Gene expression programs in PBMCs elicited by 6 major mediators of the immune response.
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Simon Waddell
PROVIDER: E-GEOD-17762 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Dissecting interferon-induced transcriptional programs in human peripheral blood cells.
Waddell Simon J SJ Popper Stephen J SJ Rubins Kathleen H KH Griffiths Michael J MJ Brown Patrick O PO Levin Michael M Relman David A DA
PloS one 20100322 3
Interferons are key modulators of the immune system, and are central to the control of many diseases. The response of immune cells to stimuli in complex populations is the product of direct and indirect effects, and of homotypic and heterotypic cell interactions. Dissecting the global transcriptional profiles of immune cell populations may provide insights into this regulatory interplay. The host transcriptional response may also be useful in discriminating between disease states, and in underst ...[more]
Similar Datasets
Project description:PBMC cell subsets were treated with 0.6 pM IFNg for 0-24h and compared to mock-treated time series. Abstract: Background Interferons are key modulators of the immune system, and are central to the control of many diseases. The response of immune cells to stimuli in complex populations is the product of direct and indirect effects, homotypic and heterotypic cell interactions. Dissecting the global transcriptional profiles of immune cell populations may provide insights into this regulatory interplay. The host transcriptional response may also be useful in discriminating between disease states, and in understanding pathophysiology. The transcriptional programs of cell populations in health therefore provide a paradigm for deconvoluting disease-associated gene expression profiles. Results: We used human cDNA microarrays to (1) compare the gene expression programs in human peripheral blood mononuclear cells (PBMCs) elicited by 6 major mediators of the immune response: interferons a, b, w and g, IL12 and TNFa; and (2) characterise the transcriptional responses of purified immune cell populations (CD4+ and CD8+ T cells, B cells, NK cells and monocytes) to IFNg stimulation. We defined a highly stereotyped response to type I interferons, while responses to IFNg and IL12 were largely restricted to a subset of type I interferon-inducible genes. TNFa stimulation resulted in a distinct pattern of gene expression. Cell type-specific transcriptional programs were identified, highlighting the pronounced response of monocytes to IFNg, and emergent properties associated with IFN-mediated activation of mixed cell populations. Conclusions: This information provides a detailed view of cellular activation by immune mediators, and contributes an interpretive framework for the detection and definition of host immune responses in a variety of disease settings. PBMC extraction and stimulation Human primary peripheral blood mononuclear cells (PBMCs) were purified from whole blood of healthy donors using Ficoll-Paque PLUS (GE Healthcare) according to manufacturers instructions. PBMCs were incubated at 1.5-2.0 x 106 cells/well for 24 h before stimulation in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum and 2 mM L-glutamine (Invitrogen) at 37C, 5% CO2. Cells were treated with 0.006 pM, 0.6 pM or 60 pM recombinant IFNg (R&D Systems), and sampled at time intervals from 0.5 h to 12 h after stimulation. Additionally, cells were treated with 0.1 % BSA/PBS alone and used for untreated (mock) control time courses. RNA extraction and amplification Total RNA was extracted in TRIzol LS (Invitrogen) followed by standard chloroform purification and isopropanol precipitation. RNA was resuspended in RNase-free water, quantitated on the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) and stored at -80C. 500 ng total RNA was amplified using the MessageAmp modified Eberwine linear amplification procedure (Applied Biosystems). All samples to be compared were extracted and amplified together. Microarray analysis 4 ug amplified RNA was labelled with Cy5-dUTP (GE Healthcare) and combined with 3 ug of Cy3-labelled reference cDNA derived from a pool of RNA derived from a panel of 11 human cell lines (Stratagene Universal Human Reference RNA). The samples were washed and concentrated using MinElute columns (Qiagen) and competitively hybridised to custom printed cDNA microarrays containing 37,632 elements for approximately 18,000 unique human genes. The hybridised slides were scanned using a GenePix 4000A microarray scanner (Axon Instruments). Comparative spot intensities were calculated from the images, and areas of poor quality excluded from further analysis using GenePix Pro 6.0 (Axon Instruments). Data were deposited in the Stanford Microarray Database. Analysis was restricted to cDNA elements with a regression correlation of > 0.6, fluorescence intensities of > 2.5 fold signal/background in Cy3 or Cy5 channels and a minimum signal intensity of > 100 in both channels for at least 80% of the arrays. The expression ratios were normalised for array variation, and the data zero-transformed using a custom-designed Microsoft Excel macro (C. Liu, Stanford). The statistical package SAM (Significance Analysis of Microarrays, version 1.15) was used to identify genes significantly differentially expressed in the normalised data sets by pairwise comparison with a minimum 2 fold cutoff at a false discovery rate of < 1% of the median. The transformed datasets were then hierarchically clustered using Cluster 2.11 and the results displayed using Treeview 1.60. A cell type comparison design experiment design type compares cells of different type for example different cell lines.
