(Inverse) Agonists of Retinoic Acid–Related Orphan Receptor γ: Regulation of Immune Responses, Inflammation, and Autoimmune Disease

Abstract

Retinoic acid–related orphan receptor γt (RORγt) functions as a liganddependent transcription factor that regulates multiple proinflammatory genes and plays a critical role in several inflammatory and autoimmune diseases. Various endogenous and synthetic RORγ (inverse) agonists have been identified that regulate RORγ transcriptional activity, including many cholesterol intermediates and oxysterols. Changes in cholesterol biosynthesis and metabolism can therefore have a significant impact on the generation of oxysterol RORγ ligands and,consequently,can control RORγt activity and inflammation. These observations contribute to a growing literature that connects cholesterol metabolism to the regulation of immune responses and autoimmune disease. Loss of RORγ function in knockout mice and in mice treated with RORγ inverse agonists results in reduced production of proinflammatory cytokines,such as IL-17A/F,and increased resistance to autoimmune disease in several experimental rodent models. Thus, RORγt inverse agonists might provide an attractive therapeutic approach to treat a variety of autoimmune diseases.

INTRODUCTION

The retinoic acid–related orphan receptor (ROR) subfamily consists of three members: RORα, RORβ, and RORγ, which are encoded by the RORA–C (or NR1F1–3) genes, respectively (1–7). Each of these receptors functions as a ligand-dependent transcription factor (8, 9). RORs regulate gene transcription by binding as monomers to retinoic acid–related orphan receptor response elements (ROREs) containing an AGGTCA consensus preceded by an A/T-rich sequence in the regulatory regions of target genes. This binding is mediated by two C₂C₂-type zinc finger motifs within the N-terminal DNA-binding domain (DBD) that are highly conserved among nuclear receptors (3, 8, 9). In the last ten years, significant breakthroughs have been made in identifying several (oxy)sterols and synthetic ligands that act as either RORγ agonists or inverse agonists (8, 9).

The RORC gene is located on chromosome 1q21.3 and encodes two isoforms in both humans and mice that arise through the use of alternative promoters (2,7–10).RORγ1 contains 518 amino acids, whereas RORγt (RORγ2) lacks the first 21 amino acids of RORγ1 and also differs from RORγ1 at the first three amino acids (MetArgThr instead of MetAspArg). Both isoforms regulate the transcription of target genes by binding a RORE consensus and recruit many of the same coactivators and corepressors (Figure 1). The existence of alternative promoters likely explains the different patterns of cell type–specific expression and the regulation of distinct physiological processes and target genes exhibited by the two isoforms. RORγ1 is expressed, often together with RORα, in many peripheral tissues, including adipose, liver, kidney, and muscle tissue, where they regulate the transcription of a number of circadian and metabolic genes (3, 6, 11–16). In contrast, the expression of RORγt is confined to several different immune cell populations, including T helper 17 (Th17) cells,lymphoid tissue inducer (LTi) cells,innate lymphoid 3 cells (ILC3s),and γδ T cells (3, 6, 17–24) (Figure 2).

Figure 1.

RORγt functions play a central intermediary role in linking the control of cholesterol biosynthesis and metabolism to the regulation of immune responses and autoimmune disease. Many endogenous oxysterols generated by the cholesterol biosynthetic pathway and cholesterol metabolism act as effective RORγt agonists, leading to the recruitment of coactivators such as NCOA1 and CBP and the transcriptional activation of RORγt target genes, including IL17, IL21, IL23R, and AHR. The increase in proinflammatory cytokines promotes inflammation and autoimmune disease. Many synthetic RORγ ligands and some oxysterols, such as 24S-OHC and several hydroxylated metabolites of vitamin D3, function as inverse agonists. Inverse agonists prevent coactivator binding, promote the recruitment of corepressors such as NCOR1 and HDAC1/2, and repress the transcription and production of proinflammatory cytokines, thereby reducing inflammation and protecting against autoimmune disease. Changes in cholesterol biosynthesis and metabolism, for example, by treatment with statins or a high-cholesterol/fat diet, affect the generation of RORγt agonists and thereby RORγt activity and the development of inflammation and autoimmune disease. Posttranslational modifications can also affect RORγt activity by changing RORγt protein stability or its ability to recruit coactivators. Abbreviations: OHC, hydroxycholesterol; ROR, retinoic acid–related orphan receptor; RORE, ROR response element.

Figure 2.

