[show abstract][hide abstract] ABSTRACT: The trafficking of cytotoxic CD8(+) T lymphocytes across the lining of the cerebral vasculature is key to the onset of the chronic neuro-inflammatory disorder multiple sclerosis. However, the mechanisms controlling their final transmigration across the brain endothelium remain unknown. Here, we describe that CD8(+) T lymphocyte trafficking into the brain is dependent on the activity of the brain endothelial adenosine triphosphate-binding cassette transporter P-glycoprotein. Silencing P-glycoprotein activity selectively reduced the trafficking of CD8(+) T cells across the brain endothelium in vitro as well as in vivo. In response to formation of the T cell-endothelial synapse, P-glycoprotein was found to regulate secretion of endothelial (C-C motif) ligand 2 (CCL2), a chemokine that mediates CD8(+) T cell migration in vitro. Notably, CCL2 levels were significantly enhanced in microvessels isolated from human multiple sclerosis lesions in comparison with non-neurological controls. Endothelial cell-specific elimination of CCL2 in mice subjected to experimental autoimmune encephalomyelitis also significantly diminished the accumulation of CD8(+) T cells compared to wild-type animals. Collectively, these results highlight a novel (patho)physiological role for P-glycoprotein in CD8(+) T cell trafficking into the central nervous system during neuro-inflammation and illustrate CCL2 secretion as a potential link in this mechanism.
[show abstract][hide abstract] ABSTRACT: The purpose of this study was the generation of central nervous system (CNS)-excluded cannabinoid receptor agonists to test the hypothesis that inhibition of spasticity, due to CNS autoimmunity, could be controlled by affecting neurotransmission within the periphery. Procedures included identification of chemicals and modeling to predict the mode of exclusion; induction and control of spasticity in the ABH mouse model of multiple sclerosis; conditional deletion of CB1 receptor in peripheral nerves; side-effect profiling to demonstrate the mechanism of CNS-exclusion via drug pumps; genome-wide association study in N2(129×ABH) backcross to map polymorphic cannabinoid drug pump; and sequencing and detection of cannabinoid drug-pump activity in human brain endothelial cell lines. Three drugs (CT3, SAB378 and SAD448) were identified that control spasticity via action on the peripheral nerve CB1 receptor. These were peripherally restricted via drug pumps that limit the CNS side effects (hypothermia) of cannabinoids to increase the therapeutic window. A cannabinoid drug pump is polymorphic and functionally lacking in many laboratory (C57BL/6, 129, CD-1) mice used for transgenesis, pharmacology, and toxicology studies. This phenotype was mapped and controlled by 1-3 genetic loci. ABCC1 within a cluster showing linkage is a cannabinoid CNS-drug pump. Global and conditional CB1 receptor-knockout mice were used as controls. In summary, CNS-excluded CB1 receptor agonists are a novel class of therapeutic agent for spasticity.-Pryce, G., Visintin, C., Ramagopalan, S. V., Al-Izki, S., De Faveri, L. E., Nuamah, R. A., Mein, C. A., Montpetit, A., Hardcastle, A. J., Kooij, G., de Vries, H. E., Amor, S., Thomas, S. A., Ledent, C., Marsicano, G., Lutz, B., Thompson, A. J., Selwood, D. L., Giovannoni, G., Baker, D. Control of spasticity in a multiple sclerosis model using central nervous system-excluded CB1 cannabinoid receptor agonists.
The FASEB Journal 01/2014; 28(1):117-130. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: Multiple sclerosis (MS) is a chronic neuro-inflammatory disorder, which is marked by the invasion of the central nervous system by monocyte-derived macrophages and autoreactive T cells across the brain vasculature. Data from experimental animal models recently implied that the passage of leukocytes across the brain vasculature is preceded by their traversal across the blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus. The correlation between the presence of leukocytes in the CSF of patients suffering from MS and the number of inflammatory lesions as detected by magnetic resonance imaging suggests that inflammation at the choroid plexus contributes to the disease, although in a yet unknown fashion. We here provide first insights into the involvement of the choroid plexus in the onset and severity of the disease and in particular address the role of the tight junction protein claudin-3 (CLDN3) in this process. Detailed analysis of human post-mortem brain tissue revealed a selective loss of CLDN3 at the choroid plexus in MS patients compared to control tissues. Importantly, mice that lack CLDN3 have an impaired BCSFB and experience a more rapid onset and exacerbated clinical signs of experimental autoimmune encephalomyelitis, which coincides with enhanced levels of infiltrated leukocytes in their CSF. Together, this study highlights a profound role for the choroid plexus in the pathogenesis of multiple sclerosis, and implies that CLDN3 may be regarded as a crucial and novel determinant of BCSFB integrity.
