Influenza timecourse

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The response of primary human monocyte-derived macrophages to infection with Influenza A virus (Udorn strain)

Time course ID: human_Monocyte-derived_macrophages_response_to_udorn_influenza_infection
Sample provider: David Hume, Kenneth Baillie, Andru Tomoiu

Introduction

The pathology of severe influenza virus infection involves a “cytokine storm”, not dissimilar to toxic shock initiated by severe bacterial infections. Although they are likely to be a major source of the cytokines in influenza virus pathology, macrophages are not generally regarded as permissive for infection by influenza virus infection. However, we and others [1] have noted that human macrophages grown in CSF1 become permissive for the virus. Like most viruses, influenza produces gene products, notably the NS1 protein, that interfere with the production of host defense proteins, notably interferon. This study aimed to compare the response of the human monocyte-derived macrophages to the response to bacterial endotoxin, assessed in parallel in this series.

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 exposed to influenza. The influenza A (A/Udorn/72 (H3N2)) was propagated in MDCK cells as described [1]. Monocyte-derived macrophages were infected at a multiplicity of 5 PFU/cell for one hour in serum-free medium supplemented with TPCK-treated trypsin. The cells were then washed two times, and incubated for 0, 2 hrs, 7 hrs or 24 hrs. In this timecourse, the 0h timepoint is taken at the end of the incubation period. Mock-infected cells were also harvested at 0 time and 24 hours to control for the effect of protease and serum-free medium.

Quality control

In each case, the cells respond morphologically to influenza with increased spreading and vacuolation and at the later time point there was visual evidence of cell death. The infected cells produced active virus, which was quantified by plaque assay. They were also stained for viral NP protein using a fluorochrome-coupled antibody. The infected macrophages induce many proinflammatory cytokines in common with LPS. By contrast to macrophages responding to LPS, the influenza-infected cells displayed induction of multiple members of the interferon A family with substantial differences between the four replicates.

Human Monocyte-derived macrophages response to udorn influenza infection.png

Figure 2: CAGE expression of marker genes in TPM.


References

[1] PMID: 22238612























































































Beginning of non-public section

Macrophage response to Udorn strain influenza timecourse

Kenneth Baillie, Malcolm Fisher, Andru Tomoiu, David Hume ...

Zenbu configurations and status


Expression profiles

MARA based network results


TSS Switching (Switch Engine)

  • TSS dynamics plots can be found here:Media:monocyteDerivedMacrophage_udornflu.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_udornflu.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_udornflu.genelist.refseq.doc and Media:monocyteDerivedMacrophage_udornflu.genelist.symbol.doc
  • BED file with regions corresponding to TSS clusters involved in TSS switching can be found here: Media:monocyteDerivedMacrophage_udornflu.switchinglist.bed.xls (please remove .xls extension after downloading)


Related samples

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References

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Quality control

https://fantom5-collaboration.gsc.riken.jp/webdav/home/arner/timecourse/time_course_main_paper_freeze_feb2013/qc_release_130226/human_Monocyte-derived_macrophages_response_to_udorn_influenza_infection/

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(Re)Mapping of the Monocyte-derived macrophages response to udorn influenza infection timecourse against the combined viral and host genomes

Initial generic mapping of 24 Monocyte-derived macrophages response to udorn influenza infection timecourse related libraries were performed as part of the F5 mapping pipeline against hg19 without regard to the specificity of the collection. Udorn influenza produces capped-transcripts that can therefore be identified by hCAGE, prompting for its remapping in order to also gain access to the dynamic of the virus promoterome/transcriptome during the timecourse.

Importantly this remapping is NOT done simply done by mapping non host mapped reads onto the viral genome, but by mapping all reads against the combined host (hg19) and viral (Influenza A virus (A/Udorn/72(H3N2)) genomes, thus making proper accurate assumptions for reads mapping qualities (mapQ)

host-caped-RNA-snatched udorn mapped tags
Influenza virus steals the first 10-14bp of cap of host RNA and therefore we can/need to rescue unmapped tags using the following procedure: We start from the sam/bam formatted unmapped read and write a looping script that

  1. trim the 1st 10bp (appending them to the tag name for later) of unmapped tags
    [a simple samtool (BAMtoSAM) | perl one-liner massaging the ascii sam formatted data | samtools (SAMtoBAM) ]
  2. map against the flu genome
    [BWA aln - samse with bam as input instead of the default fasta/fastq]
  3. trim one more base to the still unmapped tags (append it to the name)
    [samtools (BAMtoSAM and bitwise flag filter) to split mapped/unmapped | another perl one-liner to massage the sam formatted unmapped tags file | samtools (SAMtoBAM) ]
  4. repeat step 2 & 3 three time
  5. collect all flu mapped tags from step 1 to 4 and map them against the combined flu/host genomes to get meaningful mapQ values
    [samtool (merge bam) | BWA aln - Delve]


Results / Data availability


ISMARA analysis results

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

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


For more information, see ISMARA.

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