Gene Expression Profiling of the Response to Interferon Beta in Epstein-Barr-Transformed and Primary B Cells of Patients with Multiple Sclerosis

<div><p>The effects of interferon-beta (IFN-β), one of the key immunotherapies used in multiple sclerosis (MS), on peripheral blood leukocytes and T cells have been extensively studied. B cells are a less abundant leukocyte type, and accordingly less is known about the B cell-specific response to IFN-β. To identify gene expression changes and pathways induced by IFN-β in B cells, we studied the <i>in vitro</i> response of human Epstein Barr-transformed B cells (lymphoblast cell lines-LCLs), and validated our results in primary B cells. LCLs were derived from an MS patient repository. Whole genome expression analysis identified 115 genes that were more than two-fold differentially up-regulated following IFN-β exposure, with over 50 previously unrecognized as IFN-β response genes. Pathways analysis demonstrated that IFN-β affected LCLs in a similar manner to other cell types by activating known IFN-β canonical pathways. Additionally, IFN-β increased the expression of innate immune response genes, while down-regulating many B cell receptor pathway genes and genes involved in adaptive immune responses. Novel response genes identified herein, <i>NEXN</i>, <i>DDX60L</i>, <i>IGFBP4</i>, and <i>HAPLN3</i>, B cell receptor pathway genes, <i>CD79B</i> and <i>SYK</i>, and lymphocyte activation genes, <i>LAG3</i> and <i>IL27RA</i>, were validated as IFN-β response genes in primary B cells. In this study new IFN-β response genes were identified in B cells, with possible implications to B cell-specific functions. The study's results emphasize the applicability of LCLs for studies of human B cell drug response. The usage of LCLs from patient-based repositories may facilitate future studies of drug response in MS and other immune-mediated disorders with a B cell component.</p></div

Proportion of variance in gene expression is explained by a major IFN-β response component and donor-specific components.

<p>Variance component analysis was modeled with the following components: Donor - representing variance contributed by unspecified differences between donors; IFN-β exposure (<i>in vitro</i> IFN-β treated LCL sample versus the untreated sample); Age (stratified to under 40 years old or above); Gender; MS subtype (relapsing remitting, relapsing progressive, secondary progressive), Treatment status (IFN-β treatment naïve donors, or donors treated for at least 1 year at the time of sample collection); and Ethnicity (Jewish/Arab). The Residual component models all the variability that cannot be attributed to any of the explicit variance components.</p

<i>NEXN</i>, <i>DDX60L</i>, <i>IGFBP4</i> and <i>HAPLN3</i> respond transiently to IFN-β.

<p>The expression levels of <i>NEXN</i>, <i>DDX60L</i>, <i>IGFBP4</i> and <i>HAPLN3</i> in LCLs were analysed by real time RT-PCR analysis after 4 or 16 hours of exposure to 100 u/ml IFN-β. Data is presented as a box plot of the 25^th and 75^th percentiles around the median values.</p

The LCL gene expression response to IFN-β.

<p>A: Volcano plot for the IFN-β gene expression response in LCLs. Log2(fold change) of expression levels following IFN-β exposure and P-values (one-way ANOVA) are depicted for each transcript probe. The horizontal dashed line represents the threshold for the adjusted P-value of 0.05. Vertical dashed line indicates threshold of log2(fold change)>1 and <−1. Differentially expressed genes (in black) are genes with log2(fold change) ≥1 and adjusted P-value ≤0.05, novel IFN-β response genes included in RTPCR validation analyses are marked. For pathways analyses a lower threshold was employed (dotted vertical line) with log2(fold change)≥0.4 (dark grey dots). B: Venn diagram describing the proportion of differentially expressed genes in response to IFN-β [log2(fold change)≥1, adjusted P-value<0.05, bold outlined ellipse] that are novel response genes (grey shade), and a comparison to the published differentially expressed genes up-regulated in LCLs in comparison to primary B cells, based on the data of Caliskan and colleagues [log2(fold change)≥1, adjusted P-value<0.05]<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102331#pone.0102331-Caliskan1" target="_blank">[26]</a>. Definition of novel IFN-β response genes is based on the Interferome V2.01 database (accessed 19.8.13) search for the 115 IFN-β response differentially expressed genes. Numbers indicate the number of genes within each subgroup.</p

Validation of the gene expression response for four novel IFN-β response genes by real time RT-PCR.