RORγt-dependent development of T cells and ILCs from the CLP. Cells expressing RORγt are indicated. (top) Development of the indicated T cells, including CD4⁺CD8⁺ DP αβTCR⁺ T cells and γδTCR⁺ T cells in the thymus as well as αβTCR⁺ CD4⁺ Th17 cells. (bottom) Innate immune cells that also arise from the CLP, including NKp46⁺ NK cells derived from NKP cells, the three major ILC subsets derived from the ILCP, and LTi cells arising from the CHILP. Abbreviations: CHILP, common helper ILC progenitor; CLP, common lymphoid progenitor; DN, double-negative; EILP, early innate lymphoid progenitor; HSC, hematopoietic stem cell; ILC, innate lymphoid cell; ILCP, ILC progenitor; LTi, lymphoid tissue inducer; NK, natural killer; NKP, NK cell progenitor; ROR, retinoic acid–related orphan receptor; TCR, T cell receptor; Th, T helper.

Mice with impaired RORγt function are protected against the development of autoimmune diseases (23, 25).This, together with the discovery that treatment with small-molecule RORγt inverse agonists mimics the effects observed in RORγt-deficient mice, launched a search to identify additional high-affinity RORγt inverse agonists in the hope that this might lead to new therapeutic strategies in the management of several autoimmune diseases. In this review, we provide a short overview of RORγt and its (inverse) agonists and discuss their roles in regulating immune cell functions, particularly Th17 cells, and their relationship to autoimmune disease and inflammation.

ROLES OF RORγt IN IMMUNE CELLS

Th17 Differentiation

Naïve CD4⁺ T cells that exit the thymus can differentiate into several discrete T helper (Th) lineages defined primarily by their master transcription factors and the cytokines they produce (Figure 2).Th17 cells contain RORγt; produce the interleukins (ILs) IL-17A,IL-17F,and IL-22; and protect hosts against infections by extracellular bacteria and fungi. Th17-derived IL-21 also activates T follicular helper cells, which promote humoral immunity in secondary lymphoid tissue. Treatment of naïve T cells with transforming growth factor (TGF)-β together with either IL-6 or IL-1 drives Th17 differentiation by inducing Rorc expression (25–29). IL-6 induces the phosphorylation of STAT3 (pSTAT3), which then moves to the nucleus to activate the transcription of Rorc and other Th17-associated genes (30, 31). IL-1 represses the suppressor of cytokine signaling (SOCS)3, an inhibitor of pSTAT3, thereby increasing Rorc expression (32). IL-23 stabilizes Th17 cells and contributes to their pathogenicity, in part by inducing expression of the transcription factor Runx1, which in turn stimulates Rorc expression and, together with RORγt, promotes Th17 differentiation (33). TGF-β promotes Th17 differentiation in part by acting through SKI, a transcriptional repressor that forms a complex with SMAD and inhibits H3K9 acetylation and transcription at the Rorc locus (34). TGF-β signaling leads to the degradation of SKI and the consequent derepression of the Rorc locus. Upon its synthesis, RORγt is recruited to ROREs at several Th17-associated loci,including Il17a,Il17f,Irf4,and Il23r,thereby directly regulating their transcription (35). In RORγt-deficient mice, the differentiation of Th17 cells is greatly impaired as is the transcription of Th17-type inflammatory cytokines (23, 25). Many transcription factors, including RORγt, are unable to bind chromatin until it has been opened through the actions of pioneer transcription factors. BATF (36) and IRF4 (37) are pioneer transcription factors that bind cooperatively to overlapping sites in chromatin near several Th17-related genes, including Rorc and Il17. This increases chromatin accessibility at these sites and allows pSTAT3 to bind the Rorc promoter and induce its transcription (38, 39).

It is well established that differentiation into the regulatory T cell (Treg) or Th17 lineages is controlled by the level of FOXP3 and RORγt, respectively (26–28, 40, 41). Foxp3 expression and Treg development are favored in cultures containing high levels of TGF-β,IL-2,and retinoic acid (RA), whereas Rorc expression and Th17 development are promoted by low amounts of TGF-β together with proinflammatory cytokines, IL-6 and IL-1. However, recent studies have shown that these lineages are not necessarily fixed and that T cells can display substantial plasticity, with some Th17 cells expressing FOXP3 as well as RORγt. In addition to promoting the expression of genes associated with Tregs, FOXP3 can also interact with RORγt through a nuclear receptor interaction-like motif and repress Il17 promoter activation by RORγt (42). The factors that control the relative abundance of FOXP3 and RORγt, and therefore the balance of Tregs and Th17 cells, are currently an area of intense investigation.

