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

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.

Biophotonic imaging in HO-1.luc transgenic mice: real-time demonstration of gender-specific chloroform induced renal toxicity.

The metabolism of luciferin in mice transgenic for luciferase (luc) produces light that may be detected trans vivo by an intensified CCD camera (biophotonics). Thus, the generation of transgenic promoter-luciferase animals for genes regulated by specific toxic processes, coupled with real-time evaluation of site-specific gene expression may provide novel, non-invasive biomarkers which are predictive of developing toxicity in vivo. As part of a programme to evaluate the potential of biophotonics for predictive toxicology we have conducted a series of studies in HO-1.luc transgenic mice. Male and female animals were treated with chloroform (200 mg/kg, p.o., daily for 5 days) and imaged 2 and 6 h after dosing. During a 2-day washout period, female animals were treated daily with testosterone prior to repeat administration of chloroform for a further 5 days. Comparison of the in vivo response of the luciferase reporter with markers of toxicity measured ex vivo (differential gene expression of adaptive antioxidant response genes, clinical chemistry and microscopic examination) confirms the gender-specific difference in chloroform renal toxicity in HO-1.luc transgenic mice and its reversal following androgenisation of females and correlates with the expression of the endogenous haem oxygenase-1 (HO-1) gene. These studies demonstrate the capacity of biophotonics for real-time site-specific gene expression, which may be predictive of developing toxicity.

Expression of the complement C3a and C5a receptors after permanent focal ischemia: An alternative interpretation.

The receptors for the complement anaphylatoxins C3a and C5a are expressed by glial cells and neurons in normal and inflamed brain. Previous studies demonstrated modest elevations in mRNA expression of these receptors in a model of focal cerebral ischemia. Using a similar model system for both mice and rats, we report markedly different patterns of anaphylatoxin receptor mRNA expression in cerebral ischemia. C5a receptor expression was dramatically elevated within 3 h after middle cerebral artery occlusion, while C3aR expression was reduced to 25% of control animals. By 24 h post-occlusion, expression of both receptors was higher than at any other time point examined. This increased expression at late time points after occlusion is most likely the result of massive infiltration of leukocytes expressing the receptors. We also observed increased receptor mRNA expression in sham-operated animals, indicating that the procedures used for arterial occlusion affects mechanisms regulating receptor expression. This latter result highlights the importance of including this important control group in ischemic model systems for proper interpretation of changes in gene expression.