Factors regulating Th17 cells: a review

This article aims to provide a broad coverage of over 300 studies on T helper 17 (Th17) cells published mainly between 2011 and 2016, with a focus on factors negatively regulating Th17 cell differentiation and functions. During the last decade, processes underlying Th17 cell differentiation and activation, as well as Th17specific cytokines, chemokines, and transcription factors, have been characterized. Diverse modalities controlling Th17 cells range from factors modulating the state of regulatory T (Treg) cells or dendritic cells and indirectly regulating Th17 cells to cell-intrinsic factors, such as those that repress genes encoding Th17 signature cytokines, including artificial products. Since IL-17 is a major player in tissue-specific immune pathology, Th17 cells, a major source of the cytokine, have been a subject of intensive research and have been at the forefront of clinical studies. New approaches, including conditional knockout mice as well as transcriptome profiling, have revealed closely related developmental states in Th17 cells, reflecting their plasticity. For example, given that Th17 cells share a differentiation pathway with Treg cells that, in turn, control Th17 cells, the Treg/Th17 axis is important for fine-tuning the intensity of inflammatory responses. An emerging picture shows that a combination of many factors involving IL-23, IL-2, CCR6, the mammalian target of rapamycin (mTOR)-hypoxia-inducible factor (HIF) axis, metabolism (glycolysis and lipid synthesis), retinoic acid, glucocorticoids, melatonin, Wnt pathways, and salt act in synergy to regulate the Th17/Treg balance and inter-Th17 subset balance. Therapeutic interventions that can tune such balances would be efficacious when accompanied by our attentiveness to the spatial and temporal dynamics of Th17 cells. A comprehensive understanding of biochemical and cellular factors underlining these subtle regulations would give us a more integrated view that would hopefully help increase therapeutic options for many cases of autoimmune and inflammatory diseases and predisposed individuals. Correspondence to: Reiko Seki, Department of Clinical Laboratory Science, Teikyo University School of Medical Technology, 2 Kaga, Itabashi, Tokyo, 1738605 Japan, Tel: +81-3-3964-1211 ext 44558, E-mail: [email protected] Received: October 08, 2016; Accepted: November 15, 2016; Published: November 17, 2016 Introduction an overview of immunotherapy, IL-17, and Th17 biology An understanding of the cytokines responsible for autoimmune diseases has changed the concept of their treatment. Prior to the identification of IL-17, several cytokines crucial for autoimmune diseases had been identified. The treatment of autoimmune diseases, including rheumatic arthritis (RA), has been revolutionized by the advent of targeted biological agents as well as improved use of conventional drugs. Inhibitors of TNF-α have shown benefits in many patients with RA and psoriasis [1-3]. This success was followed by the development of drugs targeting IL-6 and IL-1 [2,4,5]. After the discovery and characterization of IL-17A demonstrating that it induces IL-6 secretion from synoviocytes in patients with RA [6], blockade of IL-17A has been assessed in RA, psoriasis, and other related diseases and has shown some successful results [7]. (Henceforth “IL-17” indicates IL-17A, the founding member of the IL-17 family, unless otherwise noted.) Pathological roles of IL-17 in autoimmune diseases, as well as clinical trials targeting IL-17 or IL-23, have been reviewed by Beringer et al. [7], Waisman et al. [8], and Kim et al. [3]. Roles for IL-17 in central nervous system (CNS) diseases, including multiple sclerosis (MS) and infarction [8], and in cardiovascular diseases [9] have also been discussed. To avoid redundancy, we focus on T helper 17 (Th17) biology and mechanisms that inhibit Th17 cell development and activity, with emphasis on their therapeutic relevance. Notably, Th17 is not the only cell capable of producing IL-17; other types of cells, such as γδ-T cells and innate lymphoid cells (ILCs), are likely to be the main source of IL-17 in some cases [10,11]. Upon activation by an antigen, naive CD4+ T cells proliferate and differentiate into various subsets of T helper (Th) cells, including Th1, Th2, and Th17 cells. Th17 cells develop mostly from naive T cells, produce proinflammatory cytokines IL-17A, IL-17F, and IL-22, and coordinate inflammatory responses for host defense [8,12]. Th17 cells have been shown to be important for mucosal host defense against microbial and fungal pathogens [13], but, on the other hand, are present at tissue inflammation sites and contribute to the pathogenesis of human autoimmune and chronic inflammatory disorders [12,1417]. In mice, Th17 cells express the transcription factor retinoic acidrelated orphan receptor γt (RORγt, corresponding to human RORc) as a master transcriptional regulator [18] along with the chemokine receptors CCR6 [19] and CCR2 [20]. Both RORγt and RORα, a closely related family member, are necessary for full Th17 cell development [18]. Multiple cytokines, including TGF-β, IL-6, IL-1β and IL-21 are known to induce differentiation of naive T cells to Th17 cells [12]. In particular, this differentiation can be initiated by a combination of TGF-β and IL-6 in mice [12], and is maintained by IL-23 [21,22]. It is now known that Th17 cells consist of subsets with differential inflammatory potential, ranging from a subset that is induced by TGF-β and IL-6, produces IL-10, and is a weak inducer of inflammation, to a highly inflammatory subset that produces GM-CSF/IFN-γ and is induced by IL-23 [23]. IL23R is required for effector Th17 cell responses in vivo [24], and IL-23 appears to be a promising therapeutic target [25]. The importance of pleiotropic cytokine TGF-β for Th17 (as well as inducible regulatory T [Treg] cell) development was established by early findings [26]. As the differentiation state of dendritic cells (DCs) has profound effects on Th17 differentiation, the extrinsic effect of TGF-β mediated by TGF-βsignaling in DCs is also important [27]. Seki R (2016) Factors regulating Th17 cells: a review Volume 1(4): 126-147 Biomed Res Clin Prac, 2016 doi: 10.15761/BRCP.1000122 We do not discuss IL-22, a Th17 signature cytokine, in detail, but instead suggest recent articles [28,29]. Both IL-17 and IL-22 play a central role in the pathogenesis of MS [30,31] and RA [32]. A recently proposed subset, Th22, shows similarity with Th17 [33], yet, one feature of Th22 cells is their dependency on aryl hydrocarbon receptors (AHR), rather than RORγt. Th22 cells play a pathological role in psoriasis, but their role in RA is less clear [34]. Cytokines and other factors present during T cell priming events can direct differentiation by inducing lineage-specifying transcription factors that act as master regulators. T-bet, signal transducer and activator of transcription (STAT)1, and STAT4 are the master regulators for Th1 cells; GATA3 and STAT6 direct the Th2 lineages, and STAT3 and RORγt direct differentiation of Th17 cells [18,35]. The induction of RORγt is dependent on STAT3, which is mainly activated by IL-6. PI3K/AKT signaling acts upstream to positively regulate the activation of protein kinase mammalian target of rapamycin (mTOR) C1, and this axis is a positive regulator of Th17 development [36,37]. mTORC1 positively modulates IL-17 expression through several pathways involving STAT3 and hypoxia-inducible factor 1α (HIF1α) [36,38]. This article focuses on recent reports of negative regulators that inhibit Th17 cell development or suppress their functions, with a limited coverage of the basics revealed in earlier studies. Nonetheless, given the therapeutic relevance of the overall tone of inflammation, and the indirect effect of non-T cells such as DCs, the macroscopic mechanisms governing the balance of various subsets of T cells are also important. Therefore, we begin with Th17/Treg balance, to which we aim to endow an introductory purpose. After the factors modulating Th17 development in an extrinsic manner are discussed, the regulatory factors intrinsic to T cells (i.e., without the aid of other