Innate Lymphoid Cells

Another active area of investigation is the development of innate lymphoid cells (ILCs), a class of non-B, non-T lymphocytes that are derived from the common lymphoid progenitor and possess typical lymphoid morphology but lack antigen-specific receptors and their associated molecules. There are some differences between the development of mouse ILCs and human ILCs, although the former is better understood (17) (Figure 2). An important function of ILCs is to provide a source of cytokines during the time required by antigen-specific effector T cells to develop from naïve T cells and undergo clonal expansion. The three major classes of human ILCs, as well natural killer (NK) cells, are derived from a common innate lymphoid cell progenitor (17, 24, 43) (Figure 2). There are several subsets of ILC3s and RORγt⁺ ILC3s. RORγt is selectively expressed in ILC3s that produce IL-17A,IL-17F,IL-22,granulocyte-macrophage colony-stimulating factor (GM-CSF),and tumor necrosis factor and in IL-22-producing NKp46⁺ cells (24,44,45).GATA3 is required for the development of RORγt⁺ ILC3s as well as for the other ILC subtypes (46). The transcription factor RUNX3 is also required for ILC3 development and regulates RORγt expression by inducing its transcription (47).RORγt in turn directly regulates the transcription of the aryl hydrocarbon receptor (Ahr) gene, which is required for ILC3 as well as other ILCs. ILCs are strategically located at sites of pathogen invasion, including the skin and the mucosa of the gut and lung. Although the involvement of ILCs in disease is still poorly defined, roles in infectious and inflammatory lung diseases and in the maintenance of the mucosal barrier homeostasis have emerged (44, 48). ILCs are heterogeneous and display significant plasticity. For example, ILC3s from both humans and mice can convert into ILC1-like cells (49–51). This transition follows a decrease in RORγt and a corresponding increase in T-bet and Notch signaling. Conversely, Notch ligand treatment of ILC2s induces RORγt and the consequent production of IL-17.

Lymphoid Tissue Inducer Cells

The first discovered member of the ILC3 group is the LTi cell, a type of CD4⁺CD3⁻ cell that expresses lymphotoxin α1β2. LTi cells are required for the development of secondary lymphoid organs, including Peyer’s patches and intestinal lymphoid follicles (19, 43, 52, 53). RORγt is required for LTi development, and RORγt-deficient mice consequently fail to develop secondary lymphoid organs (3, 21, 22).Rorc expression is induced by RA, which signals through RA receptors that bind to and activate the Rorc promoter (54).

Thymopoiesis

During thymopoiesis, Tcells undergo a series of maturation steps in which immature CD4⁻CD8⁻ double-negative thymocytes differentiate into maturing double-positive (DP) CD4⁺CD8⁺ cells and then into mature single-positive CD4⁺ or CD8⁺ T cells (Figure 2). RORγt is selectively expressed in DP cells and is required for their expression of the antiapoptotic gene Bcl-X_L (21, 22). DP thymocytes lacking RORγ undergo accelerated apoptosis both in vivo and in vitro due to the downregulation of Bcl-X_L. Consequently, RORγ-null mice have reduced numbers of DP cells and their descendants, including CD4⁺ and CD8⁺ Th cells.

γδ T Cells

In addition to Th17 cells, some other immune cells can also express IL-17 in a RORγt-dependent manner. This includes γδ T cells (Figure 2), which exhibit features of both adaptive and innate immune cells. In contrast to αβ T cells, which do not produce IL-17 until they differentiate into Th17 cells, γδ T cells emerge from the thymus fully capable of producing IL-17 in response to stimulation by IL-23 and IL-1β without engagement of the T cell receptor (55). This induction of IL-17 is mediated by increased RORγt expression by IL-23. The ability to rapidly produce IL-17 in response to innate cytokines allows these cells to contribute to pathogen clearance before conventional Th17 cells have had time to develop.γδ T cells have also been implicated in autoimmune disease (56). RNA for Il17a and Il17f as well as their encoded proteins, IL-17A and IL-17F, are elevated in psoriatic skin (57), and ILC3s and Th17 and γδ T cells are important sources of IL-17 (58).

REGULATION OF RORγ ACTIVITY AND FUNCTION

Several studies have demonstrated that posttranslational modifications play a critical role in modulating RORγ protein activity. RORγ is phosphorylated at multiple sites and can be ubiquitinated, sumoylated, and acetylated (9, 59–63) (Figure 1). Although the impact of the multiple RORγ phosphorylation sites on ROR activity and function has yet to be established, several studies have recently provided insights into the role of RORγt ubiquitination in RORγ activity and its regulation of Th17 cells. The HECT E3 ubiquitin ligase, ITCH, interacts with RORγ and catalyzes its K48-polyubiquitination, thereby promoting its proteasomal degradation (62). Conversely, deubiquitination of the K48-linked polyubiquitination of RORγt by the deubiquitinase ubiquitinspecific protease USP4 stabilizes RORγt and promotes its activation of Th17-associated genes, whereas inhibition of USP4 activity diminishes Th17 differentiation (64).The E3 ubiquitin ligase tumor necrosis factor receptor–associated factor 5 (TRAF5) stabilizes RORγt through K63-linked polyubiquitination (65). Accumulation of the deubiquitinating enzyme DUBA (OTUD5) negatively regulates RORγt stability and IL-17 expression in T cells by stabilizing the ubiquitin ligase UBR5, which ubiquitinates RORγt in response to TGF-β, leading to RORγt degradation and decreased IL-17 expression (66).

