Laboratory of Immune Regulation, Osaka University, World Premier International (WPI) Immunology Frontier Research Center (IFReC), 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
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Laboratory of Immune Regulation, Osaka University, World Premier International (WPI) Immunology Frontier Research Center (IFReC), 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
IL-17-producing CD4+ T cells (Th17 cells) are understood to be a distinct lineage of CD4+ T helper (Th) cells, which play an important role in the host defense, tissue inflammation and autoimmunity. The identification of Th17 cells collapsed the concept of the previously held Th1/Th2 paradigm in infection and autoimmunity. Recent studies have provided new information on the role of Th17 cells in different autoimmune diseases and the mechanisms of Th17 cell differentiation. Th17 cells contribute to the exacerbation of autoimmune disease, whereas they possess a protective aspect against microbes such as bacteria and fungi. This suggests that Th17 cells can be broadly categorized as pathogenic or non-pathogenic. Naïve CD4+ T cells can differentiate into Th17 cells in synergy with IL-6 and TGF-β, while TGF-β induces regulatory T cells (iTreg), which appear to be mutually exclusive to Th17 cells. Here we describe the detail molecular mechanism of Th17 cell differentiation, including recently identified molecules, and discuss different roles of Th17 cells in infection, inflammation and autoimmunity in a cytokine milieu.
CD4+ T cells play a pivotal role in host defense, but are also recognized to have pathogenic roles such as in autoimmunity, asthma, cancer and allergic responses (Zhu et al., 2010). On activation by co-stimulatory molecules and particular cytokines, naïve CD4+ T cells can differentiate into the distinct lineage of T helper (Th) cells with different immunological functions, including Th1, Th2, Th17 cells, and regulatory T cells (Treg).
Th1 cell polarization was driven by IL-12 and IFN-γ, and Th1 cells, which produced IFN-γ, elicited cell-mediated immunity against intracellular pathogen, whereas Th2 cells, which secreted IL-4, IL-5, and IL-13 (Abbas et al., 1996), were induced by IL-4, and involved in immune responses against extracellular parasites such as helminthes and nematodes (Pearce et al., 2002). Th1 and Th2 cells were shown to be mutually exclusive (Hwang et al., 2005). In Th1 cells, IL-12 activated Stat4 and induced IFN production, which led to the expression of master transcription factor T-box (Tbx21, T-bet). In Th2 cells, IL-4 enhanced Stat6 signaling, which upregulated the transcriptional factor GATA3. T-bet inhibited Th2 differentiation by attenuating the function of GATA3, whereas GATA3 contributed to the repression of Th1 cell lineage.
More recently, IL-17-producing T (Th17) cells, as a third of T cell lineages, have been identified (Harrington et al., 2005; Park et al., 2005). Although it was initially demonstrated that IL-23 drives Th17 cell polarization via Stat3 activation in the absence of IFN-γ, it was generally assumed that the effect of IL-23 was limited in effector and memory CD4+ T cells (Oppmann et al., 2000; Aggarwal et al., 2003; Langrish et al., 2005). Subsequently, the synergy between TGF-β and IL-6 has been shown to efficiently induce the development of Th17 cells through Smad and Stat3 pathway, respectively (Bettelli et al., 2006; Veldhoen et al., 2006). In contrast, TGF-β alone converted naïve CD4+CD25- T cells into CD4+CD25+ regulatory T cells (Chen et al., 2003).
The immunopathogenesis of infection, autoimmunity, and allergy has been extensively attributed to the concept of Th1/Th2 paradigm. However, it has been recently reported that CIA and EAE are exacerbated rather than improved under inhibition of Th1 cell-inducing condition such as deficiency of IFN-γR, IL-12 p35 or Stat1 (Iwakura and Ishigame, 2006). Cua et al. has demonstrated that IL-23 rather than IL-12 influences the critical pathogenic effect on autoimmunity such as EAE. Subsequently, by the same group, it has been shown that IL-23 promotes the development and expansion of effector CD4+ T cells, which highly produce IL-17, and IL-17 secreted from activated CD4+ T cells play an important role in various autoimmune diseases (Langrich et al., 2005).
The balance between Th17 and Treg cells is an important factor involved in the pathogenesis of autoimmunity. Sakaguchi et al. initially demonstrated that a population of CD4+CD25+ T cells derived from the thymus, known as nTreg, exhibited the inhibitory effect in immunity. TGF-β1 is required for the differentiation of both Treg and Th17 cells, whereas IL-6 suppresses Treg cell population and drives the development of Th17 cells. This suggests a dichotomy between these cells (Bettelli et al., 2007). However, it still remains to be understood how IL-6 in combination with TGF-β drives Th17 cell differentiation, and which cytokine milieu makes Th17 cells "pathogenic" in vivo. In this review, we discuss the mechanism of Th17 cell differentiation, its plasticity and pathogenicity in immune regulation, and its link with inflammatory disease and autoimmunity.
