Retinoic acid-related Orphan Receptor γ (RORγ): connecting sterol metabolism to regulation of the immune system and autoimmune disease

Abstract

Cholesterol and its metabolites are bioactive lipids that interact with and regulate the activity of various proteins and signaling pathways that are implicated in the control of a variety of physiological and pathological processes. Recent studies revealed that retinoic acid-related orphan receptors, RORα and γ, members of the ligand-dependent nuclear receptor superfamily, exhibit quite a wide binding specificity for a number of sterols. Several cholesterol intermediates and metabolites function as natural ligands of RORα and RORγ and act as agonists or inverse agonists. Changes in cholesterol homeostasis that alter the level or type of sterol metabolites in cells, can either enhance or inhibit ROR transcriptional activity that subsequently result in changes in the physiological processes regulated by RORs, including various immune responses and metabolic pathways. Consequently, this might negatively or positively impact pathologies, in which RORs are implicated, such as autoimmune disease, inflammation, metabolic syndrome, cancer, and several neurological disorders. Best studied are the links between cholesterol metabolism, RORγt activity, and their regulation of Th17 differentiation and autoimmune disease. The discovery that Th17-dependent inflammation is significantly attenuated in RORγ-deficient mice in several experimental autoimmune disease models, initiated a search for ROR modulators that led to the identification of a number of small molecular weight RORγ inverse agonists. The inverse agonists suppress Th17 differentiation and IL-17 production and protect against autoimmunity. Together, these studies suggest that RORγt may provide an attractive therapeutic target in the management of several (inflammatory) diseases.

1. Introduction

The retinoic acid-related orphan receptors alpha, beta, and gamma (RORα-γ encoded by RORA-C or NR1F1-3) constitute a subfamily of nuclear receptors [1–4]. RORs exhibit a typical nuclear receptor domain structure consisting of an N-terminal domain, a highly-conserved DNA-binding domain (DBD) consisting of two C2-C2 zinc finger motifs, a ligand-binding domain (LBD), and a hinge domain spacing the DBD and LBD [1]. The DBD of RORs recognizes ROR response elements (ROREs) consisting of the RGGTCA consensus preceded by an A/T-rich sequence. RORs regulate transcription by binding as monomers to ROREs in the regulatory regions of target genes and the recruitment of co-activators or co-repressors [5,6].

Over the last ten years, it has become evident that RORs function as ligand-dependent transcription factors. RORα/γ transcriptional activity can be modulated by various sterols and synthetic ligands that bind ROR and function either as agonists or inverse agonists [2,4,7–17]. The discovery that ROR transcriptional activity can be modulated by small (synthetic) molecules, opened the possibility that RORs may provide novel therapeutic targets in the management of various pathologies, in which RORs are implicated, such as autoimmune disease and type 2 diabetes. In addition, RORs may provide a target for various xenobiotics and thereby a mechanism by which environmental agents affect immunity and disease. This chapter provides an overview of the various links between cholesterol/sterol metabolism, RORs, and their regulation of immunity, particularly T helper 17 (Th17) cells, and their relationship to inflammatory disease.

2. ROR isoforms have different functions

The activity and function of RORs are controlled at many levels, including the generation of different isoforms. Via alternative splicing and/or alternative promoter usage the human RORA gene generates 4 isoforms (α1–4) and human RORC two isoforms, RORγ1 and RORγt (RORγ2), while human RORB encodes one protein [1,18–21]. These ROR isoforms, which differ only in their N-terminus, exhibit different patterns of expression and regulate distinct genes and physiological processes. A clear example of this is RORγ. RORγ1 is expressed in many peripheral tissues, including adipose, liver, kidney and muscle, where it regulates circadian rhythm, glucose and lipid metabolism [1,4,22–25]. In contrast, RORγt, to is selectively expressed in a variety of immune cells, including CD4⁺CD8⁺ (DP) thymocytes, T helper Th17 cells, type 3 innate lymphoid cells (ILC3), and lymphoid tissue inducer (LTi) cells, which constitute an ILC3 subtype [1,4,19,20,26–30]. RORγt regulates the survival and apoptosis of DP thymocytes and is essential for the development of ILC3 and Th17 cells, and the production of IL-17. RORγt is also required for the generation of LTi cells, which are essential for the development of peripheral lymph nodes and Peyer’s patches. RORγ-deficient mice lack Lti cells and peripheral lymph nodes [19,20]. RORγt is also expressed in several other immune cells, including subtypes of CD8+Tc17, invariant natural killer and γδT-cells, and Foxp3⁺ T regulatory cells (T_reg) [31–36]. Foxp3⁺RORγt⁺ T_regs are preferentially expressed in colon and, as Th17 cells, their induction is regulated by the gut microbiota. However, in contrast to Th17 and ILC3 cells, RORγt appears not to be a significant regulator of IL-17 expression in Foxp3⁺RORγt⁺ T_regs indicating that the control of target gene transcription by RORγ is cell context-dependent. Th17, ILC3, and Foxp3⁺RORγt⁺ T_regs all contribute to intestinal homeostasis and tolerance. Its expression in various immune cell populations with diverse functions indicates that RORγt has multiple roles in regulating immunologic homeostasis and inflammatory disease, including autoimmune disease. The complexity is further complicated by the plasticity of the various immune cell populations.

