Investigation Title Gene expression in dendritic cells stimulated with LPS in conditions allowing or inhibiting NFAT activation Comment[Submitted Name] Gene expression in dendritic cells stimulated with LPS in conditions allowing or inhibiting NFAT activation Experimental Design transcription profiling by array Experimental Design Term Source REF EFO Comment[AEMIAMESCORE] 5 Comment[SecondaryAccession] GSE15718 Comment[ArrayExpressReleaseDate] 2010-03-04 Comment[Publication DOI] 10.1038/nature08118 Comment[ArrayExpressAccession] E-GEOD-15718 Comment[MAGETAB TimeStamp_Version] 2010-08-02 14:21:18 Last Changed Rev: 13058 Experimental Factor Name STRAIN Experimental Factor Type strain_or_line Experimental Factor Term Source REF Person Last Name Zanoni Ostuni Granucci Person First Name Ivan Renato Francesca Person Mid Initials Person Email francesca.granucci@unimib.it Person Phone +390264483553 Person Fax +390264483552 Person Address University of Milano-Bicocca, Piazza della Scienza 2, Milan, Italy Person Affiliation University of Milano-Bicocca Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2010-03-04 PubMed ID 19525933 Publication DOI 19525933 Publication Author List Zanoni I, Ostuni R, Capuano G, Collini M, Caccia M, Ronchi AE, Rocchetti M, Mingozzi F, Foti M, Chirico G, Costa B, Zaza A, Ricciardi-Castagnoli P, Granucci F Publication Title CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation. Publication Status journal_article Publication Status Term Source REF The MGED Ontology Experiment Description Interleukin-2 (IL-2) is one of the molecules produced by mouse dendritic cells (DCs) after stimulation by Toll like receptor (TLR) agonists. By analogy with the events following T-cell receptor (TCR) engagement leading to IL-2 production we have observed that DC stimulation with lipopolysaccharide (LPS) induces Src-family kinase and phospholipase C (PLC)γ2 activation, influx of extracellular Ca2+ and calcineurin-dependent nuclear NFAT translocation. We have also observed that the initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. To determine the role of NFAT in LPS activated dendritic cells we have performed microarray analysis in conditions allowing or inhibiting NFAT activation. We show here that LPS-induced NFAT activation via CD14 is necessary to cause death of terminally differentiated DCs, an event that is essential for maintaining self-tolerance and preventing autoimmunity. Consequently, blocking this pathway in vivo causes prolonged DC survival and an increase in T cell priming capability. Gene expression analyses were performed using Affymetrix GeneChips in the following groups of murine bone marrow-derived dendritic cells: 1) CD14-deficient BMDCs stimulated with LPS; 2) wtBMDCs stimulated with LPS in presence of EGTA; 3) wtBMDCs stimulated with LPS. This experimental setting allowed us to select for effects due to Ca2+ fluxes and exclude the effects due to other causes, particularly the block of TRIF recruitment in CD14-deficient cells and the EGTA effects unrelated to Ca2+ chelation. Protocol Name P-GSE15718-4 P-GSE15718-3 P-GSE15718-23 P-GSE15718-21 P-GSE15718-12 P-GSE15718-20 P-GSE15718-27 P-GSE15718-17 P-GSE15718-18 P-GSE15718-16 P-GSE15718-25 P-GSE15718-19 P-GSE15718-26 P-GSE15718-9 P-GSE15718-14 P-GSE15718-24 P-GSE15718-15 P-GSE15718-22 P-GSE15718-13 P-GSE15718-2 P-GSE15718-10 P-GSE15718-5 P-GSE15718-6 P-GSE15718-7 P-GSE15718-11 P-GSE15718-8 P-GSE15718-1 Protocol Type nucleic_acid_extraction grow specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action labeling labeling hybridization image_aquisition feature_extraction feature_extraction bioassay_data_transformation Protocol Description Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. Bone marrow cells were cultured in IMDM (Euroclone) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μM 2-ME (all from Sigma-Aldrich), 10% heat-inactivated FBS (IMDM complete medium), and 10% supernatant of GM-CSF-transduced B16 tumor cells. Fresh medium was added every 3 days. After 7–10 days of culture, cells were analyzed for CD11c expression and were used in assays when >90% were CD11c positive. Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS for 2 hours. Cells were then harvested, washed in PBS and processed. Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stmulated with smooth LPS (1 μg/ml) in presence of EGTA (2 mM) for 24 hours. Cells were then harvested washed in PBS and processed. Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate), and treated with smooth LPS (1μg/ml). After 4 hours cells were harvested, washed in PBS and processed Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stmulated with smooth LPS (1 μg/ml) in presence of EGTA (2 mM) for 12 hours. Cells were then harvested washed in PBS and processed. Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS for 24 hours. Cells were then harvested, washed in PBS and processed. Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stmulated with smooth LPS (1 μg/ml) in presence of EGTA (2 mM) for 2 hours. Cells were then harvested washed in PBS and processed. Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stmulated with smooth LPS (1 μg/ml) in presence of EGTA (2 mM) for 4 hours. Cells were then harvested washed in PBS and processed. Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate), harvested 24 hours later washed in PBS and processed. Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS for 6 hours. Cells were then harvested, washed in PBS and processed. Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stmulated with smooth LPS (1 μg/ml) in presence of EGTA (2 mM) for 6 hours. Cells were then harvested washed in PBS and processed. Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS for 12 hours. Cells were then harvested, washed in PBS and processed. Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate), and treated with smooth LPS (1μg/ml). After 2 hours cells were harvested, washed in PBS and processed Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate), and treated with smooth LPS (1μg/ml). After 12 hours cells were harvested, washed in PBS and processed Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS for 4 hours. Cells were then harvested, washed in PBS and processed. Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate), and treated with smooth LPS (1μg/ml). After 24 hours cells were harvested, washed in PBS and processed Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate), harvested 24 hours later, washed in PBS and processed. Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate), and treated with smooth LPS (1μg/ml). After 6 hours cells were harvested, washed in PBS and processed DCs were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/well) and harvested 24 hours later. Biotinilated cRNA was prepared using GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinilated cRNA was prepared using GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). 10 μg of fragmented cRNAs were hybridized to the standard arrays for 16 hours at 45°C; the arrays were then washed and stained using the fluidics station. Arrays were scanned on an Affymetrix GeneChip scanner 3000. Data were processed using Affymetrix Microarray Suite 5.0. Data were processed using Affymetrix Microarray Suite 5.0 ID_REF =
VALUE = signal intensity calculated using MAS5.0, unscaled
ABS_CALL = detection call indicating if transcript was present (P), absent (A) or marginal (M)
DETECTION P-VALUE = determined by detection algorithm, indicates significance of detection call Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF The MGED Ontology The MGED Ontology The MGED Ontology SDRF File E-GEOD-15718.sdrf.txt Term Source Name NCBI Taxonomy The MGED Ontology ArrayExpress EFO The MGED Ontology Term Source File http://www.ncbi.nlm.nih.gov/Taxonomy/ http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version