2- Regulation of Th17 polarization
The mechanism of Th17 differentiation has been extensively studied with respect to the transcriptional regulation. Betteli et al. found that IL-6 but not IL-23 was a potent inducer for Th17 cell differentiation in combination with TGF-β1. It was initially shown that retinoic acid (RA)-related orphan receptor γ thymus (Rorγt) was an essential transcriptional factor for identifying a distinct lineage of CD4+ T cells (Th17 cells), which constitutively produced IL-17 in the lamina propria of the small intestine (Ivanov et al., 2006).
IL-6 mainly activates Stat3 via the Jak-Stat pathway (Kishimoto, 2005). The role of Stat3 in Th17 cell differentiation was extensively analyzed by chromatin immunoprecipitation and massive parallel sequencing (ChIP-Seq), in which STAT3 bound to the promoter of cytokine and transcriptional genes including the Rorc, IL-17, IL-17F, Ahr and IL-21 genes. Possibly Stat3 also interacted with the BATF, IRF4, and c-Maf genes (Durant et al., 2010). These transcriptional factors played an important role in Th17 cell differentiation (Ciofani et al., 2012; Yosef et al., 2013).
BATF, which is a basic leucine zipper (b-Zip) transcription factor of the AP-1 protein family, contributed to the generation of Th17 cells (Schraml et al., 2009). It has recently been reported that BATF is a multifunctional transcriptional factor, in which BATF is also required for the generation of T follicular helper (Tfh) cells but not Th1 cells and Treg cells (Betz et al., 2010; Ise et al., 2011). More recently, BATF in cooperation with IRF-4, which is also essential for the development of both Th2 and Th17 cells (Brüstle et al., 2009), has been shown to be involved in the induction of the IL10, IL-17a, and IL-21 genes in T cells (Glasmacher et al., 2012; Li et al., 2012; Tussiwand et al., 2012; Murphy et al., 2013). Although it is still not clear whether the BATF gene is directly regulated by Stat3 activation, recent studies have shown that IL-6 activates the function of BATF/IRF4 complex (Koch et al., 2013).
More recently, our group has also identified a key molecule, AT-rich interactive domain 5a (Arid5a), which is induced under Th17 cell-polarizing condition. Arid5a deficiency inhibits the differentiation of Th17 cells (unpublished data). Our previous report has shown that Arid5a positively controls IL-6 mRNA through its 3'-untranslated region (UTR). IL-6 serum level in Arid5a deficient mice was dramatically reduced after LPS injection compared to WT mice, and EAE was also ameliorated, in which the frequency of Th17 cell population was inhibited, whereas that of Th1 cells was enhanced, but not Treg cells (Masuda et al., 2013).
IL-21 and IL-23 signaling
IL-21 or IL-23 as well as IL-6 enhanced Stat3 activation in Th17 cells via IL-21R or IL-23R (Muranski et al., 2013). IL-21 has been shown to have pleiotropic effects on T cells and B cells (Leonard et al., 2005). IL-21 independent of IL-6 was able to drive naïve T cells into Th17 cells in the presence of TGF-β1 (Chen et al., 2007). Nonetheless, IL-6 activates IL-21 expression in naïve CD4+ T cells via Stat3 activation and IL-21 production is amplified under Th17 cell-inducing condition through an autocrine-loop (Zhou et al., 2007; Nurieva et al., 2007).
IL-23 is a heterodimeric cytokine composed of p19 and p40 subunits. IL-23 binds to IL-23R composed of IL-12Rβ1 and IL23R subunits (Parham et al., 2002), and mainly activates Stat3 through the Jak-stat pathway (Cesare et al., 2009). It was initially reported that IL-23 contributed to the proliferation of effector CD4+ T cells (Oppmann et al., 2000).