The ubiquitin-specific protease USP17 reduces polyubiquitination of RORγt at Lys³⁶⁰ and prevents its degradation by proteasomes, whereas downregulation of USP17 decreases RORγt protein and expression of IL-17 (67). Ubiquitination of RORγt at Lys⁴⁴⁶ inhibits its interaction with NCOA1 and negatively regulates Th17 differentiation (68).The mutation of Lys⁴⁴⁶ to Arg or the deubiquitination of Lys⁴⁴⁶ by USP15 promotes NCOA1 recruitment and Th17 differentiation.

(De)acetylation also plays a critical role in the control of RORγt expression, its transcriptional activity, and its regulation of Th17 differentiation and inflammation. RORγt transcription is dependent on STAT3; STAT3 acetylation at Lys⁶⁸⁵ by p300 is required for its nuclear translocation, transcriptional activity, induction of RORγt, and Th17 differentiation. Activation of the NAD-dependent deacetylase Sirtuin 1 (SIRT1) promotes its interaction with STAT3 and induces deacetylation at Lys⁶⁸⁵, thereby inhibiting STAT3 translocation to the nucleus and consequently reducing the transcriptional activation of RORC (69). These studies indicate that (de)acetylation plays a critical role in regulating RORC transcription and the balance between Th17 and Treg differentiation.

Another report identified a different role for SIRT1 in regulating RORγt activity (61). This study showed that the histone acetylase p300 (KAT3B) interacts with and acetylates RORγt at several positions within the DBD and impairs its interaction with RORE and thus its ability to induce Th17 differentiation and IL-17A expression. SIRT1 physically interacts with the RORγ ligand-binding domain (LBD) and promotes deacetylation of the RORγt DBD, which restores RORγt transcriptional activity and consequently promotes the generation of Th17 cells, thereby exacerbating autoimmunity (61). Recently, RORγt activity was found to be also modulated by sumoylation (70). Sumoylation of RORγt by SUMO3, but not SUMO1, at Lys³¹ catalyzed by the E3 Sumo ligase PIAS4 promotes recruitment of the lysine acetyl transferase KAT2A (GCN5) and NCOA1, which enhance RORγt transcriptional activity and stimulate Th17 differentiation. These RORγt-mediated activities were greatly diminished when Lys³¹ was mutated to Arg. Loss of SUMO3 expression was shown to significantly reduce Th17 differentiation. A recent report identified a different mechanism by which sumoylation influences RORγt activity. This study showed that SUMO-conjugating enzyme, Ubc9 (UBE2l), binds RORγt via a conserved GKAE motif and targets it for sumoylation. This subsequently leads to the recruitment of HDAC2 by RORγt and suppression of RORγt transcriptional activity and IL17 expression (71). Consistent with these observations are findings showing that sumoylation-defective Th17 cells are colitogenic upon transfer to Rag1^−/− mice.

A recent study identified an indirect way by which phosphorylation affects NCOA1–RORγ interaction and the balance between Th17 and Treg lineage determination (72). Phosphorylation of NCOA1 by protein kinase C theta (PKC-θ or PRKCQ) stimulates its interaction with RORγt, causing the stabilization of RORγt–RORE binding and the dissociation of bound FOXP3 and its subsequent proteasomal degradation. This induces a shift from Treg toward Th17 differentiation and increased IL-17A expression.

OXYSTEROLS AS ENDOGENOUS (INVERSE) AGONISTS OF RORγ

X-ray crystallography studies demonstrating that cholesterol and cholesterol sulfate are associated with the ligand-binding pocket of RORα and function as RORα agonists (73, 74) provided the first evidence for a connection between RORs and cholesterol and its derivatives. These observations suggested that cholesterol-related intermediates or metabolites might also function as ligands of RORγ and regulate its transcriptional activity and functions. Subsequent studies revealed that 20α-hydroxycholesterol (-OHC), 22R-OHC, and 25-OHC all effectively enhance the interaction between RORγ LBD and the NCOA1 coactivator peptide containing an LXXLL nuclear receptor–interacting motif (75). The naturally occurring oxysterols 7α,27-OHC, 7β,27-OHC, and 27-OHC were identified as potent and selective RORγ agonists (76) based on their ability to reverse the repression of RORγ transcriptional activity by the inverse agonist ursolic acid. In contrast, the oxysterol 7α,25-OHC was inactive, while 7α-OHC, 7β-OHC, 25-OHC, and 24S-OHC showed either weak-to-moderate agonist or inverse agonist activity (76–78).

