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Arthritogenic T cells in autoimmune arthritis
The International Journal of Biochemistry & Cell Biology 58 (2015) 92–96
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Arthritogenic T cells in autoimmune arthritis Noriko Komatsu a,b , Hiroshi Takayanagi a,b,∗ a Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan b Japan Science and Technology Agency (JST), Exploratory Research for Advanced Technology Program, Takayanagi Osteonetwork Project, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan
a r t i c l e
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Article history: Received 26 September 2014 Received in revised form 15 November 2014 Accepted 20 November 2014 Available online 28 November 2014 Keywords: Rheumatoid arthritis Regulatory T cells Th17 cells Synovial fibroblasts Osteoclasts
a b s t r a c t Autoimmune diseases, including arthritis, often result from an imbalance between regulatory T (Treg) cells and IL-17-producing (Th17) cells. Dozens of studies in mice and humans have shed light on the pathological significance of T cells in RA. Since Th17 cells play an important role in the exacerbation of inflammation and bone destruction in joints, it has been an important issue how arthritic Th17 cells arise. Th17 cells are generated in the local inflammatory milieu via cytokines produced by macrophages or synovial fibroblasts, while it is reported that Th17 cells are generated in the gut in the presence of specific commensal bacteria. A recent report showed a pathogenic Th17 cell subset with a distinct pattern of gene expression and a potent osteoclastogenic ability are converted from Foxp3+ T cells in arthritic joints. Since Foxp3+ Treg cells contain T cells which recognize self-antigens, the fate of plastic Foxp3+ T cells can be a critical determinant of autoimmunity or self-tolerance. Further analysis on the molecular basis and antigen-specificity of arthritogenic Th17 cell subsets will be helpful to establish novel therapeutic approaches and clarify how self-tolerance breaks down in autoimmune arthritis. © 2014 Published by Elsevier Ltd.
1. Introduction Rheumatoid arthritis (RA), which afflicts ∼1% of population worldwide, is one of the most common autoimmune diseases. RA is an inflammatory joint disease that ultimately leads to bone destruction (Firestein, 2003). Although the etiology of RA remains to be clarified, both genetic and environmental factors are involved in RA pathogenesis. The importance of T cells in RA is supported by their infiltration into arthritic joints, T cell-related RA-associated genes such as HLA-DR, Ptpn22 and Ccr6, the presence of autoantibodies, the efficacy of CTLA4-Ig and numerous studies from T cell-deficient animal models (Begovich et al., 2004; Lee et al., 2005; Stahl et al., 2010; Kochi et al., 2010). A recent genome-wide association study (GWAS) analysis in combination of epigenetic analysis revealed 101 RA risk loci and suggested the importance of Treg cells (Okada et al., 2014). Thus, elucidating the nature of the T cell subset implicated in the pathogenesis of RA is evidently important from both the immunological and clinical points of view. Here, we review
∗ Corresponding author at: Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: +81 3 5841 3378. E-mail address: [email protected] (H. Takayanagi). http://dx.doi.org/10.1016/j.biocel.2014.11.008 1357-2725/© 2014 Published by Elsevier Ltd.
progress regarding the origin and characteristics of arthritogenic T cells and discuss therapeutic approaches targeting these cells. 1.1. Osteoclastogenic T cells in RA RA is characterized by inflammation and the resultant bone destruction. It is important to identify arthritogenic T cells which are involved in not only inflammation but also bone destruction. Bone destruction in RA is caused by hyperactivation of osteoclastogenic bone resorption. Osteoclasts, critical bone-absorbing cells, are commonly found at the border between the bone and hyperplastic synovial tissues. Osteoclasts are differentiated from receptor activator of NF-B (RANK)-expressing monocyte/macrophage precursor cells, with their differentiation regulated by RANKL that is mainly expressed by osteoclastogenesis-supporting mesenchymal cells, such as synovial fibroblasts (Takayanagi, 2007). RANKL expression can also be detected in T cells in RA joints. Although fixed activated T cells were shown to induce osteoclatogenesis through RANKL expression (Kong et al., 1999), a subsequent study showed that activated T cells inhibit osteoclastogenesis by producing effector T cell cytokines, such as IFN-␥, that inhibit RANK signaling (Takayanagi et al., 2000). Various kinds of cytokines positively or negatively regulate osteoclast differentiation by modulating (1) RANKL expression on synovial fibroblasts and/or (2) RANK signaling in osteoclast precursor cells.