[show abstract][hide abstract] ABSTRACT: Alzheimer's disease (AD) is the most common form of dementia and marked by deposition of amyloid-β (Aβ) within the brain. Alterations of Aβ transporters at the neurovasculature may play a role in the disease process. We investigated the expression of ABC transporters P-glycoprotein (P-gp) and breast cancer related protein (BCRP) in non-neurologic controls, AD, and severe capillary cerebral amyloid angiopathy (capCAA) cases, which are characterized by deposition of Aβ within cerebral capillaries. Our data show that microvascular expression of P-gp and BCRP is strikingly decreased in capCAA-affected vessels but not in AD and control samples. Messenger RNA levels of P-gp, but not of BCRP, were downregulated in brain endothelial cells on exposure to oligomeric Aβ42, but not fibrillar Aβ42 or Aβ40. Coincubating Aβ42 together with clusterin, an amyloid-associated protein highly expressed in capCAA-affected vessels, strongly reduced levels of P-gp. In conclusion, accumulation of Aβ, in combination with clusterin, within and around cerebral capillaries, may further aggravate the disease process in AD by affecting P-gp expression. Loss of P-gp expression or activity may serve as a selective biomarker for ongoing capCAA.
Neurobiology of aging 10/2013; · 5.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: Blood-brain barrier (BBB) dysfunction is a major hallmark of many neurological diseases, including multiple sclerosis (MS). Using a genomics approach, we defined a microRNA signature that is diminished at the BBB of MS patients. In particular, miR-125a-5p is a key regulator of brain endothelial tightness and immune cell efflux. Our findings suggest that repair of a disturbed BBB through microRNAs may represent a novel avenue for effective treatment of MS.
Journal of Neuroscience 04/2013; 33(16):6857-6863. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) show great therapeutic potential for the treatment of various immune mediated diseases, including Multiple Sclerosis (MS). Systemic administration of MSCs during experimental allergic encephalomyelitis (EAE), an animal model for MS, was shown to reduce the infiltration of T cells, B cells, and macrophages into the CNS. Whether endogenous MSCs are mobilized and potentially modulate the severity of disease is not known. Here we show that during the acute phase of EAE, MSCs numbers in the bone marrow were severely reduced, which restored to control levels during the progressive phase of the disease. The number of bone marrow MSCs inversely correlated with the number of both CD4 and CD8 T cells present in the bone marrow indicating a link between activated T cells and MSC mobilization. Analysis of CD70-transgenic mice, which have a constitutively activated immune system and elevated number of activated T cells in the bone marrow, showed severely reduced number of bone marrow MSCs. Transfer of T cells that were activated through their CD27 receptor reduced the number of bone marrow MSCs dependent on IFN-y. These data provide a mechanism by which MSCs can be mobilized from the bone marrow in order to contribute to tissue repair at a distant location.
[show abstract][hide abstract] ABSTRACT: Proper function of the neurovasculature is required for optimal brain function and preventing neuroinflammation and neurodegeneration. Within this review, we discuss alterations of the function of the blood–brain barrier in neurologic disorders such as multiple sclerosis, epilepsy, and Alzheimer’s disease and address potential underlying mechanisms.
[show abstract][hide abstract] ABSTRACT: Alterations in sphingolipid metabolism are described to contribute to various neurological disorders. We here determined the expression of enzymes involved in the sphingomyelin cycle and their products in postmortem brain tissue of multiple sclerosis (MS) patients. In parallel, we investigated the effect of the sphingosine-1 receptor agonist Fingolimod (Gilenya(®)) on sphingomyelin metabolism in reactive astrocytes and determined its functional consequences for the process of neuro-inflammation. Our results demonstrate that in active MS lesions, marked by large number of infiltrated immune cells, an altered expression of enzymes involved in the sphingomyelin cycle favors enhanced ceramide production. We identified reactive astrocytes as the primary cellular source of enhanced ceramide production in MS brain samples. Astrocytes isolated from MS lesions expressed enhanced mRNA levels of the ceramide-producing enzyme acid sphingomyelinase (ASM) compared to astrocytes isolated from control white matter. In addition, TNF-α treatment induced ASM mRNA and ceramide levels in astrocytes isolated from control white matter. Incubation of astrocytes with Fingolimod prior to TNF-α treatment reduced ceramide production and mRNA expression of ASM to control levels in astrocytes. Importantly, supernatants derived from reactive astrocytes treated with Fingolimod significantly reduced transendothelial monocyte migration. Overall, the present study demonstrates that reactive astrocytes represent a possible additional cellular target for Fingolimod in MS by directly reducing the production of pro-inflammatory lipids and limiting subsequent transendothelial leukocyte migration.