Subsequently, Parham et al. found that naïve CD4+ T cells did not respond to IL-23, and express little or no IL-23R. Rather, IL-23R was induced in the process of Th17 polarization, and Th17 cell differentiation was promoted by IL-23 (Betteli et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006). Of note, IL-23 is one of the most important cytokines, which convert the Th17 cells differentiated from naïve CD4+ T cells exposed to IL-6 and TGF-β1 into the "pathogenic" Th17 (Figure 1). It has been confirmed that Th17 cells induced by IL-6 and TGF-β in the absence of IL-23 are not sufficient to induce EAE (McGeachy et al., 2007), in which IL-23 diminished the expression of IL-10 produced from such "non-pathogenic" Th17 cells. This result suggests that IL-10 is a key molecule, which distinguishes pathogenic Th17 from non-pathogenic types (Figure 2). Consistent with this, Lee et al. and Kleinewietfeld et al. have demonstrated that IL-10 expression in pathogenic Th17 is suppressed compared to non-pathogenic ones. Furthermore, IL-23 upregulated the Tbx-21 gene (encode T-bet) in pathogenic Th17 (Lee et al., 2012; Yang et al., 2009; Kleinewietfeld et al., 2013). However, it must be further investigated which molecules are critical for the generation and stability of pathogenic Th17 under IL-23 signaling or other pathways (Figure 1).
Figure 1. Pathogenic Th17 cells were induced by different patterns of cytokines or a chemical, and displayed a unique character in the expression of possible master regulators and chemokine receptors, respectively. Naïve CD4+ T cells differentiate into Th17 cells, which mainly express Rorγt, in the presence of IL-6 and TGF-β1. The Th17 cells primarily produce IL-17, and secret IL-10. IL-23 drives the expansion and proliferation of Th17 cells, and activated Th17 cells secret IL-17, IL-22, and GM-CSF as well as TGF-β3, whereas the production of IL-10 is inhibited in such a pathogenic Th17 cells. A recent study has shown that sodium chloride is a strong enhancer of Th17 cells. The high salt-induced Th17 cells display highly pathogenic, and produce GM-CSF, TNF-α, and IL-2 as well as IL-9. Moreover, it has been reported that there is a direct pathway for the generation of pathogenic Th17 cells in the presence of IL-6 and TGF-β3. In the Th17 cells, the expression of GM-CSF, IL-23R, and Tbx21 is highly upregulated, while IL-10 expression is critically attenuated. These pathogenic Th17 cells commonly express Rorγt, T-bet, and IL-23 R. The Th17 cells under high-salt conditions express serum glucocorticoid kinase 1 (SGK1) downstream of IL-23R signaling, which critically contributes to the induction of pathogenic Th17 cells. Activated Th17 cells also express some chemokine receptors, including CCR4, CCR6 and CCR10. However, it still remains to be elucidated what kinds of key molecules could become master regulators in such a pathogenic Th17 cells.
Transforming growth factor β (TGF-β) has complex roles in cell growth and development (O'Kane and Ferguson, 1997). TGF-β as well as IL-10 negatively regulates immune responses including autoimmune disease and inflammation (Letterio and Roberts, 1998; Moore et al., 2001). It is currently understood that there are at least three mammalian TGF-β isoforms (TGF-β1, 2, and 3). TGF-β is an essential factor for the generation of CD4+ CD25+ regulatory T cells (iTreg), which express the forkhead/winged helix transcription factor Foxp3 (Chen et al., 2003; Batteli et al., 2006; Hori et al., 2003). Notably, IL-2 is required for the generation of iTreg (Zheng SG et al., 2007), whereas IL-2 signaling via Stat5 inhibits the differentiation of Th17 cells in vivo and in vitro by ameliorating IL-6 signaling pathway (Liao et al., 2011; Chen et al., 2011; Laurence et al., 2007).
The TGF-β superfamily activates not only Smad-dependent but also Smad-independent pathways (Derynck and Zhang, 2003). TGFβ1 and TGFβ3 bind type II receptors on T cells, which leads to the activation of Smad1, 2, 3 and 5, although Smad2 and Smad3 are activated in a different way from the activation of Smad1 and Smad5 (Derynck and Zhang, 2003). As mentioned above, TGFβ1 induced the differentiation of Th17 cells in the presence of IL-6, although the Th17 cells did not show the pathogenicity in autoimmunity possibly due to the elevation of IL-10 production (McGeachy et al., 2007). Of note, c-Maf, which is induced by TGF-β signaling (Rutz et al., 2011), contributes to the generation of "non-pathogenic" Th17 cells, in which the expressions of Rora, Runx1, IL-1R1, Ccl6 and TNF-α are suppressed, whereas the expressions of IL-10, IL-9, Lif and CTLA-4 are enhanced (Ciofani et al., 2012; Xu et al., 2009), although it initially has been reported that c-Maf is required for the differentiation of Th17 cells (Bauquet, 2009, Nat.Immunol.). Such recent data may shed light on the question of how Th17 cells switches from pathogenic into non-pathogenic and vice versa in infection and autoimmunity (Figure 1), because non-pathogenic Th17 cells produce more IL-10 than pathogenic Th17 cells (McGeachy et al., 2007; Lee et al., 2012).
