SummaryMost cancer cells release heterogeneous populations of extracellular vesicles (EVs) containing proteins, lipids, and nucleic acids. In vitro experiments showed that EV uptake can lead to transfer of functional mRNA and altered cellular behavior. However, similar in vivo experiments remain challenging because cells that take up EVs cannot be discriminated from non-EV-receiving cells. Here, we used the Cre-LoxP system to directly identify tumor cells that take up EVs in vivo. We show that EVs released by malignant tumor cells are taken up by less malignant tumor cells located within the same and within distant tumors and that these EVs carry mRNAs involved in migration and metastasis. By intravital imaging, we show that the less malignant tumor cells that take up EVs display enhanced migratory behavior and metastatic capacity. We postulate that tumor cells locally and systemically share molecules carried by EVs in vivo and that this affects cellular behavior.

Each year about 650,000 Europeans die from stroke and a similar number lives with the sequelae of multiple sclerosis (MS). Stroke and MS differ in their etiology. Although cause and likewise clinical presentation set the two diseases apart, they share common downstream mechanisms that lead to damage and recovery. Demyelination and axonal injury are characteristics of MS but are also observed in stroke. Conversely, hallmarks of stroke, such as vascular impairment and neurodegeneration, are found in MS. However, the most conspicuous common feature is the marked neuroinflammatory response, marked by glia cell activation and immune cell influx. In MS and stroke the blood-brain barrier is disrupted allowing bone marrow-derived macrophages to invade the brain in support of the resident microglia. In addition, there is a massive invasion of auto-reactive T-cells into the brain of patients with MS. Though less pronounced a similar phenomenon is also found in ischemic lesions. Not surprisingly, the two diseases also resemble each other at the level of gene expression and the biosynthesis of other proinflammatory mediators. While MS has traditionally been considered to be an autoimmune neuroinflammatory disorder, the role of inflammation for cerebral ischemia has only been recognized later. In the case of MS the long track record as neuroinflammatory disease has paid off with respect to treatment options. There are now about a dozen of approved drugs for the treatment of MS that specifically target neuroinflammation by modulating the immune system. Interestingly, experimental work demonstrated that drugs that are in routine use to mitigate neuroinflammation in MS may also work in stroke models. Examples include Fingolimod, glatiramer acetate, and antibodies blocking the leukocyte integrin VLA-4. Moreover, therapeutic strategies that were discovered in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, turned out to be also effective in experimental stroke models. This suggests that previous achievements in MS research may be relevant for stroke. Interestingly, the converse is equally true. Concepts on the neurovascular unit that were developed in a stroke context turned out to be applicable to neuroinflammatory research in MS. Examples include work on the important role of the vascular basement membrane and the BBB for the invasion of immune cells into the brain. Furthermore, tissue plasminogen activator (tPA), the only established drug treatment in acute stroke, modulates the pathogenesis of MS. Endogenous tPA is released from endothelium and astroglia and acts on the BBB, microglia and other neuroinflammatory cells. Thus, the vascular perspective of stroke research provides important input into the mechanisms on how endothelial cells and the BBB regulate inflammation in MS, particularly the invasion of immune cells into the CNS. In the current review we will first discuss pathogenesis of both diseases and current treatment regimens and will provide a detailed overview on pathways of immune cell migr...

The choroid plexus (CP) is strategically located between the peripheral blood and the cerebrospinal fluid, and is involved in the regulation of central nervous system (CNS) homeostasis. In multiple sclerosis (MS), demyelination and inflammation occur in the CNS. While experimental animal models of MS pointed to the CP as a key route for immune cell invasion of the CNS, little is known about the distribution of immune cells in the human CP during progressive phases of MS. Here, we use immunohistochemistry and confocal microscopy to explore the main immune cell populations in the CP of progressive MS patients and non-neuroinflammatory controls, in terms of abundance and location within the distinct CP compartments. We show for the first time that the CP stromal density of granulocytes and CD8+ T cells is higher in progressive MS patients compared to controls. In line with previous studies, the CP of both controls and progressive MS patients contains relatively high numbers of macrophages and dendritic cells. Moreover, we found virtually no B cells or plasma cells in the CP. MHCII+ antigenpresenting cells were often found in close proximity to T cells, suggesting constitutive CNS immune monitoring functions of the CP. Together, our data highlights the role of the CP in immune homeostasis and indicates the occurrence of mild inflammatory processes in the CP of progressive MS patients. However, our findings suggest that the CP is only marginally involved in immune cell migration into the CNS in chronic MS.

