Macrophage influenza infection: Difference between revisions
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|TCSample_description=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.<br><br>References:<br>[1] PMID: 22238612 | |TCSample_description=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.<br><br>References:<br>[1] PMID: 22238612 | ||
|Time_Course= | |Time_Course= | ||
|category_treatment= | |category_treatment=Activation/ infection | ||
|collaborators=David Hume | |collaborators=David Hume | ||
|description=human_Monocyte-derived_macrophages_response_to_udorn_influenza_infection | |description=human_Monocyte-derived_macrophages_response_to_udorn_influenza_infection | ||
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|time_points=00hr | |time_points=00hr | ||
|time_span=24 hours | |time_span=24 hours | ||
|timepoint_design= | |timepoint_design=Activation | ||
|tissue_cell_type= | |tissue_cell_type=Monocytes | ||
|zenbu_config=http://fantom.gsc.riken.jp/zenbu/gLyphs/#config=QiyVHglCC5xm23hvG0F_oB | |zenbu_config=http://fantom.gsc.riken.jp/zenbu/gLyphs/#config=QiyVHglCC5xm23hvG0F_oB | ||
}} | }} | ||
Revision as of 12:15, 4 February 2015
| Series: | IN_VITRO DIFFERENTIATION SERIES |
|---|---|
| Species: | Human (Homo sapiens) |
| Genomic View: | Zenbu |
| Expression table: | FILE |
| Link to TET: | [{{{tet_file}}} TET] |
| Sample providers : | David Hume |
| Germ layer: | mesoderm |
| Primary cells or cell line: | primary cells |
| Time span: | 24 hours |
| Number of time points: | 4 |
| Overview |
|---|
|
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. |
| Sample description |
|---|
|
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. |
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
| 13305-142I2 | Monocyte-derived macrophages response to udorn influenza infection | 00hr00min | donor1 (868_121:Ud_0h) |
| 13306-142I3 | Monocyte-derived macrophages response to udorn influenza infection | 02hr00min | donor1 (868_121:Ud_2h) |
| 13307-142I4 | Monocyte-derived macrophages response to udorn influenza infection | 07hr00min | donor1 (868_121:Ud_7h) |
| 13308-142I5 | Monocyte-derived macrophages response to udorn influenza infection | 24hr00min | donor1 (868_121:Ud_24h) |
| 13309-142I6 | Monocyte-derived macrophages response to mock influenza infection | 24hr00min | donor1 (868_121:MI_24h) |
| 13310-142I7 | Monocyte-derived macrophages response to mock influenza infection | 00hr00min | donor4 (227_121:MI_0h) |
| 13311-142I8 | Monocyte-derived macrophages response to udorn influenza infection | 00hr00min | donor4 (227_121:Ud_0h) |
| 13312-142I9 | Monocyte-derived macrophages response to udorn influenza infection | 02hr00min | donor4 (227_121:Ud_2h) |
| 13313-143A1 | Monocyte-derived macrophages response to udorn influenza infection | 07hr00min | donor4 (227_121:Ud_7h) |
| 13315-143A3 | Monocyte-derived macrophages response to mock influenza infection | 24hr00min | donor4 (227_121:MI_24h) |
| 13316-143A4 | Monocyte-derived macrophages response to mock influenza infection | 00hr00min | donor2 (150_120:MI_0h) |
| 13317-143A5 | Monocyte-derived macrophages response to udorn influenza infection | 00hr00min | donor2 (150_120:Ud_0h) |
| 13319-143A7 | Monocyte-derived macrophages response to udorn influenza infection | 07hr00min | donor2 (150_120:Ud_7h) |
| 13320-143A8 | Monocyte-derived macrophages response to udorn influenza infection | 24hr00min | donor2 (150_120:Ud_24h) |
| 13321-143A9 | Monocyte-derived macrophages response to mock influenza infection | 24hr00min | donor2 (150_120:MI_24h) |
| 13322-143B1 | Monocyte-derived macrophages response to mock influenza infection | 00hr00min | donor3 (536_119:MI_0h) |
| 13323-143B2 | Monocyte-derived macrophages response to udorn influenza infection | 00hr00min | donor3 (536_119:Ud_0h) |
| 13324-143B3 | Monocyte-derived macrophages response to udorn influenza infection | 02hr00min | donor3 (536_119:Ud_2h) |
| 13325-143B4 | Monocyte-derived macrophages response to udorn influenza infection | 07hr00min | donor3 (536_119:Ud_7h) |
| 13326-143B5 | Monocyte-derived macrophages response to udorn influenza infection | 24hr00min | donor3 (536_119:Ud_24h) |
| 13327-143B6 | Monocyte-derived macrophages response to mock influenza infection | 24hr00min | donor3 (536_119:MI_24h) |