In contrast, Lee et al. have recently shown that TGF-β3 directly drives naïve CD4+ T cells into "pathogenic" Th17 cell in the presence of IL-6 (Lee et al., 2012). In this report, TGF-β3-induced Th17 cells highly expressed T-bet and IL-23R, and the expression of IL-10 was suppressed compared with those of TGF-β1-induced Th17 cells (Figure 1). TGF-β activated Smad1 and Smad5 rather than Smad2 and Smad3. These results suggest that Smad1 and Smad5 pathway might be important for the generation of pathogenic Th17 but not non-pathogenic cells. Further investigation of the molecular mechanism of TGF-β3 signaling is able to lead to immunotherapy specifically targeting pathogenic Th17 cells.
As mentioned above, IL-2 is a potent suppressor of Th17 cell generation. TGF-β1 inhibited IL-2 mediated Stat5 signaling (Bright et al., 1997), and also attenuated the expression of T-bet and GATA3, which resulted in elimination of Th1 or Th2 differentiation (Carsten et al., 2007). It has been also reported that TGF-β1 produced from CD4+ effector T cells, including Th17 cells, is essential for the differentiation and stabilization of Th17 cells (Gutcher et al., 2011). Thus, it seems that TGF-β1 is essential for initial Th17 polarization in vitro and in vivo. In contrast, it has been reported that TGF-β signaling is not necessary for the generation of pathogenic Th17 cells. Rather, TGF-β1 inhibited the generation of pathogenic Th17 cells (Ghoreschi et al., 2010).
Aryl hydrocarbon receptor (Ahr) signaling and other signaling pathways
Aryl hydrocarbon receptor (Ahr) is a key factor for the development of Th17 and Th22 cells. Ahr, which normally resides in the cytoplasm, is a ligand-activated transcriptional factor. On recognizing products of tryptophan metabolism as natural ligands or toxic dioxins such as TCDD, Ahr functions as a transcriptional factor. Our group and two other groups found that Ahr is induced under Th17 cell polarizing condition, and involved in autoimmunity including EAE and CIA (Kimura et al., 2008; Veldhoen et al., 2008; Quintanna et al., 2008; Nakahama et al., 2011). Recently, our group also has shown the role of miR132/212 cluster induced by Ahr in the differentiation of Th17 cells (Nakahama et al., 2013). Although it still remains to be understood how Ahr drives Th17 cell polarization in vivo, we have recently detailed role of Ahr in immune responses, including the possible mechanism of Ahr for Th17 cell differentiation (Nguyen et al., 2013). This was a unique demonstration linking environment and autoimmunity. Recently, as an environmental factor involved in autoimmunity, it has been reported that high salt diet might lead to autoimmunity, in which sodium chloride accelerates EAE through induction of Th17 cells (Kleinewietfeld et al., 2013).
Retinoic acid (RA), a vitamin A metabolite, has been shown to mediate reciprocal Th17 and Treg cell differentiation (Mucida et al., 2007). RA with TGF-β contributed to stabilization of Treg cells, and negatively regulates Th17 cell differentiation (Takahashi et al., 2012). In contrast, RA promoted effector T cells via retinoic acid receptor α (Rora) in vivo. Thus, RA controls a dichotomy between Treg and Th17 cells in a concentration-dependent manner.
The mammalian target of rapamycin (mTOR) signaling is involved in the maintenance and proliferation of Th17 cells (Chi et al., 2012). mTOR signaling is activated by TCR and/or IL-1β stimulation via Myd88 (Powell et al., 2012; Chang et al., 2013), in which IRF4 expression was regulated (Yao et al., 2013). As mentioned above, the complex of BATF and IRF4 is essential for the generation of Th17 cells. BATF is rapidly induced by TCR stimulation, and then BATF and IRF4 plays a synergistic role in setting the initial transcriptional program, including chromatin remodeling and cooperation in accessibility of the transcriptional factors to target genes such as the IL-17 gene (Ciofani et al., 2012).
IL-27 (a member of the IL-12 family of cytokines) signaling contributes to inhibition of Th17 differentiation (Pot et al., 2011). IL-27 binds to a receptor complex composed of WSX1 (IL-27R) and gp130, and in turn activates both Stat1 and Stat3. IL-27 also induces IL-10 expression in CD4+ T cells, whereas IL-27 inhibits IL-17, IL-22, and GM-CSF expression through suppression of Rorγt activation. Thus, IL-27 is a potent negative regulator of Th17 cell polarization.
