Ten top canonical pathways enriched in monocytes and T cells datasets.

1<p>Ratio indicates the number of DEGs participating in a pathway divided by the total number of molecules participating in that pathway. Data in this table was generated by IPA.</p

Interferon-Beta Induces Distinct Gene Expression Response Patterns in Human Monocytes versus T cells

<div><p>Background</p><p>Monocytes, which are key players in innate immunity, are outnumbered by neutrophils and lymphocytes among peripheral white blood cells. The cytokine interferon-β (IFN-β) is widely used as an immunomodulatory drug for multiple sclerosis and its functional pathways in peripheral blood mononuclear cells (PBMCs) have been previously described. The aim of the present study was to identify novel, cell-specific IFN-β functions and pathways in tumor necrosis factor (TNF)-α-activated monocytes that may have been missed in studies using PBMCs.</p><p>Methodology/Principal Findings</p><p>Whole genome gene expression profiles of human monocytes and T cells were compared following <i>in vitro</i> priming to TNF-α and overnight exposure to IFN-β. Statistical analyses of the gene expression data revealed a cell-type-specific change of 699 transcripts, 667 monocyte-specific transcripts, 21 T cell-specific transcripts and 11 transcripts with either a difference in the response direction or a difference in the magnitude of response. RT-PCR revealed a set of differentially expressed genes (DEGs), exhibiting responses to IFN-β that are modulated by TNF-α in monocytes, such as <i>RIPK2</i> and <i>CD83</i>, but not in T cells or PBMCs. Known IFN-β promoter response elements, such as ISRE, were enriched in T cell DEGs but not in monocyte DEGs. The overall directionality of the gene expression regulation by IFN-β was different in T cells and monocytes, with up-regulation more prevalent in T cells, and a similar extent of up and down-regulation recorded in monocytes.</p><p>Conclusions</p><p>By focusing on the response of distinct cell types and by evaluating the combined effects of two cytokines with pro and anti-inflammatory activities, we were able to present two new findings First, new IFN-β response pathways and genes, some of which were monocytes specific; second, a cell-specific modulation of the IFN-β response transcriptome by TNF-α.</p></div

Volcano plots for the differential gene expression following IFN-β treatment of monocytes and T cells.

<p>A. monocytes; B. T cells. The X axis describes the fold change in expression levels between cells treated with IFN-β relative to untreated cells, for each transcript in a log2 scale. The Y axis shows the statistical significance expressed as -log10(p-value) from the simple comparison. Transcripts with log2 difference of ≥1 and with -log10(p-value) ≥3.8, which is the equivalent of p≤0.05 after FDR adjustment, were defined as differentially expressed genes (DEGs) and are highlighted with blue for down-regulated and red for up-regulated DEGs.</p

CD38 protein expression is increased in monocytes, but not in T cells, in response to IFN-β.

<p>Flow cytometry analysis of CD38 expression in monocytes and T cells incubated with TNF-α for 2 hours and then treated with IFN-β for 40 hours. Representative results of CD38 expression with and without IFN-β treatment in A. CD14+ monocytes and B. CD3+ T cells. N≥3 for each cell type.</p

Differentially expressed genes in T cells but not in monocytes (21 in total).

1<p>Expression level in treated samples divided by expression level in untreated samples.</p>2<p>FDR adjusted p-value for IFN-β effect within each cell type.</p>3<p>FDR adjusted p-value of the two-way ANOVA for the cell-type*IFN-β interaction.</p

RT-PCR confirms differential expression between monocytes and T cells in response to IFN-β.

<p>Monocytes, T cells, and PBMCs from healthy donors were included in the validation of eight DEGs selected from the microarray data. Triangles represent monocytes, circles- T cells, and diamonds- PBMCs. Full and empty symbols represent cells pre-incubated or not with TNF-α. The horizontal bars mark the median values. The Y axis depicts the changes in expression levels in response to IFN-β as fold change (2^−ΔΔCT); *p-values<0.03 for the IFN-β response (Wilcoxon signed rank test). For difference in fold change between monocytes, T cells, and PBMCs, *p-value<0.05; **p-value <0.01 (Mann Whitney test). The mRNA levels of <i>SIGLEC10</i> were below detection level by RT-PCR in T cells, under all conditions, therefore the p-value for comparison between monocytes and T cells was calculated under the assumption of no change in expression (fold change = 1, marked by rectangle) in T cells.</p

Differentially expressed genes in both monocytes and T cells (25 out of 106 genes).

1<p>Expression level in treated samples divided by expression level in untreated samples.</p>2<p>FDR adjusted p-value for IFN-β effect within each cell type.</p>3<p>FDR adjusted p-value of the two-way ANOVA for the cell-type*IFN-β interaction.</p

Networks regulated by IFN-β in monocytes include functions of cell migration, and cellular development and proliferation.

<p>Genes were mapped to IPA-generated networks based on known gene interactions, and the networks were ranked according to the number of biological connections between the transcripts by IPA. Shown in this figure are two high-score networks (IPA score = 29 for both), which we named based on the IPA-suggested keywords: A. Cell migration; B. Cellular development and proliferation. Green nodes indicate down-regulation and red indicate up-regulation. White nodes indicate molecules that were incorporated into the network through relationships with other molecules but are not DEGs. Dotted lines represent indirect interaction, solid lines represent direct interaction. Protein symbols are explained in the symbol legend in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062366#pone-0062366-g006" target="_blank">figure 6C</a>. C. RT-PCR analyses for the genes <i>EDN1</i> from Cell migration network, and <i>IL1B</i> and <i>RXRA</i> from cellular development and proliferation network for monocytes and T cells (n = 6). The Y axis depicts the changes in RNA expression levels in response to IFN-β as fold change (2^−ΔΔCT); the fold change following the IFN-β treatment was significant for all genes in both monocytes and T cells (P-values<0.03, Wilcoxon signed rank test). *p-value <0.01 by Mann Whitney test for comparison of fold change between monocytes and T cells. Horizontal bars indicate median values for each column of data points.</p

Biological processes induced by IFN-β in monocytes and T cells.

<p>The differentially expressed transcript lists were ranked by the simple comparison p-value and fold change, and then evaluated for functional enrichment by GOrilla. Visual representations of the enriched gene ontology terms produced by GOrilla are shown for A. monocytes and B. T cells. The significance of the enrichment of the biological processes is color coded as indicated. C. Output produced by SPIKE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062366#pone.0062366-Paz1" target="_blank">[42]</a> showing enrichment of the 'Translation elongation' pathway in monocytes (p = 10⁻⁶⁵).</p

Primer sequences used in this study.

1<p>UPL – Universal Probe Library.</p