<p>The change in expression levels (2-^ΔΔ<i>C</i>_T) following four hours of IFN-β exposure is shown for four genes that are novel IFN-β response genes. The horizontal bars represent the median values. A: Comparison of the IFN-β response in LCLs from healthy controls (HC, empty symbols, n = 7) and patients with multiple sclerosis (MS, full symbols, n = 20). Fold changes for IFN-β response were significant for both groups (P-value<0.007, Wilcoxon signed rank test). No difference was observed in the IFN-β response between controls and MS LCLs, except for <i>HAPLN3</i>, for which the difference was of a small magnitude (*P-value<0.05, Mann Whitney test). B: The IFN-β response in primary B cells (n = 9) and PBMC (n = 8) from healthy controls and MS patients. All genes displayed a significant response to IFN-β for both B cells and PBMC (P-value<0.001, Wilcoxon signed rank test). Significant differences between the response in B cells and PBMCs is indicated by ** P-value = 0.0003 (Mann Whitney test). The response magnitude of B cells and LCLs differed for all genes, however, these differences were small (P-value<0.03, Mann Whitney test).</p

IFN-β activates the antimicrobial pathway and related innate immunity genes in LCLs.

<p>The pathway depicted includes the 27 genes of the of the 'antimicrobial response' pathway that showed increased activation following IFN-β exposure of LCLs (activation score Z = 2.779) by Ingenuity analysis. In addition, genes outlined in blue were manually added to the pathway based on their known participation in the innate response in B cells or other leukocytes, and include <i>TLR7</i> and <i>TLR9 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102331#pone.0102331-Poovassery1" target="_blank">[20] </a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102331#pone.0102331-Pone1" target="_blank">[74]</a>, <i>TRIM21</i> and <i>TRIM5 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102331#pone.0102331-McEwan1" target="_blank">[75]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102331#pone.0102331-Versteeg1" target="_blank">[77]</a>. Only direct connections are featured, and subcellular location of gene products is as suggested by Ingenuity knowledge base. In green- down-regulated genes; in red- up-regulated genes, color intensity is proportional to the fold change in expression levels following IFN-β exposure.</p

Networks of B cell-related genes are affected by IFN-β in LCLs.

<p>Network of genes included in the ‘Activation of Lymphocytes' function highlighted by Ingenuity analysis (Z = 2.33). Arrows points to IFN-β response genes that are not included in the Interferome database, and were included in a follow up validation analysis by quantitative RTPCR shown in D. In green- down-regulated genes; in red- up-regulated genes, color intensity is proportional to the fold change in expression levels following IFN-β exposure. A: An interaction network of CD79B, the immunoglobulin-β subunit of the B cell receptor signaling complex, with its regulators and interacting genes. The interacting nodes were limited to genes that were included in the filtered gene expression dataset, and were identified as <i>directly connected</i> to CD79B based on Ingenuity pathways analysis database. Notably, all the genes included in this network were found to be down-regulated by IFN-β (green) to some extent. Fold change and P-values are noted below each node. B: Decreased expression levels were observed for many of the genes included in the canonical B cell receptor signaling pathway in response to IFN-β. C: Quantitative reverse transcriptase RTPCR analysis of IFN-β response in primary B cells and PBMCs for genes marked in arrows in A and B. Genes were selected for validation because they were not identified by the Interferome database as IFN-β response genes, and are related to B cell function (<i>CD79B, SYK, TNFSF13B/BAFF</i>), or participate in the antimicrobial pathway depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102331#pone-0102331-g006" target="_blank">Fig. 6</a> (<i>IL27RA</i>), or are related to activation of lymphocytes (<i>LAG3, KLF2</i>). * P-value = 0.007 (Mann Whitney test).</p