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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


Primary accessions

Gene Expression Omnibus

Data deposits

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


  1. 1.

    et al. A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science 314, 1461–1463 (2006)

  2. 2.

    et al. Genome-wide scan reveals association of psoriasis with IL-23 and NF-κB pathways. Nature Genet. 41, 199–204 (2009)

  3. 3.

    et al. Genome-wide association study meta-analysis identifies seven new rheumatoid arthritis risk loci. Nature Genet. 42, 508–514 (2010)

  4. 4.

    et al. Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology. Nature 464, 1371–1375 (2010)

  5. 5.

    Interleukin-22-producing natural killer cells and lymphoid tissue inducer-like cells in mucosal immunity. Immunity 31, 15–23 (2009)

  6. 6.

    et al. The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126, 1121–1133 (2006)

  7. 7.

    et al. X-ray structure of the orphan nuclear receptor RORβ ligand-binding domain in the active conformation. EMBO J. 20, 5822–5831 (2001)

  8. 8.

    et al. Structural basis for hydroxycholesterols as natural ligands of orphan nuclear receptor RORγ. Mol. Endocrinol. 24, 923–929 (2010)

  9. 9.

    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)

  10. 10.

    et al. Generation of pathogenic TH17 cells in the absence of TGF-β signalling. Nature 467, 967–971 (2010)

  11. 11.

    et al. T helper 17 lineage differentiation is programmed by orphan nuclear receptors RORα and RORγ. Immunity 28, 29–39 (2008)

  12. 12.

    et al. A functional genomics strategy reveals Rora as a component of the mammalian circadian clock. Neuron 43, 527–537 (2004)

  13. 13.

    et al. The aryl hydrocarbon receptor links TH17-cell-mediated autoimmunity to environmental toxins. Nature 453, 106–109 (2008)

  14. 14.

    et al. TGF-β-induced Foxp3 inhibits TH17 cell differentiation by antagonizing RORγt function. Nature 453, 236–240 (2008)

  15. 15.

    et al. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med. 201, 233–240 (2005)

  16. 16.

    et al. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421, 744–748 (2003)

  17. 17.

    , & 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)

  18. 18.

    , , & The digitalis-like steroid hormones: new mechanisms of action and biological significance. Life Sci. 80, 2093–2107 (2007)

  19. 19.

    et al. Cytotoxicity of digitoxin and related cardiac glycosides in human tumor cells. Anticancer Drugs 12, 475–483 (2001)

  20. 20.

    & 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)

  21. 21.

    et al. Inhibition of the sodium/potassium ATPase impairs N-glycan expression and function. Cancer Res. 68, 6688–6697 (2008)

  22. 22.

    , & 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)

  23. 23.

    & Novel therapeutic applications of cardiac glycosides. Nature Rev. Drug Discov. 7, 926–935 (2008)

  24. 24.

    et al. Cardiotonic steroids on the road to anti-cancer therapy. Biochim. Biophys. Acta 1776, 32–57 (2007)

  25. 25.

    , , , & Reduction of digoxin to 20R-dihydrodigoxin by cultures of Eubacterium lentum. Appl. Environ. Microbiol. 51, 1300–1303 (1986)

  26. 26.

    , , , & Inactivation of digoxin by the gut flora: reversal by antibiotic therapy. N. Engl. J. Med. 305, 789–794 (1981)

  27. 27.

    et al. Cutting edge: IL-23 receptor gfp reporter mice reveal distinct populations of IL-17-producing cells. J. Immunol. 182, 5904–5908 (2009)

  28. 28.

    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)

  29. 29.

    & Endogenous digitalis: pathophysiologic roles and therapeutic applications. Nat. Clin. Pract. Nephrol. 4, 378–392 (2008)

  30. 30.

    et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 139, 485–498 (2009)

Download references


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

    • Monica W. L. Leung
    •  & Pengxiang Huang

    These authors contributed equally to this work.

    • Nicolas Manel

    Present address: Institut Curie, INSERM U932, 75005 Paris, France.


  1. Molecular Pathogenesis Program, The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, New York 10016, 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, Florida 32827, 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, Michigan 49503, USA

    • Raghu R. V. Malapaka
    •  & H. Eric Xu
  5. Howard Hughes Medical Institute, New York University School of Medicine, New York, New York 10016, USA

    • Jonathan Chow
    • , Adolfo Cuesta
    •  & Dan R. Littman


  1. Search for Jun R. Huh in:

  2. Search for Monica W. L. Leung in:

  3. Search for Pengxiang Huang in:

  4. Search for Daniel A. Ryan in:

  5. Search for Michael R. Krout in:

  6. Search for Raghu R. V. Malapaka in:

  7. Search for Jonathan Chow in:

  8. Search for Nicolas Manel in:

  9. Search for Maria Ciofani in:

  10. Search for Sangwon V. Kim in:

  11. Search for Adolfo Cuesta in:

  12. Search for Fabio R. Santori in:

  13. Search for Juan J. Lafaille in:

  14. Search for H. Eric Xu in:

  15. Search for David Y. Gin in:

  16. Search for Fraydoon Rastinejad in:

  17. Search for Dan R. Littman in:


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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Dan R. Littman.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Figures 1-14 with legends, Supplementary Methods and additional references.

About this article

Publication history






Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.