Additional studies showed that several intermediates of the cholesterol biosynthesis pathway function as endogenous ligands for RORγ (79–81) (Figure 3). Zymosterone, zymosterol, 7dehydrocholesterol, and desmosterol were among the sterols activating RORγ, while lanosterol and T-MAS showed little activity. 4α-carboxy, 4β-methyl zymosterol (4ACD8), and 25-OHC were identified as some of the most effective RORγ agonists (80). Sulfated derivatives of these sterols were about twice as effective in enhancing RORγ activity. Interestingly, the expression of the sterol sulfotransferase Sult2B1 was increased, while expression of the sterol sulfatase STS was decreased during Th17 differentiation, bringing about a shift toward more active, sulfated sterol formation that would lead to enhanced RORγt activation.

Figure 3.

Intermediates of the cholesterol biosynthetic pathway function as RORγagonists. Acetyl CoA is converted to mevalonate by HMGCR and then into farnesyl pyrophosphate and squalene via several steps, which are subsequently converted into cholesterol via multiple steps. Statins reduce cholesterol biosynthesis by inhibiting the formation of mevalonate. Zymosterol, desmosterol, zymosterone, 4ACD8, 25OHC, and 7-dehydrocholesterol are among the most effective endogenous RORγagonists. Deficiency in CYP51A1, or its inhibition by azoles, and deficiency in FDFT1 inhibit cholesterol biosynthesis and limit the availability of RORγagonists. Abbreviations: ACAT1, acetyl-CoA acetyltransferase 1; CH25H, cholesterol 25-hydroxylase; CYP51A1, cytochrome P450 family 51 subfamily A1; DHCR24, 24-dehydrocholesterol reductase; DHCR7, 7-dehydrocholesterol reductase; EBP, emopamil binding protein; FDFT1, farnesyl-diphosphate farnesyl-transferase 1; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; HMGCS1, 3-hydroxy-3-methylglutaryl-CoA synthase 1; HSD17B7, hydroxysteroid 17-βdehydrogenase 7; LSS, lanosterol synthase; NSDHL, NAD(P)-dependent steroid dehydrogenase-like; ROR, retinoic acid—related orphan receptor; SC4MOL, fatty acid hydroxylase domain containing 2; SC5D, sterol-C5-desaturase; SQLE, squalene epoxidase; TM7SF2, transmembrane 7 superfamily member 2.

In the skin,7-dehydrocholesterol can be converted by UVB to previtamin D3 and subsequently by CYP2R1, CYP27A1, and CYP27B1 to the active form 1,25-(OH)₂D3. Both can then be converted by CYP11A1 and CYP24A1 to various additional hydroxylated metabolites such as 20-OHD3 and 20,22-(OH)₂D3 (82). Alternatively, UVB can convert D3 to lumisterol (L3), which is subsequently metabolized by CYP11A1 to various hydroxylumisterols such as 20(OH)L3 (83). 1,25-(OH)₂D3, in addition to acting as an agonist of the vitamin D receptor, also functions as a weak inverse agonist of RORγ and RORα (80, 82). Several hydroxylated vitamin D3 and L3 metabolites also exhibit ROR inverse agonist activity (8, 82, 83).

MODULATION OF CHOLESTEROL METABOLISM AFFECTS RORγt ACTIVITY AND INFLAMMATION

Observations showing that various intermediates of the cholesterol biosynthetic pathway function as RORγt (inverse) agonists suggested that changes in this pathway would affect the generation of RORγ (oxy)sterol (inverse) agonists and, consequently, RORγ transcriptional activity and functions, including Th17 differentiation. Consistent with this hypothesis were findings showing that when naïve T cells differentiate into Th17 cells, the expression of many enzymes of the cholesterol biosynthetic pathway is increased, while the expression of those involved in cholesterol export is decreased (79, 84). Furthermore, inhibition of the mevalonate–cholesterol biosynthetic pathway negatively affects the differentiation and function of T cells. Statins, such as simvastatin and lovastatin, which inhibit the synthesis of cholesterol isoprenoid precursors, reduce RORγt expression and IL-17 production (79, 85) (Figure 3).Moreover, deficiency in certain cholesterol biosynthetic enzymes can have either a positive or negative effect on RORγt activity by regulating the availability of RORγt agonists. Deficiency in CYP51 or treatment with various azole fungicides (e.g., clotrimazole and hexaconazole), inhibitors of CYP51, reduces RORγ transcriptional activity and decreases Th17 differentiation and IL-17 production by inhibiting cholesterol biosynthesis and the formation of RORγt agonists (79, 80, 86) (Figure 3). This inhibition of Th17 cells could be reversed by the addition of RORγ agonists such as zymosterol, which also induces RORγt-dependent IL-17 expression in γδ T cells. The observed reduction in LTi cells and lymph node anlagen, which are also dependent on RORγt, in CYP51-deficient mice is consistent with the reduced RORγt activity in these mice (80). In addition, CYP51 ablation reduces both RORα and RORγ activities in mouse liver (87), while inhibition of CYP51 by ketoconazole reduces IL-17 production by γδ T cells and improves inflammation in imiquimod-induced psoriasis in mice (80). Likewise, squalene synthetase (FDTF1) deficiency, as well as the knockdown of SC4MOL or NSDHL, reduces RORγt transcriptional activity (Figure 3). Deletion of SC4MOL in all RORγtexpressing cells, or only in CD4⁺ T cells, showed that CD4⁺ T cells lacking SC4MOL have a greatly reduced ability to differentiate along the Th17 lineage and that this could be reversed by the addition of 4ACD8.

