Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity (original) (raw)

Nature volume 472, pages 486–490 (2011)Cite this article

Subjects

Abstract

CD4+ T helper lymphocytes that express interleukin-17 (TH17 cells) have critical roles in mouse models of autoimmunity, and there is mounting evidence that they also influence inflammatory processes in humans. Genome-wide association studies in humans have linked genes involved in TH17 cell differentiation and function with susceptibility to Crohn’s disease, rheumatoid arthritis and psoriasis1,2,3. Thus, the pathway towards differentiation of TH17 cells and, perhaps, of related innate lymphoid cells with similar effector functions4,5, is an attractive target for therapeutic applications. Mouse and human TH17 cells are distinguished by expression of the retinoic acid receptor-related orphan nuclear receptor RORγt, which is required for induction of IL-17 transcription and for the manifestation of TH17-dependent autoimmune disease in mice6. By performing a chemical screen with an insect cell-based reporter system, we identified the cardiac glycoside digoxin as a specific inhibitor of RORγt transcriptional activity. Digoxin inhibited murine TH17 cell differentiation without affecting differentiation of other T cell lineages and was effective in delaying the onset and reducing the severity of autoimmune disease in mice. At high concentrations, digoxin is toxic for human cells, but non-toxic synthetic derivatives 20,22-dihydrodigoxin-21,23-diol and digoxin-21-salicylidene specifically inhibited induction of IL-17 in human CD4+ T cells. Using these small-molecule compounds, we demonstrate that RORγt is important for the maintenance of IL-17 expression in mouse and human effector T cells. These data indicate that derivatives of digoxin can be used as chemical templates for the development of RORγt-targeted therapeutic agents that attenuate inflammatory lymphocyte function and autoimmune disease.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 51 print issues and online access

$199.00 per year

only $3.90 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

The microarray data sets are deposited in the Gene Expression Omnibus database under accession number GSE27241.