Taken together, a large number of transcriptional factors control Th17 cell differentiation, in which a balance between the activation of Stat3 and other Stat families (Stat1, 4, 5, and 6) may be an important factor for driving Th17 cell differentiation. Moreover, it is for future studies to decipher the molecular circuits and regulatory networks in the differentiation of CD4+ T cells and the plasticity between different T cell lineages, especially Th17 and Treg cells. A recent new strategy for identifying regulatory networks controlling Th17 cell differentiation and the plasticity have shown the existence of novel factors including the Mina, Fas, Pou2af1, Tsc22d3, and Fosl2 genes and dynamism among key transcriptional regulators (Ciofani et al., 2012; Yosef et al., 2013).
3- Role of Th17 cells in inflammation
It has been established that Th17 cells are critically involved in the pathogenesis of autoimmunity, gut inflammation, tissue inflammation and cancer, whereas Th17 cells contributes to protection against a variety of bacteria and fungi. Emerging data on Th17-mediated diseases suggest that different types of IL-17-producing T cells exist in vivo, which might be divided into pathogenic or non-pathogenic Th17 cells.
Th17 cells produce IL-17 (IL-17A), IL-17F, IL-17A/F heterodimers, IL-21, IL-22, and GM-CSF as well as other chemokines such as CXC cytokines (Korn et al., 2009). In an early study, IL-17A and IL-22 secreted from activated T cells were recognized as pro-inflammatory cytokines (Dumoutier et al., 2000a; Yao et al., 1995, Ouyang et al., 2008). Infante-Duarte et al. suggested that effector CD4+ T cells primed by B. burgdorferi highly produced IL-17 in inflammatory lesions, and such cell-types were distinct from Th1 and Th2 cell types (Infante-Duarte et al., 2000). Following this initial demonstration, Ye at al. reported that IL-17 secreted from CD4+ T cells protected against Klebsiella pneumonia for host defense.
Figure 2. The plasticity between Th17 cells and Treg, and various types of Th17 cells expanded by IL-23. TGF-β1 is required for the initial Th17 differentiation in the presence of IL-6. The Th17 cells produce IL-17 and IL-10. TGF-β1 also induces iTreg. In general, the plasticity of Th17 and Treg cells is tightly regulated in normal condition. However, once an antigen induces inflammation, macrophages and dendritic cells produce IL-23. IL-23 can convert non-pathogenic Th17 cells into pathogenic ones. In contrast, IL-23 can inhibit the generation of Treg cells. IL-23-induced Th17 cells convert different cytokine- producing Th17 cells. IL-6 synergized with IL-1 and IL-23 emerges IL-22 and IFN-γ- producing Th17 cells. IL-6 alone is enough to induce IL-22-producing T cells. IL-23 alone also promoted IFN-γ-producing T cells.
Role of IL-22 in Th17 cells
IL-22 as well as IL-17 plays a critical role in host defense and protecting against tissue damage. IL-22 signaling is transmitted through a heterodimeric receptor complex composed of IL-10R2 and IL-22R1 (Kotenko et al., 2001; Xie et al., 2000). IL-22R is mainly expressed in epithelial tissues, including keratinocytes, hepatocytes, and intestinal and respiratory epithelial cells, but not in immune cells (Aggawal et al., 2001; Ouyang et al., 2008; Rutz et al., 2013). The biological functions of IL-22 are known to be protective against infection and inflammation because IL-22 contributes to tissue maintenance, repair, and wound healing through the expression of anti-microbial, antiapototic proteins and proteins involved in cell proliferation via IL-22R in intestinal epithelial cells and goblet cells (Rutz et al., 2013).
IL-22 plays a protective role against bacterial infections. Neutralizing IL-22 secreted by the Th17 lineage led to the failure to clear pathogen from infected lung by K. pneumonia, and in turn the early death of infected animals (Aujla et al., 2007). In this report, IL-17A produced from Th17 cells synergized with IL-22 to protect against bacteria. The coexpression of both IL-17 and IL-22 in Th17 cells are important for expression of antimicrobial peptides, as mentioned above.
The expression pattern of IL-17 and IL-22 in CD4+ T cells, however, varies according to a cytokine milieu. IL-6 and/or IL-23 induced IL-22 expression in vitro in the absence of TGF-β (Qu et al., 2013). In contrast, TGF-β inhibited the expression of IL-22 (Zheng Y et al., 2007), whereas TGF-β is essential for the differentiation of Th17 in the presence of IL-6 (Figure 2). Moreover, IL-6 deficiency did not affect the frequency of IL-22 cell population in vivo (Zenewicz et al., 2008). These results suggest that IL-22 is produced by not only Th17 cells but also other immune cells. Therefore, not all Th17 cells might play a protective role for host defense through IL-22 production.
