Dataset Information

Stimulation of BL2 cell line with lipopolysaccharide (LPS) for 6h

ABSTRACT: Identification of genes up or down regulated in LPS stimulated samples in comparison to control samples. 3 LPS stimulated samples vs. 3 controls were analyzed

OTHER RELATED OMICS DATASETS IN: PRJNA141185

ORGANISM(S): Homo sapiens

SUBMITTER: Matthias Maneck Alexandra Schrader Dieter Kube Rainer Spang

PROVIDER: E-GEOD-29700 | ArrayExpress | 2011-07-01

SECONDARY ACCESSION(S): GSE29700PRJNA141185

REPOSITORIES: GEO, ArrayExpress

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Publications

Genomic data integration using guided clustering.

Maneck Matthias M Schrader Alexandra A Kube Dieter D Spang Rainer R

Bioinformatics (Oxford, England) 20110617 16

<h4>Motivation</h4>In biomedical research transcriptomic, proteomic or metabolomic profiles of patient samples are often combined with genomic profiles from experiments in cell lines or animal models. Integrating experimental data with patient data is still a challenging task due to the lack of tailored statistical tools.<h4>Results</h4>Here we introduce guided clustering, a new data integration strategy that combines experimental and clinical high-throughput data. Guided clustering identifies s ...[more]

PMID: 21685050

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Project description:<h4>Background</h4>Gene expression in lipopolysaccharide (LPS)-stimulated monocytes is mainly studied by quantitative real-time reverse transcription PCR (RT-qPCR) using GAPDH (glyceraldehyde 3-phosphate dehydrogenase) or ACTB (beta-actin) as reference gene for normalization. Expression of traditional reference genes has been shown to vary substantially under certain conditions leading to invalid results. To investigate whether traditional reference genes are stably expressed in LPS-stimulated monocytes or if RT-qPCR results are dependent on the choice of reference genes, we have assessed and evaluated gene expression stability of twelve candidate reference genes in this model system.<h4>Results</h4>Twelve candidate reference genes were quantified by RT-qPCR in LPS-stimulated, human monocytes and evaluated using the programs geNorm, Normfinder and BestKeeper. geNorm ranked PPIB (cyclophilin B), B2M (beta-2-microglobulin) and PPIA (cyclophilin A) as the best combination for gene expression normalization in LPS-stimulated monocytes. Normfinder suggested TBP (TATA-box binding protein) and B2M as the best combination. Compared to these combinations, normalization using GAPDH alone resulted in significantly higher changes of TNF-alpha (tumor necrosis factor-alpha) and IL10 (interleukin 10) expression. Moreover, a significant difference in TNF-alpha expression between monocytes stimulated with equimolar concentrations of LPS from N. meningitides and E. coli, respectively, was identified when using the suggested combinations of reference genes for normalization, but stayed unrecognized when employing a single reference gene, ACTB or GAPDH.<h4>Conclusions</h4>Gene expression levels in LPS-stimulated monocytes based on RT-qPCR results differ significantly when normalized to a single gene or a combination of stably expressed reference genes. Proper evaluation of reference gene stabiliy is therefore mandatory before reporting RT-qPCR results in LPS-stimulated monocytes.

Project description:BACKGROUND:An uncontrolled inflammatory response is a critical pathophysiological feature of sepsis. Mesenchymal stem cells (MSCs) induce macrophage phenotype polarization and reduce inflammation in sepsis. MSC-secreted transforming growth factor beta (TGF-?) participated in the immune modulatory function of MSCs. However, the underlying mechanism of MSC-secreted TGF-? was not fully elucidated in regulation macrophage M2-like polarization. METHODS:The paracrine effects of MSCs on macrophage polarization were studied using a co-culture protocol with LPS-stimulated RAW264.7 cells/mouse peritoneal macrophages and MSCs. The effect of TGF-? in the co-culture system was blocked by the TGF-? receptor inhibitor. To determine the role of MSC-secreted TGF-?, we used recombinant TGF-? to culture with LPS-stimulated RAW264.7 cells. In addition, we employed antibody microarray analysis to determine the mechanisms of MSC secreted TGF-? on LPS-stimulated RAW264.7 cell/mouse peritoneal macrophage M2-like polarization. Furthermore, we used an Akt inhibitor and a FoxO1 inhibitor to inhibit the Akt/FoxO1 pathway. The nuclear translocation of FoxO1 was detected by Western blot. RESULTS:MSCs induced LPS-stimulated RAW264.7 cell/mouse peritoneal macrophage polarization towards the M2-like phenotype and significantly reduced pro-inflammatory cytokine levels via paracrine, which was inhibited by TGF-? receptor inhibitor. Furthermore, we found that MSC-secreted TGF-? enhanced the macrophage phagocytic ability. The antibody microarray analysis and Western blot verified that TGF-? treatment activated the Akt/FoxO1 pathway in LPS-stimulated macrophages, TGF-?-induced FoxO1 nuclear translocation and obviously expressed in the cytoplasm, the effects of TGF-? regulatory effects on LPS-stimulated macrophage were inhibited by pre-treatment with Akt inhibitor and FoxO1 inhibitor. CONCLUSIONS:TGF-? secreted by MSCs could skew LPS-stimulated macrophage polarization towards the M2-like phenotype, reduce inflammatory reactions, and improve the phagocytic ability via the Akt/FoxO1 pathway, providing potential therapeutic strategies for sepsis.

			Action	DRS
	E-GEOD-29700.README.txt	Txt
	E-GEOD-29700.eSet.r	Other
	E-GEOD-29700.idf.txt	Idf
	E-GEOD-29700.processed.1.zip	Processed
	E-GEOD-29700.raw.1.zip	Raw