been fully characterized.

Molecular mechanisms controlling the differentiation of Th17 cells

Given that Th17 cells develop via a pathway distinct from that of Th1 and Th2 cells, it is not surprising that the molecular machinery that controls their differentiation employs components distinct from those controlling the differentiation of Th1 and Th2 cells. Here, we will briefly review the major factors that regulate the acquisition of the Th17 differentiation program at the transcriptional level.

STAT proteins

  1. Top of page
  2. Abstract.
  3. Introduction
  4. STAT proteins
  5. Conclusion
  6. Conflict of interest statement
  7. Acknowledgement
  8. References

Activation of STAT proteins is a critical initiating event in the signalling cascades of many cytokines [34]. Binding of cytokines to their receptors leads to the tyrosine phosphorylation of STAT proteins enabling STATs to dimerize and translocate to the nucleus where they will mediate changes in gene expression by targeting specific DNA elements. Although most cytokines follow this basic signalling framework, distinct cytokines differ in the specific STAT proteins that they activate. In line with the finding that IL-6 plays a pivotal role in Th17 differentiation, Stat3, the major STAT protein activated in response to IL-6, is a critical transcription factor for the generation of Th17 cells and for the production of both IL-17 and IL-21 [35–37]. Interestingly, IL-21 also activates Stat3 and this effect is likely to play a major role in the ability of IL-21 to augment the commitment of a developing Th17 cell towards the Th17 lineage or to substitute for IL-6 in the initial differentiation of Th17 cells [36]. In contrast to Stat3, STAT proteins that are activated in response to IL-2 and IFN-γ (Stat5 and Stat1 respectively) are crucial mediators of the inhibitory effects of these cytokines on Th17 differentiation [11, 26]. Stat1 has also been implicated in the ability of IL-27 to suppress Th17 differentiation [27, 28]. Consistent with the role of suppressor of cytokine signalling (SOCS) proteins in the downmodulation of JAK-STAT signalling [38, 39], SOCS-3 has been shown to inhibit the development of Th17 cells [35, 40, 41].

RORγt and RORα

Although activation of STATs is essential for the initial commitment toward specific Th effector lineages, full implementation of the transcriptional programs associated with each Th effector lineage relies on the upregulation of specific lineage-specific transcriptional regulators like T-bet for Th1 and GATA3 for Th2 cells. In the case of Th17 cells, this role is fulfilled by members of the retinoic-acid-receptor-related orphan nuclear hormone receptor family, RORγt, and, to a lesser extent, RORα [16, 17]. Expression of both RORγt and RORα is upregulated upon exposure of naïve Th cells to TGFβ and IL-6 in a manner that depends on Stat3 [26, 37, 42]. Overexpression of RORγt has been shown to be sufficient to drive IL-17 production whilst its deficiency results in impaired Th17 differentiation [43]. Interestingly, whilst the absence of RORγt by itself leads to profound impairments in IL-17 production, combined deficiencies in RORγt and RORα are required to impair the synthesis of IL-21 indicating that RORα can compensate for RORγt in this process [42]. Recent studies have shown that the function of RORγt can be inhibited by Foxp3 [18], the crucial transcription factor controlling the transcriptional program of regulatory T cells suggesting that the balance between RORγt and Foxp3 plays a major role in the commitment of Th cells toward either the Th17 or the Treg lineage.

IRF-4 and IRF-4 binding protein

Recent studies have demonstrated that IRF-4, a member of the interferon regulatory factor (IRF) family of transcription factors, is also a crucial regulator of Th17 differentiation [44]. In particular, IRF-4 was found to control the production of IL-17 via its ability to regulate the expression of RORγt. Reintroduction of RORγt in IRF-4 deficient T cells, however, did not completely rescue the defective IL-17 production exhibited by these cells. Consistent with this finding, we have recently observed that IRF-4 can also directly target the IL-17 promoter suggesting that IRF-4 employs both direct and indirect mechanisms to control IL-17 production [45]. In addition to controlling IL-17 synthesis, IRF-4 is also a critical regulator of IL-21 expression as indicated by the finding that its absence results in profound impairments in the production of IL-21 [45]. Unlike other master regulators of Th differentiation, the expression of IRF-4 is upregulated by TCR stimulation and is not restricted to a specific Th effector lineage [46, 47]. Our laboratory has recently found that a protein that interacts with IRF-4, which we termed IBP (IRF-4 Binding Protein, also known as Def6 or SLAT) [48–50], inhibits the ability of IRF-4 to upregulate the expression of IL-17 and IL-21 when Th cells are cultured in the absence of Th17 skewing conditions [45]. IBP thus appears to be a critical component of a regulatory pathway that ensures that the production of IL-17 and IL-21 occurs only when Th cells are exposed to the appropriate proinflammatory environment. Improper functioning of this regulatory pathway may play an important role in the development of autoimmunity as evidenced by the finding that mice deficient in IBP spontaneously develop autoimmune syndromes characterized by elevated production of IL-17 and IL-21 [45].

