| Series: | IN_VITRO DIFFERENTIATION SERIES |
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| Species: | Human (Homo sapiens) |
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| Genomic View: | Zenbu |
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| Expression table: | FILE |
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| Link to TET: | TET |
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| Sample providers : | David Hume, Kenneth Baillie |
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| Germ layer: | mesoderm |
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| Primary cells or cell line: | primary cells |
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| Time span: | 48 hours |
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| Number of time points: | 23 |
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| CollapseOverview
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Macrophages are effector cells of the innate immune system. Their effector function is modulated in response to a wide range of stimuli. Amongst the most studied is lipopolysaccharide (LPS) or endotoxin, the major cell wall component of gram-negative microorganisms. LPS signals through the toll-like receptor 4 (TLR4) to initiate a cascade of transcriptional changes that have been analysed in detail in mice, and which involves waves of gene regulation starting within minutes and extending over 24-48 hours [1,2]. The earliest transcriptional responses, including key pro-inflammatory cytokines, involve the activation of transcriptional elongation from poised RNApolI complexes [3]. Recent studies in the mouse have identified many of the inducible enhancers activated in response to LPS, and the production of eRNAs from these elements [4,5]. However, there are many differences in the transcriptional responses of human and mouse macrophages to LPS [6], reflecting the impact of pathogen selection, and there is also significant variation between individuals. Accordingly, we have studied the response of macrophages from three different individuals.
References:
[1] PMID: 16688168
[2] PMID: 16698233
[3] PMID: 19859064
[4] PMID: 20206554
[5] PMID: 23332752
[6] PMID: 22451944
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| ExpandSample description
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Human monocytes were obtained from anonymised donors with approval of the Human Ethics Committee of the University of Edinburgh (8/9/09). 320mls of blood was extracted from healthy human volunteers and the mononuclear cell fraction was purified using Ficoll gradient centrifugation. The CD14-positive monocytes were purified from the mononuclear cell fraction using magnetic beads (Miltenyi Biotech), and cultured on bacteriological plastic plates in medium (RPMI-1640 plus 10% foetal calf serum) in recombinant human CSF1 (a gift from Chiron) at 100ng/ml. After 7 days the monocyte-derived macrophages were stimulated with 100ng/ml of salmonella R595 LPS. Samples were taken at 0 time, then 15, 30, 45, 60, 80, 100, 120, 150, 180, 210, 240 mins; then 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 48 hours. Each time course was carried out on one preparation of cells from a single donor, and the precise temporal profile of gene expression varies somewhat between the three replicates. The system is comparable to more limited previous studies based upon microarrays and low-coverage CAGE analysis [6].
References:
[6] PMID: 22451944
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| ExpandQuality control
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In each case, the cells respond morphologically to LPS with increased spreading and vacuolation. The response to LPS can be detected through the early induction of classical inflammatory markers, TNF, IL6 and IL1B and late response genes such as IDO1 and CYP27B1 which are induced in human, but not in mouse macrophages [6]. The early response gene IFNB1, which acts in an autocrine manner to induce downstream target genes, is known to vary in its expression between individuals [7]. It was induced somewhat later in Donor 1 than in Donors 2 and 3, but the downstream response was evident in induction of known IFN targets such as MX1.
References:
[6] PMID: 22451944
[7] PMID: 24604202
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Profiled time course samples
Only samples that passed quality controls (Arner et al. 2015) are shown here. The entire set of samples are downloadable from FANTOM5 human / mouse samples