Likewise, although IL-22 played a protective role in infection by various kinds of bacteria, including C. rodentium, M. tuberculosis, and Salmonera typhimurium, the sources of IL-22 was assumed to be not from Th17 cells (Zheng et al., 2008; Schulz et al., 2008; Dhiman et al., 2009). Moreover, NK cells as well as CD4+ T cells are involved in protection against IBD by IL-22, whereas IL-17A contributed to exacerbation of IBD (Zenewicz et al., 2008). In contrast, IL-22 produced from effector Th17 cells contributed to promotion of CD-like experimental colitis (Yen et al., 2006; Strober et al., 2007). In line with these reports, Ahern et al. has demonstrated that IL-23 promotes intestinal inflammation through directly enhancing the development of effector Th17 cells, which secret IL-17, IFN-γ and possibly IL-22 (Figure 2).
Moreover, IL-22-expressing Th17 cells transferred into IL-22 deficient host mice provided protection against hepatitis, whereas IL-17 had no apparent role in liver inflammation (Zenewicz et al., 2007). However, Xu et al. and Nagata et al. have shown that IL-17 is required for the development of hepatitis. Although these reports appear to be contradictory, CD4+ T cells, which dominantly produce IL-22 rather than IL-17, might be protective against hepatitis, whereas IL-23-induced Th17 cells might have the pathogenicity in liver inflammation. In agreement with this, Zheng Y et al. has reported that the production of IL-17 and IL-22 from Th17 cells is differentially controlled, and Th17 cells enhanced by IL-23, which produce IL-22, are essential for dermal inflammation and acanthosis.
The IL-23/Th17 axis is clearly involved in various autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, psoriasis, and IBD (Iwakura and Ishigame, 2006). IL-23 plays an important role in the maintenance and expansion of Th17 cells, and makes Th17 cells "pathogenic" (Figure1). As mentioned above, although different types of effector Th17 cells reside in inflammatory lesions (Figure 2), a growing number of reports have shown that IL-17 and IL-23 critically contribute to the pathogenesis of RA, in which IL-17A and IL-17F play an important role in joint inflammation and bone erosion. Arthritis associated with psoriasis (psoriatic arthritis) is also dependent on Th17 cells activated by IL-23 (Maeda et al., 2012). The elevation of IL-23 and IL-17 were detected in synovial fluid, synovial tissues and sera of RA patients but not of osteoarthritis (Alfadhli, 2013). IL-23 is also pivotal for the onset of EAE (Cua et al., 2003). IL-23 or IL-23R deficient mice were resistant to EAE (Awasthi et al., 2009). IL-17A and IL-17F double knockout mice showed critical reduction of the development of EAE, whereas the effect of IL-17F on EAE development is less than that of IL-17A in mice (Ishigame et al., 2007). More recently, Kang et al. has reported that IL-17 is involved in perturbation of the maturation of oligodendrocyte lineage cells, which might lead to inflammation and neurodegeneration in MS.
Pathogenic Th17 cells produce GM-CSF (Figure 1), which is an important cytokine for induction of IL-23 in dendritic cells (McGeachy et al., 2011). It has been reported that neutralizing GM-CSF at the effector stage results in suppression of the further development of EAE (El-Behi et al., 2011). Thus, GM-CSF is also required for the maintenance and expansion of Th17 cells possibly through the enhancement of IL-23.
Th17 cells in host defense
Th17 cells were increased at the mucosal sites after infection (Happel et al., 2005; Mangan et al., 2006). So far, it has been demonstrated that Th17 cells are involved in protection against a variety of bacteria such as Candida albicans, Staphylococcus aureus, Citrobacter rodentium, Salmonella and Bordetella pertusis (Peck and Mellins, 2010). However, the role of IL-17 and Th17 cells in protection against Asperillus fumigatus and other fungi are diverse and controversial (Muranski et al., 2013). One of the reasons is that the inflammatory milieu for the generation of Th17 cells is different in infection by different types of fungi, in which macrophages and dendritic cells recognize different kinds of fungal pattern recognition receptors (PRRs) such as Dectin-1, Dectin-2, Mincle and MR, and produce inflammatory cytokines for Th17 polarization, including IL-6, IL-1β, TNF-α and IL-23 as well as GM-CSF (Wüthrich et al., 2012). Zielinski et al. has reported that by eliciting different cytokines respectively, C. albicans and S. aureus prime different types of IL-17-producing T cells "Th17" cells that produce IFN-γ or IL-10 respectively, which might be significant for answering the question of what makes pathogenic or non-pathogenic Th17 cells.