Th17 cells and RA

Rheumatoid arthritis is a complex inflammatory autoimmune disease that affects ∼1% of the Western population. The primary inflammatory lesion in RA affects the joints and is characterized by infiltration of the synovium by inflammatory cells, cartilage degradation and bone erosions [51]. Production of IL-17 by Th17 cells is now believed to play a crucial role in the development of these lesions as IL-17 can induce the production of proinflammatory cytokines, such as TNF-α and IL-1, the upregulation of RANK ligand and stimulate the activity of matrix metalloproteinases, matrix catabolism and bone resorption [52, 53]. Although the hallmark of RA is joint inflammation, RA patients also demonstrate evidence of a systemic inflammatory condition [54], marked by the presence of autoantibodies like rheumatoid factor and anti-cyclic citrullinated peptide (CCP) antibodies. It is intriguing to speculate that the ability of Th17 cells to produce IL-21 can play an important role in this aspect of RA pathophysiology as IL-21, in addition to augmenting the production of IL-17, is also a major regulator of IgG production and T-dependent humoral responses [55, 56].

Consistent with the idea that the cytokines produced by Th17 cells can exert functions relevant to the pathophysiology of RA, growing evidence supports the involvement of Th17 cells in this disease. Elevated production of IL-17 has been observed in murine models of RA as well as in patients affected by this disorder [57–61]. Increased levels of IL-17, furthermore, correlate with more severe joint damage [62]. Mice deficient in IL-17 are protected from the development of arthritis in CIA and blockade of IL-17 via either antibodies or soluble IL-17 receptor can ameliorate symptoms in CIA even after the arthritis is established [15, 63, 64].

A rapidly expanding literature has demonstrated that development of arthritogenic Th17 cells as well as their potential to mediate destruction is controlled by a complex set of regulatory networks. Synovial, but not peritoneal, macrophages have been found to be particularly effective at promoting Th17 differentiation suggesting that optimal generation of Th17 cells is fuelled by the inflammatory milieu present in the local microenvironment [65]. The persistent inflammatory and erosive responses that are associated with the presence of Th17 cells within the joints have been shown to be dependent on TLR-4, which presumably becomes activated upon binding endogenous ligands released upon tissue damage [66]. Surprisingly, expansion of Th17 cells was found to be augmented rather than diminished by TNFα blockade in a murine model of inflammatory arthritis [67]. Despite this paradoxical effect, TNFα blockade did prevent the accumulation of Th17 cells in the joints, an effect that is likely to contribute to the beneficial actions of TNFα inhibition in arthritis. In contrast to TNF blockade, interfering with IL-6 with an anti-IL-6R Ab suppressed the induction of Th17 cells although this treatment was ineffective if administered 2 weeks after CIA was induced in line with the idea that IL-6 plays a crucial role in the initiation rather than the maintenance of Th17 responses [68]. Interestingly, BAFF has recently been recognized as playing an important role in the production of IL-6 within the joints in CIA since localized silencing of BAFF in DCs in joint tissue suppressed development of Th17 cells and ameliorated the arthritis [69].

Consistent with the notion that amplification of Th17 responses and possibly other aspects of RA, is controlled by IL-21, blockade of the IL-21/IL-21R pathway has recently been reported to be efficacious in ameliorating disease in a murine model of RA [70]. Although a systematic analysis of the role of specific transcriptional regulators in murine models of RA has not been conducted, we have recently discovered that mice deficient in IBP spontaneously develop an RA-like syndrome characterized by elevated production of IL-17 and IL-21 in the serum and arthritic joints because of the unchecked ability of IRF-4 to drive the production of these two cytokines [45]. As IRF-4 is an important cellular target of the HTLV-I Tax oncoprotein [71], it will be important to assess whether deregulation of IRF-4 also plays a role in the autoimmune arthritis associated with the overexpression of Tax in mice [72] and possibly in the arthritis that develops in a subset of HTLVI infected patients [73].

