Transcriptomic effects of rs4845604, an IBD and allergy-associated RORC variant, in stimulated ex vivo CD4+ T cells

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.

Fig 1. Flow cytometric analysis of CXCR3 versus CCR6 of live CD4⁺ cells at day 6 of culture was used to characterize the Th1-like and Th17-like cell populations.

Representative graphical plots of cell count data from a subject of genotype group GG and genotype AA are shown, for each of the four culture conditions.

Fig 2. Characterization of the Th cell populations by flow cytometry at day 6 of cell culture under each of the four growth medium formulations: T cell maintenance medium or Th17 driving medium, in the presence of either GSK776 (GSK2794776A an inactive diastereomer) or GSK778 (GSK2794778A an inverse agonist) (n = 6, comprised of 3 subjects from each genotype group).

Box and whisker plots representing the variation of Th1-like and Th17-like cell populations as a response to genotype, growth conditions, and compound treatment (i.e. left-to-right). The observed data indicate Th17 driven medium lead to a significant decrease of the Th1-like population and GSK2794778A to a decrease in the Th17-like population (unadjusted p-values < 0.05, are indicated with an *).

Fig 3. Genotype has no significant effect on CD4+ T cell proliferation.

Cell proliferation outcomes in naïve CD4+ T cell counts following 6 days of culture, for each of the two genotypes under the four treatment conditions (i.e. Th17 driving medium or T cell maintenance medium in the presence of either GSK776 (GSK2794776A - an inactive diastereomer) or GSK778 (GSK2794778A -an inverse agonist of RORC)). Note that the cell number represents the average cell count derived from nine homozygous subjects in each genotype group. Cell counts were normalised to 1.0 x 10⁶ cells at day 0 for all subjects due to variation observed in the number of cells obtained and to facilitate the day 6 comparison (S1 supplementary materials Table 1 in S1 File). None of the observed differences between the respective genotype comparisons were reported as statistically significant (i.e. a p-value > 0.05). Data is expressed as absolute cell count and shown as means ± SD.

Fig 4. Transcriptional profiles cluster according to choice of differentiation medium.

Distance matrix and clustering: The DESeq2 dist function was used estimate sample-to-sample Euclidean distances with regard to the normalised log-transformed gene expression profile, and a heatmap generated to visualise sample similarities. The dendrograms indicate that the profiles cluster primarily by differentiation medium (i.e. T cell maintenance cultured samples cluster together (indicated by the green bar), while Th17 stimulated samples group together (indicated by the red bar) and then by subject (i.e. within each incubation culture, samples from a subject are predominantly most similar to each other). Cell colour intensity correlates with the estimated distance between the two samples in question.

Fig 5. RORC transcripts are differentially expressed in presence of differentiating medium.

Normalised of three RORC transcript counts. Each point represents the expression value observed in a subject and each box indicates the Q1, median, and Q3 quartiles for that differentiation medium group. AA & GG indicate the respective genotype of the subjects, 778 (the GSK2794778A inverse agonist) 776 (the GSK2794776A inactive diastereomer) indicate the presence of inactive and active molecules, while stim and mntn indicate TH17 and maintenance, respectively.

Fig 6. Genotype is associated with differential transcriptional effects on effects on canonical immune and metabolic signalling networks.

Canonical Pathway enrichment analysis: Ingenuity pathway enrichment analysis was completed using those gene transcripts reported as significantly differentially expressed when cells isolated from GG genotype subjects cultured for six days in T cell maintenance medium in the presence of an inactive diastereomer (GSK2794776A) were contrasted with cells isolated form AA genotype subjects and subjected to identical culture conditions. Enriched pathways were identified and ranked using Fisher’s Exact Test. The blue bars represent the -log(p-value) of these tests (i.e. longer bars represent stronger evidence of enrichment). The ratio of the number of differentially expressed genes relative to the number of genes included in the pathway is indicated by the orange line.

Fig 7. The homozygous genotype differentially effects transcriptional response in the presence of an RORC inverse agonist.

Canonical Pathway enrichment analysis: Ingenuity pathway enrichment analysis was completed using those gene transcripts reported as significantly differentially expressed when cells isolated form GG genotype subjects cultured for six days in Th17 driving medium in the presence of an RORC inverse agonist (GSK2794778A) where contrasted with cells isolated form AA genotype subjects and subjected to identical culture conditions. Enriched pathways were identified and ranked using a Fischer’s Exact Test. The blue bars represent the -log(p-value) of these tests (i.e. larger bar size equates to statistical significance of the enrichment). The ratio of the number of differentially expressed genes relative to the number of genes included in the pathway is summarised by the orange line.