Th17 cells in cancer
The role of Th17 cells in cancer displays complexity in various types of tumor immunity. Although it seems that the pathogenic role of IL-23-induced Th17 cells has been consistently documented in autoimmunity, Th17 cells in cancer display both anti-tumorigenic and pro-tumorigenic functions (Zou and Restifo, 2010). Administration of IL-23 and IL-23-producing dendritic cells, has been reported to inhibit tumor growth (Kaiga et al., 2007; Hu et al., 2006), whereas IL-23 has been shown to promote tumor incidence and growth (Langowski et al., 2006). The role of IL-17 in tumor immunity is also controversial, although the source of IL-17 is not only Th17 cells, but also other types of T cells, including CD8+ T cells (known as Tc17 cells) and Rorγt+ Foxp3+ T cells (Li and Boussiotis, 2013). IL-17 enhanced tumor growth through the promotion of tumor vasculization, especially in some immune-deficient mice (Zou and Restio, 2010). In contrast, in immunocompetent mice, IL-17 played a protective role against tumor growth (Benchetrit et al., 2002; Kryczek et al., 2009; Hirahata et al., 2001).
Although Th17 cells are present in tumor microenvironment, the number of Th17 cells represents a minor population of effector T cells (Kryczek et al., 2007), suggesting that the frequency of Th17 cell population is tightly regulated in tumor immunity. The number of Treg and Th17 cells inversely correlates in the same tumor (Zou and Restifo, 2010). It has also been reported that, in the microenvironment of ulcerative coloitis (UC) and associated colon cancer, not only Th17 cells but also IL-17+Foxp3+ T cells are detected (Kryczek et al., 2011). Thus, the character of IL-17-producing CD4+ T cells might be classified more precisely in the context of tumor immunity.
Balance between Th17 cells and Treg in autoimmunity
It seems that there is no doubt that Treg cells play a critical suppressive role in immune responses in vitro and in vivo (Shevach et al., 2009; Vignali et al., 2008). Treg cells are a potent inducer of IL-10 and TGF-β1, resulting in suppression of effector T cell functions. However, the balance between Th17 and Treg cells in vivo is dependent on the context of inflammatory disease. The existence of Treg cells does not always suppress the function of Th17 cells. Treg promoted Th17 cell development through IL-2 regulation rather than control of TGFβ1 (Pandiyan et al., 2011; Chen et al., 2011). IL-2 is a critical cytokine in deciding a dichotomy between Th17 and Treg cells. Treg cells induced by IL-2 combined with TGF-β or all-trans retinoic acid, were resistant to Th17 cells (Muchida et al., 2007; Zheng et al., 2008; Zhou et al., 2010). Conversely, IL-6 is a strong inducer of Th17 cells differentiation. Zheng has indicated that IL-6 can convert nTregs into Th17 cells, and other CD4+ T cells (Zheng, 2013). It is notable that IL-6 plays a critical role in conversion of Foxp3+ CD4+ T cells into pathogenic Th17 cells in autoimmune arthritis (Komatsu et al., 2014). Th17 cell expansion is also regulated by TGFβ1 secreted from Th17 cells itself but not Treg cells (Gutcher et al., 2011). Rather, it seems that high concentration of TGF-β reduces Th17 cell populations (Zhou et al., 2008).
The concentration of TGF-β1 is one of the important factors, which drives or inhibits the differentiation of Th17 cells (Zhou et al., 2008). High levels of TGF-β1 inhibited Th17 cell differentiation and enhanced the development of iTreg through high expression of Foxp3. In such a cytokine milieu, Foxp3 induced by TGF-β interacted with Rorγt, and in turn antagonized the function of Rorγt in CD4+ T cells, in which the expression of IL-23R, IL-22 and IL-17 was repressed (Zhou and Littmann, 2009). In contrast, at low concentrations, TGF-β1 was helpful for the generation of Th17 cells in synergy with IL-6 or IL-21 (Zhou et al., 2008), although it is still not clear how IL-6 or IL-21 overcomes the inhibitory effect of Foxp3 on Rorγt function. Runx-1 controls the differentiation of Th17 cells thorough binding both Foxp3 and Rorγt (Zhang et al., 2008). The interaction of Runx-1 with Rorγt promoted the transcription of the IL-17 gene, whereas Foxp3 inhibited Rorγt- and Runx-1-induced IL-17 expression by binding to Runx-1 (Zhang et al., 2008), suggesting that the role of Runx-1 is also dependent on the concentration of TGF-β1.