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CD4+ T cells consist of various kinds of helper T cell subsets, such as Th1, Th2 and Th17 cells. When cocultured with osteoclast precursor cells and osteoclastogenesis-supporting mesenchymal cells such as osteoblasts, Th1 and Th2 cells inhibit osteoclastogenesis via IFN-␥ and IL-4, respectively. Notably, Th17 cells were shown to induce osteoclastogenesis, mainly by upregulating RANKL expression on osteoblasts via IL-17A (hereafter IL-17), a hallmark cytokine of Th17 cells (Sato et al., 2006). IL-17 upregulates RANKL expression on synovial fibroblasts and also induces inflammatory cytokines, such as TNF-␣ and IL-6, which further upregulate RANKL expression on synovial fibroblasts and activate osteoclast precursors. In addition, Th17 cells express RANKL and do not express IFN-␥ or IL4. Taken together, Th17 cells are osteoclatogenic T cells capable of accelerating bone destruction (Takayanagi, 2007). 1.2. Th17 cells comprise an arthritogenic T cell subset Numerous studies have shown that Th17 cells play an important role in multiple immune responses, such as the elimination of pathogens and autoimmune inflammation (Miossec et al., 2009). In the inflammatory phase of arthritis, IL-17 induces migration of neutrophils to inflammatory joints and acts on a broad range of cell types to produce inflammatory cytokines, including IL-1, TNF-␣ and IL-6, chemokines and matrix metalloproteinases (Miossec et al., 2009). IL-17 also promotes germinal center formation and helps B cells to produce antibodies (Hsu et al., 2008; Wu et al., 2010). A prominent role of IL-17 in arthritis is clearly evident in mice, since deletion of IL-17 or blockade of IL-17 render mice resistant to autoimmune arthritis (Nakae et al., 2003; Lubberts et al., 2004). In RA, Th17 cells are more frequently observed in the synovial tissues of RA subjects than healthy controls (Shahrara et al., 2008). In addition, a polymorphism in CCR6, which is predominantly expressed in human Th17 cells, is associated with RA susceptibility (Stahl et al., 2010; Kochi et al., 2010). Taken together, Th17 is now recognized as an arthritogenic T cell subset which is involved in the inflammation and bone destruction that occur in arthritis. 1.3. Th1 cells in RA Diverse helper T cell subsets are present in the synovium of autoimmune arthritis. Although it was reported that the IFN-␥ expression is low in RA synovium (Firestein and Zvaifler, 1987), recent studies showed that Th1 cells are more frequently observed than Th17 cells and self-antigen-specific Th1 cells are detected in RA synovial fluid (Nistala et al., 2010; Snir et al., 2012; Ito et al., 2014). In addition, epigenetic analysis showed that demethylation of the Ifn locus is significantly enhanced in CD4+ T cells in RA synovial fluid, suggesting the skewing toward the Th1 lineage (Janson et al., 2011). As for the function, Th1-derived IFN-␥ may play a role in inflammatory responses. However, Th1 cells exert anti-osteoclastogenic effects via IFN-␥ and IFN-␥ receptor-deficient mice exhibit severer symptoms in autoimmune arthritis (Vermeire et al., 1997), suggesting that Th1 cells may not suppress the bone destruction phase in arthritic mice (Boissier et al., 1995). However, the role of Th1 cells to RA pathogenesis is still to be determined. 1.4. Th17-Th1 plasticity in RA It is now widely known that helper T cells are plastic (Nakayamada et al., 2012). Th17 cells give rise to other helper T subsets in response to the local cytokine milieu (Mukasa et al., 2010). In RA, IFN-␥+ IL-17+ T cells, which are considered to be observed in the transition state of Th17 to Th1 conversion, are detected at a higher frequency in the synovial fluid than the peripheral blood of RA patients. The frequency of IFN-␥+ IL-17+ T cells correlated to the disease activities (Nistala et al., 2010; Cosmi et al., 2011). Th17
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cells switch to IFN-␥+ IL-17+ T cells in response to IL-12 produced by synovial fibroblasts (Cosmi et al., 2011). Thus, Th17 cells are not so frequently observed in RA synovium at least partly because of plasticity of Th17 cells. In addition, Th17 cells do not proliferate well under certain inflammatory conditions (Santarlasci et al., 2012), suggesting that the low proliferative capacity of Th17 cells may also contribute to the small number of Th17 cells. The role of IFN-␥+ IL-17+ T cells in RA attracted much attention but still remains unclear. Function of diverse helper T cell subsets and the molecular basis for their plasticity should be clarified in the further studies. 1.5. Treg cells in RA Autoimmune diseases are often caused by an imbalance between Th17 cells and Treg cells. Treg cells comprise a T cell subset which plays a pivotal role in suppressing immune responses (Sakaguchi, 2004). Foxp3 is indispensable for their suppressive function. Foxp3+ Treg cells consist of thymus-derived Treg cells (tTreg) and peripherally derived Treg (pTreg) cells. tTreg cells are generated through the recognition of self-peptide/MHC complexes and are important for self-tolerance (Jordan et al., 2001). Foxp3+ pTreg cells are important for mucosal immune homeostasis, since a deficiency of Foxp3+ pTreg generation results in inflammation in the gut and lung (Josefowicz et al., 2012). In arthritis, Treg cells are protective, since transfer of CD4+ CD25+ Treg cells and deletion of Foxp3+ Treg cells result in the inhibition and exacerbation of autoimmune arthritis, respectively (Morgan et al., 2003, 2005; Nguyen et al., 2007). The accumulation of Treg cells in arthritic joints has been observed in many studies of mice and human (Miyara et al., 2011). Arthritis often proceeds even in the presence of Treg cells, possibly due to the higher number of effector T cells. In terms of their suppressive function, it is reported that inflammatory cytokines, such as TNF-␣, inhibit Treg cell function (Nie et al., 2013) and effector T cells at the site of inflammation are resistant to Treg-mediated suppression (Miyara et al., 2011; Wehrens et al., 2011), which suggests the suppressive effects of Treg cells may be inhibited under arthritic conditions, although the suppressive effects of Treg cells are inconsistent in the literature. Thus, inflammatory conditions may affect the Treg/T effector balance, which is a determinant of arthritis. It is important for the establishment of Treg cell-based therapy to stabilize the Treg cellsuppressive function, in other words, to stabilize Foxp3 expression and function, even under arthritic conditions. 1.6. The origin of Th17 cells under arthritic conditions Considering the pathological significance of Th17 cells, one of the fundamental questions is how they are generated. In the spontaneous arthritis that occurs in SKG mice, in which the strength of TCR signaling is compromised due to a ZAP70 mutation (Sakaguchi et al., 2003), C5R-expressing macrophages in the synovial tissues and spleen produce IL-6 in response to C5, which results in the local and systemic generation of Th17 cells (Hashimoto et al., 2010) (Fig. 1). A link between commensal bacteria and Th17 differentiation in arthritis was reported using the KRN B6 TCR Tg x NOD (K/BxN) arthritis model (Wu et al., 2010). Germ free K/BxN mice exhibit arthritis with an increased number of Th17 cells only after the introduction of segmented filamentous bacteria (SFB). It is suggested that Th17 cells that have differentiated in the gut in the presence of SFB migrate into the spleen, contributing to the production of autoantibodies and the K/BxN arthritis (Wu et al., 2010; Morton et al., 2014). However, as it is reported that collagen-induced arthritis (CIA), which is one of the most common RA models, develops under germ-free conditions, the role of SFB in RA is not
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Stable Foxp3+T p Foxp3+T
Plastic Foxp3+ p T
Self-tolerance
Synovial fibroblast RANKL
IL-6 IL-17
Osteoclast exFoxp3Th17 Autoimmunity Rheumatoid arthritis
Th17 Naïve T Fig. 1. Pathological significance of plastic Foxp3+ T cells.
