Dataset Information


Autoregulation of Th1-mediated inflammation by twist1 2nd part

ABSTRACT: 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. 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. 10 µg of total RNA from each cell sample was reverse transcribed using T7-(d)T24 primer and SuperScript II reverse transcriptase cDNA extraction with a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate 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) probes were subsequently hybridised with the GeneChip U74Av2 for 16 hrs at 45 oC after washing the hybridisation signals were visualised by staining with streptavidin-phycoerythrin and amplification with an anti-streptavidin antibody TH1_1w_C1; TH1_1w_C2; TH1_1w_C3TH1_4w_C1; TH1_4w_C2; TH1_4w_C3 1w: 1 week in culture; 4w: 4 weeks in culture; C1-3: Culture or Experiment No.

INSTRUMENT(S): [MG_U74Av2] Affymetrix Murine Genome U74A Version 2 Array

ORGANISM(S): Mus musculus  

SUBMITTER: Pawel Durek  




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