3. Regulation of ROR activity

The activity and function of RORs can be modulated by various posttranslational modifications (PTMs) [37]. Although the RORs are phosphorylated at multiple sites and can be acetylated, sumoylated, and ubiquitinated, the function of many of these PTMs has not yet been fully elucidated. Phosphorylation by protein kinase A (PKA) and sumoylation of RORα has been reported to positively regulate its transcriptional activity [38,39]. Sumoylation of RORα at Lys²⁴⁰ was enhanced by protein inhibitor of activated STAT (PIAS) proteins and shown to stimulate RORα transcriptional activity. The histone acetylase, p300, acetylates RORγ at several positions, while the deacetylase SIRT1 promotes deacetylation of RORγ and increases its transcriptional activity, which consequently enhances the generation of Th17 cells and promotes autoimmunity [40]. The HECT E3 ubiquitin ligase, ITCH, was found to bind RORγ and mediate its K48 poly-ubiquitination and subsequently its proteasomal degradation [41]. A recent study showed that a two-amino acid substitution in the hinge domain of RORγ (Ser⁹²Ala-Leu⁹³Ala) decreased ubiquitination at Lys⁶⁹ within the RORγ DBD; however, this ubiquitination did not affect RORγt protein stability [42]. Mice containing this mutation exhibited defective Th17 differentiation, while thymopoiesis and lymph node genesis were not affected, except for the development of Peyer’s patches. ILC3 cells development was not disrupted in these mutant mice, but cells did not produce IL-17. The mechanism of this selectivity is not fully understood and might be due to differences in promoter context of target genes or differential expression and/or recruitment of transcriptional mediators in different RORγ expressing cells.

4. Links between sterols and ROR signaling

Agonists and inverse agonists provide another mechanism to regulate ROR activity. A clear connection has been established between cholesterol intermediates/metabolites and their regulation of ROR activity. The first evidence for a link between RORs and sterols came from crystallography studies showing that cholesterol and cholesterol sulfate bind to the ligand-binding pocket of RORα and function as RORα agonists [7,43]. Subsequent studies demonstrated that several intermediates of the cholesterol biosynthesis pathway bind RORγ and enhance RORγ activity suggesting that they may function as endogenous ligands for RORγ [8,11] (Figure 1). These studies identified zymosterone, zymosterol, 7-dehydrocholesterol, and desmosterol among the most effective sterols activating RORγ, while lanosterol, T-MAS, and cholesterol, were weak agonists. Cholesterol and other sterols can be metabolized by a number of cytochrome P450 enzymes, including CYP27A1, CYP46A1, CYP7A1 and CYP7B1, that lead to formation of a wide range of oxysterols [44–46]. CYP7A1 catalyzes the formation of 7-keto- and 7α-hydroxysterols, while CYP27A1 catalyzes the formation of 25- and 27-hydroxysterols, and CYP46A1 the formation of hydroxysterols, such as 24(S)-hydroxycholesterol. The formation of 25-hydroxycholesterol can also be catalyzed by cholesterol 25-hydroxylase (Ch25h). A number of these oxysterols can act as ligands for RORα and RORγ [8–13,47–49]. These reports demonstrated that 7α- and 7β-hydroxycholesterol, 7-ketocholesterol, and 24S- and 24R-hydroxycholesterol, and 24,25-epoxycholesterol function as inverse agonists for RORα and/or RORγ, while 25-hydroxycholesterol, 27-hydroxycholesterol, 20α-hydroxycholesterol, 22R-hydroxycholesterol, 7α,27- and 7β,27-dihydroxycholesterol act as agonists of RORγ [2,10,45,50]. In addition to being hydroxylated, sterols can be sulfoconjugated by sulfotransferase SULT2B1 and generate sulfated sterols, such as cholesterol- and desmosterol sulfate, which function as RORα/γ agonists [7,8].

Figure 1. Intermediates of the cholesterol biosynthetic pathway function as RORγ agonists.

