Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS

Periodic Report Summary 1 - NEUROKINE (Initial Training Network for Neurogical disorders orchestrated by cytoKines)

Neuroinflammatory disease, in particular multiple sclerosis (MS) affects more than 700,000 people within Europe alone. European countries are at the forefront of neuroimmunological research and students in medicine and biology are greatly attracted by the field. Neuroimmunology research further impacts our understanding of the ethiopathology of other CNS-disorders including Alzheimer’s Dementia and Morbus Parkinson.
The ability of inflammatory immune cells to cause tissue damage within the nervous system is largely governed by soluble mediators including cytokines/chemokines, cytolytic molecules and growth factors. The therapeutic targeting of such mediators has proven successful for the treatment of a number of inflammatory diseases, yet is failing for the treatment of neuroinflammatory disorders. Clearly, the rules and regulatory elements, which govern inflammation and tissue injury within the CNS, differ significantly from those of other tissues and a unique CNS-focused research approach to unravel these rules is required.

In this ITN consortium, we train ER/ESRs specifically in Neuroimmunology by combining prominent laboratories in academia and industry on the task to study the communication networks between immune and CNS-resident cells. Scientifically, we focus particularly on the soluble factors mediating cell-cell communication at the immune-CNS interface. We combine expertise in molecular and cellular Neuroimmunology with Neuropathology of human and animal models of CNS inflammation. In addition, our goal is to further utilize inflammatory processes for neuroprotection.

Work performed and results:

In the first period of NeuroKine we established the consortium, initiated new collaborations between the labs and recruited the students and postdocs that are trained by the NeuroKine labs. With very few exceptions, the students and postdocs were recruited and have started their training. We have already held several NeuroKine workshops that were a great success and highly appreciated by the trainees (see below). All full members of the ITN have started their scientific tasks, as summarized below for each WP:

WP 1 Cytokines during CNS-inflammation and degeneration
Using a mouse strain that allows for the conditional deletion of the IL-23 receptor in specific cells types, we aim to understand the role that IL-23R signaling may play in distinct cellular population, which may impact on neuroinflammation. By now, we have generated mice, which lack the Il23r gene in all T cells, innate lymphoid cells, in myeloid cells, and in mature T cells in an inducible manner. In addition, we are generating reporter mice for IL-23 using the CRISPR/CAS method. Furthermore, we have cloned the IL-23p19 cDNA and have cloned a targeting vector to insert it in the ROSA26 locus, to allow for the tissue-specific expression of this cytokine. To follow IL-23R expressing cells in vivo, we have procured the IL-23R-eGFP mice and are in the process of analyzing the expression of this receptor in the development of EAE and AD.

WP2 The impact of inflammation on neural stem cells and tissue repair
Using the Nestin-floxGFPflox-TK-IRES-LacZ transgenic mouse line, we found that endogenous NPCs play a protective role in acute but not in chronic inflammatory disorders of the CNS. As a matter of fact, while ablating endogenous NPCs, EAE remains the same while ischemic middle cerebral artery occlusion (MCAO) stroke worsens significantly. At the same time, they observed that the ablation of NPCs in the Nestin-floxGFPflox-TK-IRES-LacZ mouse line - that can only be achieved before the induction of the injury model - interferes with the induction of EAE because of GCV toxicity in this particular mouse model. Thus, we generated a Nestin-CRE-ERT2-YFP mice to further studying the neurogenic response in acute inflammatory CNS injury.

WP 3 T cell-mediated inflammation of the CNS
 Using a transgenic mouse, called CamK-HA, in which the hemagglutinin (HA) of the influenza virus is expressed by the large majority of neurons of the CNS we could establish a mouse model in which autoreactive T cells, specific for HA, are able to target neurons leading to CNS inflammation. FACS analysis and histology of the CNS of these mice showed infiltration of T cells and an activation of microglia compared to control mice. Among the infiltrating T cells over 80% were CD8 positive, suggesting that these cells are the main effector in the disease course. With the help of this mouse model we will compare CNS infiltrating CD8+ T cells vs. CD8+ T cells that reside in the peripheral organs (spleen and lymph nodes) and in the tumor by RNA sequencing and flow cytometry analysis. In addition, in this model we will test different approaches for already established therapeutic strategies to block pathogenic T cells to migrate into the CNS without affecting the immune response against the tumor. We already tested FTY 720, known as Fingolimod that is a treatment for patients with multiple sclerosis. It prevents the egress of lymphocytes from the lymphatic tissue. This treatment has no curative therapeutic effect in our model. Next, we will focus on the anti-α4 integrin mAb (PS/2) that blocks the migration of T cells by binding to the integrin α4 that is necessary to cross the blood brain barrier. This treatment is also know as Natalizumab and is currently one of the most efficient treatments against MS.