cell types) are considered. Cytokines, hormones, and vitamins that negatively regulate Th17 cells include retinoic acid [39,40], IFNβ, IL-10 [41,42], IL-27, Th1 and Th2 cytokines, IFN-γ and IL-4 [43,44]. Many of these factors act, at least in part, in an extrinsic manner, i.e., mediated by DCs and other cells. On the other hand, Th17 cell-intrinsic negative regulators include Foxp3 [45], interferon regulatory factor 4 (IRF4)-binding protein (also known as Def6 or SLAT) [46], peroxisome proliferator-activated receptor γ PPAR-γ [47], liver X receptors (LXRs) [48], and STAT5 [49], which we discuss in some depth. Other negative factors include NR2F6 (Ear-2) [50], growth factor independence 1 (Gfi-1) [51], suppressor of cytokine signaling 3 (SOCS3) [52], TNF receptor–associated factor 6 (TRAF6) [53], protein kinase B (PKB)/Akt signals [54], and E26 transformation-specific sequence 1 (Ets-1) [55,56], but we only briefly mention them in related sections, as these may be integrated into some axes. For example, Ets-1 is involved in the Ets-1-IL-2 axis [56]. IL-23 signaling as a target IL-23 consists of the p40 subunit of IL-12 and an unrelated p19 peptide. There is a consensus that, in the presence of TGF-β IL-6 triggers differentiation of Th17 cells in mice. IL-23 is essential to establish and stabilize the differentiated states of Th17 cells. In support of this view, IL-23 is important in vaccination models [57]. In Khader et al.’s study, despite no involvement of IL-23 in primary resistance to Mycobacterium tuberculosis, vaccination with a defined peptide from M. tuberculosis established persistent IL-17-producing T cells in a manner dependent on IL-23 [57]. Thus-established IL-17-producing T cells, which appeared to accumulate in the lung, allowed accelerated recall response and protection against infection [58]. Setting aside evidence for IL-23 involvement, several studies have elucidated roles of memory Th17 cells in protection from several microbes. Wüthrich et al. showed the importance of Th17 cells in recall responses against several fungi, specifically, Coccidioides posadasii, Histoplasma capsulatam, and Blastomyces dermatitidis [59]. Chen et al. [60] showed the importance of memory Th17 cells established by vaccination for Klebsiella pneumoniae. In that study, Th17, but not IFN-γ was required for broader (i.e., serotype-independent) protection against K. pneumoniae. Using a baboon model of Bordetella pertussis infection, Warfel and Merkel showed the presence of IL-17-producing memory T cells and IFN-γ-producing memory T cells >2 years after infection [61]. Using an experimental autoimmune encephalomyelitis (EAE) model, Haines et al. [25] showed that memory cells were generated from IL-17+RORγt+ precursors, not noncommitted precursors. Compared to the cells on day 8 (after primary immunization), the cells on day 18 showed better Th17 phenotype stability in an IL-23-dependent manner, implying that the time length of primary immunization is critical for stability of the differentiation state of Th17 cells. Short immunization times allowed differentiation into IFN-γ-producing cells in their setting [25]. IL-23 promoted proliferation of memory Th17 cells and upregulated genes required for cell-cycle progression in Th17 cells. IL23 also induced T-bet and IFN-γ in Th17 cells. These findings implicated the therapeutic potential of blocking IL-23. Notably, tildrakizumab (MK-3222), a humanized anti-IL23p19 mAb improved psoriasis in a phase IIb randomized, placebocontrolled trial, although adverse effects, including bacterial arthritis, were reported [62]. Tregs, non-pathogenic Th17, and pathogenic Th17 After naive T cells were shown to differentiate into Th17 cells, it was noted that Treg and Th17 cells emerge from an overlapping developmental program (Figure 1) [63]. This close relationship between Treg and Th17 cells, along with the developmental plasticity of Th17 cells, led to recognition that Th17 cells play not only proinflammatory and defensive roles but also have regulatory roles. Treg cells are broadly classified into two groups: nTregs that develop in