<i>L</i>. <i>monocytogenes</i> infection modulates a number of IFN signaling pathway and IFN response genes in the blood and spleen.

<p><b>(A)</b> The IFN signaling pathway (QIAGEN Ingenuity® Pathway Analysis) was overlaid with the day 3 post infection blood genes shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150251#pone.0150251.g001" target="_blank">Fig 1A</a>. Genes (IFNαβ and JAK1) with opposite expression patterns between blood and spleen are highlighted with black circles. Red: upregulated, Blue: downregulated. <b>(B)</b> IFN response genes (type I, type II, and type I and II) associated with blood and spleen transcripts reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150251#pone.0150251.g001" target="_blank">Fig 1A and 1B</a> and the Interferome database (<a href="http://www.interferome.org/" target="_blank">www.interferome.org</a>) were quantitated.</p

Validation of several IFN regulated genes by qRT-PCR.

<p><b>(A)</b> qRT-PCR validation of several IFN regulated genes identified in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150251#pone.0150251.g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150251#pone.0150251.g005" target="_blank">5</a>. Blood (left panel), spleen (middle panel) and liver (right panel) RNA was extracted at the indicated times post <i>L</i>. <i>monocytogenes</i> infection. RNA was reverse transcribed to cDNA, and expression of the indicated genes was analyzed by qRT-PCR. Values were normalized relative to <i>Hprt1</i> expression levels (mean with SD). Data are from one experiment with four mice per group. <b>(B)</b> Ly6C⁺ monocytes and pDC in blood and spleen of uninfected and infected WT and <i>Ifnar1</i>^<i>-/-</i> mice as a percentage of total live cells. Pooled results from 3 independent experiments, mean with SEM, n = 3 per group/experiment.</p

<i>Ifnar1</i>^-/- uninfected mice show a lowered expression of several IFN regulated genes as compared with WT uninfected mice.

<p><b>(A–C)</b> The strain-associated subsets of transcripts identified from the 2-way ANOVA as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150251#pone.0150251.g003" target="_blank">Fig 3B</a> for an independent experiment were filtered to identify baseline differences (fold-ratio of 1.5 between day 3 uninfected <i>Ifnar1</i>^<b>-/-</b> to day 3 uninfected WT mice) for blood, spleen and liver. Heatmaps and a list of top three IPA® canonical pathways are shown. <b>(D)</b> Venn diagram of above detailed transcripts identifies 50 transcripts that are commonly shared between blood, spleen and liver. These transcripts map to 35 genes in IPA® and include a number of Interferome-based type I IFN responsive genes that are marked in red. <b>(E)</b> Heatmap of mean-normalised expression values for selected (<i>Irf1</i>, <i>Irf3</i>, <i>Irf7</i>, <i>Irf9</i>, <i>Stat1</i> and <i>Stat2</i>) IFN transcriptional regulator transcripts. <b>(F)</b> qRT-PCR validation of <i>Irf7</i> gene normalized relative to <i>Hprt1</i> gene in blood (left panel), spleen (middle panel) and liver (right panel) at indicated times post infection (mean with SD). Data from one experiment with four mice per group.</p

Tissue-specific transcriptional responses of WT and <i>Ifnar1</i>^-/- mice in blood, spleen and liver following <i>L</i>. <i>monocytogenes</i> infection at different time points and bacterial loads.

<p><b>(A)</b> Colony forming unit (CFU) values were assessed from the spleen and liver of C57BL/6 WT and <i>Ifnar1</i>^<b>-/-</b> mice at day 1, day 2 and day 3 following intravenous injection with 5 × 10³ of <i>L</i>. <i>monocytogenes</i> (mean with SEM, <i>n</i> = 4 or 5 mice/group). <b>(B)</b> Heatmap representations of differentially expressed transcripts at different times (days 1, 2 and 3) in blood, spleen, liver from WT and <i>Ifnar1</i>^<b>-/-</b> infected mice relative to uninfected controls (<i>p</i> < 0.05 after 2-way ANOVA with Benjamini–Hochberg multiple testing correction on transcripts passing quality control filtering, <i>n</i> = 3 or 4 mice/group). <b>(C)</b> Percentage of transcripts that were significant for infection alone, strain alone or involving a combination of strain and infection across the different tissues and the infection time course. <b>(D)</b> Venn diagrams showing temporal differences and similarities in gene expression in blood, spleen and liver after infection.</p

Canonical pathways associated with blood transcripts that are differentially expressed in <i>L</i>. <i>monocytogenes</i> infected WT versus <i>Ifnar1</i>^-/- mice against control uninfected WT mice.