2010-06-23 | E-GEOD-16253 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE7260: epithelial-mesenchymal interaction of the breast GSE7263: Effect of heterotypic interaction between breast cancer cell line MDA-MB231 and CCL-171 fibroblast Refer to individual Series
2007-11-13 | E-GEOD-7264 | biostudies-arrayexpress
Project description:These 95 arrays are the basis for Figure 2 of our manuscript "An interferon-response induced by tumor-stroma interaction in a subset of human breast cancers". Abstract:Background Communication between different cell types is a cardinal feature of multi-cellular organisms and perturbations in these cell-cell interactions are a key feature of cancer. However, little is known about the systematic effects of cell-cell interaction on global gene expression in cancer. Methods and Findings We used an ex vivo culture model to simulate tumor-stroma interaction by systematically co-cultivating breast cancer cells with stromal fibroblasts and determined associated gene expression changes with cDNA microarrays. The picture of heterotypic interaction effects that emerged from this analysis is complex, reflecting the variation in signaling capacities and responsiveness of the involved cells. A frequent and prominent response to epithelial-mesenchymal interaction was an induction of interferon-response genes, observed in 4 of the 7 breast cancer cell lines in response to co-culture with fibroblasts, but not in normal mammary epithelial cells. In response to close contact with these breast cancer cells, the fibroblasts secreted type I interferons, which, in turn, induced expression of the IRGs in the tumor cells. Immunohistochemical analysis of human breast cancer tissues showed that STAT1, the key transcriptional activator of the interferon-response genes, and itself an IRG, was expressed in a subset of the cancers, with a striking pattern of elevated expression in the cancer cells in contact with, or close proximity to, the tumor stroma ? paralleling the response seen in our ex vivo model. In vivo, expression of the interferon-response genes was remarkably coherent, providing a basis for segregation of the 295 early-stage breast cancers into two groups. Tumors with high expression levels (n=161) of IRG were associated with significantly shorter overall survival; 59 % at 10 years versus 80% for tumors with low interferon-response gene expression levels (n=134) (log-rank p=0.001). Conclusion Our results suggest that an interaction between some breast cancer cells and stromal fibroblasts can induce an interferon response, and that this response may be associated with a greater propensity for tumor progression. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Computed
2007-11-13 | E-GEOD-7260 | biostudies-arrayexpress
Project description:In response to polarization cues, cultured Caco-2 cells, a human colon adenocarcinoma-derived cell line, form a polarized epithelium resembling normal enterocytes. We investigated potential signaling mechanisms activated by Caco-2 cells that might trigger the genome-wide transcriptional reprogramming that accompanies polarization (Saaf et al, submitted-I). cDNA microarrays were used to compare the transcriptional profile of Caco-2 polarization to the gene expression profiles of normal human colon and colon tumors. The transcript profile of proliferating, non-polarized Caco-2 cells has striking parallels to the gene expression profile of human colon cancer in vivo. However, as Caco-2 cells develop polarity, the gene expression profile shifts to one more closely resembling that of normal colon tissue, suggesting that the underlying regulatory mechanisms that mediate Caco-2 cell polarization are similar to those that occur during in vivo enterocyte differentiation. We show that transcriptional re-programming of Caco-2 cells during development of cell polarity occurs in the context of signaling pathways that are regulated in a manner that is remarkably similar to those in normal intestinal development. For example, transcriptional targets of the Wnt pathway are tightly regulated during Caco-2 cell polarization, mimicking the gradient of Wnt-mediated transcription in the crypt (high expression) to villus (low expression) axis in human intestine. However, Caco-2 cells lack full-length APC necessary for normal Wnt-regulated degradation of