established (Bjork et al., 1994). It is of considerable interest to clarity the contribution of commensal bacteria to RA in further analysis. Overall, Th17 cells are generated at the sites of inflammation, secondary lymphoid tissues and gut microbial environment under arthritic conditions. However, since there are only few reports and these studies were based on particular RA animal models, the origin of Th17 cells in arthritis at present has not been fully elucidated. 1.7. Arthritogenic Th17 cells differentiated from Foxp3+ T cells Since Foxp3+ Treg cells contain T cells which recognize selfantigens, they would be expected to regulate Foxp3 expression in order to maintain self-tolerance. The stability of Foxp3+ Treg cells under pathological conditions is controversial (Rubtsov et al., 2010; Zhou et al., 2009). It has been reported that Foxp3+ T cells consist of Foxp3-stable CD25hi cells and Foxp3-unstable CD25lo cells (Miyao et al., 2012; Komatsu et al., 2009). However, the pathogenic significance of the latter population in vivo has been unclear. Recently, we tested the pathogenic relevance of plastic Foxp3+ T cells (Komatsu et al., 2014). When transferred into lymphoreplete WT mice with CIA, CD25lo Foxp3+ T cells lose Foxp3 expression and differentiate into Th17 cells (hereafter exFoxp3Th17 cells). Fate mapping analysis showed exFoxp3Th17 cells accumulate in arthritic joints. DNA methylation analysis demonstrated that they are not derived from thymic Foxp3+ Treg cells nor conventional activated T cells which transiently express Foxp3 during the course of their differentiation. It is suggested that pTreg cells may be the origin of exFoxp3Th17 cells, although further analysis is required. In vitro analysis showed arthritic synovial fibroblasts trigger the conversion of Foxp3+ T cells into Th17 cells by the expression of IL-6. Interestingly, exFoxp3Th17 cells express genes distinct from naïve CD4+ T-derived Th17 cells, in particular, a higher expression of Sox4, CCR6, CCL20, IL-23R and RANKL, and a lower expression of TNF-␣ and IL-10, indicating that exFoxp3Th17 cells a distinct Th17 subset. Notably, exFoxp3Th17 cells induce osteoclastogenesis in the presence of arthritic synovial fibroblasts three times more potently than Th17 cells do, indicating that exFoxp3Th17 cells are the most potent osteoclastogenic T cells ever reported. The relative contribution of Th17-derived RANKL and synovial fibroblast-derived RANKL to Th17-mediated osteoclastogenesis in RA remains unclear. In this coculture system, synovial fibroblasts were shown to be the major source of RANKL, suggesting that exFoxp3Th17 cells induce osteoclastogenesis mainly by
unregulating RANKL on synovial fibroblasts, while T cell RANKL contributes less. As for in vivo pathogenesis, the transfer of CD25lo Foxp3+ T cells from collagen-immunized mice exacerbated arthritis, suggesting that CD25lo Foxp3+ T cells contain T cells which recognize selfantigen and convert into self-reactive arthritogenic Th17 cells after losing Foxp3. Importantly, Foxp3+ IL-17+ T cells, which are considered to be in a transition state, are frequently observed in the synovial tissues of active but not inactive inactive RA subjects, which suggest a pathogenic role for plastic Foxp3+ T cells in RA. Thus, it has been demonstrated that the arthritogenic Th17 cells exacerbate inflammation and bone destruction are differentiated from plastic Foxp3+ T cells (Komatsu et al., 2014). So why do these potentially ‘harmful’ cells need to exist? It is possible that immune system may require ‘flexibility’ to adapt to changes in microenvironments, since continuous immune suppression sometimes results in deleterious effects, such as the progression of cancer and infectious diseases. It will be an important issue to determine whether ‘bona fide’ Treg cells bearing self-reactivity are the origin of arthritogenic Th17 cells at the single cell level. 1.8. Antigen specificity of arthritogenic T cells The specific antigens of the arthritogenic T cells in RA have not been identified yet, although anti-citrullinated peptide antibodies (ACPA) and rheumatoid factors are widely used for the purpose of disease diagnosis. Both citrullinating enzymes, such as PADI4 (Suzuki et al., 2003), as well as substrates such as collagen and vimentin (Burkhardt et al., 2005; Vossenaar et al., 2004), are highly detected in arthritic synovial tissues, suggesting the abundance of ACPA immune complexes (ICs) in the arthritic joints. Transfer of ACPA ICs induce bone loss and exacerbate autoimmune arthritis in mice (Harre et al., 2012; Kuhn et al., 2006), supporting the pathogenicity of ACPA. These studies also suggest that self-antigens which are not joint-specific, but nonetheless are abundant in joints, may be among the relevant pathogenic antigens. Likewise, studies using CIA and the spontaneous arthritis of K/BxN mice suggest that T cells specific for ubiquitous self-antigens are able to induce arthritis when they are activated by the innate immune system. In line with this, a recent study identified the ubiquitously expressed 60S ribosomal protein L23a (RPL23A) as one of the self-antigens in the SKG mice. Humoral and cellular immune responses against RPL23A were also observed in RA patients (Ito et al., 2014). Studies on how T cells elicit local immune responses against autoantigens
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using this mice model will contribute to our understanding for RA pathogenesis. Overall, studies in humans and mice have shown the pathogenic importance of self-reactive T cells. This may support the pathogenic significance of Th17 cells derived from Foxp3+ T cells because they contain self-reactive T cells. 1.9. Th17-synovial fibroblast interaction The inflamed joints that develop in RA are characterized by a hyperplasia of the synovial tissues in which synovial fibroblasts are hyper-proliferating and hyper-activated. Accumulating studies have shown arthritic synovial fibroblasts contribute to the Th17-mediated pathogenic events. Synovial fibroblasts recruit CCR6-expressing Th17 cells into the inflammatory sites via the ligand CCL20, generate Th17 cells via IL-6 and promote the proliferation of T cells via IL-7 (Hirota et al., 2007a,b; Sawa et al., 2006). Notably, arthritic synovial fibroblasts trigger the generation of exFoxp3Th17 cells and osteoclastogenesis by exFoxp3Th17 cells, which highlights substantial role of local Th17-synovial fibroblast interaction in the pathogenesis of autoimmune arthritis (Komatsu et al., 2014). In turn, the IL-17 produced by Th17 cells acts on synovial fibroblasts so as to produce CCL20, IL-6 and RANKL (Hirota et al., 2007a,b; Ogura et al., 2008; Kotake et al., 1999). These findings indicate that the Th17-synovial fibroblast local interaction acts as a vicious circle in arthritis.
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T cell-synovial fibroblast interaction amplifies the local immune dysregulation, which suggests a substantial contribution of local immune-mesenchymal cell interactions in autoimmune diseases. Studies on this interaction will provide mechanical insight into the process by which self-tolerance collapses in various autoimmune diseases. Further analysis is required to clarify which Th17 cell subset mainly contributes to the pathogenesis of the various autoimmune diseases, and these investigation need to be carried out in both a qualitative and quantitative manner. This will help identify the arthritogenic subsets among the RA Th17 cells that are characterized by both plasticity and heterogeneity, leading to the establishment of new therapies against autoimmune arthritis. Th17 cells derived from plastic Foxp3+ T cells (exFoxp3Th17 cells) have been shown to comprise a novel Th17 cell subset with a distinct pattern of gene expression and arthritogenic properties. The fate of plastic Foxp3+ T cells may be a key determinant of the Treg/Th17 balance that is critically involved in the self-tolerance and autoimmunity.
Acknowledgements We would like to thank Kazuo Okamoto and Shin-ichiro Sawa for helpful discussion.
References 1.10. Therapeutic approaches As a Treg/Th17 imbalance is often involved in the pathogenesis of autoimmune diseases, the molecules and signaling pathways which regulate this balance are attractive therapeutic targets. The blockade of IL-6 or TNF-␣ increases the Treg/Th17 ratio, suggesting that the efficacy may be partly due to the improved Treg/Th17 balance (Samson et al., 2012; Nie et al., 2013). IL-2/STAT5 signaling and retinoic acid increase, while IL-6/STAT3 and AKT/mTOR signaling decrease the Treg/Th17 ratio (Yang et al., 2011; Elias et al., 2008; Wan et al., 2011; Haxhinasto et al., 2008). Transcription factors which are indispensable for Th17 differentiation, such as ROR␥t and IB, may also be promising targets (Solt et al., 2011; Huh et al., 2011; Okamoto et al., 2010). JAK inhibitors are effective in RA and murine arthritis in part due to an inhibition of Th17 and Th1 differentiation (Maeshima et al., 2012; Yamaoka and Tanaka, 2014). Anti-IL-17A blockade is potently effective in psoriasis arthritis. It is also effective in RA, although to a lesser extent than murine arthritis (Hueber et al., 2010; Genovese et al., 2010, 2013). This may be due to species differences in the dependency of the Th17 function on IL-17A. It is possible that patients are treated with anti-IL-17A antibody when IL-17A is no longer responsible for the pathogenesis. Human Th17 cells are plastic and heterogeneous, which is suggested by the presence of Foxp3+ IL-17+ cells and IFN-␥+ IL-17+ cells. It is important to evaluate the roles of these subpopulations in arthritis and identify the arthritogenic Th17 cell subpopulation. The molecules which govern the plasticity of these T cell subsets can be good therapeutic targets. It is needed to identify the characteristics of arthritogenic Th17 cells and the molecular basis of their generation and antigen-specificity. The pathogenic antigens will also be highly useful for generating antigen-specific Treg cells from naïve T cells and effector T cells. 2. Concluding remarks In RA, Th17 cells function as the principal drivers of both inflammation and bone destruction. The Th17 cells derived from Foxp3+ T cells comprise a novel Th17 cell subset with a distinct pattern of gene expression and arthritogenic properties. The