References

  1. Duerr, R. H. et al. A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science 314, 1461–1463 (2006)
    Article ADS CAS Google Scholar
  2. Nair, R. P. et al. Genome-wide scan reveals association of psoriasis with IL-23 and NF-κB pathways. Nature Genet. 41, 199–204 (2009)
    Article CAS Google Scholar
  3. Stahl, E. A. et al. Genome-wide association study meta-analysis identifies seven new rheumatoid arthritis risk loci. Nature Genet. 42, 508–514 (2010)
    Article CAS Google Scholar
  4. Buonocore, S. et al. Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology. Nature 464, 1371–1375 (2010)
    Article ADS CAS Google Scholar
  5. Colonna, M. Interleukin-22-producing natural killer cells and lymphoid tissue inducer-like cells in mucosal immunity. Immunity 31, 15–23 (2009)
    Article CAS Google Scholar
  6. Ivanov, I. I. et al. The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126, 1121–1133 (2006)
    Article CAS Google Scholar
  7. Stehlin, C. et al. X-ray structure of the orphan nuclear receptor RORβ ligand-binding domain in the active conformation. EMBO J. 20, 5822–5831 (2001)
    Article CAS Google Scholar
  8. Jin, L. et al. Structural basis for hydroxycholesterols as natural ligands of orphan nuclear receptor RORγ. Mol. Endocrinol. 24, 923–929 (2010)
    Article CAS Google Scholar
  9. 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, 1207–1213 (2007)
    Article CAS Google Scholar
  10. Ghoreschi, K. et al. Generation of pathogenic TH17 cells in the absence of TGF-β signalling. Nature 467, 967–971 (2010)
    Article ADS CAS Google Scholar
  11. Yang, X. O. et al. T helper 17 lineage differentiation is programmed by orphan nuclear receptors RORα and RORγ. Immunity 28, 29–39 (2008)
    Article CAS Google Scholar
  12. Sato, T. K. et al. A functional genomics strategy reveals Rora as a component of the mammalian circadian clock. Neuron 43, 527–537 (2004)
    Article CAS Google Scholar
  13. Veldhoen, M. et al. The aryl hydrocarbon receptor links TH17-cell-mediated autoimmunity to environmental toxins. Nature 453, 106–109 (2008)
    Article ADS CAS Google Scholar
  14. Zhou, L. et al. TGF-β-induced Foxp3 inhibits TH17 cell differentiation by antagonizing RORγt function. Nature 453, 236–240 (2008)
    Article ADS CAS Google Scholar
  15. Langrish, C. L. et al. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med. 201, 233–240 (2005)
    Article CAS Google Scholar
  16. Cua, D. J. et al. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421, 744–748 (2003)
    Article ADS CAS Google Scholar
  17. Paula, S., Tabet, M. R. & Ball, W. J., Jr Interactions between cardiac glycosides and sodium/potassium-ATPase: three-dimensional structure–activity relationship models for ligand binding to the E2-Pi form of the enzyme versus activity inhibition. Biochemistry 44, 498–510 (2005)
    Article CAS Google Scholar
  18. Nesher, M., Shpolansky, U., Rosen, H. & Lichtstein, D. The digitalis-like steroid hormones: new mechanisms of action and biological significance. Life Sci. 80, 2093–2107 (2007)
    Article CAS Google Scholar
  19. Johansson, S. et al. Cytotoxicity of digitoxin and related cardiac glycosides in human tumor cells. Anticancer Drugs 12, 475–483 (2001)
    Article CAS Google Scholar
  20. Price, E. M. & Lingrel, J. B. Structure-function relationships in the Na,K-ATPase α subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme. Biochemistry 27, 8400–8408 (1988)
    Article CAS Google Scholar
  21. Zavareh, R. B. et al. Inhibition of the sodium/potassium ATPase impairs N-glycan expression and function. Cancer Res. 68, 6688–6697 (2008)
    Article CAS Google Scholar
  22. 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, 641–649 (2008)
    Article CAS Google Scholar
  23. Prassas, I. & Diamandis, E. P. Novel therapeutic applications of cardiac glycosides. Nature Rev. Drug Discov. 7, 926–935 (2008)
    Article CAS Google Scholar
  24. Mijatovic, T. et al. Cardiotonic steroids on the road to anti-cancer therapy. Biochim. Biophys. Acta 1776, 32–57 (2007)
    CAS PubMed Google Scholar
  25. Robertson, L. W., Chandrasekaran, A., Reuning, R. H., Hui, J. & Rawal, B. D. Reduction of digoxin to 20R-dihydrodigoxin by cultures of Eubacterium lentum . Appl. Environ. Microbiol. 51, 1300–1303 (1986)
    CAS PubMed PubMed Central Google Scholar
  26. Lindenbaum, J., Rund, D. G., Butler, V. P., Jr, Tse-Eng, D. & Saha, J. R. Inactivation of digoxin by the gut flora: reversal by antibiotic therapy. N. Engl. J. Med. 305, 789–794 (1981)
    Article CAS Google Scholar
  27. Awasthi, A. et al. Cutting edge: IL-23 receptor gfp reporter mice reveal distinct populations of IL-17-producing cells. J. Immunol. 182, 5904–5908 (2009)
    Article CAS Google Scholar
  28. Luci, C. et al. Influence of the transcription factor RORγt on the development of NKp46+ cell populations in gut and skin. Nature Immunol. 10, 75–82 (2009)
    Article CAS Google Scholar
  29. Bagrov, A. Y. & Shapiro, J. I. Endogenous digitalis: pathophysiologic roles and therapeutic applications. Nat. Clin. Pract. Nephrol. 4, 378–392 (2008)
    Article CAS Google Scholar
  30. Ivanov, I. I. et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 139, 485–498 (2009)
    Article CAS Google Scholar

Download references

Acknowledgements

We thank C. Shamu at ICCB-Longwood for screening small-compound libraries. We also thank the New York Cord Blood Center for providing samples, the Developmental Studies Hybridoma Bank for β-tubulin antibody, the NYU Cancer Institute Genomics Facility for expert assistance with microarray experiments, V. Kuchroo and M. Oukka for the IL-23R GFP reporter mice, T. Iwaki, C. Thummel and R. Dasgupta for plasmids, M. Garabedian for R1881 and plasmids, D. Mangelsdorf for dafachronic acid and plasmids, G. Diehl and M. Sellars for reading the manuscript, M. Menager and J. Johnson for sharing peripheral blood mononuclear cells, and members of the D.R.L. laboratory for their suggestions. Supported by the Jane Coffin Childs Memorial Funds (J.R.H.), the Howard Hughes Medical Institute (D.R.L.) and National Institutes of Health grants F32GM0860552 (M.R.K.), RO1GM058833 (D.Y.G.), RO1GM067659 (D.Y.G), 2RO1GM55217 (F.R.) and RO1AI080885 (D.R.L.).

