Reassessment of Blood Gene Expression Markers for the Prognosis of Relapsing-Remitting Multiple Sclerosis

Despite considerable advances in the treatment of multiple sclerosis, current drugs are only partially effective. Most patients show reduced disease activity with therapy, but still experience relapses, increasing disability, and new brain lesions. Since there are no reliable clinical or biological markers of disease progression, long-term prognosis is difficult to predict for individual patients. We identified 18 studies that suggested genes expressed in blood as predictive biomarkers. We validated the prognostic value of those genes with three different microarray data sets comprising 148 patients in total. Using these data, we tested whether the genes were significantly differentially expressed between patients with good and poor courses of the disease. Poor progression was defined by relapses and/or increase of disability during a two-year follow-up, independent of the administered therapy. Of 110 genes that have been proposed as predictive biomarkers, most could not be confirmed in our analysis. However, the G protein-coupled membrane receptor GPR3 was expressed at significantly lower levels in patients with poor disease progression in all data sets. GPR3 has therefore a high potential to be a biomarker for predicting future disease activity. In addition, we examined the IL17 cytokines and receptors in more detail and propose IL17RC as a new, promising, transcript-based biomarker candidate. Further studies are needed to better understand the roles of these receptors in multiple sclerosis and its treatment and to clarify the utility of GPR3 and IL17RC expression levels in the blood as markers of long-term prognosis

Multiple Sclerosis: Modulation of Toll-Like Receptor (TLR) Expression by Interferon-β Includes Upregulation of TLR7 in Plasmacytoid Dendritic Cells

<div><p>Interferon-β is an established treatment for patients with multiple sclerosis (MS) but its mechanisms of action are not well understood. Viral infections are a known trigger of MS relapses. Toll-like receptors (TLRs) are key components of the innate immune system, which sense conserved structures of viruses and other pathogens. Effects of interferon-β on mRNA levels of all known human TLRs (TLR1-10) and the TLR adaptor molecule MyD88 were analyzed in peripheral blood mononuclear cells (PBMCs) of healthy donors by quantitative real-time PCR and by transcriptome analysis in PBMCs of 25 interferon-β-treated patients with relapsing-remitting MS. Regulation of TLR protein expression by interferon-β was investigated by flow cytometry of leukocyte subsets of healthy subjects and of untreated, interferon-β-, or glatiramer acetate-treated patients with MS. Interferon-β specifically upregulated mRNA expression of TLR3, TLR7, and MyD88 and downregulated TLR9 mRNA in PBMCs of healthy donors as well as in PBMCs of patients with MS. Plasmacytoid dendritic cells (pDCs) were identified as the major cell type responding to interferon-β with increased expression of TLR7 and MyD88 protein. In line with this, expression of TLR7 protein was increased in pDCs of interferon-β-treated, but not untreated or glatiramer acetate-treated patients with MS. Interferon-β-induced upregulation of TLR7 in pDCs is of functional relevance since pre-treatment of PBMCs with interferon-β resulted in a strongly increased production of interferon-α upon stimulation with the TLR7 agonist loxoribine. Flow cytometry confirmed pDCs as the cellular source of interferon-α production induced by activation of TLR7. Thus, upregulation of TLR7 in pDCs and a consequently increased activation of pDCs by TLR7 ligands represents a novel immunoregulatory mechanism of interferon-β. We hypothesize that this mechanism could contribute to a reduction of virus-triggered relapses in patients with MS.</p></div

Demographic and clinical details of patients shown in Figure 5.

<p>CIS, clinically isolated syndrome; RRMS, relapsing-remitting multiple sclerosis; EDSS, expanded disability status scale; i.m., intramuscular; s.c., subcutaneous.</p

Differential regulation of TLR mRNA expression in human PBMCs by interferon-β.

<p>PBMCs from healthy donors were incubated with 1000 U/ml interferon-β. Untreated cells served as control. After 6 hours of treatment RNA was isolated and qRT-PCR performed with primers for TLR1-10, MyD88, and MX1. Results (mean±SD) from <i>n</i> = 3 independent experiments with PBMCs from different healthy donors are shown as fold-change to unstimulated controls. The dotted line marks the expression level in unstimulated control PBMCs, which was set to 1. Statistical significance of differences was assessed by paired <i>t</i>-test. *<i>p</i><0.05; **<i>p</i><0.01; n.d., not detectable.</p

Interferon-β upregulates TLR7 and MyD88 protein expression in plasmacytoid dendritic cells <i>in vitro</i>.

<p>(<b>A, B, C, D</b>) PBMCs were either incubated with 1000 U/ml interferon-β or left untreated. (<b>A</b>) After 24 hours cells were stained with antibodies to TLR7 and MyD88 as well as isotype controls and analyzed by flow cytometry. Mean fluorescence intensity (MFI) was determined. Results are presented as mean±SD (MFI^{TLR7 or MyD88} – MFI^isotype) from <i>n</i> = 3 healthy donors for each cell type and TLR. (<b>B</b>) Representative dot plots and histogram plots of TLR7 and MyD88 expression in pDCs. (<b>C</b>) Percentages of each cell population of all live cells are depicted with each dot representing one healthy donor. (<b>D</b>) Representative dot plots display the gating strategy for pDCs. Statistical significance was assessed by paired <i>t</i>-test. IFN-β, interferon-β; *<i>p</i><0.05; **<i>p</i><0.01.</p

Regulation of TLR, MyD88, and MX1 mRNA expression in interferon-β-treated patients with MS.

<p>Expression levels of TLR1-10, MyD88, and MX1 mRNA were determined in PBMCs of 25 patients with RRMS by Affymetrix microarray analysis immediately before and one month after start of therapy with interferon-β-1b. Boxplots show medians, upper and lower quartiles as well as outliers, and graphically display the spread (interquartile range) of the pre-processed microarray data. Statistical significance of gene expression changes in response to therapy was assessed by Wilcoxon signed-rank test. <i>p</i>-values <0.01 are indicated.</p

Dose- and time-dependent expression of TLR mRNA in PBMCs in response to interferon-β.

<p>(<b>A</b>) PBMCs from healthy donors were incubated with 1000 U/ml interferon-β. Control cells were left untreated. After 6, 24, and 48 hours RNA was isolated and qRT-PCR performed with primers for the indicated genes. (<b>B</b>) PBMCs were stimulated with 0, 10, 100, or 1000 U/ml interferon-β. After 6 hours (TLR6, TLR9, TLR10) or 24 hours (TLR3, TLR7, MyD88, MX1) RNA was isolated and qRT-PCR analysis performed. Results (mean±SD) from <i>n</i> = 3 independent experiments with PBMCs from different healthy donors are shown as fold-change to unstimulated controls. Statistical significance was assessed by one-way ANOVA with Tukey's post test. IFN-β, interferon-β; *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p