Transcription profiling by array of human dendritic cells exposed to mesenchymal stem cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Luca Beltrame
PROVIDER: E-MTAB-286 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:To assess the effect of carbon nanotubes substrated on dendritic cell (DCs) properties, DCs were generated from human monocytes of healthy donors as previously described (Aldinucci A et al, 2010). In particular, isolated monocytes were cultured for 6 days in medium supplemented with GM-CSF (1000U/ml) and IL-4 (1000U/ml), in presence or absence of multi-walled carbon nanotubes (MWCNT); then DCs were activated by 24 hours of incubation with LPS (1ug/ml). RNA extracted from floating and CNT adherent DCs were then used for transcriptional profilingthen used
2015-04-01 | E-MEXP-3560 | biostudies-arrayexpress
Project description:Human monocyte-derived dendritic cells (moDCs) have been used as an in vitro model for studying tolerance and immunity. However, the underlying metabolic states of tolerogenic (dexamethasone and vitamin D3-treated), immature and immunogenic (mature, LPS-treated) moDCs have not been completely characterized. Through transcriptomic analyses, we determined that tolerogenic moDCs exhibit augmented catabolic pathways with respect to oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO) and glycolysis. Functionally, tolerogenic moDCs showed the highest mitochondrial membrane potential, production of reactive oxygen species and superoxide, and increased mitochondrial spare respiratory capacity. Tolerogenic and mature moDCs manifested differential FAO gene expression with FAO activity being significantly higher in tolerogenic and immature moDCs than in mature. In addition, tolerogenic and mature moDCs demonstrated similar levels of glycolytic rate, but not glycolytic capacity and reserve, which were more pronounced in tolerogenic and immature moDCs. Finally, tolerogenic and immature moDCs, but not mature moDCs, showed high plasticity to compensate the intracellular ATP content after inhibition of different energetic metabolic pathways. Overall, tolerogenic moDCs exhibit a metabolic signature of increased, stable OXPHOS programing and high plasticity for metabolic adaptation. These findings provide a framework for future research of metabolic properties of human DCs. Total RNA of sixteen samples from four moDC types (tolerogenic, LPS-tolerogenic, immature and mature) were extracted by Trizol (Invitrogen) followed by a clean-up procedure using RNeasy Micro Kit (Qiagen). All RNA samples had an integrity number ≥9.6 assessed by Agilent Bioanalyzer. Total RNA samples were amplified using TargetAmp™ and the biotinilated cRNA was prepared by Nano-g™ Biotin-aRNA Labeling Kit for the Illumina® System (Epicentre). After the hybridization to the Illumina Human HT-12 v4 Beadchips for 17 h at 58°C, the arrays were washed, stained (Illumina Wash Protocol) and then scanned using BeadArray Scanner 500GX. Array data were extracted at the probe level without background correction using Illumina GenomeStudio software. These raw data were quantile normalized and log2 transformed. Technical replicates were obtained from the hybridization in duplicate of three samples. Pearson correlation analysis showed high correlation between the technical replicates (r>0.99). Differentially expressed genes (DEGs) were identified using Limma16 with Benjamini-Hochberg multiple testing correction (p<0.05). DEGs were further clustered into different groups according to the patterns of expression change among the different moDC types using STEM software17. The analysis was performed in R v.2.12.2 (http://www.R-project.org) with Bioconductor 2.12 (http://www.bioconductor.org) and enabled by Pipeline Pilot (www.accelrys.com).
2015-05-08 | E-GEOD-52894 | biostudies-arrayexpress
Project description:Dendritic cells (DCs) are the most potent antigen (Ag)-presenting cells. Whereas immature DCs down-regulate T cell responses to induce/maintain immunological tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact and vesicle exchange. Transfer of nanovesicles (<100nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle microRNAs (miRNAs), and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, an hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, as they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and post-transcriptional regulation between DCs. The study has analyzed the microRNA content of 4 samples of immature exosomes, 4 samples of matures exosomes, 2 samples of immature bone-marrow-derived DCs, and 2 samples of mature bone marrow-derived DCs.