the thymus and iTregs that are induced in peripheral organs by TGF-β. Thus, both iTreg and Th17 cells can be induced from naive CD4+ T cells in the periphery upon antigen stimulation and exposure to TGF-β. The notion that Treg and Th17 have a reciprocal (mutually exclusive) relationship [39] leads us to surmise that subtle modulations of Th17-iTreg cell balance can exert immense effects on the outcome of therapeutic interventions. Physiologically, switching differentiation between Treg and Th17 cells is mainly regulated by IL-6 [53]. TGF-β signaling alone leads to Foxp3 expression and induction of Treg, but costimulation with IL-6 can suppress Foxp3 and, therefore, release RORγt from inhibition by Foxp3, promoting Th17 development. Th17 cells can be derived from Treg cells if appropriate conditions are provided. In Veldhoen et al.’s study, in the presence of DC and ligands for TLR3, 4, or 9, coculture of naive CD4+ T cells with Tregs resulted in the development of Th17 cells [64]. In another study, TGF-β likely produced by Tregs and DCs, was a key cytokine that promoted Th17 differentiation from naive CD4 T cells in the presence of dectin-1 agonists [65]. Later, Xu et al. used Foxp3-IRES-GFP knock-in mice, which ensured that only CD4+CD25+Foxp3+ cells were used, and showed that these cells undergo self-induced Th17 differentiation [66]. Treg cells not only expressed TGF-β but also induced DCs to produce increased amounts of TGF-β [66]. A differentiation pathway from the Seki R (2016) Factors regulating Th17 cells: a review Volume 1(4): 126-147 Biomed Res Clin Prac, 2016 doi: 10.15761/BRCP.1000122 Foxp3+ passing through the Foxp3+/IL-17+ double positive stage and then to the IL-17 single positive stage was also observed [66]. Thus, Treg cells can differentiate into Th17 cells in a manner dependent on TGF-β. Several studies have focused on the relevance of IL-2 in Th17iTreg balance. IL-2 is a cytokine that normally suppresses Th17 cell differentiation and function. Later, rather than TGF-β production by Treg cells, IL-2 depletion by Treg cells was proposed to be the key factor promoting early stages in Th17 development. Using an in vivo Candida albicans infection model, Pandiyan et al. found that the effect of Treg cells on the induction of IL-17 production from responding CD4+ T cells is dependent on consumption of IL-2 by Treg cells at early time points [67]. Further, using a system in which diphtheria toxin can kill Foxp3+ Tregs at a specific stage, Chen et al. showed that Treg cells promote Th17 cell development in vivo and this is mediated by consumption of IL-2. Strikingly, their analysis with TGFb knockout mice conditional to Foxp3 expression showed that Treg cell production of TGF-β was not required for Th17 induction in vivo [68]. Cejas et al. showed that TRAF6-deficient mice exhibited enhanced Th17 cell differentiation, and this was at least partly explained by the finding that TRAF6-deficient CD4+ T cells showed lower expression levels of IL-2 compared to those of wild-type CD4+ T cells [53]. Thus, it is possible that negative regulation by IL-2 is playing a central role in suppressing Th17 cell differentiation in many unknown cases. In any case, Pandiyan et al. [67] and Chen et al. [68] showed that Treg cells are likely to promote priming of Th17 in vivo, although further evaluation appears to be necessary to establish the significance of Tregs as a source of TGF-β in vivo. The role of TGF-β in Th17 development is still controversial for human T cells [69]. Effects of IL-2 are also discussed in the following section. Thus, several findings indicated or suggested derivation of Th17 cells or Foxp3+IL-17+ “double positive” cells from Foxp3+ Treg cells. Of clinical importance, fate-mapping analysis by Komatsu et al. showed that Th17 cells arise from Foxp3+ Treg cells by the loss of Foxp3 expression in the presence of synovial fibroblast-derived IL-6 in collagen-induced arthritis (CIA) model mice, suggesting that T cell plasticity, combined with the inflammatory rheumatic environment, facilitates Th17 polarization, altering the balanced Treg/Th17 ratio [70]. In another study using peripheral blood from patients with RA, Th17 cells were enriched with Helios-producing Foxp3IL2RAcells, suggestive of nTreg cells that had presumably lost suppressive capability [71]. Notably, Helios expression is indicative of the recent thymic origin of the cells, and IL-2RAis abundantly expressed in Treg cells. Thus, in patients with RA, nTreg cells appear to have anomalously high chances of transdifferentiating into IL-17-producing cells. Ueno et al. observed that the prevalence of circulating double positive (IL-17+ Foxp3+) CD4+ T cells is increased in patients with inflammatory bowel diseases (IBDs) [72]. Basu et al. further showed that IL-1 signaling represses SOCS3, a molecule that normally inhibits STAT3. This was suggested to be the molecular basis for IL-1βdependent increases in phosphorylated STAT3 and alterations of the STAT3/STAT5 balance resulting in Th17 generation, even in retinoic acid-mediated iTreg induction that is predominant in the normal intestine [63]. It is recognized that Th17 cells have functional plasticity, but can Th17 cells transdifferentiate into Tregs? By using a triple reporter mouse model that reports expression of IL-17A, IL-10, and Foxp3 genes, Gagliani et al. [73] showed that Th17 cells generated during Staphylococcus aureus infection can be converted into IL-10high Foxp3lo Tr-1-like cells. Besides being positive for lymphocyte-activation gene 3 (LAG-3) and negative for CCR6, the latter cells (referred to as Tr-1exTh17 cells) showed features of Tr-1 in transcriptome analyses. TGF-β promoted Th17 to Tr-1 conversion [73]. AHR ligand 6-formylindolo[3,2-b]carbazole (FICZ) also promoted Th17 to Tr-1 cell conversion [73]. Thus, Th17 cells can transdifferentiate into regulatory cells. Recent notable reports include those of Gaublomme et al. [74] and Wang et al. [75]. Reflecting the functional diversity of Th17, in vitro polarized Th17 cells can either cause severe autoimmune responses upon adaptive transfer (“pathogenic,” polarized with IL-1β + IL-6 + IL-23) or have little or no effect in inducing autoimmune responses (“non-pathogenic,” polarized with TGF-β + IL-6) [76,77]. RNA-seq was performed for single CD4+IL-17+cells isolated in vivo from EAE model mice. Significant cellular variation was observed, and in vivo Th17 showed cell states were progressively changed from the lymph nodes (LNs) to the central nervous system (CNS) [74]. The in vitro Th17 cells were also analyzed after activation under non-pathogenic (TGF-β + IL-6) or pathogenic (IL-1β+ IL-6 + IL-23) conditions. The profiles of these sets of cells formed a spectrum with distinctions and similarities when compared with the profile of the in vivo Th17 [74]. Interestingly, pathogenic Th17 cells expressed T-bet, GM-CSF, and IL-23R, for example, while non-pathogenic Th17 cells expressed IL-10. Exposure of non-pathogenic Th17 cells to IL-23 converted them to a pathogenic phenotype (Figure 1). In humans, Th17 cells that coproduce IL-17 and IFN-γ are generated upon infection with C. albicans, and this state of Th17 appears to be similar to that of pathogenic Th17 cells [75]. Further, in humans, Th17 cells that coproduce IL-17 with IL-10 are induced upon S. aureus infection [78], and this state is more similar to the non-pathogenic Th17 cells [75]. Wang et al. further reported CD5L/ AIM expression in non-pathogenic, but not in pathogenic, Th17 cells. CD5L behaved as a functional switch; its loss converted non-pathogenic Th17 cells into pathogenic Th17 cells. CD5L inhibits this conversion in a manner mediated by modulation of the intracellular lipidome, such as maintaining a high ratio of poly-unsaturated fatty acids (PUFA)/ saturated fatty acids (SFA) and restriction of cholesterol synthesis, and, thereby, ligand availability of RORγt. Notably, cholesterol synthesis is considered to be linked to the production of RORγt ligands, including oxysterol [79]. Thus, it is reasonable to consider that lipid metabolism plays important roles in T cell-mediated immunity, helping Th17 cells adapt to protective, as well as inflammatory, immune responses.