Abstract
RORγt is an isoform of RORC, preferentially expressed in Th17 cells, that functions as a critical regulator of type 3 immunity. As murine Th17-driven inflammatory disease models were greatly diminished in RORC knock-out mice, this receptor was prioritised as an attractive therapeutic target for the treatment of several autoimmune diseases. Human genetic studies indicate a significant contributory role for RORC in several human disease conditions. Furthermore, genome-wide association studies (GWAS) report a significant association between inflammatory bowel disease (IBD) and the RORC regulatory variant rs4845604. To investigate if the rs4845604 variant may affect CD4+ T cell differentiation events, naïve CD4+ T cells were isolated from eighteen healthy subjects homozygous for the rs4845604 minor (A) or major (G) allele). Isolated cells from each subject were differentiated into distinct T cell lineages by culturing in either T cell maintenance medium or Th17 driving medium conditions for six days in the presence of an RORC inverse agonist (to prevent constitutive receptor activity) or an inactive diastereomer (control). Our proof of concept study indicated that genotype had no significant effect on the mean number of naïve CD4 T cells isolated, nor the frequency of Th1-like and Th17-like cells following six days of culture in any of the four culture conditions. Analysis of the derived RNA-seq count data identified genotype-driven transcriptional effects in each of the four culture conditions. Subsequent pathway enrichment analysis of these profiles reported perturbation of metabolic signalling networks, with the potential to affect the cellular detoxification response. This investigation reveals that rs4845604 genotype is associated with transcriptional effects in CD4+ T cells that may perturb immune and metabolic pathways. Most significantly, the rs4845604 GG, IBD risk associated, genotype may be associated with a differential detoxification response. This observation justifies further investigation in a larger cohort of both healthy and IBD-affected individuals.
Highlights
RORC is a ligand-dependent nuclear hormone receptor with validated functional roles in a wide variety of biological processes, including: cellular immunity, steroid and glucose metabolism, and the peripheral circadian rhythm [1,2]
Healthy volunteers previously recruited to the Clinical Unit Cambridge (CUC) database were genotyped for rs4845604 (Chr1: 151801680), and nine subjects homozygous for the minor (A) allele were age and gender matched with nine subjects homozygous for the major (G) allele
Naïve CD4+ T cells were purified from peripheral blood mononuclear cells (PBMCs) of each subject, and an unpaired t-test used to compare the baseline population means of each genotype: see the Pre-study protocol optimisation section in S1 supplementary materials for further details in S1 File
Summary
RORC is a ligand-dependent nuclear hormone receptor with validated functional roles in a wide variety of biological processes, including: cellular immunity, steroid and glucose metabolism, and the peripheral circadian rhythm [1,2]. Studies reported that some cholesterol metabolites are likely to be endogenous ligands of RORγt [2,6]. These combined characteristics suggest that RORγt may, in part, link type 3 immunity with the metabolic system. The observation that Th17-driven inflammatory disease models were greatly attenuated in RORC knock-out mice [7] positioned this receptor as an attractive therapeutic target for the treatment of several autoimmune diseases. RORC inverse agonists that suppress Th17 differentiation were identified [8,9] and their therapeutic potential evaluated in human clinical trials [10]
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