2011-10-24 | E-GEOD-33179 | biostudies-arrayexpress
Project description:Dendritic cells (DCs) are the sentinels of the mammalian immune system and they undergo a complex maturation process mediated by activation upon pathogen detection. Recent studies described the analysis of activated DCs by transcriptional profiling, but translation regulation was never taken in account. Therefore, the nature of the mRNAs being translated at various stages of DC activation was determined with the help of translational profiling, which is the sucrose gradient fractionation of polysomal-bound mRNAs combined to microarrays analysis. Total and polysomal-bound mRNA populations were compared in immature (0h) and LPS-stimulated (4h and 16h) human monocyte-derived DCs with the help of Affymetrix microarrays. Biostatistical analysis indicated that 296 mRNA molecules are translationally regulated during DC-activation. The most abundant biological process among the regulated mRNAs was protein biosynthesis, indicating the existence of a negative feedback loop regulating translation. Interestingly, a cluster of 17 ribosomal proteins were part of the regulated mRNAs, indicating that translation may be fine-tuned by particular components of the translational machinery. Our observations highlight the importance of translation regulation during the immune response, and may favour the identification of novel gene clusters or protein networks relevant for immunity. Our study also provides information on the possible absence of correlation between gene expression and real protein production in DCs. To identify translationally regulated mRNA molecules, gene expression derived from the polysome-bound mRNAs was compared by Affymetrix microarrays analysis to the gene expression derived from unfractionated total mRNAs derived from whole-cell lysates, as recently described on several reports (Johannes 1999, Rajasekhar 2003, Bushell 2006, Lü 2006, Parent 2008). Polysomal RNA (P) and total RNA (T) were isolated from MoDCs generated from four different blood donors. Since three timepoints (0h, 4h and 16h) were chosen for each blood donor and RNA type, twenty-four RNA samples were totally analyzed by microarrays.
2009-12-30 | E-GEOD-14000 | biostudies-arrayexpress
Project description:Human MSCs were cocultured for 18 hours with macrophages which had been differentiated from human peripheral blood-derived monocytes by PMA and sequentially stimulated by 2 μg/mL LPS (InvivoGen) for 3 hours and 5 mM ATP (InvivoGen) for 45 minutes. Normal MSCs without macrophage coculture and MSCs cocultured with activated macrophages were assayed by miRNA arrays.
2020-07-28 | E-MTAB-9418 | biostudies-arrayexpress
Project description:In order to detect the microRNA expression profile of in vitro generated dendritic cells , purified monocytes from PBMCs were used as dendritic cell (DCs) precursors and were cultured in medium with cocktail for differentiation and maturation to immature dendritic cells (iDCs) and mature dendritic cells (mDCs). microRNA samples were isolated from precursor, iDCs and mDCs and used for microarray-based microRNAs expression profiles. To generate enough amount of immature DC (iDCs) and mature DCs (mDCs), monocytes were differentiated with GM-CSF and rhIL-4 for 2 days and maturated in the presence of TNF-α, IL-1β, IL-6 and PGE2 for another 2 days. With the anticipation to insight developmental-stage-specific microRNAs with potential functions related to monocyte derived DCs, global microRNAs expression profiling was set using microarray technology.microRNA expression profiles were performed in triplicate independent experiments starting for 3 groups of precursor, iDC and mDC generated from different blood donors.
2013-01-28 | E-GEOD-15036 | biostudies-arrayexpress
Project description:In order to detect the gene expression profile of in vitro generated dendritic cells , purified monocytes from PBMCs were used as dendritic cell (DCs) precursors and were cultured in medium with cocktail for differentiation and maturation to immature dendritic cells (iDCs) and mature dendritic cells (mDCs). Total RNA samples were isolated from precursor, iDCs and mDCs and used for microarray-based gene expression profiles. To generate enough amount of immature DC (iDCs) and mature DCs (mDCs), monocytes were differentiated with GM-CSF and rhIL-4 for 2 days and maturated in the presence of TNF-α, IL-1β, IL-6 and PGE2 for another 2 days. With the anticipation to insight developmental-stage-specific genes with potential functions related to monocyte derived DCs, global gene expression profiling was set using microarray technology.gene expression profiles were performed in triplicate independent experiments starting for 3 groups of precursor, iDC and mDC generated from different blood donors.
