Andreas Junker

CCL19 is constitutively expressed in the CNS, up-regulated in neuroinflammation, active and also inactive multiple sclerosis lesions

CCL19 and CCL21 bind to CCR7, which is crucial for both inducing an immune response and establishing immunological tolerance. We report that in the normal human brain CCL19, but not CCL21, is transcribed, and detectable as a protein in tissue lysates and in cerebrospinal fluid. In both active and inactive multiple sclerosis (MS) lesions CCL19 transcripts were elevated. In cerebrospinal fluid from MS and OIND patients CCL19 protein was increased. In relapsing-remitting and secondary progressive MS patients CCL19 correlated with intrathecal IgG production. This study suggests that CCL19 plays a role in both the physiological immunosurveillance of the healthy CNS and the pathological maintenance of immune cells in the CNS of MS patients.

The emerging role of microRNAs in multiple sclerosis

Several hundred microRNAs (miRNAs) fine-tune the expression of approximately half of all human genes. Recent studies have revealed that miRNA profiles in blood cells become altered in multiple sclerosis (MS), and that active and inactive MS lesions have distinct miRNA expression patterns. The dysregulated miRNAs in MS lesions seem to be associated with astrocytes and infiltrating immune cells, and might unleash local macrophages through downregulation of the self-recognition signal CD47. The expression of miRNA-326 in blood cells has been reported to increase during relapses. This miRNA promotes T helper 17 cell differentiation and is highly abundant in active MS lesions. miRNAs are needed for maintenance of the myelin sheath, and the absence of such molecules results in axonal damage in mice. miRNA-219 and other miRNAs promote oligodendrocyte differentiation. Here, we discuss the possible contribution of miRNAs to MS pathogenesis. An improved understanding of this contribution should help to identify novel therapeutic targets and biomarkers for this disease.

Extracellular Matrix in Multiple Sclerosis Lesions: Fibrillar Collagens, Biglycan and Decorin are Upregulated and Associated with Infiltrating Immune Cells

Extracellular matrix (ECM) proteins can modify immune reactions, e.g. by sequestering or displaying growth factors and by interacting with immune and glial cells. Here we quantified by quantitative polymerase chain reaction (qPCR) expression of 50 ECM components and 34 ECM degrading enzymes in multiple sclerosis (MS) active and inactive white matter lesions. COL1A1, COL3A1, COL5A1 and COL5A2 chains were induced strongly in active lesions and even more in inactive lesions. These chains interact to form collagen types I, III and V, which are fibrillar collagens. Biglycan and decorin, which can decorate fibrillar collagens, were also induced strongly. The fibrillar collagens, biglycan and decorin were largely found between the endothelium and astrocytic glia limitans in the perivascular space where they formed a meshwork which was closely associated with infiltrating immune cells. In active lesions collagen V was also seen in the heavily infiltrated parenchyma. Fibrillar collagens I and III inhibited in vitro human monocyte production of CCL2 (MCP‐1), an inflammatory chemokine involved in recruitment of immune cells. Together, ECM changes in lesions with different activities were quantified and proteins forming a perivascular fibrosis were identified. Induced fibrillar collagens may contribute to limiting enlargement of MS lesions by inhibiting the production of CCL2 by monocytes.

Compartmentalization of inflammation in the CNS: A major mechanism driving progressive multiple sclerosis ☆

In multiple sclerosis (MS) the CNS is not only the target of the pathological immune response, but the CNS itself becomes an immunological compartment during the course of the disease. This comprises (i) inflammation beyond classical white matter lesions, (ii) intrathecal Ig production with oligoclonal bands, (iii) an environment fostering immune cell persistence, (iv) follicle-like aggregates in the meninges, (v) a disruption of the blood-brain barrier also outside of active lesions, which allows influx of autoantibodies possibly promoting demyelination or axonal injury and influx of fibrinogen driving inflammation.

Pathophysiology of translational regulation by microRNAs in multiple sclerosis

This review describes miRNAs regulated in the blood or in brain lesions of MS patients in the context of their previously described functions in physiology and pathophysiology.

Expression of Herpes Simplex Virus 1-Encoded MicroRNAs in Human Trigeminal Ganglia and Their Relation to Local T-Cell Infiltrates

ABSTRACT Herpes simplex type 1 (HSV-1) is a neurotropic virus which establishes lifelong latency in human trigeminal ganglia (TG). Currently, two nonexclusive control mechanisms of HSV-1 latency are discussed: antiviral CD8+ T cells and viral microRNAs (miRNAs) encoded by the latency associated transcript (LAT). We investigate here to what extent these mechanisms may contribute to the maintenance of HSV-1 latency. We show that only a small proportion of LAT+ neurons is surrounded by T cells in human TG. This indicates that viral latency in human TG might be controlled by other mechanisms such as viral miRNAs. Therefore, we assessed TG sections for the presence of HSV-1 miRNA, DNA, and mRNA by combining LAT in situ hybridization, T-cell immunohistochemistry, and single cell analysis of laser-microdissected sensory neurons. Quantitative reverse transcription-PCR (RT-PCR) revealed that LAT+ neurons with or without surrounding T cells were always positive for HSV-1 miRNAs and DNA. Furthermore, ICP0 mRNA could rarely be detected only in LAT+ neurons, as analyzed by single-cell RT-PCR. In contrast, in LAT− neurons that were surrounded by T cells, neither miRNAs nor the DNA of HSV-1, HSV-2, or varicella-zoster virus could be detected. These data indicate that the majority of LAT+ neurons is not directly controlled by T cells. However, miRNA expression in every latently infected neuron would provide an additional checkpoint before viral replication is initiated.

MicroRNA regulation in experimental autoimmune encephalomyelitis in mice and marmosets resembles regulation in human multiple sclerosis lesions.

Here we demonstrate that miRNA regulation in marmoset (Callithrix jacchus) and C57/BL6 mouse EAE lesions largely resembles miRNA regulation in active human MS lesions. Detailed quantitative PCR analyses of the most up- and downregulated miRNAs of active human MS lesions in dissected lesions from marmoset EAE brains and inflamed spinal cords of EAE mice revealed that the conserved and highly regulated miRNAs, miRNA-155, miRNA-142-3p, miRNA-146a, miRNA-146b and miRNA-21, turned out to be similarly upregulated in marmoset and mouse EAE lesions.

Prospects of Transcript Profiling for mRNAs and MicroRNAs Using Formalin‐Fixed and Paraffin‐Embedded Dissected Autoptic Multiple Sclerosis Lesions

The elaboration of novel pathogenic aspects of multiple sclerosis (MS) requires the analysis of well‐defined stages of lesion development. However, specimens of certain stages and lesion types are either present in small brain biopsies, insufficient in size for further molecular studies or available as formalin‐fixed and paraffin‐embedded (FFPE) material only. Therefore, application of current molecular biology techniques to FFPE tissue is warranted. We compared FFPE and frozen tissue by using quantitative polymerase chain reaction and report: (1) FFPE material is highly heterogeneous regarding the utility for transcript profiling of mRNAs; well‐preserved FFPE samples had about a 100‐fold reduced sensitivity compared with frozen tissue, but gave similar results for genes of sufficient abundance; (2) FFPE samples not suitable for mRNA analysis are still highly valuable for miRNA quantification; (3) the length of tissue fixation greatly affects utility for mRNA but not for miRNA analysis; (4) FFPE samples can be processed via a hot water bath for dissection of defined lesion areas; and (5) in situ hybridization for proteolipid protein (PLP) helps to identify samples not suitable for mRNA amplification. In summary, we present a detailed protocol how to use autoptic FFPE tissue for transcript profiling in dissected tissue areas.

CTLA4 as Immunological Checkpoint in the Development of Multiple Sclerosis.

We investigated a patient who developed multiple sclerosis (MS) during treatment with the CTLA4‐blocking antibody ipilimumab for metastatic melanoma. Initially he showed subclinical magnetic resonance imaging (MRI) changes (radiologically isolated syndrome). Two courses of ipilimumab were each followed by a clinical episode of MS, 1 of which was accompanied by a massive increase of MRI activity. Brain biopsy confirmed active, T‐cell type MS. Quantitative next generation sequencing of T‐cell receptor genes revealed distinct oligoclonal CD4+ and CD8+ T‐cell repertoires in the primary melanoma and cerebrospinal fluid. Our results pinpoint the coinhibitory molecule CTLA4 as an immunological checkpoint and therapeutic target in MS. Ann Neurol 2016;80:294–300

Transcript profiling of different types of multiple sclerosis lesions yields FGF1 as a promoter of remyelination

Chronic demyelination is a pathological hallmark of multiple sclerosis (MS). Only a minority of MS lesions remyelinates completely. Enhancing remyelination is, therefore, a major aim of future MS therapies. Here we took a novel approach to identify factors that may inhibit or support endogenous remyelination in MS. We dissected remyelinated, demyelinated active, and demyelinated inactive white matter MS lesions, and compared transcript levels of myelination and inflammation-related genes using quantitative PCR on customized TaqMan Low Density Arrays. In remyelinated lesions, fibroblast growth factor (FGF) 1 was the most abundant of all analyzed myelination-regulating factors, showed a trend towards higher expression as compared to demyelinated lesions and was significantly higher than in control white matter. Two MS tissue blocks comprised lesions with adjacent de- and remyelinated areas and FGF1 expression was higher in the remyelinated rim compared to the demyelinated lesion core. In functional experiments, FGF1 accelerated developmental myelination in dissociated mixed cultures and promoted remyelination in slice cultures, whereas it decelerated differentiation of purified primary oligodendrocytes, suggesting that promotion of remyelination by FGF1 is based on an indirect mechanism. The analysis of human astrocyte responses to FGF1 by genome wide expression profiling showed that FGF1 induced the expression of the chemokine CXCL8 and leukemia inhibitory factor, two factors implicated in recruitment of oligodendrocytes and promotion of remyelination. Together, this study presents a transcript profiling of remyelinated MS lesions and identified FGF1 as a promoter of remyelination. Modulation of FGF family members might improve myelin repair in MS.