Suppression of TH17 differentiation and autoimmunity by a synthetic ROR ligand

Journal name:
Nature
Volume:
472,
Pages:
491–494
Date published:
DOI:
doi:10.1038/nature10075
Received
Accepted
Published online

T-helper cells that produce interleukin-17 (TH17 cells) are a recently identified CD4+ T-cell subset with characterized pathological roles in autoimmune diseases1, 2, 3. 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 type4, 5, 6, 7. 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 TH17 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 TH17 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 TH17 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 TH17 cell differentiation and function, and indicate that this novel class of compound has potential utility in the treatment of autoimmune diseases.

At a glance

Figures

  1. SR1001 is a selective ROR[agr] and ROR[ggr] inverse agonist.
    Figure 1: SR1001 is a selective RORα and RORγ inverse agonist.

    a, Structure of SR1001 and T0901317. b, GAL4–LXRα, GAL4–RORα and GAL4–RORγ co-transfection assays in HEK293 cells comparing T0901317 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). c.p.m., counts per minute. 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, Alpha screen assay indicating that SR1001 dose-dependently inhibits the recruitment of a TRAP220 NR box 2 peptide to the LBD of RORγ (n = 3). All error bars denote s.e.m.

  2. SR1001 modulates the expression of ROR target genes by decreasing co-activator recruitment.
    Figure 2: SR1001 modulates the expression of ROR target genes by decreasing co-activator recruitment.

    a, Il17a, Rora and Rorc mRNA expression in EL4 cells treated with control (C) or mouse RORα/γ siRNA, and vehicle (DMSO) or SR1001 (10μM, 24h) (n = 3). Protein expression of RORα and RORγ is shown. *, P<0.05; ***, P<0.005. Error bars denote s.e.m. b, c, ChIP-reChIP assay in EL4 cells showing that SR1001 reduces RORα-dependent (b) and RORγ-dependent (c) recruitment of SRC2 and promotes the 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.

  3. SR1001 inhibits the expression of cytokines expressed by TH17 cells.
    Figure 3: SR1001 inhibits the expression of cytokines expressed by TH17 cells.

    Il17a, Il17f, Il21 and Il22 mRNA expression in splenocytes differentiated under TH17 polarizing conditions in the presence of vehicle (DMSO) or SR1001 (5μM) for 5 days. Messenger RNA expression levels are normalized to Gapdh (n = 3). Error bars denote s.e.m.

  4. SR1001 inhibits TH17 cell development and IL-17A secretion.
    Figure 4: SR1001 inhibits TH17 cell development and IL-17A secretion.

    a, IL-17 expression in splenocytes cultured under TH17 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 naive murine CD4+ T cells normalized to vehicle control (n = 3). c, IL-17A secretion from splenocytes cultured under TH17 polarizing conditions with SR1001 (5μM) for 3days (n = 3). d, Intracellular IL-17A expression in human PBMCs cultured for 24h with vehicle or SR1001 (5μM) (n = 3). e, Treatment with SR1001 suppresses the clinical severity of EAE. Vehicle (circles, n = 12) or SR1001 (25mgkg−1) (triangles, n = 10). f, Il17a, Il21 and Il22 mRNA expression from spinal cords of sham-operated (IFA, incomplete Freund’s adjuvant), vehicle control (V) or drug treated (SR1001) mice. (n = 4). All error bars denote s.e.m. *, P<0.05; **, P<0.01; ***, P<0.001.

