Friday, May 3, 2024
5/3/2024
TH17 cells are a subset of CD4+ T cells that are important for immune responses to extracellular pathogens and fungi, however, they can become dysregulated and lead to autoimmunity and chronic inflammatory conditions, such as inflammatory bowel disease (IBD). TH17 cells secrete a myriad of pro-inflammatory cytokines, including IL-17A, IL- 17F, IL-21, and IL-22, which give them a dynamic range of pathogenic potential. This could explain why IL-17A/IL- 17R-neutralizing antibodies have had limited success in treating IBD, while targeting cell-intrinsic factors that broadly regulate the TH17 cell phenotype may be more effective. Modulating the development of TH17 cells through intracellular transcription factors (TFs) like NRs provides an approach to lessen multiple components of the inflammatory response responsible for IBD, avoiding some of the pitfalls of current therapies. Our preliminary data is consistent with previously published literature describing the orphan nuclear receptor NR2F6 as a negative regulator of pro-inflammatory cytokines implicated in IBD, including IL-17A and TNF¿. Genetic experiments have demonstrated loss of NR2F6 enhances T cell effector responses, leading to increased sensitivity to autoimmune and chronic inflammatory diseases, making it an attractive target for therapy. NRs act as ligand regulated TFs that can activate or repress transcription of target genes through recruitment of various co-factor molecules. Additionally, these co-factors can recruit chromatin remodelers, adding another layer of transcriptional regulation. Little is known about NR2F6’s transcriptional and ligand-regulated function at the molecular level. The overarching goal of this project is to elucidate NR2F6’s mechanism of transcriptional regulation in TH17 cells and determine how this may influence TH17-mediated diseases, including IBD. My first aim is to enumerate NR2F6’s transcriptional function in TH17 cells. This will be done through integration of NR2F6 overexpression models using CUT&RUN footprint analysis and RNAseq to identify genes potentially modulated by NR2F6; RNAseq and ATACseq of NR2F6 knockout TH17 cells will be used to identify NR2F6 target genes and its capacity to remodel chromatin at these gene loci. Co-immunoprecipitation (CoIP) studies will determine the co-factors NR2F6 recruits to regulate transcription. Integration of this Multi-OMICS data will provide a comprehensive mechanism for NR2F6’s transcriptional activity. My second aim is to define how small molecules affect NR2F6 transcriptional activity and function in TH17 cells. Previous efforts by our lab have identified a synthetic NR2F6 ligand. RNAseq and multi- parametric flow cytometry will be used to characterize and validate the effects of treatment with this ligand. Additionally, Co-IP proteomic studies will identify ligand-dependent changes in NR2F6 co-factor recruitment that may lead to changes in function. We hypothesize that NR2F6 serves as a critical negative regulator of TH17 cell effector responses and NR2F6 small molecule ligands can be used to further explore its biology.
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