Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

PBMC response to B. pertussis filamentous hemagglutinin stimulation in vitro

ABSTRACT: Filamentous hemagglutinin (FHA), Bordetella pertussis' key adhesin, is both present at the cell surface and released in the extracellular milieu. To comprehend the role free FHA plays in the pathogenic process, we analyzed the global transcriptional changes in human peripheral blood mononuclear cells (PBMC) after exposure to that purified virulence factor. This analysis was undertaken with four different FHA preparations (FHA-1, FHA-2, FHA-3, and FHA-4), purified from four different B. pertussis strains. Cultures comprising 3x106 PBMC were stimulated for 0.5, 2, 4, and 6 hours with 5 ug/ml of each purified B. pertussis FHA. Control PBMC cultures were simultaneously treated with similar volumes of elution buffers (Mock-1 and Mock-2), and sampled at the same time points. The array experiment was composed of 27 samples. Most of the transcripts known to be part of the "common host-transcriptional response" that mediates inflammation were among the 1,235 array elements (representing 683 known unique genes) showing a greater than 3-fold change in transcript abundance between FHA-treated and untreated cells. We also identified an FHA-specific signature not observed in cells stimulated with B. pertussis LPS or heat-killed B. pertussis. This response, mounted by 13 genes, largely (69%, 9/13 genes) interferon (IFN)-regulated genes. Further analysis revealed that 18.3% (125 out of 683) of the FHA-responsive genes were in fact regulated by IFN. These included several major players of the antiviral IFN type I response, as well as three key components of the ISGylation pathway. Induction of the ubiquitin-like protein ISG15 and its specific protease USP18 was confirmed at the mRNA level by relative real-time polymerase chain reaction (RT-PCR). Western-blot analysis demonstrated the presence of both free ISG15 and several ISGylated conjugates in FHA-stimulated PBMC cell lysate. Intracellular FACS analysis provided further evidence that monocytes and a natural-killer (NK)-enriched cell population are the primary producers of ISG15 after FHA stimulation. Our results revealed new activities of free B. pertussis FHA: activation of the host IFN and ISGylation pathways. The consequences of such activation are yet to be resolved, but we hypothesize that FHA utilizes the ISGylation pathway or USP18 to regulate the IFN response, which in turn sensitizes the host to the infection and induces cell death. Groups of assays that are related as part of a time series. Infection: treatment with purified B. pertussis filamentous hemagglutinin (FHA) at 0.005 mg/mL Time: Samples were collected after various treatment time (hours) Keywords: time_series_design Computed

ORGANISM(S): Homo sapiens

SUBMITTER: Christine Dieterich

PROVIDER: E-GEOD-8802 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

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.

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

Dataset Information

PBMC response to B. pertussis filamentous hemagglutinin stimulation in vitro

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets