Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome response after addition of individual agonists of TLR4 (MPLA) and NOD2 (MDP) receptors to THP-1 cells or its combination

ABSTRACT: Using microarray analysis, we explored the differences in gene expression profiles between individual and combined stimulation of Toll-like receptor 4 (TLR4) and Nucleotide oligomerization domain (NOD)-like receptor (NOD2) in THP-1 cells. Analysis was performed 3 hours post addition of TLR4 agonist MPLA and the NOD2 agonist MDP to THP-1 cells. The results provide the detailed molecular profile of the the genetic response to individual and combined stimulation of TLR4 and NOD2 receptors THP-1 cells (Invivogen) were seeded in 3-cm culture dishes at 1x10^6 cells per dish in RPMI medium. Next day, cells were treated with MDP (20Î¼g/ml) and MPLA (1Î¼g/ml) individually or in combination or left untreated.

ORGANISM(S): Homo sapiens

SUBMITTER: Amir Tukhvatulin

PROVIDER: E-GEOD-79900 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We have begun to approach gd T cells more as prospective innate cells than as conventional T cells. Recent results indicated that purified gd T cells are primed directly in response to pathogen associated molecular patterns (PAMPs) to better respond to secondary signals and increase expression of chemokine and activation-related genes. In microarray and real time PCR analyses of RNA derived from bovine and human gd T cells, transcripts encoding Nod2 were repeatedly amplified. Nod2 is the intracellular receptor for muramyl dipeptide (MDP), a subunit of PGN, functions in regulating innate activities, and was thought to be expressed primarily in APCs. Given our repeated detection of Nod2 transcripts in gd T cells, the specific direct response of gd T cells to MDP was analyzed by microarray, real time PCR, proteome array and in a functional priming assay. The results indicate a subtle activation in response to MDP akin to priming, and suggest a unique mechanism for differential gene expression. Experiment Overall Design: PBLs from calf 129 were stained with GD3.8 directly conjugated to FITC, washed, and sorted on a Vantage SE cell sorter (BD Immunocytometry Systems) as previously described. PBLs from calves 150 and 151 were stained with GD3.8, and anti-Mouse IgG magnetic beads and sorted with MACS magnetic bead system (Miltenyi Biotec) as previously described to a purity of >98%. Sorted cells were rested overnight, stimulated for 4 hours then lysed in Buffer RLT and genomic DNA sheared using Qiashredder columns then frozen at -80oC. RNA was extracted following the manufacturerâs protocol for RNeasy (Qiagen) column purification, assessed on a Bioanalyzer 2100 (Agilent Technologies), and amplified using the Affymetrix One-cycle protocol with approximately 1.7micrograms of total RNA as described in the GeneChipÂ® Expression Analysis Technical Manual (June 2004). Hybridizations to GenechipÂ® Bovine Genome Arrays (Affymetrix) were performed with 15 mg biotin labeled cRNA. Washing and staining was performed in the GeneChipÂ® Fluidics Station 450 using the Midi_euk2v3 protocol. Chip scans were performed on the Affymetrix GeneChipÂ® Scanner 3000. GeneChipÂ® Operating Software (GCOS v.1.1, Affymetrix) 19;20 was used for data collection.

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Transcriptome response after addition of individual agonists of TLR4 (MPLA) and NOD2 (MDP) receptors to THP-1 cells or its combination

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