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

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

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

Increased TLR7 and MyD88 protein expression in plasmacytoid dendritic cells of interferon-β-treated patients with MS.

<p>Expression of TLR7 and MyD88 in pDCs of 9 untreated, 7 interferon-β-, and 7 glatiramer acetate-treated patients with CIS/RRMS was analyzed by flow cytometry. Results are expressed as MFI^{TLR7 or MyD88} – MFI^isotype with each dot representing one individual. Empty symbols represent female and filled symbols male individuals. Statistical significance of differences was assessed by Mann Whitney U test. GA, glatiramer acetate; *<i>p</i><0.05.</p

Interferon-β pre-treatment leads to strongly increased production of interferon-α upon TLR7 stimulation.

<p>(<b>A</b>) PBMCs from healthy donors were either incubated with 1000 U/ml interferon-β or left untreated for 12 hours. Subsequently, TLR7 was stimulated with 0,1 mM loxoribine for additional 6 hours. Amounts of cytokines and chemokines were determined in cell culture supernatants by bead-based immunoassays. Results shown are mean±SD from <i>n</i> = 3 healthy donors. For assessment of statistical significance, the values of loxoribine-untreated cells were subtracted from values of loxoribine-treated cells for both, cells with and without interferon-β pre-treatment, and the resulting differences compared by paired t-test. * <i>p</i><0,05. (<b>B</b>, <b>C</b>) Cells were treated as in (<b>A</b>), but brefeldin A was added for the final 3 hours of stimulation before the intracellular expression of interferon-α was analyzed by flow cytometry. Representative dot plots showing (<b>B</b>) the percentage of CD303+ cells (pDCs) of all interferon-α positive cells and (<b>C</b>) the percentage of pDCs positive for interferon-α for one donor representative of <i>n</i> = 4. IFN-α, interferon-α; TNF-α, tumor necrosis factor-α; IFN-β, interferon-β; MIP-1α, macrophage inflammatory protein-1α, IP-10, interferon-γ-induced protein 10; IL, interleukin, n.d., not detected.</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