4- Therapy (treatment of Th17-dependent autoimmunity)
A great number of recent studies have revealed that the biology of Th17 cells in mice is broadly common with phenomena in humans (Tesmer et al., 2008; Jong et al., 2010). Th17 cells in humans play an important role in the pathogenesis of rheumatoid arthritis, psoriasis, asthma, inflammatory bowel disease (IBD) and transplantation rejection. Accordingly, blocking pro-inflammatory cytokines, including IL-6, IL-1β, IL-23, and IL-17 as well as GM-CSF, will lead to the abatement of tissue inflammation, gut inflammation and autoimmunity.
Several anti-IL-17A monoclonal antibodies, including secukinumab and ixekizumab, and anti-IL-17A receptor monoclonal antibody, brodalumab have been treated to patients in the process of phase II clinical trials (Kellner et al., 2013). Secukinumab is most likely to be clinically efficacious in RA. An anti-IL-1β monoclonal antibody, gevokizumab is currently being investigated in a Phase â
¡ clinical program. An anti-IL-12/IL-23 monoclonal antibody, usutekinumab has been demonstrated the high efficacy in the treatment of patients with psoriasis (Krueger et al., 2007). An anti-GM-CSF monoclonal antibody, mavrilimumab for treatment of rheumatoid arthritis has shown promising results under phase II clinical trial (Burmester et al., 2013). In the future, emerging data will establish the efficacy of these monoclonal antibodies against several autoimmune diseases such as rheumatoid arthritis.
IL-6 is involved in the initial differentiation of Th17 cells (Betteli et al., 2006). In mice, blockade of IL-6 pathway has been shown to result in a decrease of the frequency of Th17 cell population (Nowell et al., 2009; Serada et al., 2009). A humanized anti-IL-6 receptor antibody (Tocilizumab, TCZ) displayed a remarkable protective effect in patients with rheumatoid arthritis as well as Castleman's disease and juvenile idiopathic arthritis (Genovese et al., 2008; Tanaka et al., 2012). Although the biological effect of TCZ on human autoimmune disease is complex (Tanaka et al., 2013), Samon et al. have demonstrated TCZ affects the IL-6/Th17 axis in patients with rheumatoid arthritis, in which the ratio of Th17 cells to Treg cells was significantly reduced. Therefore, administration of IL-6 blockade (TCZ) might be highly efficacious against not only such diseases as mentioned above, but also Th17 cell-dependent diseases, including IBD and psoriasis, and cancer.
On the contrary, although elevation of IL-6 level in inflammatory lesions leads to the exacerbation of autoimmunity, it remains to be understood why IL-6 is overproduced in autoimmune disease such as rheumatoid arthritis. A recent report has shown that posttranscriptional regulation of IL-6 production is essential for immune homeostasis. Zc3h12a (known as Regnase-1, MCPIP) constitutively degrades level of IL-6 mRNA trough binding to its 3'UTR, whereas Zc3h12a deficiency led to spontaneous autoimmunity (Matsushita et al., 2009). Moreover, our recent study has shown that Arid5a controls IL-6 level in vivo through stabilization of IL-6 mRNA. Arid5a deficient mice are resistant to EAE, in which the frequency of Th17 cell population is dramatically reduced (Masuda et al, 2013, PNAS). Notably, Arid5a counteracted the destabilization effect of Zc3h12a (Masuda et al., 2013). Consequently, imbalance between Arid5a and Zc3h12a in vivo might be involved in autoimmunity. Given that administration of TCZ is efficacious against several autoimmune diseases, the monoclonal antibody targeting Arid5a might be also clinically useful.
Since IL-17-producing CD4+ T cells have been shown to be involved in various types of inflammatory diseases, our understanding of the pathogenesis of such diseases as rheumatoid arthritis, multiple sclerosis, and psoriasis, has been dramatically improved since the original concept of a Th1/Th2 paradigm. However, emerging data on Th17 cells have revealed that IL-17-producing CD4+ T cells are not simple cell population. Th17 cells are classified into pathogenic or non-pathogenic ones. IL-23 is a critical factor, which enhances the pathogenecity of Th17 cells. Recent data suggests that TGF-β3 and sodium chloride as well as IL-23 are involved in the generation of pathogenic Th17 cells. In contrast, TGFβ1 secreted from Treg cells inhibits the conversion into pathogenic Th17 cells by attenuating IL-23 production from activated macrophages and dendritic cells, whereas TGF-β1 is essential for the initial induction of Th17 cells. Moreover, the concentration of TGFβ1 is essential for the plasticity of Th17 and Treg cells. Taken together, Th17 cells play a pathogenic or protective role in infection and inflammatory disease in an antigen-dependent manner. Consequently, to identify which molecules and signaling pathways are critical for the generation of pathogenic Th17 cells, will lead to the development of more efficacious therapeutic drugs for Th17 cell-dependent inflammatory disease.