Open Access Research article

A systems-based approach to analyse the host response in murine lung macrophages challenged with respiratory syncytial virus

Laxmi Iyer Ravi1, Liang Li12, Richard Sutejo1, Hui Chen1, Pui San Wong3, Boon Huan Tan3 and Richard J Sugrue12*

Author Affiliations

1 Division of Molecular Genetics and Cell Biology, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore

2 Singapore-MIT Alliance for Research & Technology (SMART), Centre for Life Sciences, 28 Medical Drive, Singapore 117456, Singapore

3 Detection and Diagnostics Laboratory, DSO National Laboratories, 27 Medical Drive, Singapore, 117510, Singapore

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BMC Genomics 2013, 14:190  doi:10.1186/1471-2164-14-190

Published: 18 March 2013

Additional files

Additional file 1: Figure S1:

Antigenic characterization of lung macrophage cells. (A) PMΦ cells were labelled using anti-CD11b and anti-F4/80 and (B) AMΦ cells were labelled with anti-CD11b and anti-CD11c and examined using immunofluorescence (IF) microscopy or bright field microscopy (BF) (at magnification x10). (C) The PMΦ cells exhibited phagocytic activity. Polystyrene latex beads (2.0-μm diameter) (Sigma) were coated with BSA (Sigma Aldrich) as described previously (May et al., 2000). Briefly, the beads were washed three times in PBS, incubated with BSA (10mg/ml) at 4°C overnight with gentle rotation, and then washed to remove excess BSA at room temperature for 1 hour with rotation, and then washed again with PBS. The PMΦ cells were plated on cover slips placed and the latex beads were added at approximately 20 particles/cell for 30 minutes at 330C. Non-internalized beads were removed by washing with PBS, the cells fixed using 3% PFA in PBS and visualized using an inverted fluorescent microscope (Nikon) (objective x100).

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Additional file 2: Figure S2:

Ultrastructural analysis of RSV-infected PMΦ cells by electron microscopy. Mock and RSV-infected MΦ cells at 24 hour post-infection (hpi) were processed for (A) scanning electron microscopy (SEM) or (B) transmission electron microscopy (TEM). Representative images are shown. Membrane ruffling and protrusions on the surface of the MΦ cells imaged by SEM and TEM respectively are highlighted (black arrows). SEM, magnification at x5,000;TEM, magnification at x40,000.

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Additional file 3: Figure S3:

Infectious virus particles are not produced in RSV-infected macrophages (A) The total RNA was extracted from RSV-infected MΦ cells at 2.5 and 24 hpi and the vRNA levels estimated by qPCR as described in methods. This is the average of 3 measurements and p < 0.05. (B) The tissue culture supernatant (TCS) from mock-infected or RSV-infected HEp2 cells or PMΦ cells was harvested at 24 hpi and used to infect HEp2 monolayers. At 24 hpi the presence of infected cells in the HEp2 cell monolyer was stained using anti-RSV and viewed by fluorescence microscopy (anti-RSV) and bright field microscopy (BF) (objective x10).

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Additional file 4: Table S1:

Pathway enrichment analysis based on global macrophage host genes showing changes in gene expression following RSV infection at 4 and 24 hpi. Macrophages were infected with RSV at two different time points and IPA version 2012 software was applied for pathway analysis. The 10 most significant canonical pathways enriched by global gene expression (p-value ≤ 0.05 and FC ≥ 2) at 4 hpi and 24 hpi are listed. Corresponding p-values, gene numbers and individual genes are also indicated.

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Additional file 5: Table S2:

Pathway enrichment analysis based on macrophage host genes showing up-regulated and down-regulated gene expression following infection with RSV at 4 and 24 hpi. Macrophages were infected with RSV at two different time points and IPA version 2012 software was applied for pathway analysis. Significant canonical pathways enriched by differentially up-regulated or down-regulated genes (p-value ≤ 0.05 and FC ≥ 2) at 4 hpi and 24 hpi were listed. Corresponding p-values, gene numbers and individual genes were also represented. The pathway in bold indicated that it is commonly enriched at both 4 hpi and 24 hpi.

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Additional file 6: Figure S4:

UV-treatment inactivates RSV infectivity. The RSV inoculum was either non-treated (RSV-NT) or UV-treated (RSV-UVT) and used to infect either HEp2 cells and at 24 hours post-infection the cells were stained using anti-RSV and viewed using a Nikon eclipse 80i fluorescence microscope (objective x10).

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Additional file 7: Figure S5:

Cytokine induction in the murine alveolar macrophage (AMФ) cell preparation. The AMФ cell preparation was either mock-infected (Mock) or infected using a RSV inoculum that was either non-treated (RSV) or UV-inactivated (RSV(UV)). The levels of pro-inflammatory cytokines in the tissue culture supernatant (TCS) was measured at 24 hrs post-infection (hpi). In each case the data shown were obtained from triplicate measurements (p < 0.05) and representative data from two independent experiments is shown.

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Additional file 8: Figure S6:

Validation in the differential gene expression of selected genes in RSV-infected MΦ cells using qPCR. The relative mRNA levels in mock and RSV-infected MΦ cells of (i) 2’,5’-oligoadenylate synthase 2 (OAS2), radical S-adenosyl methionine domain containing 2 (RSAD2), RANTES, Interferon-β (IFNβ) and 2’,5’-oligoadenylate synthase-like genes (OASL) (insets show expression levels in mock-infected cells) and (ii) TAP1, CD40. The average values obtained from three independent measurements (p < 0.05) and representative data from one experiment.

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Additional file 9: Table S3:

Primer and probes sequences designed for real-time qPCR. Primer sequences and UPL probes (Roche) used for real-time qPCR validation . RSV N gene, IFN-β1: interferon β; OAS2: 2’, 5’-oligoadenylate synthase 2; OASL: 2’, 5’-oligoadenylate synthase-like; RSAD2: radical S-adenosyl methionine domain containing 2; HMGCR: 3-hydroxy-3-methyl-glutaryl-CoA reductase; CH25H: cholesterol 25-hydroxylase; TAP1: Antigen peptide transporter 1; CD40:CD40; RANTES (CCL5); EF: elongation factor.

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