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Medicine in focus
Arthritogenic T cells in autoimmune arthritis Noriko Komatsu a,b , Hiroshi Takayanagi a,b,∗ a Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan b Japan Science and Technology Agency (JST), Exploratory Research for Advanced Technology Program, Takayanagi Osteonetwork Project, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan
a r t i c l e
i n f o
Article history: Received 26 September 2014 Received in revised form 15 November 2014 Accepted 20 November 2014 Available online 28 November 2014 Keywords: Rheumatoid arthritis Regulatory T cells Th17 cells Synovial fibroblasts Osteoclasts
a b s t r a c t Autoimmune diseases, including arthritis, often result from an imbalance between regulatory T (Treg) cells and IL-17-producing (Th17) cells. Dozens of studies in mice and humans have shed light on the pathological significance of T cells in RA. Since Th17 cells play an important role in the exacerbation of inflammation and bone destruction in joints, it has been an important issue how arthritic Th17 cells arise. Th17 cells are generated in the local inflammatory milieu via cytokines produced by macrophages or synovial fibroblasts, while it is reported that Th17 cells are generated in the gut in the presence of specific commensal bacteria. A recent report showed a pathogenic Th17 cell subset with a distinct pattern of gene expression and a potent osteoclastogenic ability are converted from Foxp3+ T cells in arthritic joints. Since Foxp3+ Treg cells contain T cells which recognize self-antigens, the fate of plastic Foxp3+ T cells can be a critical determinant of autoimmunity or self-tolerance. Further analysis on the molecular basis and antigen-specificity of arthritogenic Th17 cell subsets will be helpful to establish novel therapeutic approaches and clarify how self-tolerance breaks down in autoimmune arthritis. © 2014 Published by Elsevier Ltd.
1. Introduction Rheumatoid arthritis (RA), which afflicts ∼1% of population worldwide, is one of the most common autoimmune diseases. RA is an inflammatory joint disease that ultimately leads to bone destruction (Firestein, 2003). Although the etiology of RA remains to be clarified, both genetic and environmental factors are involved in RA pathogenesis. The importance of T cells in RA is supported by their infiltration into arthritic joints, T cell-related RA-associated genes such as HLA-DR, Ptpn22 and Ccr6, the presence of autoantibodies, the efficacy of CTLA4-Ig and numerous studies from T cell-deficient animal models (Begovich et al., 2004; Lee et al., 2005; Stahl et al., 2010; Kochi et al., 2010). A recent genome-wide association study (GWAS) analysis in combination of epigenetic analysis revealed 101 RA risk loci and suggested the importance of Treg cells (Okada et al., 2014). Thus, elucidating the nature of the T cell subset implicated in the pathogenesis of RA is evidently important from both the immunological and clinical points of view. Here, we review
∗ Corresponding author at: Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: +81 3 5841 3378. E-mail address: [email protected] (H. Takayanagi). http://dx.doi.org/10.1016/j.biocel.2014.11.008 1357-2725/© 2014 Published by Elsevier Ltd.
progress regarding the origin and characteristics of arthritogenic T cells and discuss therapeutic approaches targeting these cells. 1.1. Osteoclastogenic T cells in RA RA is characterized by inflammation and the resultant bone destruction. It is important to identify arthritogenic T cells which are involved in not only inflammation but also bone destruction. Bone destruction in RA is caused by hyperactivation of osteoclastogenic bone resorption. Osteoclasts, critical bone-absorbing cells, are commonly found at the border between the bone and hyperplastic synovial tissues. Osteoclasts are differentiated from receptor activator of NF-B (RANK)-expressing monocyte/macrophage precursor cells, with their differentiation regulated by RANKL that is mainly expressed by osteoclastogenesis-supporting mesenchymal cells, such as synovial fibroblasts (Takayanagi, 2007). RANKL expression can also be detected in T cells in RA joints. Although fixed activated T cells were shown to induce osteoclatogenesis through RANKL expression (Kong et al., 1999), a subsequent study showed that activated T cells inhibit osteoclastogenesis by producing effector T cell cytokines, such as IFN-␥, that inhibit RANK signaling (Takayanagi et al., 2000). Various kinds of cytokines positively or negatively regulate osteoclast differentiation by modulating (1) RANKL expression on synovial fibroblasts and/or (2) RANK signaling in osteoclast precursor cells.