Impairment of the protective properties of the blood-brain barrier (BBB) is a key event during numerous neurological diseases, including multiple sclerosis (MS). Under these pathological conditions, the specialized brain endothelial cells (BECs) lose their protective function leading to neuroinflammation and neurodegeneration. To date, underlying mechanisms for this loss of function remain unclear. Endothelial to mesenchymal transition (EndoMT) is a dynamic process by which endothelial cells (ECs) dedifferentiate into mesenchymal cells and as a result lose their specific phenotype and function. As yet, little is known about the involvement of this process in the impaired function of the BECs under pathological conditions such as MS. Interestingly, several signaling pathways that can induce EndoMT are also involved in different central nervous system (CNS) pathologies associated with BBB dysfunction. In this review, we first discuss the structure and function of the BBB highlighting the changes that occur during MS. Next, we will summarize recent findings on the pathways underlying EndoMT, and finally, we will discuss the potential role of EndoMT during BBB dysfunction in neurological disorders. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.

Cell migration depends on the dynamic formation and turnover of cell adhesions and is tightly controlled by actomyosin contractility and local Ca2+ signals. The divalent cation channel TRPM7 (Transient Receptor Potential cation channel, subfamily Melastatin, member 7) has recently received much attention as a regulator of cell adhesion, migration and (localized) Ca2+ signaling. Overexpression and knockdown of TRPM7 affects actomyosin contractility and the formation of cell adhesions such as invadosomes and focal adhesions, but the role of TRPM7-mediated Ca2+ signals herein is currently not understood. Using Total Internal Reflection Fluorescence (TIRF) Ca2+ fluorometry and a novel automated analysis routine we have addressed the role of Ca2+ in the control of invadosome dynamics in N1E-115 mouse neuroblastoma cells. We find that TRPM7 promotes the formation of highly repetitive and localized Ca2+ microdomains or “Ca2+ sparking hotspots” at the ventral plasma membrane. Ca2+ sparking appears strictly dependent on extracellular Ca2+ and is abolished by TRPM7 channel inhibitors such as waixenicin-A. TRPM7 inhibition also induces invadosome dissolution. However, invadosome formation is (functionally and spatially) dissociated from TRPM7-mediated Ca2+ sparks. Rather, our data indicate that TRPM7 affects actomyosin contractility and invadosome formation independent of Ca2+ influx.

Trafficking of myelin-reactive CD4+ T-cells across the brain endothelium, an essential step in the pathogenesis of multiple sclerosis (MS), is suggested to be an antigen-specific process, yet which cells provide this signal is unknown. Here we provide direct evidence that under inflammatory conditions, brain endothelial cells (BECs) stimulate the migration of myelin-reactive CD4+ T-cells by acting as non-professional antigen presenting cells through the processing and presentation of myelin-derived antigens in MHC-II. Inflamed BECs internalized myelin, which was routed to endo-lysosomal compartment for processing in a time-dependent manner. Moreover, myelin/MHC-II complexes on inflamed BECs stimulated the trans-endothelial migration of myelin-reactive Th1 and Th17 2D2 cells, while control antigen loaded BECs did not stimulate T-cell migration. Furthermore, blocking the interaction between myelin/MHC-II complexes and myelin-reactive T-cells prevented T-cell transmigration. These results demonstrate that endothelial cells derived from the brain are capable of enhancing antigen-specific T cell recruitment.DOI: http://dx.doi.org/10.7554/eLife.13149.001

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Meningeal inflammation strongly associates with demyelination and neuronal loss in the underlying cortex of progressive MS patients, thereby contributing significantly to clinical disability. However, the pathological mechanisms of meningeal inflammation-induced cortical pathology are still largely elusive. By extensive analysis of cortical microglia in post-mortem progressive MS tissue, we identified cortical areas with two MS-specific microglial populations, termed MS1 and MS2 cortex. The microglial population in MS1 cortex was characterized by a higher density and increased expression of the activation markers HLA class II and CD68, whereas microglia in MS2 cortex showed increased morphological complexity and loss of P2Y12 and TMEM119 expression. Interestingly, both populations associated with inflammation of the overlying meninges and were time-dependently replicated in an in vivo rat model for progressive MS-like chronic meningeal inflammation. In this recently developed animal model, cortical microglia at 1-month post-induction of experimental meningeal inflammation resembled microglia in MS1 cortex, and microglia at 2 months post-induction acquired a MS2-like phenotype. Furthermore, we observed that MS1 microglia in both MS cortex and the animal model were found closely apposing neuronal cell bodies and to mediate pre-synaptic displacement and phagocytosis, which coincided with a relative sparing of neurons. In contrast, microglia in MS2 cortex were not involved in these synaptic alterations, but instead associated with substantial neuronal loss. Taken together, our results show that in response to meningeal inflammation, microglia acquire two distinct phenotypes that differentially associate with neurodegeneration in the progressive MS cortex. Furthermore, our in vivo data suggests that microglia initially protect neurons from meningeal inflammation-induced cell death by removing pre-synapses from the neuronal soma, but eventually lose these protective properties contributing to neuronal loss.