Macrophage LPS

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The response of primary human monocyte-derived macrophages to lipopolysaccharide

Time course ID: human_Monocyte-derived_macrophages_response_to_LPS
Sample provider: David Hume, Kenneth Baillie, Geoff Faulkner, Lynsey Fairbairn, Malcolm Fisher

Introduction

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.

Samples

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].

Quality control

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.

Human Monocyte-derived macrophages response to LPS.png

Figure 1: CAGE expression of marker genes in TPM


References

[1] PMID: 16688168

[2] PMID: 16698233

[3] PMID: 19859064

[4] PMID: 20206554

[5] PMID: 23332752

[6] PMID: 22451944

[7] PMID: 24604202























































































Beginning of non-public section

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Zenbu configurations and status

Gene expression profiles


MARA based network results


Self-organising maps File:LPS.pdf

TSS Switching (Switch Engine)

  • TSS dynamics plots can be found here:Media:monocyteDerivedMacrophage_LPS.plots.pdf (it is recommended to download these files instead of viewing in browser as they are large in size)
  • These figures only show those genes for which Switch Engine has detected TSS switching to occur. For now, only the first and last time points are being compared to define a switch.
    • One gene per page, RefSeq and Gene Symbol identifiers on top. Each panel is a separate TSS associated with the gene. TPM expression on y-axis and time on x-axis. Colored points correspond to specific expression, with colors referring to different replicates (key on top). Replicates with a * next to the name are those that have missing data from the currently available release of the DPI clustered and normalized TPM matrix. Such missing data were replaced by imputed values. Black lines are the mean trajectories across replicates. Purple vertical bars correspond to the time points being compared for switch definition. Magnitude of switch is expressed in each panel under TSS identifier with approximate 95% confidence interval in brackets. Note: some confidence intervals may not be accurate or may be missing due to the small sample size. TSS identifiers colored GREEN show an increasing TSS, those colored RED show decreasing TSS, WHITE show no statistically significant change.
    • TSS switch defined as the simultaneous presence of one or more increasing, together with one or more decreasing TSSs per gene. At least one increase and at least one decrease has to be statistically significant (alpha approximately 5%). At least one of the two time points being compared has to be greater than 5 tpm. At least 2 replicates have to agree to call an increasing/decreasing trajectory. TSSs have to be at least 250 base pairs away from one another to call a switch.
  • A summary file can be found here: Media:monocyteDerivedMacrophage_LPS.summary.xls
    • The file contains the following columns: RefSeq ID, Gene Symbol, TSS ID, X{value}: mean tpm expression at {value} time point, Delta: change in tpm expression from first to last time point, FC: fold change in tpm expression from first to last time point, DeltaSum: net change in tpm expression for this gene (summed across all TSS in this gene), Balance: ratio of increasing to decreasing tpm expression TSSs per gene (e.g. a balance value of 1 means that the total increase in tpm expression in increasing TSSs is equal to the total decrease in tpm expression in decreasing TSSs), Chromosome, Strand, Start position of TSS cluster, Stop position of TSS cluster, DBTSS: distance between TSSs relative to NA value.
  • A list of genes in which TSS switching occurs can be found here: Media:monocyteDerivedMacrophage_LPS.genelist.refseq.doc and Media:monocyteDerivedMacrophage_LPS.genelist.symbol.doc
  • BED file with regions corresponding to TSS clusters involved in TSS switching can be found here: Media:monocyteDerivedMacrophage_LPS.switchinglist.bed.xls (please remove .xls extension after downloading)


Related samples

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References

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Quality control

ISMARA analysis results

All samples: http://ismara.unibas.ch/timecourses/macrophageLPS/ismara_report/index.html

Replicate averaged: http://ismara.unibas.ch/timecourses/macrophage-lps-avgd/averaged_report/index.html

For more information, see ISMARA.

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