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Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type, DDX5 knockout, RORc knockout, RmrpG262T knockin T helper 17 cell Transcriptomes [RNA-seq]

ABSTRACT: Purpose: The goals of this study are to compare in vitro polarized T helper 17 cell transcriptome profiling (RNA-seq) in different genetic background. Methods: Th17 mRNA profiles of 96hrs in vitro cultured T helper 17 cells from wild-type and mutant mice were generated by deep sequencing, using Illumina RapidRun. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) and TopHat followed by CuffDiff. qRT–PCR validation was performed using SYBR Green assays. Results: Among the 325 genes that were significantly dysregulated in DDX5-deficient T cells, approximately 40% were previously identified as RORγt targets in Th17 cells. 96 RORγt-dependent Th17 cell genes co-regulated by Rmrp together with DDX5. Conclusions: Our study suggest that the DDX5-Rmrp axis is critical for expression of a critical subset of the RORγt transcriptional targets in Th17 cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE70108 | GEO | 2015/12/21

SECONDARY ACCESSION(S): PRJNA287657

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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Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type, DDX5 knockout, RORc knockout, RmrpG262T knockin T helper 17 cell Transcriptomes [RNA-seq]

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