Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcrioptome analysis of human primary macrophages stimulated by bacterial endotoxin lipopolysaccharide (LPS), and (1,3)-b-glucans, curdlan and glucan from bakerM-BM-4s yeast (GBY)

ABSTRACT: We performed gene expression microarray experiments to compare the global transcriptional response induced by b-glucans and LPS with their secretomes. We identified 1683, 767 and 1447 genes with over two-fold increase or decrease in LPS-, curdlan- or GBY-stimulated macrophages, respectively. We show that both LPS and b-glucan induces significant gene expression changes in macrophages, but only b-glucans activate a robust protein secretion. The gene expression of human primary macrophage cells was measured at 6 hours after exposure the cells to 1 M-BM-5g LPS or 10 M-BM-5g Curdlan or 100 M-BM-5g GBY (glucan from bakerM-BM-4s yeast), also 0-group was included. Three independent experiments were performed using three different donors for each experiment.

ORGANISM(S): Homo sapiens

SUBMITTER: Tiina M-CM-^Vhman

PROVIDER: E-GEOD-32282 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

Dectin-1 pathway activates robust autophagy-dependent unconventional protein secretion in human macrophages.

Öhman Tiina T Teirilä Laura L Lahesmaa-Korpinen Anna-Maria AM Cypryk Wojciech W Veckman Ville V Saijo Shinobu S Wolff Henrik H Hautaniemi Sampsa S Nyman Tuula A TA Matikainen Sampsa S

Journal of immunology (Baltimore, Md. : 1950) 20140507 12

Dectin-1 is a membrane-bound pattern recognition receptor for β-glucans, which are the main constituents of fungal cell walls. Detection of β-glucans by dectin-1 triggers an effective innate immune response. In this study, we have used a systems biology approach to provide the first comprehensive characterization of the secretome and associated intracellular signaling pathways involved in activation of dectin-1/Syk in human macrophages. Transcriptome and secretome analysis revealed that the dect ...[more]

PMID: 24808366

Similar Datasets

Project description:Acanthamoeba castellanii (Ac) and macrophages share structural, morphological, physiological and biochemical similarities, including the ability to phagocyte a myriad of microorganisms in the environments they live. Whereas there is voluminous information on phagocytic receptors of macrophages, for Ac, the understanding of how the recognition of extracellular microorganisms works still awaits elucidation. Recently, our group described mannose-binding proteins expressed on the surface of the trophozoites. However, as soluble mannose did not inhibit entirely the process, other interactions might be possible. In the present work, we aimed to characterize the potential Ac proteins able to recognize the polysaccharide β-1,3-glucan on fungal surfaces. Our data demonstrate that Ac could bind curdlan or laminarin on its surface as detected by Dectin-1-Fc and fluorescent conjugate, suggesting the presence of β-1,3-glucan binding molecules. Optical tweezers detected higher adhesion affinity of laminarin or curdlan coated beads to A. castellanii (characteristic time of 46.9 s and 43.9 s, respectively) in comparison control beads (BSA or dextran-coated). In agreement, a H. capsulatum (Hc) G217B having β-1,3-glucan as the most external layer strongly adhered to Ac (characteristic time of 5.3 s), whereas Hc G186A, an α-1,3-glucan expressing strain, displayed much lower adhesion forces (characteristic time of 83.6 s). The specificity of our system was confirmed with addition of soluble β-1,3-glucan, which inhibited dramatically the adhesion of Hc G217B to Ac (characteristic time of 38,5 s). By indirect ELISA, the biotinylated extract of Ac showed higher binding to Hc G217B surface than Hc G186A, as similar results were observed when using Dectin-1-Fc. By interaction assays, association rates to Ac and RAW macrophages were twice higher for Hc G217B when compared to Hc G186A. Inhibitions with mannose, or its combinations with curdlan or laminarin demonstrated inhibitions higher than 50% during Ac and Hc G217B interaction. For RAW macrophages, the combinations mannose + laminarin and mannose + curdlan had inhibition of 64.4% and 51.5%, respectively, in the interaction with Hc G217B. The killing assay show that for A. castellanii, there was a decrease in the number of viable fungi when either laminarin and curdlan were added, which is similar to results observed with macrophages, suggesting the participation of this receptor for fungal entrance and survival within phagocytes. Proteomics identified several proteins with the capacity to bind β-1,3-glucans, including a membrane integral component (L8HDD6) displaying a legume lectin domain and also belonging to the Concanavalin A-like lectin/glucanase domain superfamily. By the demonstrated binding specificity of this receptor, our data reinforce other pathways of fungal recognition and suggests to a possible parallel or even divergent evolution, between A. castellanii and macrophages.