beta-catenin. Biochemical analysis indicates that regulation of the Wnt pathway occurs in the nucleus at the level of activation of target genes by the beta-catenin-TCF complex, revealing a novel additional mechanism by which intestinal cells may regulate Wnt signaling during their maturation. In addition, other signaling pathways including Notch, BMP, Hedgehog, and growth factor, were temporally regulated during Caco-2 cell polarization. Surprisingly, modulation of these signaling pathways in Caco-2 cells occurs in the absence of morphogen gradients and interactions with stromal cells characteristic of enterocyte differentiation in situ. This dataset contains gene expression profiles of 9 normal colon samples and 15 colon tumor samples. Samples of tumor and normal colon mucosa were collected from colon cancer resection from Department of Surgery, Queen Mary Hospital University of Hong Kong. Tissue was frozen in liquid nitrogen within 30 min of resection. Nonneoplastic mucosa from colon was dissected free of muscle and histologically confirmed to be tumor free by frozen section. Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) from each tissue sample and processed for microarray hybridization. A disease state experiment design type is where the state of some disease such as infection, pathology, syndrome, etc is studied. Disease State: Tumor/Normal colon samples Using regression correlation
2007-08-17 | E-GEOD-7466 | biostudies-arrayexpress
Project description:Background MeCP2, methyl-CpG-binding protein 2, binds to methylated cytosines at CpG dinucleotides, as well as to unmethylated DNA, and affects chromatin condensation. MECP2 mutations in females lead to Rett syndrome, a neurological disorder characterized by developmental stagnation and regression, loss of purposeful hand use and speech, stereotypic hand movements, deceleration of brain growth, autonomic dysfunction and seizures. Most mutations occur de novo during spermatogenesis. Located at Xq28, MECP2 is subject to X inactivation, and affected females are mosaic. Rare hemizygous males suffer from a severe congenital encephalopathy. Methods To identify pathways mis-regulated by MeCP2 deficiency, microarray-based global gene expression studies were carried out on cerebellum of Mecp2 mutant mice. We compared transcript levels in mutant/wildtype male sibs of two different MeCP2-deficient mouse models at 2, 4 and 8 weeks of age. Increased transcript levels were evaluated by real-time quantitative RT-PCR. Chromatin immunoprecipitation assays were used to document in vivo MeCP2 binding to promoter regions of candidate target genes. Results In the mutants, several hundred genes showed altered expression levels. Twice as many were increased than decreased, and only 27 genes were differentially expressed at more than one time point. The number of misregulated genes was 30% lower in mice with an exon 3 deletion (Mecp2tm1.1Jae) than in mice with a larger deletion (Mecp2tm1.1Bird). Between the mutants, few misregulated genes overlapped at each time point. Real-time quantitative RT-PCR assays validated increased transcript levels for four genes: Irak1, interleukin-1 receptor-associated kinase 1; Fxyd1, phospholemman, associated with Na, K-ATPase; Reln, encoding reelin, an extracellular signaling molecule essential for neuronal lamination and synaptic plasticity; and Gtl2/Meg3, an imprinted maternally expressed non-translated RNA that serves as a host gene for C/D box snoRNAs and microRNAs. Chromatin immunoprecipitation assays documented in vivo MeCP2 binding to promoter regions of Fxyd1, Reln, and Gtl2. Conclusions Transcriptional profiling of cerebellum failed to detect significant global changes in Mecp2-mutant mice. Increased transcript levels of Irak1, Fxyd1, Reln, and Gtl2 may contribute to the neuronal dysfunction in MeCP2-deficient mice and individuals with Rett syndrome. Our data provide testable hypotheses for future studies of the regulatory or signaling pathways that these genes act on. A genetic modification design type is where an organism(s) has had genetic material removed, rearranged, mutagenized or added, such as knock out. Computed
2007-08-15 | E-GEOD-8774 | biostudies-arrayexpress