Author information

Author notes

  1. Nicolas Manel
    Present address: Present address: Institut Curie, INSERM U932, 75005 Paris, France.,
  2. Monica W. L. Leung and Pengxiang Huang: These authors contributed equally to this work.

Authors and Affiliations

  1. Molecular Pathogenesis Program, The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, 10016, New York, USA
    Jun R. Huh, Monica W. L. Leung, Jonathan Chow, Nicolas Manel, Maria Ciofani, Sangwon V. Kim, Adolfo Cuesta, Fabio R. Santori, Juan J. Lafaille & Dan R. Littman
  2. Sanford-Burnham Medical Research Institute at Lake Nona, 6400 Sanger Road, Orlando, 32827, Florida, USA
    Pengxiang Huang & Fraydoon Rastinejad
  3. Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA ,
    Daniel A. Ryan, Michael R. Krout & David Y. Gin
  4. Laboratory of Structural Sciences, Van Andel Research Institute, Grand Rapids, 49503, Michigan, USA
    Raghu R. V. Malapaka & H. Eric Xu
  5. Howard Hughes Medical Institute, New York University School of Medicine, New York, 10016, New York, USA
    Jonathan Chow, Adolfo Cuesta & Dan R. Littman

Authors

  1. Jun R. Huh
    You can also search for this author inPubMed Google Scholar
  2. Monica W. L. Leung
    You can also search for this author inPubMed Google Scholar
  3. Pengxiang Huang
    You can also search for this author inPubMed Google Scholar
  4. Daniel A. Ryan
    You can also search for this author inPubMed Google Scholar
  5. Michael R. Krout
    You can also search for this author inPubMed Google Scholar
  6. Raghu R. V. Malapaka
    You can also search for this author inPubMed Google Scholar
  7. Jonathan Chow
    You can also search for this author inPubMed Google Scholar
  8. Nicolas Manel
    You can also search for this author inPubMed Google Scholar
  9. Maria Ciofani
    You can also search for this author inPubMed Google Scholar
  10. Sangwon V. Kim
    You can also search for this author inPubMed Google Scholar
  11. Adolfo Cuesta
    You can also search for this author inPubMed Google Scholar
  12. Fabio R. Santori
    You can also search for this author inPubMed Google Scholar
  13. Juan J. Lafaille
    You can also search for this author inPubMed Google Scholar
  14. H. Eric Xu
    You can also search for this author inPubMed Google Scholar
  15. David Y. Gin
    You can also search for this author inPubMed Google Scholar
  16. Fraydoon Rastinejad
    You can also search for this author inPubMed Google Scholar
  17. Dan R. Littman
    You can also search for this author inPubMed Google Scholar

Contributions

J.R.H., J.J.L., H.E.X., D.Y.G., F.R. and D.R.L. designed the experiments. J.R.H. and D.R.L. wrote the manuscript with input from the co-authors. J.R.H. developed the screen and executed it with assistance from J.C. and A.C. F.R.S. developed the serum-free system for S2 cell culture. M.C. performed the ChIP experiments, J.R.H., N.M. and S.V.K. performed the T cell culture experiments, and J.R.H. and M.W.L.L. did in vivo compound tests and the follow-up analyses. P.H. did in vitro competition and circular dichroism assays, R.R.V.M. performed ALPHA screen assays, and D.A.R. and M.R.K. synthesized and purified digoxin derivatives.

Corresponding author

Correspondence toDan R. Littman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-14 with legends, Supplementary Methods and additional references. (PDF 2412 kb)

PowerPoint slides

Rights and permissions

About this article

Cite this article

Huh, J., Leung, M., Huang, P. et al. Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity.Nature 472, 486–490 (2011). https://doi.org/10.1038/nature09978

Download citation

Editorial Summary

A new class of immunomodulator

The nuclear receptors RORα and RORγt (retinoic acid receptor-related orphan receptors α and γt) are essential for the development of TH17 cells, the T-helper cells that produce interleukin-17. Two groups report the identification of RORγt inhibitors, compounds that could have potential in the treatment of autoimmune diseases. Huh et al. used a chemical screen in an insect-cell-based reporter system to identify the cardiac glycoside digoxin and various derivatives as inhibitors of the transcriptional activity of RORγt. Through this mechanism, these compounds block the differentiation of TH17 cells in mice, and inhibit interleukin-17 production in vitro in human T cells. Solt et al. describe a synthetic ligand, named SR1001, that functions as an inverse agonist for RORα and RORγt, and show that it blocks TH17 development in vitro and inhibits experimental encephalomyelitis in mice.

Associated content