Shown is a schematic view of the Bloch cholesterol biosynthetic pathway. In peroxisomes, acetyl CoA is converted to mevalonate by HMGCR, a step that is inhibited by statins, and then via multiple steps into farnesyl pyrophosphate, which is then converted into squalene in the endoplasmic reticulum. This is subsequently converted into cholesterol via the steps indicated. Several cholesterol intermediates can act as RORγ agonists, but zymosterol and desmosterol exhibit the highest activity. Deficiency in Fdft1 or Cyp51A1, enzymes acting upstream in the cholesterol biosynthetic pathway, inhibit the synthesis of cholesterol and the availability of RORγ agonists. DHCR24 can convert 7-dehydrodesmosterol to 7-dehydrocholesterol, which acts as an agonist of RORγ. ACAT1, Acetyl-CoA Acetyltransferase 1; HMGCS1, 3-Hydroxy-3-Methylglutaryl-CoA Synthase 1; HMGCR, 3-Hydroxy-3-Methylglutaryl-CoA Reductase; FDT1, Farnesyl-Diphosphate Farnesyltransferase 1; SQLE, Squalene Epoxidase; LSS, Lanosterol Synthase; TM7SF2, Transmembrane 7 Superfamily Member 2; FAXDC2/SC4MOL, Fatty Acid Hydroxylase Domain Containing 2; NSDHL, NAD(P) Dependent Steroid Dehydrogenase-Like; HSD17B7, Hydroxysteroid 17-Beta Dehydrogenase 7; EBP, Emopamil Binding Protein; SC5D, Sterol-C5-Desaturase; DHCR7, 7-Dehydrocholesterol Reductase; DHCR24, 24-Dehydrocholesterol Reductase.

Triterpenes, which are produced by many plants, fruits, herbs, and fungi, are structurally closely related to sterols (Figure 2). Several triterpenes have been reported to bind RORγ and function as natural RORγ ligands. Among them, ursolic acid, which functions as an RORγ inverse agonist [51] and ganoderone A, an antiviral triterpenoid produced by the fungus Ganoderma pfeifferi, which was recently identified as a very potent RORγ agonist [16](Figure 2).

Figure 2.

Chemical structure of two sterol RORγ agonists, cholesterol sulfate, 25-hydroxycholesterol, and two triterpenes, ganoderone A and ursolic acid, which act as an RORγ agonist and RORγ inverse agonist, respectively.

As was demonstrated for the binding of cholesterol sulfate to the RORα(LBD) [7], X-ray crystal structure analysis confirmed the interaction of cholesterol sulfate and hydroxysterols with the ligand binding pocket of RORγ [9,16,52,53]. Studies examining the crystal structures of apo (unbound) RORγ(LBD) showed that, in the absence of ligand, RORγ is in an active conformation that is capable of recruiting co-activators [9,16,52]. Study of X-ray structures of agonist-bound RORγ(LBD) further revealed that binding of agonists stabilizes the active conformation of RORγ and its interaction with coactivators, whereas most inverse agonists do the opposite. The hydrogen bond between Hε2-His⁴⁷⁹ in helix 11 and OH-Tyr⁵⁰² in helix 12 (referred to as His-Tyr lock) plays a critical role in stabilizing the active conformation of RORγt. These studies further indicated that this stabilization involves the formation of different bonds with RORγ [9,16,52]. For example, 25-hydroxycholesterol stabilizes H12 by interacting with Tyr⁵⁰² indirectly via a water-mediated hydrogen bond with its 25-OH group leaving the His-Tyr lock in place, whereas ganoderone A holds helix 12 (H12) in the agonist position through the formation of two strong hydrogen bonds with His⁴⁷⁹ and Tyr⁵⁰², thereby eliminating the His-Tyr lock. It is conceivable that such differences in agonist-RORγ interaction might bring about different changes in RORγ conformation that affect the specificity by which RORγ recruits co-activator complexes and activates target gene transcription. Consequently, this might affect the regulation of RORγ functions as well.

5. Vitamin D and lumisterol metabolites and RORs

Recently, several vitamin D3 and lumisterol metabolites were identified that function as weak RORα/γ inverse agonists [15,53–55]. Previtamin D3, and subsequently vitamin D3, are formed in the skin from 7-dehydrocholesterol upon photochemical transformation by UVB. In the classical pathway, CYP2R1 and CYP27B1 convert vitamin D3 to 1,25(OH)D3, the active form that binds the vitamin D receptor (VDR), but also acts as a weak inverse agonist of RORγ [11,15,53](Figure 3). In an alternative pathway in the skin, CYP11A1 catalyzes the formation of additional D3 metabolites, including 20(OH)D3, 22(OH)D3, 20,23(OH)D3, 20,22(OH)D3 and 17,20,23(OH)D3 [54,56]. Some of these metabolites are further hydroxylated by CYP27B1 at C1α, by CYP24A1 and CYP27A1 at C24, C25, and C26 [54,56,57]. Several of the D3 metabolites, including 20(OH)D3, 20,23(OH)₂D3, 1,20,25(OH)₃D3, 17,20,23(OH)₃D3, 1,20(OH)₂D3, and 20,26(OH)₂D3 were able to inhibit RORE-mediated transactivation as well as the interaction between the RORα/γ LBD with an LXXLL coactivator peptide [11,15,53]. Molecular modeling using established crystal structures of RORα and RORγ LBDs, further showed that these inverse agonists exhibit high docking scores supporting interaction of these vitamin D3 metabolites with the ligand binding pocket of RORα/γ [15,53]. These findings suggest these noncalcemic D3 metabolites, which are produced endogenously, can act as RORα/γ inverse agonists and modulate their activity and functions. These studies indicate that, in addition to VDR, RORs may provide an alternative pathway by which D3 and its metabolites regulate physiological processes. Previtamin D3 can also undergo photoisomerization to lumisterol (L3) under the influence of UVB [56], which then can undergo sequential hydroxylations by CYP11A1 producing 20(OH)L3, 22(OH)L3, and 20,22(OH)₂L3 [55](Figure 3). Cell-based and in vitro analyses demonstrated that several hydroxylumisterols exhibit inverse agonist activity, while molecular modeling supported the hypothesis that they act as ligands of RORα and RORγ [55].