[show abstract][hide abstract] ABSTRACT: Neuroinflammation contributes to a wide range of disorders of the central nervous system (CNS). Of the available anti-inflammatory drugs, only glucocorticoids have shown central efficacy in CNS-related disorders, such as multiple sclerosis (MS). However, their side effects are dose limiting. To optimally improve the therapeutic window of methylprednisolone, we enhanced its CNS delivery by using pegylated liposomes conjugated to the brain-targeting ligand glutathione. In healthy rats, plasma circulation and brain uptake were significantly increased after encapsulating methylprednisolone in glutathione pegylated (GSH-PEG) liposomes. Furthermore, the efficacy of GSH-PEG liposomal methylprednisolone was investigated in rats with acute experimental autoimmune encephalomyelitis (EAE), an animal model of MS; rats received treatment (10mg/kg; i.v. injection), before disease onset, at disease onset, or at the peak of disease. Free methylprednisolone and non-targeted pegylated (PEG) liposomal methylprednisolone served as control treatments. When treatment was initiated at disease onset, free methylprednisolone showed no effect, while GSH-PEG liposomal methylprednisolone significantly reduced the clinical signs to 42±6.4% of saline control. Moreover, treatment using GSH-PEG liposomes was significantly more effective compared to PEG liposomes. Our findings hold promise for MS treatment and warrant further investigations into this brain delivery system for the treatment of neuroinflammation.
Journal of Controlled Release 06/2012; · 7.63 Impact Factor
[show abstract][hide abstract] ABSTRACT: The sphingosine 1-phosphate (S1P) receptor modulator FTY720P (Gilenya®) potently reduces relapse rate and lesion activity in the neuroinflammatory disorder multiple sclerosis. Although most of its efficacy has been shown to be related to immunosuppression through the induction of lymphopenia, it has been suggested that a number of its beneficial effects are related to altered endothelial and blood-brain barrier (BBB) functionality. However, to date it remains unknown whether brain endothelial S1P receptors are involved in the maintenance of the function of the BBB thereby mediating immune quiescence of the brain. Here we demonstrate that the brain endothelial receptor S1P5 largely contributes to the maintenance of brain endothelial barrier function.
We analyzed the expression of S1P5 in human post-mortem tissues using immunohistochemistry. The function of S1P5 at the BBB was assessed in cultured human brain endothelial cells (ECs) using agonists and lentivirus-mediated knockdown of S1P5. Subsequent analyses of different aspects of the brain EC barrier included the formation of a tight barrier, the expression of BBB proteins and markers of inflammation and monocyte transmigration.
We show that activation of S1P5 on cultured human brain ECs by a selective agonist elicits enhanced barrier integrity and reduced transendothelial migration of monocytes in vitro. These results were corroborated by genetically silencing S1P5 in brain ECs. Interestingly, functional studies with these cells revealed that S1P5 strongly contributes to brain EC barrier function and underlies the expression of specific BBB endothelial characteristics such as tight junctions and permeability. In addition, S1P5 maintains the immunoquiescent state of brain ECs with low expression levels of leukocyte adhesion molecules and inflammatory chemokines and cytokines through lowering the activation of the transcription factor NFκB.
Our findings demonstrate that S1P5 in brain ECs contributes to optimal barrier formation and maintenance of immune quiescence of the barrier endothelium.
Journal of Neuroinflammation 06/2012; 9:133. · 4.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: The increased incidence of auto-inflammatory and autoimmune diseases in the developed countries seems to be caused by an imbalance of the immune system due to the lack of proper regulation. Helminth parasites are well known modulators of the immune system and as such are of great interest for the treatment of these disorders. Clinical studies showed that administration of eggs of the pig nematode Trichuris suis to patients with inflammatory bowel disease reduces the disease severity. Here we demonstrate that treatment with soluble products from the nematodes T. suis and Trichinella spiralis induces significant suppression of symptoms in murine experimental autoimmune encephalomyelitis, a validated animal model for multiple sclerosis. These data show that infection with live nematodes is not a prerequisite for suppression of inflammation. To translate these results to the human system, the effects of soluble products of T. suis, T. spiralis and Schistosoma mansoni on the phenotype and function of human dendritic cells (DCs) were compared. Our data show that soluble products of T. suis, S. mansoni and T. spiralis suppress TNF-α and IL-12 secretion by TLR-activated human DCs, and that T. suis and S. mansoni, but not T. spiralis, strongly enhance expression of OX40L. Furthermore, helminth-primed human DCs differentially suppress the development of Th1 and/or Th17 cells. In conclusion, our data demonstrate that soluble helminth products have strong immunomodulatory capacities, but might exert their effects through different mechanisms. The suppressed secretion of pro-inflammatory cytokines together with an upregulation of OX40L expression on human DCs might contribute to achieve this modulation.