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CD4+ T cells consist of various kinds of helper T cell subsets, such as Th1, Th2 and Th17 cells. When cocultured with osteoclast precursor cells and osteoclastogenesis-supporting mesenchymal cells such as osteoblasts, Th1 and Th2 cells inhibit osteoclastogenesis via IFN-␥ and IL-4, respectively. Notably, Th17 cells were shown to induce osteoclastogenesis, mainly by upregulating RANKL expression on osteoblasts via IL-17A (hereafter IL-17), a hallmark cytokine of Th17 cells (Sato et al., 2006). IL-17 upregulates RANKL expression on synovial fibroblasts and also induces inflammatory cytokines, such as TNF-␣ and IL-6, which further upregulate RANKL expression on synovial fibroblasts and activate osteoclast precursors. In addition, Th17 cells express RANKL and do not express IFN-␥ or IL4. Taken together, Th17 cells are osteoclatogenic T cells capable of accelerating bone destruction (Takayanagi, 2007). 1.2. Th17 cells comprise an arthritogenic T cell subset Numerous studies have shown that Th17 cells play an important role in multiple immune responses, such as the elimination of pathogens and autoimmune inflammation (Miossec et al., 2009). In the inflammatory phase of arthritis, IL-17 induces migration of neutrophils to inflammatory joints and acts on a broad range of cell types to produce inflammatory cytokines, including IL-1, TNF-␣ and IL-6, chemokines and matrix metalloproteinases (Miossec et al., 2009). IL-17 also promotes germinal center formation and helps B cells to produce antibodies (Hsu et al., 2008; Wu et al., 2010). A prominent role of IL-17 in arthritis is clearly evident in mice, since deletion of IL-17 or blockade of IL-17 render mice resistant to autoimmune arthritis (Nakae et al., 2003; Lubberts et al., 2004). In RA, Th17 cells are more frequently observed in the synovial tissues of RA subjects than healthy controls (Shahrara et al., 2008). In addition, a polymorphism in CCR6, which is predominantly expressed in human Th17 cells, is associated with RA susceptibility (Stahl et al., 2010; Kochi et al., 2010). Taken together, Th17 is now recognized as an arthritogenic T cell subset which is involved in the inflammation and bone destruction that occur in arthritis. 1.3. Th1 cells in RA Diverse helper T cell subsets are present in the synovium of autoimmune arthritis. Although it was reported that the IFN-␥ expression is low in RA synovium (Firestein and Zvaifler, 1987), recent studies showed that Th1 cells are more frequently observed than Th17 cells and self-antigen-specific Th1 cells are detected in RA synovial fluid (Nistala et al., 2010; Snir et al., 2012; Ito et al., 2014). In addition, epigenetic analysis showed that demethylation of the Ifn locus is significantly enhanced in CD4+ T cells in RA synovial fluid, suggesting the skewing toward the Th1 lineage (Janson et al., 2011). As for the function, Th1-derived IFN-␥ may play a role in inflammatory responses. However, Th1 cells exert anti-osteoclastogenic effects via IFN-␥ and IFN-␥ receptor-deficient mice exhibit severer symptoms in autoimmune arthritis (Vermeire et al., 1997), suggesting that Th1 cells may not suppress the bone destruction phase in arthritic mice (Boissier et al., 1995). However, the role of Th1 cells to RA pathogenesis is still to be determined. 1.4. Th17-Th1 plasticity in RA It is now widely known that helper T cells are plastic (Nakayamada et al., 2012). Th17 cells give rise to other helper T subsets in response to the local cytokine milieu (Mukasa et al., 2010). In RA, IFN-␥+ IL-17+ T cells, which are considered to be observed in the transition state of Th17 to Th1 conversion, are detected at a higher frequency in the synovial fluid than the peripheral blood of RA patients. The frequency of IFN-␥+ IL-17+ T cells correlated to the disease activities (Nistala et al., 2010; Cosmi et al., 2011). Th17
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cells switch to IFN-␥+ IL-17+ T cells in response to IL-12 produced by synovial fibroblasts (Cosmi et al., 2011). Thus, Th17 cells are not so frequently observed in RA synovium at least partly because of plasticity of Th17 cells. In addition, Th17 cells do not proliferate well under certain inflammatory conditions (Santarlasci et al., 2012), suggesting that the low proliferative capacity of Th17 cells may also contribute to the small number of Th17 cells. The role of IFN-␥+ IL-17+ T cells in RA attracted much attention but still remains unclear. Function of diverse helper T cell subsets and the molecular basis for their plasticity should be clarified in the further studies. 1.5. Treg cells in RA Autoimmune diseases are often caused by an imbalance between Th17 cells and Treg cells. Treg cells comprise a T cell subset which plays a pivotal role in suppressing immune responses (Sakaguchi, 2004). Foxp3 is indispensable for their suppressive function. Foxp3+ Treg cells consist of thymus-derived Treg cells (tTreg) and peripherally derived Treg (pTreg) cells. tTreg cells are generated through the recognition of self-peptide/MHC complexes and are important for self-tolerance (Jordan et al., 2001). Foxp3+ pTreg cells are important for mucosal immune homeostasis, since a deficiency of Foxp3+ pTreg generation results in inflammation in the gut and lung (Josefowicz et al., 2012). In arthritis, Treg cells are protective, since transfer of CD4+ CD25+ Treg cells and deletion of Foxp3+ Treg cells result in the inhibition and exacerbation of autoimmune arthritis, respectively (Morgan et al., 2003, 2005; Nguyen et al., 2007). The accumulation of Treg cells in arthritic joints has been observed in many studies of mice and human (Miyara et al., 2011). Arthritis often proceeds even in the presence of Treg cells, possibly due to the higher number of effector T cells. In terms of their suppressive function, it is reported that inflammatory cytokines, such as TNF-␣, inhibit Treg cell function (Nie et al., 2013) and effector T cells at the site of inflammation are resistant to Treg-mediated suppression (Miyara et al., 2011; Wehrens et al., 2011), which suggests the suppressive effects of Treg cells may be inhibited under arthritic conditions, although the suppressive effects of Treg cells are inconsistent in the literature. Thus, inflammatory conditions may affect the Treg/T effector balance, which is a determinant of arthritis. It is important for the establishment of Treg cell-based therapy to stabilize the Treg cellsuppressive function, in other words, to stabilize Foxp3 expression and function, even under arthritic conditions. 1.6. The origin of Th17 cells under arthritic conditions Considering the pathological significance of Th17 cells, one of the fundamental questions is how they are generated. In the spontaneous arthritis that occurs in SKG mice, in which the strength of TCR signaling is compromised due to a ZAP70 mutation (Sakaguchi et al., 2003), C5R-expressing macrophages in the synovial tissues and spleen produce IL-6 in response to C5, which results in the local and systemic generation of Th17 cells (Hashimoto et al., 2010) (Fig. 1). A link between commensal bacteria and Th17 differentiation in arthritis was reported using the KRN B6 TCR Tg x NOD (K/BxN) arthritis model (Wu et al., 2010). Germ free K/BxN mice exhibit arthritis with an increased number of Th17 cells only after the introduction of segmented filamentous bacteria (SFB). It is suggested that Th17 cells that have differentiated in the gut in the presence of SFB migrate into the spleen, contributing to the production of autoantibodies and the K/BxN arthritis (Wu et al., 2010; Morton et al., 2014). However, as it is reported that collagen-induced arthritis (CIA), which is one of the most common RA models, develops under germ-free conditions, the role of SFB in RA is not
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Stable Foxp3+T p Foxp3+T
Plastic Foxp3+ p T
Self-tolerance
Synovial fibroblast RANKL
IL-6 IL-17
Osteoclast exFoxp3Th17 Autoimmunity Rheumatoid arthritis
Th17 Naïve T Fig. 1. Pathological significance of plastic Foxp3+ T cells.