Deficiency in the scavenger receptor cysteine-rich family member CD5L, which regulates lipid homeostasis, enhances the expression of several cholesterol biosynthetic enzymes, including CYP51A1 and SC4MOL, and subsequently increases availability of RORγt agonists and IL-17 expression in pathogenic Th17 cells (8, 88). The transcription factor SREBF2, which increases cholesterol biosynthesis by enhancing the transcription of several cholesterol biosynthetic genes, including HMGCR and HMGCS, contributes to a proinflammatory response (89) that might in part be due to increased synthesis of RORγt agonists. These observations further suggest that stimulation of cholesterol biosynthesis by a high-fat or hypercholesterolemic diet might result in higher levels of RORγt agonists,Th17 differentiation,and the production of proinflammatory cytokines.This is consistent with a report showing that apolipoprotein E–deficient mice, which have elevated levels of cholesterol, exhibit an increased susceptibility to type II collagen-induced arthritis that is in part due to the activation of Th17 inflammatory responses (90).Together, these studies indicate that RORγ transcriptional activity and RORγ-regulated physiological functions can be modulated by changes in the cholesterol biosynthetic pathway. Thus, RORγt functions as a mediator that plays a central role in linking the control of cholesterol metabolism with regulation of the immune system; immune responses; and, consequently, autoimmune disease (8, 81) (Figure 1).

INVERSE AGONISTS

The discovery that RORγ functions as a ligand-dependent transcription factor, together with findings showing that the loss of RORγ function protects against autoimmune disease, initiated a search for small-molecule inverse agonists that would inhibit RORγt transcriptional activity as well as proinflammatory responses regulated by RORγt, protect against autoimmune diseases, and provide a new therapeutic approach in managing these diseases. High-throughput screens led to the discovery of several series of RORγ (inverse) agonists.The cardiac drug digoxin and its derivatives β-acetyldigoxin 20,22-dihydrodigoxin-21,23-diol [Dig(dhd)] and digoxin-21-salicylidene [Dig(sal)] were among some of the first synthetic RORγ-selective inverse agonists identified (91) (Figure 4). In contrast to its inverse agonist effect, a recent study reported that, at low concentrations, digoxin exhibited agonist activity in HepG2 cells (92).

Figure 4.

Chemical structures of several retinoic acid–related orphan receptor (ROR) inverse agonists: digoxin (91), SR1001 (a RORα and RORγ inverse agonist) and SR2211 (135), ursolic acid (95), VTP-43742 (141), GSK2981278 (124), BIO-0554019 (129), TMP778 and TMP920 (35), JNJ-54271074, Cpd-1 (123), and A213 (126). The structures of a sulfonamide inverse agonist antagonist (121) and the agonist ganoderone A (96) are also shown.

In another study, the LXR agonist T0901317 was identified as an inverse agonist of both RORα and RORγ but not of RORβ (93).As a result of this discovery, a series of chemical derivatives were synthesized and their effect on ROR activity analyzed. This led to the identification of several additional (inverse) agonists, including the RORα and RORγ inverse agonist SR1001 and agonist SR1078 and the selective RORγ inverse agonist SR2211 (94). Several natural products have been reported to act as RORγ ligands. Ursolic acid, a pentacyclic triterpenoid found in many plants, acts as a RORγ-selective inverse agonist, whereas ganoderone A, an antiviral triterpenoid produced by the fungus Ganoderma pfeifferi, acts as a very potent RORγ agonist (95, 96) (Figure 4).

Subsequent studies by many different laboratories led to the discovery of a number of additional small-molecule RORγ (inverse) agonists (35, 97–113) (Figure 4). For a more comprehensive review of studies that identified and characterized RORγt inverse agonists, the reader is referred to several sources (8, 9, 94, 114–117).

The configuration of H12 plays a critical role in determining the activator or repressor state of nuclear receptors. Structure analysis of the RORγ LBD in complex with several oxysterols and chemical ligands showed that agonists stabilize the active position of H12, which becomes tightly packed against the LBD, thereby allowing it to interact with the LXXLL motif of coactivators, such as NCOA1/2 (8, 9, 75, 80, 91, 96, 97, 118, 119) (Figure 1). Agonists can bind to and regulate RORγ activity through different mechanisms. Many agonists act via a canonical mechanism in which they bind to the ligand-binding pocket of RORγ, allowing for the recruitment of coactivators and consequent transcriptional activation of RORγ target genes (75, 76, 96) (Figure 1). However,in certain instances,agonist-bound RORγ can also bind corepressors such as nuclear receptor interacting protein 1 (NRIP1,also referred to as RIP140),thereby repressing transcription (96).

