Suppression of TH17 differentiation and autoimmunity by a synthetic ROR ligand

Abstract

T-helper cells that produce interleukin-17 (T(H)17 cells) are a recently identified CD4(+) T-cell subset with characterized pathological roles in autoimmune diseases. The nuclear receptors retinoic-acid-receptor-related orphan receptors α and γt (RORα and RORγt, respectively) have indispensible roles in the development of this cell type. Here we present SR1001, a high-affinity synthetic ligand-the first in a new class of compound-that is specific to both RORα and RORγt and which inhibits T(H)17 cell differentiation and function. SR1001 binds specifically to the ligand-binding domains of RORα and RORγt, inducing a conformational change within the ligand-binding domain that encompasses the repositioning of helix 12 and leads to diminished affinity for co-activators and increased affinity for co-repressors, resulting in suppression of the receptors' transcriptional activity. SR1001 inhibited the development of murine T(H)17 cells, as demonstrated by inhibition of interleukin-17A gene expression and protein production. Furthermore, SR1001 inhibited the expression of cytokines when added to differentiated murine or human T(H)17 cells. Finally, SR1001 effectively suppressed the clinical severity of autoimmune disease in mice. Our data demonstrate the feasibility of targeting the orphan receptors RORα and RORγt to inhibit specifically T(H)17 cell differentiation and function, and indicate that this novel class of compound has potential utility in the treatment of autoimmune diseases.

Figure 1. SR1001 is a selective RORα and RORγ inverse agonist

a, Structure of SR1001 and T0901317 (T1317). b, GAL4-LXRα, GAL4-RORα, and GAL4-RORγ cotransfection assays in HEK293 cells comparing T1317 to SR1001 (n=8). c, Competition radioligand binding assays illustrating the direct binding of SR1001 to the LBD of RORα and RORγ relative to [³H]25-hydroxycholesterol (n=4). d, SR1001 dose-dependently inhibits an Il17 promoter-driven luciferase construct in the presence of RORα or RORγt in HEK293 cells. Results are normalized to vehicle (DMSO) control (n=4). e, AlphaScreen assay indicating SR1001 dose-dependently inhibits the recruitment of a TRAP220 NR box 2 peptide to the LBD of RORγ (n=3). Error bars denote sem.

Figure 2. SR1001 modulates the expression of ROR target genes by decreasing coactivator recruitment

a, Il17a, Rora, and Rorc mRNA expression in EL4 cells treated with control (C), or mouse RORα/γ siRNA, vehicle (DMSO), or SR1001 (10µM, 24 hours) (n=3). Protein expression of RORα and RORγ is shown. *P<0.05; ***P<0.005. b, ChIP-reCHIP assay in EL4 cells illustrating that SR1001 reduces b, RORα- and c, RORγ-dependent recruitment of SRC-2 and promotes recruitment of NCoR to the Il17 promoter. d, Illustration of the HDX kinetics of peptic peptides derived from the RORγ LBD.. Cyan indicates an increase in protection to exchange; yellow represents a decrease in protection to exchange. Error bars denote sem.

Figure 3. SR1001 inhibits the expression of cytokines expressed by T_H17 cells

Il17a, Il17f, Il21, and Il22 mRNA expression in splenocytes differentiated under T_H17 polarizing conditions in the presence of vehicle (DMSO) or SR1001 (5µM) for 5 days. mRNA expression levels are normalized to Gapdh (n=3). Error bars denote sem.

Figure 4. SR1001 inhibits T_H17 cell development and IL-17A secretion

a, IL-17 expression in splenocytes cultured under T_H17 polarizing conditions with vehicle control (DMSO) or SR1001 (5µM). Graphs represent the average percentage of IL-17A expressing cells normalized to vehicle control (n=3). b, IL-17 expression in differentiated purified naïve murine CD4⁺ T cells normalized to vehicle control (n=3). c, IL-17A secretion from splenocytes cultured under T_H17 polarizing conditions with SR1001 (5µM) for 3 days (n=3). d, Intracellular IL-17A expression in hPBMCs cultured for 24 hours with vehicle or SR1001 (5µM) (n=3). e, Treatment with SR1001 suppresses the clinical severity of EAE: vehicle (●, n=12) or SR1001 (25 mg kg⁻¹) (▲, n=10). f, Il17a, Il21, and Il22 mRNA expression from spinal cords of sham-operated (IFA), vehicle control (V), or drug treated (SR1001) mice. (n=4). Error bars denote sem. ***P <0.001; **P <0.01; and *P <0.05.