Figure 3. Hydroxylated derivatives of vitamin D function as weak RORα/RORγ inverse agonists.

After absorption of UVB the RORγ agonist, 7-dehydrocholesterol, is phototransformed to vitamin D (D3) and lumisterol (L3). Vitamin D is activated through sequential hydroxylation at C25 and C1α to produce 1,25 (OH)2D3 that, in addition of acting on vitamin D receptor (VDR), also serves as an inverse agonist of RORs. Vitamin D3 and lumisterol can also be hydroxylated by CYP11A1 to produce hydroxylumisterols or hydroxyderivatives of vitamin D that can further be hydroxylated by other CYPs to produce hydroxyderivatives that can also act as inverse agonists of RORs. Weak inverse agonists shown in red; 7DHC, RORγ agonist 7-dehydrocholesterol.

6. Disruption of cholesterol biosynthesis and RORα/γ activity

The discovery that various sterols function as ligands, predicted that alterations in sterol homeostasis that change the intracellular pool of ROR (inverse) agonists, would affect ROR transcriptional activity and as a consequence the physiological as well as pathological processes in which RORs are implicated. This hypothesis was strongly supported by studies examining the effect of disruption of cholesterol biosynthesis on RORγt-dependent regulation of Th17 differentiation and gene expression.

RORγt is required for the differentiation of naïve T cells into Th17 cells and for the transcriptional activation of Il17A/F, IL21, and Il23R (Figure 4A). Interestingly, during Th17 differentiation, the expression of several cholesterol biosynthetic genes (e.g., FDFT1, LSS, and DHCR24) are enhanced, whereas that of genes involved in cholesterol metabolism (e.g., CYP7A1, CYP27A1) and efflux (e.g., ABCA1, ABCG1) are decreased [8]. In addition, Th17 differentiation is accompanied with an increase in the expression of the sulfotransferase SULT2B1 and a decrease in sterol sulfatase (STS) expression [8]. A positive correlation between IL-17 levels and the expression of several cholesterol biosynthesis-related genes was also revealed by transcriptome analysis of RNA isolated from normal and psoriatic skin biopsies [58]. Together, these findings are consistent with the concept that increased cholesterol biosynthetic gene expression would enhance the availability of RORγt agonists and subsequently stimulate RORγt activity, Th17 differentiation, and IL-17 production, and consequently promote inflammatory disease.

Figure 4. Changes in cholesterol/sterol homeostasis alter RORγ transcriptional activity and subsequently the regulation of gene expression, physiological functions and disease, in which RORγ is involved.

A. The formation of RORγt agonists by the cholesterol biosynthetic pathway leads to activation of RORγt, which is required for Th17 differentiation and the expression of target genes, such as Il17, Il21, and Il23R. The HMG-CoA reductase inhibitors, statins, inhibit the generation of RORγt agonists resulting in reduced RORγt activation and Th17 differentiation. Statins also reduce RORγt expression and Th17 differentiation by inhibiting STAT3, which is required for RORγt transcription. The scavenger receptor CD5L is expressed at high levels in nonpathogenic Th17 cells, where it suppresses cholesterol biosynthesis and the formation of RORγt agonists. It becomes expressed at low levels in pathogenic Th17 cells leading to increased cholesterol biosynthesis, generation of RORγt agonists, and pathogenicity of Th17 cells. B. Inhibition of CYP51A1 by azoles, deficiency in CYP51A1 or FDFT1 inhibit cholesterol biosynthesis, thereby reducing the availability of RORγ agonists and RORγt activation. Statins and CD5L have a negative effect on cholesterol biosynthesis and RORγt activation. This subsequently reduces Th17 differentiation and Il17 expression, and suppresses the development of inflammatory disease. Other mechanisms that affects the generation of RORγt agonists are CYP27A1 deficiency, which reduces the generation of 27-hydroxycholesterol. Induction of ABCG1/A1 expression by LXRs increase cholesterol efflux and potentially reduce the availability of RORγt agonists and Il17 expression, whereas ApoE-deficiency and high fat/cholesterol diet (HFD) have the opposite effect by increasing cholesterol levels.