2013-01-28 | E-GEOD-15075 | biostudies-arrayexpress
Project description:T helper type 2 (Th2) responses are induced by protease allergens and helminthes. However the molecular mechanisms that initiate Th2 responses are poorly understood. To obtain insight into this mechanism, we performed a microarray analysis of lymph node DCs stimulated in vitro with the protease allergen papain, or with LPS, a Th1 inducing stimulus. Key words: Th2 response, LPS, dendritic cells, Papain CD11c+ DCs were isolated from the lymph nodes of C57BL/6 mice, and cultured in vitro (1x106 DCs per ml) with 500 3T3-CD40L fibroblasts, either alone, or in the presence of papain (25 µg/ml) or LPS (1 µg/ml). 4h and 17h later, the cells were harvested and RNA isolated and processed for microarray analyses. RNA was extracted and processed from freshly isolated LN DCs. For a given time point, the expression profile of DCs treated with papain or LPS, were compared to that of untreated DCs
2010-05-05 | E-GEOD-21602 | biostudies-arrayexpress
Project description:Abstract Two major dendritic cell (DC) subsets have been described in the islets of mice: The immunogenic CD8α-CD11b+ DCs and the tolerogenic CD8α+CD103+ DCs. We have recently reported on reduced numbers of the minor population of tolerogenic CD8α+CD103+ DCs in the pancreas of 5 week old pre-diabetic non-obese diabetic (NOD) mice. Aim: To analyze also the larger subset of CD11c+CD8α- DCs isolated from the pancreas of pre-diabetic NOD mice 1) for maturation and tolerance inducing molecules found abnormally expressed on CD8α+CD103+ DCs, and 2) for genome-wide gene expression to further elucidate abnormalities in underlying gene expression networks. Methods: CD11c+CD8α- DCs were isolated from 5 week old C57BL/6 and NOD pancreas. Expression of cell surface markers including CD86, CCR5, CD11b, CD103, Clec9a, CD24 and CD200R3 were measured by FACS. Genome-wide gene expression by microarray was assessed during the steady state and after in vitro LPS stimulation. Results: The steady state pancreatic CD11c+ CD8α- DCs during the pre-diabetic stage showed: 1) A reduced expression of several gene networks important for the prime functions of the cell, such as for cell renewal, immune stimulation and immune tolerance induction, for migration and for the provision of growth factors for beta cell regeneration. This general deficiency state was corroborated by a reduced in vivo proliferation (BrdU incorporation) of the cells and the reduced expression in FACS analysis of CD86, CCR5, CD103, Clec9a, CD24 and CD200R3 on the cells. 2) A hyper reactivity of these cells to LPS correlated with an enhanced pro-inflammatory state characterized by altered expression of a number of classical pro-inflammatory factors and cytokines. Conclusion: The NOD CD11c+CD8α- DCs seem to be Janus-faced depending on the conditions: Deficient in steady state with reduced immune stimulation capabilities also for tolerance induction; over-inflammatory with a molecular profile suggesting a preferential stimulatory capacity for Th1 cells when encountering a Pathogen-Associated Molecular Pattern (PAMP) in the form of LPS. We used microarray gene expression analysis to explain the abnormal expression of several cell surface markers involved in tolerace, migration and maturation in the steady-state and to measure the effect of a PAMP such as LPS We isolated RNA from FACS sorted CD11c+CD8α- DCs in 10 pooled pancreases from pre-diabetic NOD and non-diabetic C57BL/6 mice at 5 weeks. In addition, we treated in another experiment the isolated pancreas DCs with LPS (and PBS), incubated for 18h and measured gene expression. We compared gene expression between strains NOD vs C57BL/6 under steady-state and after in-vitro LPS/PBS stimulation.
2014-07-01 | E-GEOD-45028 | biostudies-arrayexpress
Project description:Dendritic cells (DCs) are a special class of leukocytes able to activate both innate and adaptive immune responses. They interact with microbes through germline-encoded pattern-recognition receptors (PRRs), which recognize molecular patterns expressed by various microorganisms. Upon antigen binding, PRRs instruct DCs for the appropriate priming of natural killer cells, followed by specific T-cell responses. Once completed the effector phase, DCs reach the terminal differentiation stage and eventually die by apoptosis. We have observed that following lipopolysaccharide (LPS)-stimulation the initiation of the apoptotic pathway in DCs is due the activation of NFAT proteins. Indeed, LPS induces Src-family kinase and phospholipase C (PLC)γ2 activation, influx of extracellular Ca2+ and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. According with this observation CD14-deficient DCs do not die following LPS stimulation. Nevertheless, CD14-deficient DC death following LPS activation can be restored by co-stimulating DCs with LPS and thapsigargin. Thapsigargin empties the intracellular calcium stores by blocking calcium pumping into the sarcoplasmic and endoplasmic reticulum and thereby activates plasma membrane calcium channels. This, in turn, allows an influx of calcium into the cytosol and NFAT activation. To identify the NFAT controlled apoptosis genes in LPS activated DCs we have performed a kinetic microarray analysis (0, 48 and 60 h) in conditions allowing or inhibiting NFAT activation. Four genes have been selected: Nur77, Gadd45g, Ddit3/Gadd153/Chop-10 and Tia1. To identify apoptosis genes selectively modulated by NFAT, a comparative kinetic (time points 0, 48 and 60 h) microarray analysis was performed in the following conditions: 1) wild type bone marrow derived DCs (wtBMDCs) stimulated with LPS; 2) CD14-deficient BMDCs stimulated with LPS; 3) wtBMDCs stimulated with LPS in presence of thapsigargin; 4) CD14-deficient BMDCs stimulated with LPS in presence of thapsigargin.
2010-03-03 | E-GEOD-15720 | biostudies-arrayexpress