Abstract: Data mining published microarray experiments require that expression profiles are directly comparable. We performed linear global normalization on the data of 1967 Affymetrix U74av2 microarrays, i.e. the transcriptomes of >100 murine tissues or cell types. The mathematical transformation effectively nullifies inter-experimental or inter-laboratory differences between microarrays. The correctness of expression values was validated by quantitative RT-PCR. Using the database we analyze components of the aryl hydrocarbon receptor (AHR) signaling pathway in various tissues. We identified lineage and differentiation specific variant expression of AHR, ARNT, and HIF1alpha in the T-cell lineage and high expression of CYP1A1 in immature B cells and dendritic cells. Performing co-expression analysis we found unorthodox expression of the AHR in the absence of ARNT, particularly in stem cell populations, and can reject the hypothesis that ARNT2 takes over and is highly expressed when ARNT expression is low or absent. Furthermore the AHR shows no co-expression with any other transcript present on the chip. Analysis of differential gene expression under 308 conditions revealed 53 conditions under which the AHR is regulated, numerous conditions under which an intrinsic AHR action is modified as well as conditions activating the AHR even in the absence of known AHR ligands. Thus meta-analysis of published expression profiles is a powerful tool to gain novel insights into known and unknown systems.

Abstract: The ligand-activated aryl hydrocarbon receptor (AHR) is known to modulate many genes in a highly cell-specific manner, either directly or indirectly via secondary effects. In contrast, little is known about the effects of AHR deficiency on gene expression balance. We compared the transcriptome of CD4 T cells from AHR-/- mice and wild-type mice; 390 genes, many of them immunotypic, were deregulated in AHR-deficient CD4 cells. TCDD-induced transcriptome changes correlated with the AHR expression level in immune cells. However, there was little overlap in AHR-dependent transcripts found in T lineage cells or dendritic cells. Our results demonstrate flexible gene accessibility for the AHR in immune cells. The idea of a universal battery of AHR-responsive genes is not tenable.

Abstract: T cell maturation into TCRalphabeta(+) or TCRgammadelta(+) cells from common immature CD4(-)CD8(-)(DN) precursors occurs in the thymus, and is controlled through ordered regulation of genes. The aryl hydrocarbon receptor (AHR), a latent cytoplasmic transcription factor, affects thymocyte maturation and differentiation at several stages, also including DN cells. We analyzed in murine fetal thymus organ cultures (FTOC) the outcome of AHR-signaling and found a higher frequency of DN TCRgammadelta(+) cells in the presence of the AHR-activating ligand TCDD. We detected a novel population of CD25(int/lo)CD44(hi) cells associated with preferential emigration and a TCRgammadelta(+) T cell fate of thymocytes. Sorted DN TCRgammadelta(+) emigrants could proliferate if IL-2 was available. Moreover, they suppressed the proliferation of co-cultivated, activated CD4(+) T cells. Gene expression profiles of purified DN emigrants from TCDD*FTOC revealed 295 modulated genes, 10% of which are genes of the immune system. For instance, RAG-1, TdT, and Gfi-1 were downregulated, yet genes indicative of mature thymocytes were upregulated. In conclusion, we have detected changes in the differentiation programme of fetal DN thymocytes after ligand-activation of the AHR. In particular, we observed a higher frequency of DN TCRgammadelta(+) cells with high emigration potential, and possible regulatory functions.

Abstract: The aryl hydrocarbon receptor (AHR) is a ligand-dependent member of the PAS-bHLH-family of nuclear receptors. Anthropogenic ligands include environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Over-activation of the AHR causes thymus atrophy and immunosuppression. Signaling via the AHR changes the thymocyte differentiation program at several checkpoints, in particular within the CD4-CD8- double-negative (DN) thymocyte subset. Here, we show that AHR over-activation led to the preferential emigration of DN thymocytes to the periphery and accumulation in the spleen. Some of these recent thymic emigrants (RTE) had a novel "activated immature" phenotype (CD3-TCRbeta-CD25+/intCD44-CD45RB+/intCD62L+CD69- cells). Gene expression profiling of DN RTE revealed 15 genes that were up-regulated more than threefold by TCDD, including the S100A9 gene. Exposure of S100A9 null mice to TCDD showed a role for this protein in AHR-mediated thymic egress.