Several studies have demonstrated that disruption of the cholesterol biosynthesis pathway has a negative effect on RORγt activation and Th17 differentiation (Figure 4A and B). The cholesterol-lowering drugs, statins, bind to and inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR), which catalyzes the conversion of HMG-CoA to mevalonic acid, one of the first steps in cholesterol biosynthesis. Statins have been shown to inhibit Th17 differentiation and the expression of inflammatory cytokines [59]. This appears to involve several mechanisms including up-regulation of suppressor of cytokine secretion (SOCS3 and -7) and reduced activation of STAT3 and RORγt expression. In addition, the reduction in cholesterol biosynthesis by statins might diminish the generation of RORγt agonists, thereby reducing RORγt activation, RORγt-mediated Th17 differentiation, and activation of Il17 (Figure 4A). The formation of squalene by squalene synthase (Fdft1) is another early step in the cholesterol biosynthetic pathway. A study examining the effect of disruption of squalene synthesis on RORγt activation demonstrated that the RORγ-mediated transactivation of a RORE-driven reporter was greatly reduced in a squalene synthase (Fdft1)-deficient cell line, SXLT, while exogenous expression of Fdft1 or the addition of squalene restored RORγt-mediated transactivation of the reporter [11] (Figure 4B).

Disruption of 14α-demethylase cytochrome P450, Cyp51a1 (lanosterol 14α-demethylase), either in Cyp51a1-deficient mice or in cells treated with Cyp51a1 inhibitors, such as azole-type fungicides, resulted in decreased synthesis of zymosterol and desmosterol and a significant reduction in RORγ-mediated transactivation and consequently in Th17 differentiation and IL-17 expression [8,11,60](Figure 4B). Consistent with this are similarities in the phenotype between Cyp51a1- and RORγ-deficient mice. Branchial lymph node anlagen were absent in 75% of Cyp51a1^−/− mice and the number of IL17RA⁺ and CD4⁺ Lti cells was reduced. A recent study of a hepatocyte-specific knockout of Cyp51a1 in mice, referred to as H^Cyp51−/−, showed development of progressive liver injury and fibrosis [61]. Hepatic cholesterol biosynthesis was disrupted in these mice leading to lower levels of hepatic and blood cholesterol, as well as several cholesterol intermediates, including zymosterol and desmosterol, which act as ROR agonists. This reduced agonist levels are likely responsible for the observed reduction in RORα and RORγ transcriptional activity. In addition, in some of alterations in hepatic gene expression in H^Cyp51−/− mice overlapped with ROR target genes consistent with the concept that some of these changes are due to reduced RORα/γ activation.

7. Regulation of RORγ activity by other sterol metabolic pathways

Cytochrome P450 enzymes play a key role in the regulation of various aspects of cholesterol homeostasis and many catalyze the formation of oxysterols, several of which act as (inverse) antagonists of RORγt. CYP27A1 catalyzes the formation of 27-hydroxysterols, including 27-hydroxycholesterol, 7α, 27- and 7β, 27-dihydroxycholesterol. These hydroxysterols, which were reported to be preferentially produced by mouse Th17 cells, all act as RORγt agonists and stimulate Th17 differentiation [10]. The production of 27-hydroxycholesterols in Cyp27a1-deficient Th17 cells was greatly diminished as was the number of IL-17 producing cells in Cyp27a1-deficient mice suggesting that 27-hydroxysterols are an important source of RORγt agonists in these cells (Figure 4B). However, these findings appear at odds with another study showing that enzymes involved in oxysterol formation are expressed at low levels during Th17 differentiation [8].

One might expect that hypercholesterolemic conditions lead to an increase in endogenous sterol levels and availability of ROR agonists. This would subsequently results in increased activation of RORγt and promote, for example, Th17 differentiation and the production of the inflammatory cytokine IL-17A. Low cholesterol diet might do the opposite. This hypothesis is supported by several reports. Apolipoprotein E (ApoE)-deficient mice have increased levels of total cholesterol and LDL-cholesterol. These mice spontaneously develop atherosclerosis and show an increased susceptibility to type II collagen-induced arthritis [62]. Since Th17 cells and the production of inflammatory cytokines, such as IL17A, play a critical role in the development of these pathologies, the increased susceptibility to these diseases might be in part related to the elevated blood levels of cholesterol that lead to an increase in RORγ agonists and Th17 cells [62](Figure 4B). A different study reported that patients with chronic hepatitis C, when placed on a normocaloric, low cholesterol diet showed a significant reduction in Th17 cells and IL-17 levels, which might at least in part be related to reduced RORγt activation [63].

