Multiple Sclerosis . MicroRNA . Treatment Response . Autoantibodies Prognostic Markers . Autoreactive B Cells

Clinical Neuroimmunology

Molecular and immunological analysis of Multiple Sclerosis

Our research focuses the molecular and immunological analysis of multiple sclerosis(MS), an inflammatory, demyelinating central nervous system (CNS) disease.We have two main research lines: 1) genomic investigations (including genetic, transcriptional and protein expression analysis) and 2) studies on B cell involvement in MS pathogenesis. Both approaches provide tools and markers for immunomonitoring of current and newly emerging treatments.

Immune regulation by microRNAs in MS
MicroRNAs (miRNAs), small non-coding RNA molecules, which modulate gene expression of > 50 % of all protein-encoding genes, and are key regulators of a wide variety of biological processes, e.g. cell proliferation, differentiation, apoptosis and organ development. Our cellular miRNA studies in immune cells from MS patients have revealed distinct expression profiles compared with those in healthy volunteers. We have also shown that natalizumab, the treatment for relapsing-remitting MS, has diverse effects on miRNA expression. We uncovered recently a specific effect of natalizumab on the expression of miR-126 and miR-10 and their potential target, POU2AF1, an important regulator of the transcription factor Spi-B, which binds to unique sequences of the JC virus and plays a critical role in driving virus activity (Meira et al., 2014, 2016, Fig. 1). Natalizumab treatment has been associated with the development of progressive multifocal leukoencephalopathy(PML), a severe opportunistic infection of the CNS caused by reactivation of the latent JC virus. We are presently evaluating the expression of miR-126/10and POU2AF1 as biomarkers for a PML risk in MS patients treated with natalizumab. Another focus of our research is extracellular miRNAs, stored in extracellular vesicles (EVs), in serum and CSF from MS patients. Our aim is to get new insights into the functional role of EVs in immune regulation and cell-to-cell communication in MS.

B cells and their targets in MS
B-cells have a major role in the pathogenesis of MS. Depletion of B-cells leads to a remarkable amelioration of the disease. The mechanisms by which B-cells impact MS are however incompletely understood. Our research focuses on the identification of novel B-cell autoantigens and the characterization of the interaction of autoaggressive B-cells with the CNS. We could show that antibodies against nativemyelin oligodendrocyte glycoprotein (MOG) identify a subset of patients with neuromyelitisoptica (Pröbstel et al., 2015). Antibodies against the potassium channelKIR4.1 have been suggested as a biomarker in MS. Using eukaryotic expression of this protein we could show that the prominently described antibody reactivities are directed against other proteins than KIR4.1 and that the assay as published is not useful for clinical practice (Pröbstel et al., 2016, Fig 2). Currently, weare using transgenic animals to better characterize the pathogenic mechanisms of B cells in the animal model of MS. We identified the co-capture of membrane antigens by the B cell receptor as a key step in initiating an autoimmune response.This work will be continued in analyzing the capacity of B cell to migrate to peripheral tissues and harvest their cognate and non-cognate antigens from the tissue.This phenomenon of membrane capture will also be used to identify novel autoantigens in MS.

Immunomonitoring of new treatments and biomarker research
The mode of action of many current disease modifying treatments is often not well understood. We aim to get a better understanding of the mode of action and there by identify candidates for treatment response biomarkers. Currently, we are recruiting MS patients that start treatment with dimethyl fumarate. Immune cells of the blood as well as serum will be analyzed for immunological markers, and miRNA/RNA expression profiles that could potentially correlate with the clinical response to treatment. Combination of these biomarkers with a standardized clinical and neuroradiologic assessment will hopefully provide means to better characterize the heterogeneous MS patient populations and to predict the disease course and the treatment response.

Fig. 1: Transcriptional expression of POU2AF1 (A) and miR-10b (B) in untreated, natalizumab treated RRMS (1–24mo; >24mo) and natalizumab associated PML patients. Relative expression levels (median with interquartile range) are depicted. ***p<0.001; **p<0.01, *p<0.05.

Fig. 2: KIR4.1 protein ELISA: 141 patients with clinically isolated syndrome (CIS, n = 82) or multiple sclerosis (MS, n = 59) and 131 controls (other (non-inflammatory) neurological diseases (OND) n = 48, neurodegenerative diseases (ND) n = 48, other inflammatory neurological diseases (OIND) n = 35). Anti-KIR4.1 reactivity is expressed as the mean optical density (OD) of duplicate measurements. The distribution of OD KIR4.1 protein by group is shown in notched box-and-whisker plots, each accompanied by histograms of the same data. The dashed horizontal line represents a cut-off value of 0.628 (5 standard deviations (SD) above the mean of an unblinded OND control group1, n = 10). Statistical comparison between the groups revealed no significant differences (Kruskal-Wallis rank sum test for five groups: p = 0.16).