Transcriptional Regulators of T Helper 17 Cell Differentiation in Health and Autoimmune Diseases

T helper (Th) 17 cells are a subtype of CD4 T lymphocytes characterized by the expression of retinoic acid-receptor (RAR)-related orphan receptor (ROR)γt transcription factor, encoded by gene Rorc. These cells are implicated in the pathology of autoimmune inflammatory disorders as well as in the clearance of extracellular infections. The main function of Th17 cells is the production of cytokine called interleukin (IL)-17A. This review highlights recent advances in mechanisms regulating transcription of IL-17A. In particular, we described the lineage defining transcription factor RORγt and other factors that regulate transcription of Il17a or Rorc by interacting with RORγt or by binding their specific DNA regions, which may positively or negatively influence their expression. Moreover, we reported the eventual involvement of those factors in Th17-related diseases, such as multiple sclerosis, rheumatoid arthritis, psoriasis, and Crohn's disease, characterized by an exaggerated Th17 response. Finally, we discussed the potential new therapeutic approaches for Th17-related diseases targeting these transcription factors. The wide knowledge of transcriptional regulators of Th17 cells is crucial for the better understanding of the pathogenic role of these cells and for development of therapeutic strategies aimed at fighting Th17-related diseases.

However, a dysregulated activity of Th17 cells has been associated to autoimmune diseases, such as multiple sclerosis (MS), rheumatoid arthritis, psoriasis, and Crohn's disease (8, 16).
Given the relevance of Th17 cells in both physiological and pathological contexts, numerous studies investigated the molecular mechanisms regulating the transcriptional program of Th17 cells.
Majority of the Th17 transcription factors were discovered and validated through analysis of IL-17A expression in mice deficient for specific transcription factors, and mice containing a GFP reporter cDNA knocked-in at the site for initiation of the translation of specific transcription factors (17)(18)(19)(20)(21). Similarly, the in vitro expression of IL-17A was assessed in cells cotransfected with constructs overexpressing the specific transcription factors and reporter constructs containing regions upstream of the Il17a transcription start site (17,19). More recently, modern technologies, such as chromatin immunoprecipation (ChIP) and single-cell RNA-sequencing, were allowed to better explore the functions of transcription factors in Th17 cells (22)(23)(24). However, although the expression of Th17 transcription factors was validated in human Th17 cells, most of the studies demonstrating their regulatory mechanism were performed in murine cells.
The first transcription factor discovered, designated as the "lineage defining transcription factor of Th17 cells, " is RORγt, which is essential and sufficient to induce Th17 lineage fate in both human and mouse cells (5, 17,25).
However, succeeding studies revealed that multiple transcriptional regulators contribute to full Th17 differentiation program through several mechanisms, including binding to specific regions of Il17a and Rorc genes, or interacting and synergizing with RORγt, or facilitating the recruitment of other proteins on Il17a or Rorc promoters.
Collectively, Th17 transcriptional regulators may contribute to Th17 functions in physiological and pathological contexts. Thus, in this review, we reported recent advances on the molecular mechanisms directly regulating transcription of Il17a and Rorc. Moreover, we discussed their involvement in autoimmune disorders associated to an exaggerated Th17 response. Finally, we discussed the recent therapeutic approaches targeting Th17 transcriptional regulators in Th17-related autoimmune diseases.

RETINOIC ACID-RECEPTOR-RELATED ORPHAN RECEPTOR (ROR) TRANSCRIPTION FACTORS IN TH17 CELLS
The retinoic acid-related orphan nuclear receptors (RORs) belong to a superfamily of ligand regulated transcription factors (26,27). ROR transcription factors bind DNA response elements, called ROR response elements (ROREs) (26,28), consisting of the consensus core motif AGGTCA preceded by a 5 ′ A/T-rich sequence located into regulatory regions of target genes (27).
The interaction of ROR factors with their specific ligands allows recruitment of cofactor proteins, which leads to the transcription of their target genes (29).
Interestingly, RORγt is expressed in thymocytes at the doublepositive stage of T cell development, but is absent in mature thymocytes and in mature naive T cells in spleen and peripheral lymph nodes (33). In 2006, RORγt has been detected in IL-17producing T cells (17), and it has been shown to play a central role in Th17 differentiation (17,34).
Precursors or derivatives of cholesterol, such as desmosterol (35) and oxysterols (36), respectively, have been identified as activator ligands of RORγt, while bile acid synthesized from cholesterol called 3-oxoLC is an inhibitory ligand of RORγt (37).
RORγt regulates Il17a transcription by binding RORE sequences present in the 2-kb promoter fragment upstream of the transcription start site (38). In addition, the conserved non-coding sequences (CNS)2 (also called CNS5) located in the vicinity of the Il17a gene (approximately 5-kb upstream of promoter) (39) contains two ROREs, which are also conserved in human (39,40). It has been demonstrated that RORγt binds CNS2 of the Il17a gene ( Figure 1) and mediates Il17a transcription by controlling the chromatin remodeling. In fact, CNS2 is also bound by p300 and JmjC domain-containing protein (JMJD)3 that mediate permissive histone acetylation (41,42) and remove repressive histone marker H3K27me3 (43)(44)(45), respectively, resulting in hyperacetylation of histone H3 (46,47). Moreover, CNS2 interacts with Il17a promoter by forming a loop, and brings CNS2-associated histone remodeling enzymes to the promoter for the activation of Il17a transcription (39).
Given the high similarity of DNA-binding domains between RORα4 and RORγt, they activate Il17a transcription through the same molecular mechanism (40) (Figure 1).
However, RORα4 and RORγt are not sufficient to generate and specify the full Th17 program.
In fact, transcriptional regulators of RORγt, as well as other transcription factors that interact with RORγt, or bind the promoter or the intergenic regions of the Il17a locus, play a crucial role in the generation of Th17 cells.

OTHER TRANSCRIPTIONAL REGULATORS OF RORC AND IL17A
The transcription of RORγt is initiated by activation of the promoter RORC2 into the Rorc locus. RORC2 promoter contains nuclear factor of activated T cells (NFAT)-binding sequences, specific for NFAT and nuclear factor (NF)-kB proteins. Recently, it has been reported that the p65 NF-kB subunit and NFATc2 bind human Rorc promoter and promote a permissive chromatin conformation at RORC2 regulatory regions (49). Consistently, it has been reported that two NF-kB proteins, c-Rel and p65, activate the murine Rorc promoter (50).
Interestingly, the nuclear protein inhibitor of kB (IκB)ζ, which belongs to the IkB kinases and regulates activation of NF-kB pathway, binds CNS2 elements in Il17a locus (Figure 1), thus leading to an efficient recruitment of transcriptional coactivators with histone acetylase activity (18) and promoting Il17a expression without modulating expression of Rorc and Rora (51,52).
CNS2 region of Il17a is bound by another transcriptional regulator called Runt-related transcription factor (RUNX)1, whose effect is dependent on RORγt. In fact, it has been demonstrated that RUNX1 interacts with RORγt to potentiate Il17a expression and is required for the full effect of RORγt on Il17a expression (38) (Figure 1). Additionally, RUNX1 plays a role in Th17 differentiation, independently of RORγt, by binding the promoter of the gene encoding RORγt through three conserved RUNX1-binding sites (53) (Figure 1).
Hypoxia-inducible factor (HIF)-1α is a key metabolic sensor (19,54), which binds hypoxia response element (HRE, a conserved HIF)-1α-binding site) located in the proximal region of the Rorc promoter, in both human and mouse (19). Moreover, HIF-1α might physically associate with RORγt, serving as a coactivator for RORγt, thus contributing to Il17a expression without direct DNA binding on Il17a locus (19) (Figure 1). Further studies discovered that HIF-1α activates target genes by recruiting the factor p300, which possesses histone acetyltransferase activity and acetylates histones to "open" the chromatin structure (55). Indeed, the colocalized binding of RORγt, HIF-1α, and p300 occurs at the promoter of the Il17a gene (19).
Signal transducer and activator of transcription (STAT) 3 is another transcription factor regulating RORγt, and IL-17A (56) by interacting with the Stat-binding domains into the Rorc first intron, the Il17a promoter, and the intergenic region of the Il17a locus (Figure 1) (56)(57)(58). Moreover, STAT3 regulates positive epigenetic modifications, increasing permissive H3K4me3 marks on its target genes, including Rorc, Rora, and another gene encoding for transcriptional regulator of Th17 cells, called basic leucine zipper ATF-like transcription factor (BATF) (56).
BATF forms a heterodimer with JunB, and binds to the Il17a promoter as well as two conserved intergenic elements in the Il17a locus in Th17 cells (Figure 1). Interestingly, BATF synergizes with RORγt by binding to an overlapped conserved region recognized by RORγt into Il17a gene (20). Furthermore, the complex JunB and BATF also promotes the transcription of Rorc and Rora (58, 59) (Figure 1).
Genome-wide JunB-DNA binding analysis, using ChIP sequencing with anti-JunB antibody, revealed that JunB colocalizes in Th17 cells with another transcription factor, called interferon regulatory factor (IRF)4, involved in Th17 differentiation (21). In fact, IRF4 targets sequences enriched for activating protein 1 (AP-1)-IRF composite elements (AICEs) located into regulatory elements of the Il17a promoter (58, 60), which are cobound by BATF, an AP-1 factor (61). Thus, IRF4 and BATF bind cooperatively to structurally divergent AICEs to promote IL-17A activation in Th17 cells (61). Importantly, not only Il17a locus but also Il21, Il22, and Il23r loci contain one or more coincident binding peaks for IRF4 and BATF that were positioned in promoters and/or intronic regions, and ChIP assays verified the binding to these regions of both IRF4 and BATF complexed with JunB (61). The Kruppel-like factor (KLF)4 is another factor involved in the direct regulation of IL-17A, as demonstrated by ChIP analysis. In fact KLF4 binds the Il17a promoter and induces IL-17A expression, independently of RORγt (62).
Altogether, this information reveals a complex interconnected network of transcriptional regulators that finely regulates generation of Th17 cells.
The timing of transcriptional events leading to the full Th17 differentiation remains enigmatic. However, the transcriptional regulators activated upon T cell receptor engagement, such as NFAT, likely initiate the differentiation process by inducing RORγt transcription, and up-regulating receptor for polarizing cytokines, whose ligation leads to activation of other transcription factors. Among them, BATF, IRF4, and STAT3 are considered initiator transcription factors (24,63). In fact, BATF and IRF4 are responsible of initial chromatin accessibility in Il17a locus and, with STAT3, of initiation of the transcriptional program that is then globally tuned by the lineage-specific transcription factor RORγt, which plays a pivotal deterministic role at key loci (24,63). Then, RUNX1, HIF1a, and IκBζ can be considered cooperators of ROR nuclear receptors.
Importantly, there is high interconnectivity among transcription factors, including positive feedback loops reinforcing expression of initiator transcription factors BATF, IRF4, and STAT3 (24).
However, a negative feedback loop mediated by c-Maf, which is induced by initiator transcription factors, may limit Th17 response. In particular, c-Maf is a transcriptional regulator that, in Th17 cells, functions as a negative regulator, attenuating the expression of pro-inflammatory loci (e.g., Batf, Rora, Runx1, Il1r1, Ccr6, and Tnf ) and positively regulating few loci linked to attenuating inflammation (e.g., Il9, Il10, Lif, and Ctla4). Another transcription factor known to limit Th17 response is Fosl2 exerting antagonistic effect to BATF, by competing for the same binding sites and by directly repressing BATF (24). STAT1 and STAT5 are known to inhibit Th17 polarization by directly binding Rorc or Il17a loci. In particular, STAT5 represses IL-17A induction by binding the Il17a locus, removing accessible histone marks, and displacing STAT3 occupancy (64, 65); STAT1 has been shown to bind upstream of the Rorc locus in human Hela cells (66).