<p>Top IPA® canonical pathways that are associated with differentially expressed blood day 1, day 2 and day 3 transcripts shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150251#pone.0150251.g003" target="_blank">Fig 3B</a> and that pass a further 1.5-fold change filter ratio between <b>(A)</b> WT infected to WT uninfected; <b>(B)</b> KO infected to WT uninfected; and <b>(C)</b> (WT infected to WT uninfected) as compared to (KO infected to WT uninfected). Percent pathway modulation relative to each dataset and pathway size is indicated in red for up-regulated and blue for down-regulated genes. Pathway rank for pathways passing <i>p<0</i>.<i>05</i> after Fisher’s Exact test at each time-point is marked. <b>(D and E)</b> Detailed heat map of differentially expressed genes found in the <b>(D)</b> Interferon Signaling Pathway; and <b>(E)</b> Antigen Presentation Pathway.</p

Transcriptional response following <i>L</i>. <i>monocytogenes</i> infection of WT mice in blood and spleen.

<p><b>(A, B)</b> Heatmaps of the significantly regulated blood and spleen transcripts at day 3 following intravenous injection of C57BL/6 WT mice with 5 × 10³ of <i>L</i>. <i>monocytogenes</i> (<i>p</i> < 0.01 after unpaired <i>t</i>-test with Benjamini–Hochberg multiple testing correction on transcripts passing quality control filtering, <i>n</i> = 10 mice/group). The top five QIAGEN Ingenuity® Pathway Analysis (IPA®) canonical pathways by significance (Fisher’s exact test) for upregulated (red bars) or downregulated (blue bars) genes are listed below the heatmaps. <b>(C)</b> qRT-PCR data on selected transcripts normalized relative to <i>Hprt1</i> expression levels (mean with SD, <i>n</i> = 5 mice/group). Samples are from the same experiment as the microarray data. <b>(D)</b> CD3⁺Thy1⁺ cells in blood and spleen as a percentage of total live cells. Pooled results are from triplicates of 3 independent experiments (mean with SEM, <i>n</i> = 3 mice/group/experiment).</p

Canonical pathways associated with blood transcripts that are differentially expressed in <i>L</i>. <i>monocytogenes</i> infected <i>Ifnar1</i>^-/- versus WT mice against control uninfected <i>Ifnar1</i>^-/- mice.

<p>Top IPA® canonical pathways that are associated with differentially expressed blood day 1, day 2 and day 3 transcripts from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150251#pone.0150251.g003" target="_blank">Fig 3B</a> and pass a further 1.5-fold change filter ratio between <b>(A)</b> WT infected to KO uninfected; <b>(B)</b> KO infected to KO uninfected; and <b>(C)</b> (KO infected to KO uninfected) as compared to (WT infected to KO uninfected). Percent pathway modulation relative to each dataset and pathway size is indicated in red for up-regulated and blue for down-regulated genes. Pathway rank for pathways passing <i>p<0</i>.<i>05</i> after Fisher’s Exact test at each time-point is marked. <b>(D and E)</b> Detailed heat map of differentially expressed genes found in the <b>(D)</b> Interferon Signaling Pathway; and <b>(E)</b> Antigen Presentation Pathway.</p

IFN response genes show differential early and delayed expression patterns following <i>L</i>. <i>monocytogenes</i> infection in WT and <i>Ifnar1</i>^-/- mice.

<p><b>(A)</b> Heatmaps of IFN-response genes (type I, type II, and type I and II) associated with blood, spleen and liver transcripts reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150251#pone.0150251.g003" target="_blank">Fig 3B</a> and the Interferome database are shown. Total numbers of IFN-response genes identified across each of the three tissues and their time-course distribution are shown at far right. <b>(B)</b> Venn diagrams showing temporal differences and similarities in the expression of IFN-response genes in blood, spleen and liver. <b>(C)</b> Weighted molecular scores of the identified IFN-response genes calculated relative to uninfected WT mice. <b>(D)</b> The distribution of IFN-response genes within blood, spleen and liver of infected and uninfected mice at individual times post infection.</p