CD5-like (CD5L/AIM), a member of the scavenger receptor cysteine-rich superfamily, is highly expressed in non-pathogenic Th17 cells, where it regulates lipid synthesis and modulates intracellular lipid homeostasis, but is down-regulated in pathogenic Th17 cells (Fig. 4A)[64]. CD5L reduces the content of polyunsaturated fatty acid (PUFA) in favor of saturated fatty acids (SFA) and limits cholesterol biosynthesis. The low expression of CD5L in pathogenic Th17 cells increases cholesterol biosynthesis and the generation of RORγt agonists, and promotes their pathogenicity. Similarly, deficiency in CD5L in mice leads to increased PUFA content and expression of several cholesterol biosynthetic enzymes, such as Cyp51a1 and Sc4mol, in Th17 cells leading to increased availability of RORγt agonists (Figure 4B). Consequently, this leads to RORγt-dependent transactivation of Il17 and Il23r promoters, thereby promoting a shift from non-pathogenic to pathogenic Th17 cells.

RORα/γ activity might be influenced by a number of other cholesterol regulatory pathways, including sterol regulatory element binding protein 2 (SREBP2) and the LXR nuclear receptors [45,65–67]. The transcription factor SREBP2 regulates cholesterol biosynthesis in part by increasing the transcription of several genes involved in cholesterol biosynthesis, including HMGCR and HMGCS, thereby enhancing the availability of RORγ agonists [66,67]. Activation of the T cell receptor (TCR) pathway results in activation of SREBP in favor of sterol-sulfate and cholesterol synthesis and might synergize with RORγ in promoting Th17 differentiation and IL-17 synthesis [8,68]. LXRs play a critical role in cholesterol homeostasis by regulating the expression of ABCA1 and ABCG1, which mediate cholesterol efflux [45,65]. Thus, LXR agonists reduce cholesterol levels in cells thereby reducing the availability of RORα/γ agonists (Figure 4B).

8. Regulation of sterol metabolism by RORs

In addition to being regulated by sterols, RORs themselves regulate the expression of a number of genes involved in cholesterol transport and sterol metabolism. The hepatic expression of ABCA1 and ABCG1, encoding reverse cholesterol transporters, is reduced in the liver of RORα-deficient mice [69]. In HUVEC endothelial cells and the monocytic cell line, THP-1, ABCA1 was found to be directly regulated by RORα [70]. Treatment with an inverse RORα agonist was shown to decrease plasma cholesterol levels and suppress atherosclerosis development in mice, which appeared in part to be related to increased ABCG5/G8 expression and cholesterol excretion by the intestine [71]. The hepatic expression of sterol 12α-hydroxylase, CYP8B1, and 7α hydroxylase, CYP7B1, which are involved in the conversion of cholesterol into bile acids and the regulation of hepatic and serum cholesterol levels, have been reported to be directly regulated by RORα and/or RORγ [22,25,72]. RORα regulates CYP8B1 by directly binding to an RORE in its promoter and through the recruitment of the co-activator, CBP [72].

In addition, RORα/γ have been implicated in the circadian regulation of several 3β-hydroxysteroid dehydrogenases and sterol sulfotransferases [22,25]. RORα/γ positively regulate the hepatic expression of the hydroxysteroid sulfotransferase, Sult1B1, and steroid/bile acid sulfotransferase, Sult2A1 [22,73,74]. Sult2A1 transcription was shown to be regulated directly by RORα and RORγ [73]. In macrophages, RORα regulates cholesterol 25-hydroxylase (Ch25h), which converts cholesterol to 25-hydroxycholesterol, and promotes phagocytosis [50]. Regulation of sterol metabolic genes by RORs may enhance or reduce the synthesis of agonists or inverse agonist thereby modulating RORγ activity, thereby creating a positive or negative feedback loop. Whether the regulation of sterol metabolic genes by RORs affects the availability of ROR (inverse) agonists and therefore ROR activation, needs further study.

9. Synthetic RORγ ligands

Genetic deficiency of RORγt in mice greatly affects Th17 cell differentiation and Il17 expression and protects mice against Th17-dependent inflammation in several experimental autoimmune disease models [75–78]. This, together with the discovery that RORs act as ligand-dependent transcription factors, prompted a search for synthetic ROR inverse agonists with therapeutic potential. Screens of chemical libraries for ROR modulators identified several series of synthetic small molecular weight molecules that function either as an agonist or inverse agonist [2,17,48,79–91]. Recently, a synthetic RORγ agonist, SR0987, was also described [92]. Chemical structures of several synthetic ROR inverse agonists are shown in Figure 5. For a more comprehensive overview, we refer to several recent reviews [2,4,12–14].

Figure 5. Chemical structures of several RORγ inverse agonists.

SR1001 (Scripps Institute) is a dual RORα/RORγ inverse agonist, while all others are RORγ-selective inverse agonists.

X-ray crystallography of the RORγ(LBD) in complex with various inverse agonists suggested the existence of distinct classes [16,93,94]. Most inverse agonists bind the canonical ligand binding pocket of RORγ; however, these interactions can be distinguished on basis of the formation of different bonds with RORγ. In one class, binding of the inverse agonist causes repositioning of H12 that disrupts interaction with co-activators. Binding of a second class of inverse agonists involves a “water trapping” mechanism, in which H12 remains in the active conformation and still allows interaction with co-activator peptides [16]. A different study revealed still another type of antagonism [93]. Indazole-type inverse agonists, such as MRL-871, do not interact with the canonical, orthosteric ligand binding pocket, but with a different, distal, allosteric binding site in RORγ(LBD). MRL-871 reorients H12 and actively blocks binding of cofactors. Molecular studies revealed two additional modes of action by which inverse agonists can inhibit RORγ transcriptional activity. The inverse agonists TMP778, TMP920, and GSK805 interact physically with the canonical ligand-binding pocket of RORγt and inhibit Th17 differentiation; however, interaction with TMP920 disrupts RORγt binding to DNA, whereas TMP778- and GSK805-bound RORγt are still associated with target genes and actually stabilized RORγt binding to a number of new genomic sites, suggesting that RORγ binding to DNA remains largely intact [17]. Similarly, the inverse agonist Cpd1 did not affect the binding of RORγ to ROREs (Guntermann, 2017 #1276}. Binding of RORγt to the Il17a and Il23r promoter was found to be associated with a more open chromatin structure as indicated by H3AcK9/K14 acetylation and H3K4me3 methylation at these loci. This histone methylation and acetylation is downregulated in the presence of Cpd1 suggesting a less open chromatin structure.

10. RORγ as therapeutic target

Treatment of cells and rodents with synthetic RORγ inverse agonists induces many of the same effects as observed in RORγ null mice, including inhibition of Th17 differentiation, RORE-mediated transactivation, activation of the Il17 transcription, and IL-17 production [2,4,17,19,20,95–98]. Moreover, as observed in RORγ null mice, RORγ inverse agonists greatly alleviate Th17-dependent inflammation in several experimental autoimmune disease models in rodents [17,51,76,89,96,98–102]. The dual RORα/RORγ inverse agonist SR1001 and the RORγ-selective inverse agonists, ursolic acid, digoxin, MRL-248, TMP778, TMP920, have been reported to ameliorate Th17-mediated experimental autoimmune encephalomyelitis (EAE) in mice and rats [17,51,103–106]. Treatment with inverse RORγ agonists Bio-0554019, GSK2981278, TMP778, VTP-43742, JNJ-54271074 attenuated skin inflammation in imiquimod-induced psoriatic model as well as in other psoriatic models, the IL-23-injection model and K5.Stat3C transgenic mice [98–100,105,107–109]. Administration of the inverse RORα/RORγ inverse agonist SR1001 to NOD mice, a model of type 1 diabetes, significantly reduced diabetes incidence and insulitis [101], while treatment of obese diabetic mice with SR1555 caused a significant reduction in fat mass and improved insulin sensitivity [110]. A different study showed that Cpd1 reduced hypersensitivity responses in a methylated bovine serum albumin–induced skin inflammation model in rats [102]. The inverse agonists, JNJ-54271074 and SR2211, have been reported to inhibit inflammation in a collagen-induced arthritis mouse model [89,99] and Cpd1 in antigen-induced arthritis in rats [102]. In an experimental murine model, treatment with TMP778 markedly alleviated chronic graft-versus-host disease (cGvHD), which represents a major complication of allogeneic stem cell transplantation, and may provide a new therapeutic approach in the management of cGvHD [111].

It is to be expected that treatment with RORγ inverse agonists would also affect the function of RORγ in other RORγ⁺ immune cells, including DP thymocytes, ILC3s, RORt⁺T_regs, and RORt⁺γδ. Treatment of mouse thymocytes with the inverse agonists was shown to down-regulate Bcl2l1 expression and to increase apoptosis, while treatment of mice or rats with, respectively, the RORγ inverse agonists MRL-248 or Cpd1, causes a reduction in the number of total thymocytes and perturbs DP thymocyte survival [97,105], as observed in RORγ null mice [19,20]. Moreover, administration of MRL-248 in mice resulted in skewed cell receptor alpha (TCRα) gene rearrangements and limited T cell repertoire diversity [105]. In a myelin oligodendrocyte glycoprotein (MOG)-induced EAE model, MRL-248 reduced the number of MOG-specific CD4⁺ T cells and delayed autoimmune EAE progression. Although ILC3s can be pro-inflammatory, RORγt⁺ ILC3s play a critical role in intestinal immunity and protect against infection-mediated inflammation [112]. In response to infection with Citrobacter rodentium, Th17 cell responses and intestinal inflammation were significantly inhibited in mice treated with the inverse agonist GSK805, while cytokine expression in ILC3s, including IL-17, was largely maintained [113]. Thus RORγ inverse agonists might provide an attractive strategy to treat inflammatory bowel disease.

The discovery that treatment with small molecular weight RORγ inverse agonists protects against autoimmune disease in several experimental rodent models initiated a number of clinical trials [109,114,115]. Vitae Pharmaceuticals/Allergan successfully finished its Phase 1 trial with VTP-43742 (Figure 5) and a Phase 2a trial showed statistical significant efficacy in psoriatic patients [115]. These studies suggest that RORγt inverse agonist might provide an attractive therapeutic alternative in the management of inflammatory autoimmune disease.

11. Lymphoma concerns

Ueda et al. [116] reported that RORγ-deficient mice develop thymic lymphomas within three months of age that was confirmed in a later study [117]. Knockout of RORγ in adult mice using conditional RORγ knockout mice also leads to thymic lymphoma development; however, in mice, in which the Rorc locus was incompletely deleted, RORγ-deficient thymocytes were rapidly replaced by wild type thymocytes [117]. These findings raised concerns whether therapeutic treatment with RORγ antagonists increases the risk of developing T cell lymphoma. Chronic treatment of rats with the RORγ inverse agonist Cpd1 was recently shown to cause progressive thymic changes that resemble those observed in RORγ-deficient mice [97]. This study further showed that one rat developed preneoplastic, hypertrophic lesions in the cortical thymus. Although this may not occur in humans, these studies point at an apparent risk of T cell lymphoma with chronic RORγ inverse agonist treatment at least in rodents.

Although these concerns may be real, the observed diversity in modes of interaction of inverse agonists with RORγ(LBD) provides hope that future studies might be able to identify inverse agonists that target certain biological processes more specifically thereby minimizing possible side effects of therapeutic treatment [16,17,93]. A recent study [42] showing that a two-amino-acid substitution in RORγt disrupts Th17 differentiation, but not thymocyte development, is consistent with this prospect.

12. Further considerations

Multiple T helper cell lineages and immune cell types contribute to immunopathology. As mentioned above RORγt is expressed in a number of immune cell subtypes with pathogenic as well as protective functions, including Th17, RORt⁺T_regs, RORt⁺γδ, and ILC3s. For example, Th17 cells have a pro-inflammatory role in the development of autoimmune disease, but are also key players in host defense against extracellular pathogens [78,118]. In the gut, a delicate balance exists between anti- and pro-inflammatory mechanisms that maintain the epithelial barrier integrity and control intestinal homeostasis and inflammation. This involves complex, mutual interactions between the gut microbiota and different immune cells, such as Th17, RORt⁺T_regs, RORt⁺γδ, and ILC3s (reviewed in [31,35,112,119]). How RORγt inverse agonists affect these interactions is not yet fully understood.

Adding to this complexity is that microbiota, metabolism, and immunity, are also regulated by the circadian clock [120–122] and in several tissues ROR expression is under the control of the circadian clock and expressed in a rhythmic pattern [4,123]. In addition, RORs themselves participate in the regulation of circadian clock genes [4,123–125]. Together, these findings suggest that timing of administering inverse agonists to treat autoimmune disease could be important and that the greatest therapeutic benefits might be obtained at the circadian time of optimal RORγt expression.

One also needs to take into account that changes in sterol homeostasis influences the activity of many other signaling pathways [45,66]. Cholesterol is critical for membrane fluidity of the plasma membrane and can interact with and regulate the activity of many other proteins, including G protein-coupled receptors, such as GPR183 [126] and Smoothened (SMO), a component of Hedgehog signaling [127], and several other nuclear receptors, such as LXRs [45,128]. Several (inverse) agonists have been reported to interact with both LXRs and RORs. It is therefore likely that the regulation of physiological processes by RORs intersects with many of the other cholesterol/sterol mediated regulatory signaling pathways.

13. Concluding remarks

An increasing number of studies have demonstrated that RORγt has multiple functions in immunity and immunopathology. Autoimmune disease is a complex disease in which metabolism, the microbiome, circadian clock, and multiple cell immune cells play mutually dependent roles. Further studies are needed to understand the precise molecular mechanisms underlying these interrelationships and how loss of RORγt function affects these interactions and immunopathologies. The discovery that loss of RORγt function protects against autoimmune disease and the identification of synthetic RORγ inverse agonists that protect against autoimmune disease in several experimental rodent models has made RORγ a conceivable therapeutic target. Successful phase 1 and phase 2 clinical trials suggest that RORγ inverse agonists may offer an attractive alternative therapy for autoimmune disease. To what extent the risk of developing lymphoma is a concern needs further study. Discovery of inverse agonists that target RORγt-regulated physiological processes more specifically may get around certain side effects. Like RORγt, RORα is also expressed in a number of immune cells, including Th17 and ILC2s; however, its role in immunity and immunopathology is less well understood. Identification of high affinity RORα (inverse) agonists will not only increase our understanding of the role of RORα in immunity and immune disease, but may also uncover its therapeutic potential.