Th17 cells and SLE

Systemic lupus erythematosus (SLE) is a prototypical systemic autoimmune disorder characterized by hypergammaglobulinaemia, autoantibody production and multi-organ involvement, which commonly affects the kidneys [3, 74, 75]. Th17 cells are beginning to be implicated in the pathogenesis of SLE. Increased levels of IL-17 have been detected in the serum of SLE patients in some studies [76–78]. Interestingly, recent work has demonstrated that the increased IL-17 production that is detected in SLE patients results from excessive IL-17 synthesis by CD4+ T cells and from expansion of CD3+CD4CD8 double negative (DN) T cells [33]. This group, furthermore, demonstrated that IL-17 and IL-23 can be detected in the kidneys of patients with lupus nephritis suggesting that these cytokines may contribute to renal damage. Consistent with these findings, studies in SNF1 mice, a spontaneous murine model of SLE, revealed increased numbers of autoreactive Th17 cells, which infiltrated the kidneys [79]. Although the exact role played by IL-17 in SLE has not been fully elucidated, IL-17 was recently shown to be critical for the formation of autoreactive germinal centres in BXD2 mice, a strain that develops a lupus-like syndrome [80]. This effect was believed to be secondary to the ability of IL-17 to control the migration of B cells leading to their prolonged retention within the germinal centre where they presumably could receive enhanced help from T cells. Interestingly, downregulation of IL-17 production by T cells has been shown to correlate with the amelioration of murine lupus after treatment with either low-dose peptide tolerance therapy or nasal anti-CD3 antibodies [79, 81].

The ability of Th17 cells to produce IL-21 is also likely to play a key role in the pathophysiology of SLE given the major role exerted by this cytokine on the control of humoral responses. In line with this notion, IL-21 polymorphisms have been recently associated with SLE [82]. Although the role of IL-21 was not directly investigated in the SNF1 and BXD2 mice, enhanced levels of IL-21 have been detected in other spontaneous murine models of lupus like BXSB-Yaa and sanroque mice [83, 84]. Blockade of the IL-21/IL-21R pathway, furthermore, has recently been reported to be efficacious in ameliorating disease in MRL/lpr mice [85] Consistent with the fact that familial aggregation of RA and SLE has been reported [86, 87], we have found that, in selected strains of mice, instead of RA-like arthritis, the absence of IBP can result in the development of a lupus-like syndrome [88], which is again likely because of the deregulated ability of IRF-4 to drive the production of IL-17 and IL-21. These findings suggest that deregulation of the mechanisms that properly control IL-17 and IL-21 production may represent a common pathogenic mechanism that underlies the development of both RA and SLE.

Conclusion

  1. Top of page
  2. Abstract.
  3. Introduction
  4. STAT proteins
  5. Conclusion
  6. Conflict of interest statement
  7. Acknowledgement
  8. References

Evidence is emerging that inappropriate regulation of Th17 cells plays a fundamental role in the development of many autoimmune diseases. In particular, recent studies support the notion that deregulated production of IL-17 and IL-21, cytokines produced by Th17 cells, may participate in the pathogenesis of both RA and SLE. Knowledge of the pathways and molecular networks responsible for the regulation of this T helper cell subset is accumulating at a rapid pace. This information will undoubtedly be critical for the development of innovative therapeutic strategies aimed at targeting autoimmune diseases like RA and SLE.

Acknowledgement

  1. Top of page
  2. Abstract.
  3. Introduction
  4. STAT proteins
  5. Conclusion
  6. Conflict of interest statement
  7. Acknowledgement
  8. References

Research support was provided by NIH grant R01 HL-62215, the Lupus Research Institute and the Alliance for Lupus Research.

References

  1. Top of page
  2. Abstract.
  3. Introduction
  4. STAT proteins
  5. Conclusion
  6. Conflict of interest statement
  7. Acknowledgement
  8. References