Binding inverse agonists induces a conformational change in the RORγ LBD that leads to the displacement of coactivators and recruitment of corepressors such as NCOR1/2 and the histone deacetylases HDAC1/2 (8, 95, 96). In the canonical mechanism, inverse agonists, including some (oxy)sterols, TMP778, TMP920, and GSK805, interact with the RORγ ligand-binding pocket but suppress RORγ activity in two different ways. TMP778- and GSK805-bound RORγt are still able to bind ROREs in the promoter regions of target genes, whereas TMP920-bound RORγt is no longer capable of interacting with ROREs (35) (Figure 4). However, certain inverse agonists suppress RORγ activity via a noncanonical mechanism. For example, MRL-871 does not interact with the canonical ligand-binding pocket but with a distal,allosteric binding site,causing a change in the RORγ LBD conformation that prevents recruitment of coactivators (120). Small changes in the chemical structure of oxysterols or synthetic RORγ ligands can determine whether they act as inverse agonists or agonists, as reported for several tertiary sulfonamide RORγ modulators (121) (e.g., see Figure 4).

Administration of RORγ inverse agonists mimics many of the same effects observed in RORγ-deficient mice, including inhibition of Th17 differentiation and repression of the transcription of Th17 signature genes such as IL17A,IL17F,and IL23R (6,35,75,76,91,95,122–126) (Figure 1). In contrast to the suppression of Th17 signature genes, inverse agonists enhance the expression of signature genes from other CD4⁺ lineages, including Treg cells (35). The latter indicates that inverse agonists cause a shift toward Treg differentiation, which is consistent with the concept that the Th17 and Treg lineages are reciprocally controlled by the levels of RORγt and FOXP3. The repression of Il17A and Il23R transcription correlates with several epigenetic changes at their promoter regions, including a reduction in H3AcK9/14 and H3K4m3, transcription-permissive acetylation, and methylation signatures (25, 127). In addition to the effects on Th17 cells, treatment with inverse agonists also mimics other phenotypes seen in RORγ-null mice. For example, inverse agonists inhibit the stimulation of RORγt-dependent IL-17 in γδ T cells by IL-23 and IL-1β (125, 128,129). In addition, treatment of isolated CD4⁺CD8⁺ DP thymocytes with the inverse agonist Cpd 1 induces a significant increase in apoptosis, as has been observed in RORγ-null mice (21, 22, 123).

RORγt, INVERSE AGONISTS, AND INFLAMMATORY DISEASE

Mice deficient in RORγt function have been shown to be protected against multiple autoimmune diseases (23, 25). Several RORγt-dependent IL-17⁺ immune cells, including Th17, ILC3, and γδ T cells, contribute to different degrees to the development and progression of autoimmune diseases (56, 57, 130, 131). Multiple sclerosis (MS) is a disease of the central nervous system, involving demyelination of and consequent damage to nerve cells. Experimental autoimmune encephalomyelitis (EAE) has been the most widely used animal model of MS to study the role of RORγt, and it has proven to be extremely informative for uncovering the pathogenic mechanisms that drive MS, including the role of RORγt. Th17 cells are thought to be of central importance in this disease because the Th17-produced proinflammatory cytokines IL-17A/F, IL-23, and IL-22 play a critical role in the pathogenicity of EAE as well as in other autoimmune diseases (132, 133). This is supported by studies demonstrating that antibodies against IL-17A and IL-23 or their receptors are effective in the treatment of several autoimmune diseases. Mice lacking RORγ are significantly protected from EAE (23, 25), type II collagen-induced arthritis, and IL-23-induced psoriasis-like lesions (126, 128). RORγ-null mice also displayed reduced numbers of eosinophils and CD4⁺ lymphocytes in an ovalbumin-induced model of allergic airway inflammation (134). Together, these studies raised the prospect that RORγ (inverse) agonists might be useful in the management of allergic and autoimmune diseases (35,76,80,135,136).This was confirmed by several studies demonstrating that small-molecule RORγt inverse agonists ameliorate disease in several rodent models of autoimmunity. Thus, treatment with the RORα/RORγ inverse agonist SR1001 and RORγ-selective inverse agonists, including ursolic acid, digoxin, MRL-248, TMP778, TMP920, Cpd 1, and dehydrodiconiferyl alcohol, suppresses IL17 transcription and IL-17 production and significantly reduces the severity of EAE (35, 91, 95, 137, 138) (Figure 1).