WP4 Mechanisms of tissue injury in inflammatory demyelinating diseases in humans and non-human primates. Many different cytokine-controlled immunological activities appear to be involved in the induction of demyelination and tissue injury in human inflammatory demyelinating diseases. They include direct T-cell mediated cytotoxicity, injury by specific autoantibody-mediated cytopathic mechanisms and mechanisms of innate immunity. However, cellular sources and target cell subsets of pro- and anti-inflammatory cytokines in MS remain incompletely defined, as are the mechanisms by which EBV, the prime suspect virus in triggering the pathogenesis of MS, may promote disease. Within WP 4 a large repository of human autopsy and biopsy tissue of well-characterized lesions of inflammatory demyelinating disease (e.g. MS, ADEM, Balo’s disease or NMO) and of various normal and disease controls are available. We have extensively used this material during the last years to define key pathways of lesion pathogenesis in MS and neuromyelitis optica and also have used an unbiased approach using genome wide microarrays for analysis of gene expression in carefully microdissected MS lesion areas. In parallel to the human tissue collection, WP4 employs a well-characterized non-human primate preclinical EAE model. The common marmoset (Callithrix jacchus) is a small-sized outbred non-endangered Neotropical primate that provides several relevant models of the human neuroinflammatory disease MS, dependent on the antigen- adjuvant combination used both white matter and grey matter lesions resemble human MS pathology. In a model induced with recombinant human MOG in CFA, a therapeutic antibody against the p40 subunit shared by IL-12 and IL-23 limits EAE development, suggesting an important pathogenic role of Th1 and Th17 cells in this model. In addition, depletion of B-cells by an anti-CD20 antibody effectively prevented EAE, strongly reducing CNS inflammation and demyelination.

WP 5 Unbiased examination of the transcriptome of patient biopsy material through NextGeneration sequencing. Genome wide microarray studies have been performed in microdissected white and grey matter lesions comparing different activity stages of the lesions and comparing the results with those obtained in human inflammatory, neurodegenerative and normal controls. These studies provided compelling data on the importance of oxidative injury and mitochondrial damage in active MS lesions, which were then validated by immunocytochemistry for key markers in a large set of MS and control tissue. The mircoarray data have been made public in respective international data bases (NCBI’s Gene Expression Omnibus; GEO accession numbers GSE32915 and GSE32645). The analysis of the data regarding oxidative and mitochondrial injury have been performed prior to the start of the NeuroKine project, due to delay between the application and actual funding and have been summarized in a recent review article, which provides a comprehensive account on the mechanisms of tissue injury in the transition between early relapsing and progressive MS. Within the NeuroKine project we then focused on mechanisms of inflammation in MS lesions in comparison to inflammatory and neurodegenerative human controls. Based on the available microarray data we focused on the phenotype, activation status and functional polarization of T- and B-lymphocytes within the lesions. In comparison to rapidly evolving inflammatory lesions seen in acute disseminated leukoencephalomyelitis, neuromyelitis optica and virus induced brain diseases we found a moderate proliferation rate of T-cells and B-cells in MS lesions and this was even the case in lesions of progressive MS. CD8 positive T-cells showed the most profound proliferation rate and activation status in MS, and this was different from NMO, in which a higher contribution of CD4+ T-cells was seen. We have then established the technology to study T- and B-cell polarization through the expression of functionally relevant transcription factors and the respective data are currently evaluated by quantitative analysis of MS and control lesions at different activity stages. Our studies so far indicate a unique signature of lymphocyte activation and polarization in MS in comparison to other human inflammatory brain diseases, which may in part explain differences in the response to anti-inflammatory treatment between these conditions.

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