Transcription profiling of mouse splenic thymocytes reveals autoregulation of Th1-mediated inflammation by twist1
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ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Joachim Grün
PROVIDER: E-GEOD-11556 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor κB (NF-κB)-dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We could show that twist1 is expressed by activated T helper (Th) 1 effector memory cells. Induction of twist1 in Th cells is dependent on NF-κB, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 is transient following T-cell receptor engagement, and increases upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression is characteristic of repeatedly restimulated effector memory Th cells and thus of the pathogenic memory Th cells of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohnâ??s disease or ulcerative colitis express high levels of twist1. Expression of twist1 in Th1 lymphocytes limits the expression of the cytokines interferon-γ, IL-2 and tumor necrosis factor-α, and ameliorates Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis. In order to identify the effect of twist1 expression on the function of Th cells, twist1 was ectopically expressed and the transcriptome was compared to empty-virus infected control cells. In addition, this experiment allows for the identification of genes regulated by the transcription factor twist1. Experiment Overall Design: Genes differentially expressed upon ectopic twist1 overexpression. Splenic DO11.10 cells were activated in vitro with the cognate peptide ova327-339 in the presence of 1ng/ml IL-12 and 1ng/ml IL-2. On day 2 cells were infected with control virus, or twist1-encoding virus. On day 5 cells were sorted according to expression of the viral marker gene gfp. Cells were restimulated for four hours with PMA/ionomycin. The transcriptional profiles of duplicates of cultures were compared using Affymetrix Murine Genome (MG) 430A 2.0 GeneChip arrays.
2008-06-18 | E-GEOD-11533 | biostudies-arrayexpress
Project description:Gene expression profiling of repeatedly activated compared to recently activated Th1 cells to identify genes that play a role in chronic inflammatory disorders and may qualify as diagnostic or therapeutic targets; ; Upon activation under appropriate costimulatory conditions, naive T helper (Th) cells differentiate into Th2 or Th17 cells, each characterized by the expression of specific effector cytokines. In response to a repeated stimulation with antigen, Th cells develop a stable memory for the expression of those cytokines as well as for other secreted or membrane-associated factors. The stable memory for the expression of proinflammatory effector functions may explain the resistance of Th effector cells to conventional immunosuppressive therapy, and the inability of immunosuppression to cure chronic inflammation. The imprinting of the functional memory is based on epigenetic modifications and expression of distinct transcription factors. In this project, we compare the transcriptomes of once and repeatedly activated murine Th1 cells, to identify genes that induce and maintain the functional memory and control the persistence of pathogenic memory Th1 cells. This in turn might help to discriminate pathogenic versus protective cells in immunopathology and present novel targets for the diagnosis and therapy of chronic inflammatory disease. Experiment Overall Design: Genes differentially expressed in once versus four times stimulated Th1 cells. In vitro polarization of murine naïve DO11.10 T cells towards Th1 direction (5 ng/ml recombinant murine IL-12, 5 μg/ml anti-IL-4 antibody) with antigenic stimulation (ova323-339 and irradiated splenic APCs). The transcriptional profiles of resting one week old Th1 (Th1 1w) cells and resting 4 week old Th1 (Th1 4w) cells were compared using Affymetrix Murine Genome (MG) U74V2A GeneChip arrays. Experiment Overall Design: 10 µg of total RNA from each cell sample was reverse transcribed using T7-(d)T24 primer and SuperScript II reverse transcriptase Experiment Overall Design: cDNA extraction with a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate Experiment Overall Design: Biotinylated cRNA was transcribed with the MEGAscript high yield transcription kit (Ambion), fragmented, and the hybridization cocktail was prepared (15 µg fragmented biotin-labeled cRNA spiked with Eukaryotic Hybridization control) Experiment Overall Design: probes were subsequently hybridised with the GeneChip U74Av2 for 16 hrs at 45 oC Experiment Overall Design: after washing the hybridisation signals were visualised by staining with streptavidin-phycoerythrin and amplification with an anti-streptavidin antibody Experiment Overall Design: TH1_1w_C1; TH1_1w_C2; TH1_1w_C3TH1_4w_C1; TH1_4w_C2; TH1_4w_C3 Experiment Overall Design: 1w: 1 week in culture; 4w: 4 weeks in culture; C1-3: Culture or Experiment No.
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Project description:The aim of this study was to characterize the basal expression of OC, MOD and SV of newborn rats (3-5 day old) and its changes by shortâterm cultivation (24 hrs) and mild hypoxia (5 hrs). Three varieties of tissue samples were analyzed (i) Freshly prepared tissue, used as control; (ii) cultures held under normoxic conditions and (iii) cultures exposed during cultivation to 5 h hypoxia. The gene expression was analyzed using the Affymetrix GeneChip RN-U34. The functional specificity of the cochlea regions are well reflected by the gene expression pattern. For example, the basal expression of Organ of Corti is characterized by genes involved in potassium and calcium homeostasis. The MOD is characterized by genes involved in regulation of maturation of neuronal cells. The SV is characterized by genes involved in ion homeostasis and blood flow. Cultivation dramatically changes the gene expression. In contrast, mild hypoxia showed only small changes. The changes are interpreted as stress response. We used this analysis as screening approach to detect significant changes in gene expression and used the RT-PCR analyses of independent sister cultures for confirmation. Correlation analysis showed close relations of the expression levels when aliquots of the biological material were compared. However, comparisons of the expression levels received by microarray and by RT-PCR analysis of independent sister samples resulted in 70-90 % in similar predictions. This data indicate that possible discrepancies has its basis in biological sampling rather than factors of methodical errors on the level of RT-PCR. Experiment Overall Design: Pooled samples of the following cochlea preparations were studied: Experiment Overall Design: (i) Native preparations of Organ of Corti, modiolus and stria vascularis from newborn rats used as controls; (ii) Organotypic cultures of these preparations under normoxic conditions (24 h); (iii) Cultures which were exposed to 5 h hypoxia during cultivation. Samples were collected for total RNA immediately after preparation of the inner ear structures (native samples) or 24 h after onset of cultivation (normoxia, hypoxia).
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