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

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

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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.

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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.

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Gene Expression Omnibus

Data deposits

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

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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.).

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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

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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.

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Correspondence to Dan R. Littman.

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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

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