TH17-RELATED TRANSCRIPTIONAL REGULATORS IN AUTOIMMUNE DISEASES
Given the crucial role of Th17 cells in autoimmune disorders, the altered expression of Th17 transcriptional regulators may be related to a persistent Th17 cell response typical of diseases, such as psoriasis, rheumatoid arthritis, Crohn's disease, and MS (16). The role of the transcription factors activating a Th17 response has been mainly investigated in the murine model of MS, the experimental autoimmune encephalomyelitis (EAE), where deletion of each specific Th17 transcription factor reduced the disease (17-21, 40, 62, 67, 68). However, the potential involvement of such transcription factors in human autoimmune diseases, as well as their expression in immune cells from patients, has not been largely investigated.
It has been reported that the levels of phosphorylated STAT3 (pSTAT-3) in lymphocytes are up-regulated in MS patients during relapse compared to healthy donors and MS patients in remission phase. Moreover, pSTAT-3 levels positively correlate with magnetic resonance imaging data, indicating that STAT3 activation is associated to disease activity (69). In contrast, the expression of RORγt analyzed at transcriptional (70) and protein level (71) does not differ between MS patients and healthy donors.
However, the activity of RORγt is ligand regulated and the putative natural ligands of RORγt are molecules of the cholesterol pathway. In this context, it has been reported that levels of oxysterols in relapsing-remitting MS patients were associated with conversion to secondary progressive-MS (72).
Moreover, an aberrant activation of STAT3 was found in intestinal T cells of Crohn's disease patients compared to healthy donors (73); the expression of IRF-4 was significantly increased in inflammatory cells of psoriasis patients than that in healthy controls (74); HIF-1α was found strongly expressed by immune cells in the intimal layer of the synovium in rheumatoid arthritis patients (75). However, the lack of correlations with clinical parameters in most part of these studies does not permit the definition of the role of the enhanced expression of those transcriptional regulators in human diseases.
Additionally, genetic abnormalities in Th17 transcriptional regulators may favor Th17 cell response and may influence susceptibility to autoimmune diseases. However, few studies demonstrate association between gene variants of Th17 transcription factors and Th17-related diseases. For instance, single-nucleotide polymorphisms (rs734232) affecting the consensus-binding site for RUNX1, or Runx1 itself, are associated with susceptibility to rheumatoid arthritis and psoriasis (76)(77)(78), while Stat3 gene was identified as risk locus for Crohn's disease and MS (79,80).
Clinical studies testing the actual clinical efficacy and eventual side effects are active or completed. For instance, the oral compound VTP-43742 demonstrated efficacy through the reduction of clinical scores in psoriasis patients (NCT02555709). However, clinical data also showed liver toxicity, and VTP-43742 has been replaced with a new improved molecule VTP-45489 (Table 1). Similarly, other early clinical agents like GSK-2981278, JTE-151, JNJ-3534, ABBV-553, TAK-828, and AZD-0284 were either discontinued or suspended for further development ( Table 1) (99). Currently, novel RORγt inhibitors are   Table 1).
Another promising target among Th17 transcription factors is HIF-1α. To date, the most advanced HIF pathway-targeted pharmaceuticals in terms of clinical development are cellpermeable prolyl hydroxylase inhibitors, evaluated for treatment of anemia. A number of HIF inhibitors have been developed also for cancer therapy (97) and are considered promising novel treatments for rheumatoid arthritis (101), such as the 2-benzoyl-phenoxy acetamide that acts as anti-arthritic agent in an experimental adjuvant induced arthritis rat model (98) ( Table 1). However, none of the compounds targeting HIF-1α has been assessed in clinical trials for rheumatoid arthritis.
STAT3 is another potential drug target currently used for cancer therapy given its aberrant activation in many human tumors (102). Concerning Th17-related diseases, the small STAT3 inhibitor STA-21 has been tested on psoriasis patients in a nonrandomized study, and psoriatic lesions in six of the eight patients showed improvement after topical STA-21 treatment for 2 weeks (NCT01047943) ( Table 1) (103). However, this effect is likely related to the inhibition of epidermal keratinocyte proliferation, rather than to immune cell activity (103).
Collectively, these data indicate that Th17 transcriptional regulators are promising targets for Th17-related diseases. However, given their broad expression in different cell types, it is crucial to develop inhibitors highly specific for immune cells to minimize off-target effects.

CONCLUSIONS
Since the discovery of Th17 cells, remarkable advances in the understanding of Th17 response have been reported. In particular, the study of the mechanisms regulating the transcription of Rorc and Il17a genes has advanced our understanding of the generation of Th17 cells. Moreover, small molecules interfering with these mechanisms provide promising results in pre-clinical research and clinical trials. Future studies further detailing the transcriptional program of Th17 cells could lead to the identification of pathways or regulators that are specifically activated during diseases. Advances in these points are critical for the development of new compounds that target more accurately the pathogenic effect of Th17 cells, and that could become new therapeutic strategies in Th17-related diseases.

AUTHOR CONTRIBUTIONS
AC drafted the manuscript. EV critically reviewed the manuscript and finalized the manuscript for submission. AC and EV approved the final version.