Inflammation-induced TRPV4 channels exacerbate blood-brain barrier dysfunction in multiple sclerosis.

BACKGROUND: Blood-brain barrier (BBB) dysfunction and immune cell migration into the central nervous system (CNS) are pathogenic drivers of multiple sclerosis (MS). Ways to reinstate BBB function and subsequently limit neuroinflammation present promising strategies to restrict disease progression. However, to date, the molecular players directing BBB impairment in MS remain poorly understood. One suggested candidate to impact BBB function is the transient receptor potential vanilloid-type 4 ion channel (TRPV4), but its specific role in MS pathogenesis remains unclear. Here, we investigated the role of TRPV4 in BBB dysfunction in MS. MAIN TEXT: In human post-mortem MS brain tissue, we observed a region-specific increase in endothelial TRPV4 expression around mixed active/inactive lesions, which coincided with perivascular microglia enrichment in the same area. Using in vitro models, we identified that microglia-derived tumor necrosis factor-α (TNFα) induced brain endothelial TRPV4 expression. Also, we found that TRPV4 levels influenced brain endothelial barrier formation via expression of the brain endothelial tight junction molecule claudin-5. In contrast, during an inflammatory insult, TRPV4 promoted a pathological endothelial molecular signature, as evidenced by enhanced expression of inflammatory mediators and cell adhesion molecules. Moreover, TRPV4 activity mediated T cell extravasation across the brain endothelium. CONCLUSION: Collectively, our findings suggest a novel role for endothelial TRPV4 in MS, in which enhanced expression contributes to MS pathogenesis by driving BBB dysfunction and immune cell migration.

Transfer of Cellular Content from the Allogeneic Cell-Based Cancer Vaccine DCP-001 to Host Dendritic Cells Hinges on Phosphatidylserine and Is Enhanced by CD47 Blockade.

DCP-001 is a cell-based cancer vaccine generated by differentiation and maturation of cells from the human DCOne myeloid leukemic cell line. This results in a vaccine comprising a broad array of endogenous tumor antigens combined with a mature dendritic cell (mDC) costimulatory profile, functioning as a local inflammatory adjuvant when injected into an allogeneic recipient. Intradermal DCP-001 vaccination has been shown to be safe and feasible as a post-remission therapy in acute myeloid leukemia. In the current study, the mode of action of DCP-001 was further characterized by static and dynamic analysis of the interaction between labelled DCP-001 and host antigen-presenting cells (APCs). Direct cell-cell interactions and uptake of DCP-001 cellular content by APCs were shown to depend on DCP-001 cell surface expression of calreticulin and phosphatidylserine, while blockade of CD47 enhanced the process. Injection of DCP-001 in an ex vivo human skin model led to its uptake by activated skin-emigrating DCs. These data suggest that, following intradermal DCP-001 vaccination, local and recruited host APCs capture tumor-associated antigens from the vaccine, become activated and migrate to the draining lymph nodes to subsequently (re)activate tumor-reactive T-cells. The improved uptake of DCP-001 by blocking CD47 rationalizes the possible combination of DCP-001 vaccination with CD47 blocking therapies.

Inflammation of the choroid plexus in progressive multiple sclerosis: accumulation of granulocytes and T cells.

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+ antigen-presenting 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.

Setmelanotide, a Novel, Selective Melanocortin Receptor-4 Agonist Exerts Anti-inflammatory Actions in Astrocytes and Promotes an Anti-inflammatory Macrophage Phenotype.

To date, available treatment strategies for multiple sclerosis (MS) are ineffective in preventing or reversing progressive neurologic deterioration, creating a high, and unmet medical need. One potential way to fight MS may be by limiting the detrimental effects of reactive astrocytes, a key pathological hallmark for disease progression. One class of compounds that may exert beneficial effects via astrocytes are melanocortin receptor (MCR) agonists. Among the MCR, MC4R is most abundantly expressed in the CNS and several rodent studies have described that MC4R is-besides neurons-expressed by astrocytes. Activation of MC4R in astrocytes has shown to have potent anti-inflammatory as well as neuroprotective effects in vitro, suggesting that this could be a potential target to ameliorate ongoing inflammation, and neurodegeneration in MS. In this study, we set out to investigate human MC4R expression and analyze its downstream effects. We identified MC4R mRNA and protein to be expressed on astrocytes and observed increased astrocytic MC4R expression in active MS lesions. Furthermore, we show that the novel, highly selective MC4R agonist setmelanotide ameliorates the reactive phenotype in astrocytes in vitro and markedly induced interleukin-6 and -11 production, possibly through enhanced cAMP response element-binding protein (CREB) phosphorylation. Notably, stimulation of human macrophages with medium from astrocytes that were exposed to setmelanotide, skewed macrophages toward an anti-inflammatory phenotype. Taken together, these findings suggest that targeting MC4R on astrocytes might be a novel therapeutic strategy to halt inflammation-associated neurodegeneration in MS.

Reduced Angiopoietin-Like 4 Expression in Multiple Sclerosis Lesions Facilitates Lipid Uptake by Phagocytes via Modulation of Lipoprotein-Lipase Activity.

Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS) characterized by the presence of focal demyelinated plaques. Sufficient clearance of myelin and cellular debris is one of the requirements for proper tissue repair and remyelination. The mechanisms underlying the clearance of such debris by phagocytes are not fully understood, but recent findings suggest a prominent role for lipoprotein-lipase (LPL) in this process. Here, we demonstrate that angiopoietin-like 4 (ANGPTL4), a potent inhibitor of LPL, is abundantly expressed in astrocytes in control white matter tissue and its expression is markedly reduced in active MS lesions. We provide evidence that ANGPTL4 inhibits the uptake of myelin-derived lipids by LPL-immunoreactive phagocytes. Taken together, our data suggest that the strong reduction in astrocytic ANGPTL4 expression in active demyelinating MS lesions enables phagocytes to adequately clear myelin debris, setting the stage for remyelination.

In Vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior.

Most 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.

TRPM7 maintains progenitor-like features of neuroblastoma cells: implications for metastasis formation.

Neuroblastoma is an embryonal tumor derived from poorly differentiated neural crest cells. Current research is aimed at identifying the molecular mechanisms that maintain the progenitor state of neuroblastoma cells and to develop novel therapeutic strategies that induce neuroblastoma cell differentiation. Mechanisms controlling neural crest development are typically dysregulated during neuroblastoma progression, and provide an appealing starting point for drug target discovery. Transcriptional programs involved in neural crest development act as a context dependent gene regulatory network. In addition to BMP, Wnt and Notch signaling, activation of developmental gene expression programs depends on the physical characteristics of the tissue microenvironment. TRPM7, a mechanically regulated TRP channel with kinase activity, was previously found essential for embryogenesis and the maintenance of undifferentiated neural crest progenitors. Hence, we hypothesized that TRPM7 may preserve progenitor-like, metastatic features of neuroblastoma cells. Using multiple neuroblastoma cell models, we demonstrate that TRPM7 expression closely associates with the migratory and metastatic properties of neuroblastoma cells in vitro and in vivo. Moreover, microarray-based expression profiling on control and TRPM7 shRNA transduced neuroblastoma cells indicates that TRPM7 controls a developmental transcriptional program involving the transcription factor SNAI2. Overall, our data indicate that TRPM7 contributes to neuroblastoma progression by maintaining progenitor-like features.

TRPM7 triggers Ca2+ sparks and invadosome formation in neuroblastoma cells.

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.