The inverse agonist GSK2981278, a very effective, selective inhibitor of RORγ activity, attenuates inflammation in an imiquimod-induced, psoriasis-like mouse model when treated topically (124). GSK2981278 significantly reduced the levels of several proinflammatory cytokines, including IL-17A/F, IL-22, and IL-1β, in treated skin and reduced epidermal hyperplasia and skin thickness. Topical treatment with GSK2981278 also inhibited Th17-type cytokine production and IL-17-driven inflammation in human tissue–based assays and skin explants from psoriatic patients (124). However, in a human randomized, double-blind trial, topical treatment with GSK2981278 did not change psoriatic infiltrate thickness or the level of several Th17-type genes such as IL17A/F (139), possibly because longer and more frequent treatment or systemic treatment is needed to be effective. Another RORγ inverse agonist, BIO-0554019, inhibited imiquimodinduced skin inflammation when applied as a cream (129).Subcutaneous treatment with TMP776 also attenuated imiquimod-induced skin inflammation (125). In both cases, this correlated with the inhibition of IL17A/F expression in Th17 cells as well as γδ T cells, which are critical in psoriasis (140). Two different studies showed that oral administration of the inverse agonists VTP-43742 and A213 attenuated psoriatic lesions in mouse models (126, 141). A213 mitigated phorbol ester–induced psoriasis and IL-23-induced dermatitis in K5.Stat3C transgenic mice that constitutively expressed active Stat3 in epidermal cells, and VTP-43742 statistically significant efficacy in psoriatic patients in a phase 2a trial carried out by Vitae Pharmaceuticals (142). A different RORγt inverse agonist, JNJ-54271074, has also been reported to decrease IL-23-induced psoriasis-like skin inflammation (128), and using a translational model of human epidermal and skin-homing T cells from psoriatic patients, JNJ-54271074 inhibited streptococcus extract–induced IL-17A/F.

Using a model of antigen (methylated BSA)-induced arthritis in rats, oral gavage with the RORγt inverse agonist Cpd 1 reduced paw swelling and numbers of IL-17-producing cells in a dose-dependent manner, consistent with Cpd 1–mediated suppression of pathogenic T cell development (127). A different RORγt inverse agonist, JNJ-54271074, suppressed IL-17A production in peripheral blood mononuclear cells (PBMCs) from human rheumatoid arthritis patients and suppressed pathologic scores in collagen-induced arthritis in mice (128). Similarly, the inverse agonist SR2211 has also been reported to diminish inflammation in a collagen-induced arthritis mouse model (143), while MRL-248 and MRL-367 were effective in reducing Th17 function in PBMCs isolated from patients with rheumatoid arthritis (144).

Crohn’s disease (CD) is a chronic and relapsing disease of the intestine that affects more than 4 million people worldwide. While management of this disease has improved in recent years, there is still a need for more effective therapies. However, blocking IL-17 has generally proven to be ineffective, and in some CD patients it has led to worse pathology, possibly because of an increased incidence of intestinal infections (145). The orally available RORγ inhibitor GSK805 diminished inflammation and tissue damage in the colon in response to infection with Citrobacter rodentium (146). Although RORγt promotes the development of both Th17 cells and ILC3s, GSK805 reduced the number of IL-17A-producing cells in the colon lamina propria and mesenteric lymph node without affecting the number of IL-17-producing ILC3s. GSK805 also attenuated spontaneous colonic inflammation in Il10^−/− mice as well as intestinal inflammation resulting from CBir1 T cell transfer. In addition, GSK805 inhibited the number Th17 cells and IL-17 expression in tissues resected from the colon of CD patients (146). Similarly, oral administration of the RORγt inhibitor BI119 improved clinical signs in a colitis model of CD4⁺CD45RB^high T cell transfer into immune-deficient, X-linked, severe combined immunodeficiency (SCID) mice (147). Together, these studies suggest that RORγt inverse agonists might provide an attractive therapeutic approach to treat inflammatory bowel disease and other chronic inflammatory diseases.

While the inhibition of RORγ-dependent transcription has tremendous potential for treating a wide variety of autoimmune diseases, RORγ-deficient mice have a high incidence of developing thymic lymphomas that rapidly metastasize to the liver and spleen (148). Lymphoblastic lymphomas also develop in a conditional knockout mouse model in which the immune system develops normally, with RORγ being deleted when animals reach adulthood (149). In addition, long-term treatment of rats with an inverse agonist (Cpd 1) caused thymic preneoplastic hyperplasia similar to that observed in RORγ-null mice (123,148).It remains to be seen whether long-term treatment with RORγt inverse agonists also causes lymphoma in humans, but if so, this deleterious effect of such compounds might preclude their long-term use. Additional studies will be needed to assess these risks and to weigh them against the great promise short-term inhibition of RORγ has already shown in multiple autoimmune diseases.