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

Abstract

CD4(+) T helper lymphocytes that express interleukin-17 (T(H)17 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 T(H)17 cell differentiation and function with susceptibility to Crohn's disease, rheumatoid arthritis and psoriasis. Thus, the pathway towards differentiation of T(H)17 cells and, perhaps, of related innate lymphoid cells with similar effector functions, is an attractive target for therapeutic applications. Mouse and human T(H)17 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 T(H)17-dependent autoimmune disease in mice. 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 T(H)17 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.

Figure 1). Digoxin binds to RORγ and inhibits its transcriptional activity

a, Chemical structure of digoxin. b, Digoxin demonstrates dose-dependent inhibition of RORγ transcriptional activity in the Drosophila S2 cell luciferase reporter system. Ratio of firefly to Renilla luciferase activity is shown as relative luciferase unit (RLU) on the y-axis. c, Digoxin (10 μM) selectively inhibits RORγ dependent transcriptional activity without affecting that of RORα, DHR3, or VP16. Percentages of relative luciferase units compared to DMSO-treated reporter cells are shown on the y-axis. Error bars indicate standard deviation. d, In vitro competition assay. Recombinant human RORγ LBD was loaded with fluorescently-labeled 25-hydroxycholesterol in the presence of the indicated concentrations of digoxin, and fluorescence polarization was measured.

Figure 2). Digoxin inhibits mouse Th17 cell differentiation and ameliorates Th17-mediated autoimmune disease

a, Flow cytometry of intracellular staining for IL-17a and IFN-γ in sorted naïve T cell populations (from RORγt^fl/fl mice following transduction with control-IRES-GFP (WT) or CRE-IRES-GFP (KO) retrovirus) activated and expanded in the presence of mouse Th17 polarizing cytokines. DMSO or 10 μM digoxin was added at 6 hours after viral transduction on day 1 and GFP expressing cells were gated for analysis on day 5. b, Two-dimensional hierarchical clustering of the 67 genes (including redundant probe sets and genes of unknown function) identified to be significantly affected by 2-way ANOVA analysis (DMSO versus digoxin treament, p<0.05). Each row corresponds to a gene and each column corresponds to an experimental sample. c, FACS-sorted naïve T cells were transduced with retroviral vectors encoding murine RORα-IRES-GFP or RORγt-IRES-GFP on Day 1 (16 h after TCR stimulation) and GFP expressing cells were gated for anyalsis on Day 5. DMSO or 10 μM digoxin was added 6–8 h after transduction. d, EAE disease course in B6 wild-type mice that were IP injected with either DMSO or digoxin (40 μg/mouse) every day starting from day 2 after disease induction with MOG(35-55)/CFA. Shown is averaged curve shape from seven experiments (10 or 20 mice were used per trial). * indicates statistical significance (p<0.05). Error bars represent standard deviation. e, f, Th1 and Th17 cells in spinal cord of EAE mice treated with DMSO or digoxin. Lymphocytes were isolated on day 21 after disease induction. The cells were stimulated for 4h with PMA/Ionomycin and stained for surface markers and intracellular cytokines. Representative FACS plots (gated on CD45⁺CD11b⁻CD4⁺ cells) from mice from each group are shown (top). T cells isolated from spinal cords of DMSO (n=7) or digoxin treated mice (n=7) were stained intracellularly for IFN-γ or IL-17a. Statistical analysis was by a two-tailed unpaired Student's t test; NS, not significant and p=0.014 (bottom).

Figure 3). 20,22-Dihydrodigoxin-21,23-diol and digoxin-25-salicylidene inhibit human Th17 cell differentiation

a, Chemical structures of 20,22-dihydrodigoxin-21,23-diol and digoxin-21-salicylidene. b, Flow cytometry of the production of IL-17a and IFN-γ by human naïve cord blood T cells cultured for six days in the presence of IL-2, IL-23, and IL-1β, with various concentrations of TGF-β. DMSO, Dig(dhd), or Dig(sal) at indicated concentrations (μM) was added 16h after cytokine addition.

Figure 4). RORγt activity is important for maintenance of mouse and human Th17 cells

a, b, Flow cytometry of intracellular staining for IL-17a and IFN-γ by CD4⁺ T cells. Mononuclear cells were collected from draining lymph nodes of wild-type or IL-23R-GFP knock-in heterozygous mice 7 days after MOG(35-55)/CFA injection. Cells were cultured for four more days with MOG(35-55) peptide and exogenous IL-23 or IL-12, in the presence of DMSO or 10 μM digoxin. Without pre-immunization, addition of IL-23 and MOG(35-55) peptide to culture did not lead to de novo Th17 cell differentiation (data not shown). Digoxin treatment suppressed expansion of in vivo differentiated Th17 cells, assayed by IL-23R reporter GFP expression (a) or by IL-17a production (b). c, d, Human naïve (CD45RA⁺CD3⁺CD4⁺) or memory (CD45RO⁺CD45RA⁻ CD3⁺CD4⁺CCR6⁺CD161⁺) cells were purified from healthy donor peripheral blood samples and were cultured in the presence of IL-1β, IL-23 and IL-2 for 6 days with or without 40 μM Dig(dhd). Intracellular staining for IFN-γ or IL-17a in memory CD4⁺ T cells from multiple donors (n=11) in the presence of IL-1β, IL-23, and IL-2, assessed on day 6. c, Representative FACS plots from one donor are shown. d, Each symbol indicates a separate donor. Statistical analysis was by a two-tailed unpaired Student's t test; IL-17a⁻ IFN-γ⁺, not significant and IL-17a⁺IFN-γ^+/−, p=0.02.