Project description:We analyzed effects of monkeypox (MPX) and vaccinia (VAC) infection on cultured human cells. Our custom-designed arrays contain >18,000 human genes as well as genes for each open reading frame of MPX and VAC. A reference experiement design type is where all samples are compared to a common reference. Compound Based Treatment: Chemical used for treating infected cells Infection: Virus used for infection Cell Line: Cell line used for infection Time: Time cells were harvested after infection Keywords: reference_design Experiments used primary human monocytes with mock infection, VAC-Western Reserve, VAC-NYBOH, MPX-Zaire, gamma irradiated (killed) MPX-Zaire, and Ebola-Zaire (EBOV) (deposited March 2004). The next set of experiments used primary human monocytes (Mf7.29) and primary human fibroblasts (S100) with mock infection, VAC-Western Reserve, MPX-Zaire, and gamma irradiated MPX-Zaire (g-MPX) (deposited August 2004). The final set used primary human monocytes (Mph), primary human fibroblasts (NHDF), and HeLa cells with mock infection, VAC-Western Reserve, MPX-Zaire, and gamma irradiated MPX-Zaire (g-MPX). Some cells were treated with ionomycin + PMA (I+P), cycloheximide (CHX) or hydroxyurea (HU). (deposited November 2005). Total RNA from cells was harvested at each timepoint, preserved in Trizol, Trizol-extracted total RNA, 1 round amplified (Ambion MessageAmp I), directly labeled by Cy5 incorporation during reverse transcription of amplified RNA. Each sample was hybridized versus a common reference (Stratagene Universal Human Reference RNA plus a mix of poxvirus transcripts from all timepoints). The reference was1 round of amplification (Ambion MessageAmp I), directly labeled by Cy3 incorporation during reverse transcription of amplified reference RNA. Cy3 and Cy5 samples were hybridized to a custom human-poxvirus array. Fluorescent images of hybridized microarrays were acquired using the GenePix 4000B and GenePix 4200AL microarray scanners.
2008-07-17 | E-GEOD-11234 | biostudies-arrayexpress
Project description:Treatment of PBMC with 0.6 pM cytokine from 0-24h Abstract: Background Interferons are key modulators of the immune system, and are central to the control of many diseases. The response of immune cells to stimuli in complex populations is the product of direct and indirect effects, homotypic and heterotypic cell interactions. Dissecting the global transcriptional profiles of immune cell populations may provide insights into this regulatory interplay. The host transcriptional response may also be useful in discriminating between disease states, and in understanding pathophysiology. The transcriptional programs of cell populations in health therefore provide a paradigm for deconvoluting disease-associated gene expression profiles. Results: We used human cDNA microarrays to (1) compare the gene expression programs in human peripheral blood mononuclear cells (PBMCs) elicited by 6 major mediators of the immune response: interferons a, b, w and g, IL12 and TNFa; and (2) characterise the transcriptional responses of purified immune cell populations (CD4+ and CD8+ T cells, B cells, NK cells and monocytes) to IFNg stimulation. We defined a highly stereotyped response to type I interferons, while responses to IFNg and IL12 were largely restricted to a subset of type I interferon-inducible genes. TNFa stimulation resulted in a distinct pattern of gene expression. Cell type-specific transcriptional programs were identified, highlighting the pronounced response of monocytes to IFNg, and emergent properties associated with IFN-mediated activation of mixed cell populations. Conclusions: This information provides a detailed view of cellular activation by immune mediators, and contributes an interpretive framework for the detection and definition of host immune responses in a variety of disease settings. PBMC extraction and stimulation Human primary peripheral blood mononuclear cells (PBMCs) were purified from whole blood of healthy donors using Ficoll-Paque PLUS (GE Healthcare) according to manufacturers instructions. PBMCs were incubated at 1.5-2.0 x 106 cells/well for 24 h before stimulation in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum and 2 mM L-glutamine (Invitrogen) at 37C, 5% CO2. Cells were treated with 0.006 pM, 0.6 pM or 60 pM recombinant IFNg (R&D Systems), and sampled at time intervals from 0.5 h to 12 h after stimulation. Additionally, cells were treated with 0.1 % BSA/PBS alone and used for untreated (mock) control time courses. RNA extraction and amplification Total RNA was extracted in TRIzol LS (Invitrogen) followed by standard chloroform purification and isopropanol precipitation. RNA was resuspended in RNase-free water, quantitated on the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) and stored at -80C. 500 ng total RNA was amplified using the MessageAmp modified Eberwine linear amplification procedure (Applied Biosystems). All samples to be compared were extracted and amplified together. Microarray analysis 4 ug amplified RNA was labelled with Cy5-dUTP (GE Healthcare) and combined with 3 ug of Cy3-labelled reference cDNA derived from a pool of RNA derived from a panel of 11 human cell lines (Stratagene Universal Human Reference RNA). The samples were washed and concentrated using MinElute columns (Qiagen) and competitively hybridised to custom printed cDNA microarrays containing 37,632 elements for approximately 18,000 unique human genes. The hybridised slides were scanned using a GenePix 4000A microarray scanner (Axon Instruments). Comparative spot intensities were calculated from the images, and areas of poor quality excluded from further analysis using GenePix Pro 6.0 (Axon Instruments). Data were deposited in the Stanford Microarray Database. Analysis was restricted to cDNA elements with a regression correlation of > 0.6, fluorescence intensities of > 2.5 fold signal/background in Cy3 or Cy5 channels and a minimum signal intensity of > 100 in both channels for at least 80% of the arrays. The expression ratios were normalised for array variation, and the data zero-transformed using a custom-designed Microsoft Excel macro (C. Liu, Stanford). The statistical package SAM (Significance Analysis of Microarrays, version 1.15) was used to identify genes significantly differentially expressed in the normalised data sets by pairwise comparison with a minimum 2 fold cutoff at a false discovery rate of < 1% of the median. The transformed datasets were then hierarchically clustered using Cluster 2.11 and the results displayed using Treeview 1.60. A compound treatment design type is where the response to administration of a compound or chemical (including biological compounds such as hormones) is assayed.
2010-06-23 | E-GEOD-16251 | biostudies-arrayexpress
Project description:CELL CULTURE: The human intestinal epithelial cell line Caco-2 (obtained from Dr. Stanley Falkow, Stanford University) was used to study the genome-wide expression program underlying the transition from cell proliferation to establishment of a polarized epithelial layer. Cells were first grown in sparse culture on plastic p15 dishes to generate a population of "contact naive" cells. These cells were combined and plated at confluency on Costar filter inserts. The day cells were plated on filters is designated the zero time-point. Cells were cultured for 26 days, during which time they develop structural and functional polarity. Media was changed every other day. Triplicate samples were harvested at eleven time-points over the time course for microarray analysis. The entire time course was performed twice. Three control arrays are included in this data set; (1) to determine how the gene-expression patterns in Caco-2 cells grown on a plastic dish compares to the expression profiles identified in Caco-2 cells grown on Costar filter inserts (microarray shcu182, data not shown), (2) to tested whether feeding the cells at different time-points had an effect on the Caco-2 gene-expression program. All samples taken (except at the day zero time-point) were fed with fresh media one day prior to harvesting the sample. During TC3 and TC4, we performed two test arrays analyzing the transcriptional program in Caco-2 cells that were fed with fresh cell culture media 4h prior to taking the 7D time-point (microarrays shcu177 and shcu178, indicated with * in table S1). Groups of assays that are related as part of a time series. See additional information on overall design in Series supplementary file. ABSTRACT: Although there is considerable evidence implicating post-translational mechanisms in the development of epithelial cell polarity, little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized the temporal program of gene expression during cell-cell adhesion-initiated polarization of human Caco-2 cells in tissue culture, which