established (Bjork et al., 1994). It is of considerable interest to clarity the contribution of commensal bacteria to RA in further analysis. Overall, Th17 cells are generated at the sites of inflammation, secondary lymphoid tissues and gut microbial environment under arthritic conditions. However, since there are only few reports and these studies were based on particular RA animal models, the origin of Th17 cells in arthritis at present has not been fully elucidated. 1.7. Arthritogenic Th17 cells differentiated from Foxp3+ T cells Since Foxp3+ Treg cells contain T cells which recognize selfantigens, they would be expected to regulate Foxp3 expression in order to maintain self-tolerance. The stability of Foxp3+ Treg cells under pathological conditions is controversial (Rubtsov et al., 2010; Zhou et al., 2009). It has been reported that Foxp3+ T cells consist of Foxp3-stable CD25hi cells and Foxp3-unstable CD25lo cells (Miyao et al., 2012; Komatsu et al., 2009). However, the pathogenic significance of the latter population in vivo has been unclear. Recently, we tested the pathogenic relevance of plastic Foxp3+ T cells (Komatsu et al., 2014). When transferred into lymphoreplete WT mice with CIA, CD25lo Foxp3+ T cells lose Foxp3 expression and differentiate into Th17 cells (hereafter exFoxp3Th17 cells). Fate mapping analysis showed exFoxp3Th17 cells accumulate in arthritic joints. DNA methylation analysis demonstrated that they are not derived from thymic Foxp3+ Treg cells nor conventional activated T cells which transiently express Foxp3 during the course of their differentiation. It is suggested that pTreg cells may be the origin of exFoxp3Th17 cells, although further analysis is required. In vitro analysis showed arthritic synovial fibroblasts trigger the conversion of Foxp3+ T cells into Th17 cells by the expression of IL-6. Interestingly, exFoxp3Th17 cells express genes distinct from naïve CD4+ T-derived Th17 cells, in particular, a higher expression of Sox4, CCR6, CCL20, IL-23R and RANKL, and a lower expression of TNF-␣ and IL-10, indicating that exFoxp3Th17 cells a distinct Th17 subset. Notably, exFoxp3Th17 cells induce osteoclastogenesis in the presence of arthritic synovial fibroblasts three times more potently than Th17 cells do, indicating that exFoxp3Th17 cells are the most potent osteoclastogenic T cells ever reported. The relative contribution of Th17-derived RANKL and synovial fibroblast-derived RANKL to Th17-mediated osteoclastogenesis in RA remains unclear. In this coculture system, synovial fibroblasts were shown to be the major source of RANKL, suggesting that exFoxp3Th17 cells induce osteoclastogenesis mainly by
unregulating RANKL on synovial fibroblasts, while T cell RANKL contributes less. As for in vivo pathogenesis, the transfer of CD25lo Foxp3+ T cells from collagen-immunized mice exacerbated arthritis, suggesting that CD25lo Foxp3+ T cells contain T cells which recognize selfantigen and convert into self-reactive arthritogenic Th17 cells after losing Foxp3. Importantly, Foxp3+ IL-17+ T cells, which are considered to be in a transition state, are frequently observed in the synovial tissues of active but not inactive inactive RA subjects, which suggest a pathogenic role for plastic Foxp3+ T cells in RA. Thus, it has been demonstrated that the arthritogenic Th17 cells exacerbate inflammation and bone destruction are differentiated from plastic Foxp3+ T cells (Komatsu et al., 2014). So why do these potentially ‘harmful’ cells need to exist? It is possible that immune system may require ‘flexibility’ to adapt to changes in microenvironments, since continuous immune suppression sometimes results in deleterious effects, such as the progression of cancer and infectious diseases. It will be an important issue to determine whether ‘bona fide’ Treg cells bearing self-reactivity are the origin of arthritogenic Th17 cells at the single cell level. 1.8. Antigen specificity of arthritogenic T cells The specific antigens of the arthritogenic T cells in RA have not been identified yet, although anti-citrullinated peptide antibodies (ACPA) and rheumatoid factors are widely used for the purpose of disease diagnosis. Both citrullinating enzymes, such as PADI4 (Suzuki et al., 2003), as well as substrates such as collagen and vimentin (Burkhardt et al., 2005; Vossenaar et al., 2004), are highly detected in arthritic synovial tissues, suggesting the abundance of ACPA immune complexes (ICs) in the arthritic joints. Transfer of ACPA ICs induce bone loss and exacerbate autoimmune arthritis in mice (Harre et al., 2012; Kuhn et al., 2006), supporting the pathogenicity of ACPA. These studies also suggest that self-antigens which are not joint-specific, but nonetheless are abundant in joints, may be among the relevant pathogenic antigens. Likewise, studies using CIA and the spontaneous arthritis of K/BxN mice suggest that T cells specific for ubiquitous self-antigens are able to induce arthritis when they are activated by the innate immune system. In line with this, a recent study identified the ubiquitously expressed 60S ribosomal protein L23a (RPL23A) as one of the self-antigens in the SKG mice. Humoral and cellular immune responses against RPL23A were also observed in RA patients (Ito et al., 2014). Studies on how T cells elicit local immune responses against autoantigens