Project description:IThe transcription factor (TF) interferon regulatory factor 8 (IRF8) controls both developmental and inflammatory stimulus-inducible genes in macrophages, but the mechanisms underlying these two different functions are largely unknown. One possibility is that these different roles are linked to the ability of IRF8 to bind alternative DNA sequences. We found that IRF8 is recruited to distinct sets of DNA consensus sequences before and after lipopolysaccharide (LPS) stimulation. In resting cells, IRF8 was mainly bound to composite sites together with the master regulator of myeloid development PU.1. Basal IRF8M-bM-^@M-^SPU.1 binding maintained the expression of a broad panel of genes essential for macrophage functions (such as microbial recognition and response to purines) and contributed to basal expression of many LPS-inducible genes. After LPS stimulation, increased expression of IRF8, other IRFs, and AP-1 family TFs enabled IRF8 binding to thousands of additional regions containing low-affinity multimerized IRF sites and composite IRFM-bM-^@M-^SAP-1 sites, which were not premarked by PU.1 and did not contribute to the basal IRF8 cistrome. While constitutively expressed IRF8-dependent genes contained only sites mediating basal IRF8/PU.1 recruitment, inducible IRF8-dependent genes contained variable combinations of constitutive and inducible sites. Overall, these data show at the genome scale how the same TF can be linked to constitutive and inducible gene regulation via distinct combinations of alternative DNA-binding sites. Bone marrow cells isolated from C57BL/6 or BXH2/TyJ mice were plated in 10 cm plates in 5ml of BM-medium (high glucose DMEM supplemented with 20% low endotoxin fetal bovine serum, 30% L929-conditioned medium, 1% glutamine, 1%, Pen/Strep, 0.5% Sodium Pyruvate, 0.1% M-NM-2-mercaptoethanol). Cultures were fed with 2.5 ml of fresh medium every two days. Macrophages were subjected to different treatment (see individual samples for details). Total RNA was extracted from 5x106 cells using RNAeasy kit (Qiagen). Libraries were prepared from 1-2 M-BM-5g of RNA, after oligo-dT selection, using the TruSeq RNA sample preparation kit (Illumina).

Project description:A comprehensive in vitro assessment of two commercial metal oxide nanoparticles, TiO2 and ZnO, was performed using human monocyte-derived macrophages (HMDM), monocyte-derived dendritic cells (MDDC), and T cell leukemia-derived cell line (Jurkat). TiO2 nanoparticles were found to be non-toxic whereas ZnO nanoparticles caused dose-dependent cell death. Subsequently, global gene expression profiling was performed to identify signaling pathways underlying the cytotoxicity caused by ZnO nanoparticles. Analysis was done with doses, 1M-BM-5g/ml and 10M-BM-5g/ml after 6 and 24 hours of exposure. Interestingly, 2703 genes were significantly differentially expressed in HMDM upon exposure to 10M-BM-5g/ml ZnO nanoparticles, while in MDDCs only 12 genes were affected. In Jurkat cells, 980 genes were differentially expressed. It is noteworthy that the gene expression of metallothioneins was upregulated in all the three cell types. In addition to the common ZnO-inducible changes, a notable proportion of the genes were regulated in a cell type-specific manner. Using a panel of ZnO nanoparticles, we obtained an additional support that the cellular response to ZnO nanoparticles is caused by particle dissolution. Gene ontology analysis revealed that the top biological processes disturbed in HMDM and Jurkat cells were regulating cell death and growth. In addition, genes controlling immune system development were affected. Bioinformatics assessment showed that the top human disease category associated with ZnO-responsive genes in both HMDM and Jurkat cells was cancer. Overall, the study revealed novel genes and pathways for mediating ZnO nanoparticle-induced toxicity and demonstrated the value of assessing nanoparticle responses through combined transcriptomics and bioinformatics approach. Altogether 90 samples were analyzed originating from three biological replicates of HMDM, MDDC or Jurkat cells containg untreated control cells, ZnO or TiO2 treated cells with doses 1M-BM-5g/ml and 10M-BM-5g/ml. The sample timepoints were 6 hours and 24 hours.

Project description:To determine characterize human B cells that express IL-10 on a molecular level, we compared the global gene expression of primary CD19pos B cells secreting IL-10 or not, upon activation with anti-CD40, IL-4 and CpG for 2 days. Human B cells from healthy donors were sorted according to CD19 expression by magnetic cell separation. The cells were stimulated at 2.5E06 cells/ml by activating anti-CD40 mAb (1 M-BM-5g/ml, clone 82111, RnD Systems), recombinant human IL-4 (10 ng/ml, Immunotools) and CpG2006 (3 M-BM-5g/ml) for 2 days following restimulation for 3 h with phorbol myristate acetate (PMA, 10 ng/ml) and ionomycin (1 M-BM-5g/ml). Staining of viable IL-10 secreting human CD19pos B cells was performed by the IL-10 capture assay according the manufacturers instructions (Miltenyi Biotec, n=2). Finally, the cells were sorted into IL-10posCD69pos and IL-10negCD69pos cells by flow cytometric cell sorting on a BD Aria II sorter (Becton Dickinson). Dead cells were excluded using propidium iodide. Total RNA was extracted using the RNeasy Mini kit (Qiagen). The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Double-stranded complementary RNA was synthesized from 1 M-BM-5g total RNA using Message AmpII Biotin (Ambion, USA). Fifteen micrograms of fragmented cRNA of each sample were hybridized to four HG-U133A plus 2.0 GeneChips (Affymetrix). Hybridization was performed in a Hybridization Oven 640, and chips were washed and stained in the Fluidics Station 400 (both Affymetrix). Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software, version 1.4, both Affymetrix. All relevant GCOS data of quality checked microarrays were analyzed with High Performance Chip Data Analysis (HPCDA, unpublished), using the BioRetis database (www.bioretis-analysis.de), as described and validated previously.

Dataset Information

Transcrioptome analysis of human primary macrophages stimulated by bacterial endotoxin lipopolysaccharide (LPS), and (1,3)-b-glucans, curdlan and glucan from bakerM-BM-4s yeast (GBY)

Publications

Dectin-1 pathway activates robust autophagy-dependent unconventional protein secretion in human macrophages.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure