Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Expression data from study on IL33 signaling in Dusp5 deficient eosinophils

ABSTRACT: Dusp5 regulates ERK phosporylation following IL-33 receptor ligation in cultured eosinophils. Dusp5 deficient eosinophils show increased ERK phosphorylation, and as a result are less apoptotic. Since ERK stimulation results in downstream activation of transcription factors, we are utilizing a microarray approach to find alterations in gene expression to uncover potential mechanisms for increased cell survival. Day 12 bone marrow derived eosinophils from Dusp5+/+ and Dusp5-/- mice (5 mice per genotype) were left untreated (media only) or IL-33 (25ng/ml) stimulated for 4 hours. Treated cells were washed with ice cold PBS and cell pellets were directly lysed in RLT buffer (Qiagen) followed by immediate RNA isolation (RNEasy plus mini kit with genomic DNA eliminator columns). Sample group names: Group 1- Dusp5+/+ Untreated (N=5) Group 2- Dusp5-/- Untreated (N=5) Group 3- Dusp5+/+ IL-33 4hrs (N=5) Group 4- Dusp5-/- IL-33 4hrs (N=5) Sample Names: Group 1- BM Eos unstimulated WT-1....WT-5 Group 2- BM Eos unstimulated Dusp5-/- -1......Dusp5-/- -5 Group 3- BM Eos IL-33 4hrs WT-1....WT-5 Group 4- BM Eos IL-33 4hrs Dusp5-/- -1......Dusp5-/- -5

ORGANISM(S): Mus musculus

SUBMITTER: Derek Holmes

PROVIDER: E-GEOD-62999 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Objectives: Eosinophils cultured with all-trans RA (ATRA) and 9-cis RA showed dramatically induced cell survival, and the efficacy of RAs (10-6 M) was similar to that of IL-5 (1 ng/ml), the most critical cytokine for eosinophil activation. Using a gene microarray, we studied mRNA profile regulated by RAs. Methods: Peripheral venous blood was obtained from subjects with mild eosinophilia. Eosinophils were isolated by sedimentation with 6% dextran followed by centrifugation on 1.088 Percoll (Pharmacia, Uppsala, Sweden) density gradients. The cells were further purified by negative selection using anti-CD16 immunomagnetic beads and a MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). Purified eosinophils were resuspended in RPMI 1640 medium (Gibco, Grand Island, NY) with 10% FCS, and incubated with vehicle, 10-6 M of 9-cis RA, or ATRA in 1% human serum albumin-coated plates, each 4 Ã 106 cells, for 4 h. Then cells were lysed, and total RNA was isolated using Isogen (Nippon Gene, Tokyo, Japan) as per the manufacturerâs instructions. RNA was repurified with phenol-chloroform extraction and ethanol precipitation. For each cell culture condition, the total RNA from three donors was pooled, enabling us to acquire adequate amounts of RNA for microarray analysis. Results: There was little variation between the samples treated by 9-cisRA and ATRA. The comparison between vehicle control and 9-cisRA, or vehicle control and ATRA, identified an increase (greater than twofold change over control) in 19 and 11 transcripts, respectively. The number of decreased transcripts (less than 0.5-fold) resulting fromby 9-cisRA or ATRA exposure wasere 19 and 8 transcripts, respectively. In these transcripts, the expression changes relative to vehicle control were smaller in withthe ATRA compared with 9-cis RA, suggesting that 9-cis RA induced gene transcription more effectively rather than ATRA. Of note, among the ten genes with the biggestmost decrease relative tofrom vehicle control, we found an apoptosis- related gene, caspase-3. Conclusions: We screened several genes involved in RA-induced prolonged eosinophil survival. Purified peripheral blood eosinophils were incubated with vehicle, 10-6 M of 9-cis RA, or ATRA in 1% human serum albumin-coated plates, each 4 Ã 106 cells, for 4 h. Then cells were lysed, and total RNA was isolated. RNA was repurified with phenol-chloroform extraction and ethanol precipitation. For each cell culture condition, the total RNA from three donors was pooled, enabling us to acquire adequate amounts of RNA for microarray analysis.

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Expression data from study on IL33 signaling in Dusp5 deficient eosinophils

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