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using this mice model will contribute to our understanding for RA pathogenesis. Overall, studies in humans and mice have shown the pathogenic importance of self-reactive T cells. This may support the pathogenic significance of Th17 cells derived from Foxp3+ T cells because they contain self-reactive T cells. 1.9. Th17-synovial fibroblast interaction The inflamed joints that develop in RA are characterized by a hyperplasia of the synovial tissues in which synovial fibroblasts are hyper-proliferating and hyper-activated. Accumulating studies have shown arthritic synovial fibroblasts contribute to the Th17-mediated pathogenic events. Synovial fibroblasts recruit CCR6-expressing Th17 cells into the inflammatory sites via the ligand CCL20, generate Th17 cells via IL-6 and promote the proliferation of T cells via IL-7 (Hirota et al., 2007a,b; Sawa et al., 2006). Notably, arthritic synovial fibroblasts trigger the generation of exFoxp3Th17 cells and osteoclastogenesis by exFoxp3Th17 cells, which highlights substantial role of local Th17-synovial fibroblast interaction in the pathogenesis of autoimmune arthritis (Komatsu et al., 2014). In turn, the IL-17 produced by Th17 cells acts on synovial fibroblasts so as to produce CCL20, IL-6 and RANKL (Hirota et al., 2007a,b; Ogura et al., 2008; Kotake et al., 1999). These findings indicate that the Th17-synovial fibroblast local interaction acts as a vicious circle in arthritis.
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T cell-synovial fibroblast interaction amplifies the local immune dysregulation, which suggests a substantial contribution of local immune-mesenchymal cell interactions in autoimmune diseases. Studies on this interaction will provide mechanical insight into the process by which self-tolerance collapses in various autoimmune diseases. Further analysis is required to clarify which Th17 cell subset mainly contributes to the pathogenesis of the various autoimmune diseases, and these investigation need to be carried out in both a qualitative and quantitative manner. This will help identify the arthritogenic subsets among the RA Th17 cells that are characterized by both plasticity and heterogeneity, leading to the establishment of new therapies against autoimmune arthritis. Th17 cells derived from plastic Foxp3+ T cells (exFoxp3Th17 cells) have been shown to comprise a novel Th17 cell subset with a distinct pattern of gene expression and arthritogenic properties. The fate of plastic Foxp3+ T cells may be a key determinant of the Treg/Th17 balance that is critically involved in the self-tolerance and autoimmunity.
Acknowledgements We would like to thank Kazuo Okamoto and Shin-ichiro Sawa for helpful discussion.
References 1.10. Therapeutic approaches As a Treg/Th17 imbalance is often involved in the pathogenesis of autoimmune diseases, the molecules and signaling pathways which regulate this balance are attractive therapeutic targets. The blockade of IL-6 or TNF-␣ increases the Treg/Th17 ratio, suggesting that the efficacy may be partly due to the improved Treg/Th17 balance (Samson et al., 2012; Nie et al., 2013). IL-2/STAT5 signaling and retinoic acid increase, while IL-6/STAT3 and AKT/mTOR signaling decrease the Treg/Th17 ratio (Yang et al., 2011; Elias et al., 2008; Wan et al., 2011; Haxhinasto et al., 2008). Transcription factors which are indispensable for Th17 differentiation, such as ROR␥t and IB, may also be promising targets (Solt et al., 2011; Huh et al., 2011; Okamoto et al., 2010). JAK inhibitors are effective in RA and murine arthritis in part due to an inhibition of Th17 and Th1 differentiation (Maeshima et al., 2012; Yamaoka and Tanaka, 2014). Anti-IL-17A blockade is potently effective in psoriasis arthritis. It is also effective in RA, although to a lesser extent than murine arthritis (Hueber et al., 2010; Genovese et al., 2010, 2013). This may be due to species differences in the dependency of the Th17 function on IL-17A. It is possible that patients are treated with anti-IL-17A antibody when IL-17A is no longer responsible for the pathogenesis. Human Th17 cells are plastic and heterogeneous, which is suggested by the presence of Foxp3+ IL-17+ cells and IFN-␥+ IL-17+ cells. It is important to evaluate the roles of these subpopulations in arthritis and identify the arthritogenic Th17 cell subpopulation. The molecules which govern the plasticity of these T cell subsets can be good therapeutic targets. It is needed to identify the characteristics of arthritogenic Th17 cells and the molecular basis of their generation and antigen-specificity. The pathogenic antigens will also be highly useful for generating antigen-specific Treg cells from naïve T cells and effector T cells. 2. Concluding remarks In RA, Th17 cells function as the principal drivers of both inflammation and bone destruction. The Th17 cells derived from Foxp3+ T cells comprise a novel Th17 cell subset with a distinct pattern of gene expression and arthritogenic properties. The
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