develop structural and functional polarity similar to that of enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell-cell contacts between neighboring cells. Expression of a cluster of genes involved in cell proliferation switched off and concomitantly the expression of genes related to functional specializations of polarized epithelial cells was induced. Induction of these latter genes correlated with formation of important structural and functional features related to enterocyte differentiation and establishment of structural and functional cell polarity. Coordinated expression of genes encoding components of functional cell structures were often observed, indicating temporal control of expression and assembly of multiprotein complexes. Changes in gene expression patterns during Caco-2 cell differentiation in vitro paralleled those in terminally differentiating enterocytes migrating up the intestinal crypt-villus axis in vivo, despite the lack of stromal cells and gradients of morphogens in this tissue culture model. Keywords: time_series_design Computed
2007-08-16 | E-GEOD-7442 | biostudies-arrayexpress
Project description:These 12 arrays are the basis for Figure 1 of the "An interferon-response induced by tumor-stroma interaction in a subset of human breast cancers" manuscript. Figure 1: Effect of heterotypic interaction between breast cancer cell line MDA-MB231 and CCL-171 fibroblast. Biologically independent replicates of the mono-cultured fibroblast CCL-171, the breast cancer cell line MDA-MB231 and the mixed co-culture of CCL-171 and MDA-MB231 were grown for 48h at low serum conditions and characterized by DNA microarray hybridization. Hierarchical clustering of a total of 4333 elements that display a greater than 3-fold variance in expression in more than 3 different experimental samples. Data from individual elements or genes are represented as single rows, and different experiments are shown as columns. Red and green denote expression levels of the samples. The intensity of the color reflects the magnitude of the deviation from baseline. Unsupervised hierarchical clustering of the experiments grouped the biological replicates together. Gene expression varied considerably between fibroblast and MDA-MB231 as expected for cells of mesenchymal or epithelial origin respectively. The co-culture profile showed mainly intermediate expression levels. However, the vertical black bar marks a cluster of genes induced in all co-cultures compared to both mono-cultures indicating that they are induced by heterotypic interaction Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Computed
2007-11-13 | E-GEOD-7263 | biostudies-arrayexpress
Project description:Core binding factor (CBF) leukemias, characterized by either inv(16)/t(16;16) or t(8;21), constitute acute myeloid leukemia (AML) subgroups with favorable prognosis. However, there exists substantial biological and clinical heterogeneity within these cytogenetic groups, which is not fully reflected by the current classification system. To improve the molecular characterization we profiled gene expression in a large series (n=93) of AML patients with CBF leukemia [inv(16) n=55, t(8;21) n=38]. By unsupervised hierarchical clustering we were able to define a subgroup of CBF cases (n=35) characterized by shorter overall survival times (P=0.03). While there was no obvious correlation with fusion gene transcript levels, FLT3 tyrosine kinase domain, KIT, and NRAS mutations, the newly defined inv(16)/t(8;21)-subgroup was associated with elevated white blood cell counts and FLT3 internal tandem duplications (P=0.011 and P=0.026, respectively). Supervised analyses of gene expression suggested alternative cooperating pathways leading to transformation. In the ?favorable? CBF leukemias anti-apoptotic mechanisms and deregulated mTOR-signaling, and in the newly defined ?unfavorable? subgroup aberrant MAPKinase-signaling and chemotherapy-resistance mechanisms might play a role. While the leukemogenic relevance of these signatures remains to be validated, their existence nevertheless supports a prognostically relevant biological basis for the heterogeneity observed in CBF leukemia. A reference experiement design type is where all samples are compared to a common reference. Using regression correlation
2007-08-09 | E-GEOD-8653 | biostudies-arrayexpress