Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity


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

Buy this article

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

Figure 1: Digoxin binds to RORγ and inhibits its transcriptional activity.
Figure 2: Digoxin inhibits mouse T H 17 cell differentiation and ameliorates T H 17-mediated autoimmune disease.
Figure 3: Dig(dhd) and Dig(sal) inhibit human T H 17 cell differentiation.
Figure 4: RORγt activity is important for maintenance of mouse and human T H 17 cells.

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.


  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


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

Authors and Affiliations



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 to Dan 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

Reprints 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).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing