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Chemical Inhibition of the RORγt-dependent Transcriptional Network in Th17 cells [ChIP-Seq]

ABSTRACT: RORγt is a transcription factor required for T helper 17 (Th17) cell development. We identified three RORγt-specific inhibitors that suppress Th17 cell responses including Th17 cell-mediated autoimmune disease. We systemically characterized RORγt binding data in the presence and absence of drug with corresponding whole-transcriptome sequencing for wild-type and RORγt-deficient cells. RORγt is central in a densely interconnected regulatory network, acting both as a direct activator of genes important for Th17 cell differentiation and as a direct repressor of genes from other T-cell lineages. The three inhibitors identified here reversed both of these modes of action, but to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target-loci, the two more potent inhibitors affected transcription predominantly without removing DNA-binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity.

ORGANISM(S): Mus musculus

PROVIDER: GSE56019 | GEO | 2014/04/17

SECONDARY ACCESSION(S): PRJNA242231

REPOSITORIES: GEO

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Project description: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 psoriasis1-3. 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 (Dig(dhd)) and digoxin-21-salicylidene (Dig(sal)), specifically inhibited induction of IL-17 in human CD4+ T cells. Using these small molecule compounds, we demonstrated that RORγt is imporant for the maintenance of IL-17 expression in mouse and human effector T cells. These data suggest that derivatives of digoxin can be used as chemical probes for development of RORγt-targeted therapeutic agents that attenuate inflammatory lymphocyte function and autoimmune disease. We performed gene expression profiling with total RNA samples isolated from DMSO- or digoxin-treated wildtype or RORγt-deficient cells cultured in Th17 conditions. Total RNA was extracted with TRIzol. RNA was labeled and hybridized to GeneChip Mouse Genome 430 2.0 arrays following the Affymetrix protocols. Data were analyzed in GeneSpring GX11.5.

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Chemical Inhibition of the RORγt-dependent Transcriptional Network in Th17 cells [ChIP-Seq]

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