References

  1. McGeachy, M. J. & Cua, D. J. Th17 cell differentiation: The long and winding road. Immunity 28, 445453 (2008)
  2. Bettelli, E., Korn, T., Oukka, M. & Kuchroo, V. K. Induction and effector functions of TH17 cells. Nature 453, 10511057 (2008)
  3. Littman, D. R. & Rudensky, A. Y. Th17 and regulatory T cells in mediating and restraining inflammation. Cell 140, 845858 (2010)
  4. Yang, X. X. O. et al. T helper 17 lineage differentiation is programmed by orphan nuclear receptors RORα and RORγ. Immunity 28, 2939 (2008)
  5. Ivanov, I. I. et al. The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126, 11211133 (2006)
  6. Ivanov, I. I., Zhou, L. & Littman, D. R. Transcriptional regulation of Th17 cell differentiation. Semin. Immunol. 19, 409417 (2007)
  7. Manel, N., Unutmaz, D. & Littman, D. R. The differentiation of human TH-17 cells requires transforming growth factor-β and induction of the nuclear receptor RORγt. Nature Immunol. 9, 641649 (2008)
  8. Kumar, N. et al. The benzenesulfonamide T0901317 is a novel RORα/γ inverse agonist. Mol. Pharmacol. 77, 228236 (2010)
  9. Zhang, F. P., Meng, G. X. & Strober, W. Interactions among the transcription factors Runx1, RORγt and Foxp3 regulate the differentiation of interleukin 17-producing T cells. Nature Immunol. 9, 12971306 (2008)
  10. Ichiyama, K. et al. Foxp3 inhibits RORγt-mediated IL-17A mRNA transcription through direct interaction with RORγt. J. Biol. Chem. 283, 1700317008 (2008)
  11. Wang, Y. et al. Modulation of retinoic acid receptor-related orphan receptor α and γ activity by 7-oxygenated sterol ligands. J. Biol. Chem. 285, 50135025 (2010)
  12. Chopra, A. R. et al. Absence of the SRC-2 coactivator results in a glycogenopathy resembling Von Gierke’s disease. Science 322, 13951399 (2008)
  13. Jin, L. H. et al. Structural basis for hydroxycholesterols as natural ligands of orphan nuclear receptor RORγ. Mol. Endocrinol. 24, 923929 (2010)
  14. Xu, J., Wagoner, G., Douglas, J. C. & Drew, P. D. Liver X receptor agonist regulation of Th17 lympocyte function in autoimmunity. J. Leukoc. Biol. 86, 401409 (2009)
  15. Xu, J. H., Racke, M. K. & Drew, P. D. Peroxisome proliferator-activated receptor-α agonist fenofibrate regulates IL-12 family cytokine expression in the CNS: relevance to multiple sclerosis. J. Neurochem. 103, 18011810 (2007)
  16. Delerive, P., Chin, W. W. & Suen, C. S. Identification of Reverbα as a novel RORα target gene. J. Biol. Chem. 277, 3501335018 (2002)
  17. Wada, T. et al. Identification of oxysterol 7α-hydroxylase (Cyp7b1) as a novel retinoid-related orphan receptor α (RORα) (NR1F1) target gene and a functional cross-talk between RORα and liver X receptor (NR1H3). Mol. Pharmacol. 73, 891899 (2008)
  18. Wang, J., Yin, L. & Lazar, M. A. The orphan nuclear receptor Rev-erbα regulates circadian expression of plasminogen activator inhibitor type 1. J. Biol. Chem. 281, 3384233848 (2006)
  19. Okamoto, K. et al. IκBζ regulates TH17 development by cooperating with ROR nuclear receptors. Nature 464, 13811385 (2010)
  20. Raghuram, S. et al. Identification of heme as the ligand for the orphan nuclear receptors REV-ERBα and REV-ERBβ. Nature Struct. Mol. Biol. 14, 12071213 (2007)
  21. Lighvani, A. A. et al. T-bet is rapidly induced by interferon-γ in lymphoid and myeloid cells. Proc. Natl Acad. Sci. USA 98, 1513715142 (2001)
  22. Tamauchi, H. et al. Evidence of GATA-3-dependent Th2 commitment during the in vivo immune response. Int. Immunol. 16, 179187 (2004)
  23. Chalmers, M. J. et al. Probing protein ligand interactions by automated hydrogen/deuterium exchange mass spectrometry. Anal. Chem. 78, 10051014 (2006)
  24. Pascal, B. D., Chalmers, M. J., Busby, S. A. & Griffin, P. R. H. D. Desktop: An integrated platform for the analysis and visualization of H/D exchange data. J. Am. Soc. Mass Spectrom. 20, 601610 (2009)
  25. Zhang, Z. Q. & Smith, D. L. Determination of amide hydrogen-exchange by mass-spectrometry—a new tool for protein-structure elucidation. Protein Sci. 2, 522531 (1993)

Download references

Author information

Affiliations

  1. Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, Florida 33458, USA

    • Laura A. Solt,
    • Naresh Kumar,
    • Yongjun Wang,
    • Janelle L. Lauer,
    • Jin Liu,
    • Monica A. Istrate,
    • Patrick R. Griffin &
    • Thomas P. Burris
  2. The Scripps Research Institute Molecular Screening Center, The Scripps Research Institute, Jupiter, Florida 33458, USA

    • Naresh Kumar,
    • Monica A. Istrate,
    • Dušica Vidović,
    • Stephan C. Schürer &
    • Patrick R. Griffin
  3. Department of Chemistry, The Scripps Research Institute, Jupiter, Florida 33458, USA

    • Philippe Nuhant &
    • William R. Roush
  4. The Translational Research Institute, The Scripps Research Institute, Jupiter, Florida 33458, USA

    • Theodore M. Kamenecka &
    • Patrick R. Griffin
  5. Center for Computational Science University of Miami, Miami, Florida 33136, USA

    • Dušica Vidović &
    • Stephan C. Schürer
  6. Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA

    • Jihong Xu,
    • Gail Wagoner &
    • Paul D. Drew

Contributions

P.R.G. and T.P.B. conceived the project. L.A.S., P.R.G. and T.P.B. planned the project. Medicinal chemistry was planned and performed by P.N., T.M.K. and W.R.R. Biochemical and cell based assays were performed by L.A.S., N.K., Y.W., J.L. and M.A.I. Molecular modelling was performed by D.V. and S.C.C. The EAE model was designed and performed by J.X., G.W. and P.D.D. HDX studies were performed by J.L.L. The manuscript was written by L.A.S. and T.P.B.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Figures (720K)

    The file contains Supplementary Figures 1-9 with legends.

Additional data