Unknown,Transcriptomics,Genomics,Proteomics

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T lymphocyte activation gene identification by coregulated expression on DNA microarrays

ABSTRACT: High-capacity methods for assessing gene function have become increasingly important because of the increasing number of newly identified genes emerging from large-scale genome sequencing and cDNA cloning efforts. We investigated the use of DNA microarrays to identify uncharacterized genes specifically involved in human T cell activation. Activation of human peripheral blood T lymphocytes induced significant changes in hundreds of transcripts, but most of these were not unique to T cell activation. Variation of experimental parameters and analysis techniques allowed better enrichment for gene expression changes unique to T cell activation. Best results were achieved by identification of genes that were most highly coregulated with the T-cell-specific transcript interleukin 2 (IL2) in a "compendium" of experiments involving both T cells and other cell types. Among the genes most highly coregulated with IL2 were many genes known to function during T cell activation, together with ESTs of unknown function. Four of these ESTs were extended to novel full-length clones encoding T-cell-regulated proteins with predicted functions in GTP metabolism, cell organization, and signal transduction.

ORGANISM(S): Homo sapiens

SUBMITTER: Mao Mao

PROVIDER: E-GEOD-902 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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T lymphocyte activation gene identification by coregulated expression on DNA microarrays.

Mao Mao M Biery Matt C MC Kobayashi Sumire V SV Ward Terry T Schimmack Greg G Burchard Julja J Schelter Janell M JM Dai Hongyue H He Yudong D YD Linsley Peter S PS

Genomics 20040601 6

High-capacity methods for assessing gene function have become increasingly important because of the increasing number of newly identified genes emerging from large-scale genome sequencing and cDNA cloning efforts. We investigated the use of DNA microarrays to identify uncharacterized genes specifically involved in human T cell activation. Activation of human peripheral blood T lymphocytes induced significant changes in hundreds of transcripts, but most of these were not unique to T cell activati ...[more]

PMID: 15177553

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Project description:MicroRNAs have well-established roles in eukaryotic host responses to viruses and extracellular bacterial pathogens. In contrast, microRNA responses to invasive bacteria have remained unknown. Here, we report cell type-dependent microRNA regulations upon infection of mammalian cells with the enteroinvasive pathogen, Salmonella Typhimurium. Murine macrophages strongly up-regulate NF-κB associated microRNAs; strikingly, these regulations which are induced by the bacterial lipopolysaccharides (LPS) occur and persist regardless of successful host invasion and/or replication, or whether an inflammatory response is mounted, suggesting that microRNAs belong to the first line of anti-bacterial defence. However, a suppression of the global immune regulator miR-155 in endotoxintolerant macrophages revealed that microRNA responses also depend on the status of infected cells. This study identifies the let-7 family as the common denominator of Salmonella regulated microRNAs in macrophages and epithelial cells, and suggests that repression of let-7 relieves cytokine IL-6 and IL-10 mRNAs from negative post-transcriptional control. Our results establish a paradigm of microRNA-mediated feed-forward activation of inflammatory factors when mammalian cells are targeted by bacterial pathogens. The murine macrophage cell-line RAW 264.7 was exposed to wild-type Salmonella typhimurium SL1344 or to the corresponding knockout strain lacking both major genomic loci associated with Salmonella pathogenicity ("SPI1" and "SPI2"). To induce infectivity overnight cultures of Salmonella were dilluted 1:100 and grown to OD2. Host cells at a density of approximately 10e6 per well of a 6-well plate were infected at an MOI of 1. Upon initial incubation for 30 min cells were further incubated for 24 h in RPMI supplemented with gentamicin (10 µg / ml). As determined by a gentamicin protection and plating assay the SPI1/2 double knockout strain is deficient in both invasion and intracellular replication. We compared mouse mRNA abundances between cells infected with wild-type ("24WT") or SPI1/2 knockout Salmonella ("24SPI1/2") and non-infected, mock-treated controls ("24C"). Both strains mounted a similar inflammatory response as judged from the abundance of pro-inflammatory genes compared to the controls. These data indicate, that macrophages can mount a full inflammatory response to extracellular bacteria, that is not further enhanced or attenuated by the sensing of intracellular bacteria. Murine RAW 264.7 cells were rendered endotoxin-tolerant by pre-exposure to heat-killed Salmonella typhimurium SL1344 ("HKS") at an MOI of 10 for 20 h. 5 days upon removal of the HKS stimulus cells were exposed to wild-type Salmonella typhimurium SL1344 or to the corresponding knockout strain lacking both major genomic loci associated with Salmonella pathogenicity ("SPI1" and "SPI2"). To induce infectivity overnight cultures of Salmonella were dilluted 1:100 and grown to OD2. Host cells at a density of approximately 10e6 per well of a 6-well plate were infected at an MOI of 1. Upon initial incubation for 30 min cells were further incubated for 24 h in RPMI supplemented with gentamicin (10 µg / ml). As determined by a gentamicin protection and plating assay the SPI1/2 double knockout strain is deficient in both invasion and intracellular replication. The wild-type Salmonella strain invades and replicates in endotoxin-tolerant RAW 264.7 cells, though the intracellular replication rate is significantly reduced (by roughly 40 %) compared to naive RAW 264.7 cells. We compared mouse mRNA abundances between cells infected with wild-type ("wt") and non-infected, mock-treated controls ("Mock") or between wild-type ("wt") and SPI1/2-knockout ("SPI1/2") infected cells. As apparent from the Microarray data the wild-type Salmonella strain induces a typical inflamatory response in endotoxin-tolerant RAW 264.7 cells (as judged from the fold-induction of NFkB-inducible genes). As expected however, mRNAs corresponding to known inflammatory genes are significantly less abundant in response to the non-invasive SPI1/2 knockout strain, compared to wild-type infection. These data indicate, that endotoxin-tolerance renders macrophages largely insensitive to extracellular, but not to intracellular bacteria. ***This submission represents the mRNA component of the study

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T lymphocyte activation gene identification by coregulated expression on DNA microarrays

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T lymphocyte activation gene identification by coregulated expression on DNA microarrays.

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