[show abstract][hide abstract] ABSTRACT: ATP-binding cassette (ABC) transporters are highly expressed by brain endothelial cells that form the blood-brain barrier (BBB). These efflux pumps play an important role in maintaining brain homeostasis as they actively hinder the entry of unwanted blood-derived compounds into the central nervous system (CNS). Consequently, their high activity at the BBB has been a major hurdle for the treatment of several brain diseases, as they prevent numerous drugs to reach their site of action within the brain. Importantly, recent data indicate that endogenous substrates for ABC transporters may include inflammatory mediators, such as prostaglandins, leukotrienes, cytokines, chemokines, and bioactive lipids, suggesting a potential role for ABC transporters in immunological responses, and more specifically in inflammatory brain disorders, such as multiple sclerosis (MS). In this review, we will give a comprehensive overview of recent findings that illustrate this novel role for ABC transporters in neuro-inflammatory processes. Moreover, we will provide first insights into underlying mechanisms and focus on the importance for bioactive lipids, in particular platelet-activating factor, herein. A thorough understanding of these events may form the basis for the development for selective treatment modalities to dampen the neuro-inflammatory attack in MS and thereby reducing tissue damage.
[show abstract][hide abstract] ABSTRACT: Homeostasis of the brain is dependent on the blood-brain barrier (BBB). This barrier tightly regulates the exchange of essential nutrients and limits the free flow of immune cells into the CNS. Perturbations of BBB function and the loss of its immune quiescence are hallmarks of a variety of brain diseases, including multiple sclerosis (MS), vascular dementia, and stroke. In particular, diapedesis of monocytes and subsequent trafficking of monocyte-derived macrophages into the brain are key mediators of demyelination and axonal damage in MS. Endothelin-1 (ET-1) is considered as a potent pro-inflammatory peptide and has been implicated in the development of cardiovascular diseases. Here, we studied the role of different components of the endothelin system, i.e., ET-1, its type B receptor (ET(B)) and endothelin-converting enzyme-1 (ECE-1) in monocyte diapedesis of a human brain endothelial cell barrier. Our pharmacological inhibitory and specific gene knockdown studies point to a regulatory function of these proteins in transendothelial passage of monocytes. Results from this study suggest that the endothelin system is a putative target within the brain for anti-inflammatory treatment in neurological diseases.
Journal of Neurochemistry 07/2011; 121(5):730-7. · 3.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: Adenosine triphosphate-binding cassette efflux transporters are highly expressed at the blood-brain barrier and actively hinder passage of harmful compounds, thereby maintaining brain homoeostasis. Since, adenosine triphosphate-binding cassette transporters drive cellular exclusion of potential neurotoxic compounds or inflammatory molecules, alterations in their expression and function at the blood-brain barrier may contribute to the pathogenesis of neuroinflammatory disorders, such as multiple sclerosis. Therefore, we investigated the expression pattern of different adenosine triphosphate-binding cassette efflux transporters, including P-glycoprotein, multidrug resistance-associated proteins-1 and -2 and breast cancer resistance protein in various well-characterized human multiple sclerosis lesions. Cerebrovascular expression of P-glycoprotein was decreased in both active and chronic inactive multiple sclerosis lesions. Interestingly, foamy macrophages in active multiple sclerosis lesions showed enhanced expression of multidrug resistance-associated protein-1 and breast cancer resistance protein, which coincided with their increased function of cultured foamy macrophages. Strikingly, reactive astrocytes display an increased expression of P-glycoprotein and multidrug resistance-associated protein-1 in both active and inactive multiple sclerosis lesions, which correlated with their enhanced in vitro activity on astrocytes derived from multiple sclerosis lesions. To investigate whether adenosine triphosphate-binding cassette transporters on reactive astrocytes can contribute to the inflammatory process, primary cultures of reactive human astrocytes were generated through activation of Toll-like receptor-3 to mimic the astrocytic phenotype as observed in multiple sclerosis lesions. Notably, blocking adenosine triphosphate-binding cassette transporter activity on reactive astrocytes inhibited immune cell migration across a blood-brain barrier model in vitro, which was due to the reduction of astrocytic release of the chemokine (C-C motif) ligand 2. Our data point towards a novel (patho)physiological role for adenosine triphosphate-binding cassette transporters, suggesting that limiting their activity by dampening astrocyte activation may open therapeutic avenues to diminish tissue damage during multiple sclerosis pathogenesis.
[show abstract][hide abstract] ABSTRACT: Normal neuronal functioning is dependent on the blood-brain barrier. This barrier is confined to specialized brain endothelial cells lining the inner vessel wall, and tightly controlling transport of nutrients, efflux of potentially harmful molecules and entry of immune cells into the brain. Loss of blood-brain barrier function is an early and significant event which contributes to inflammation in the brain and subsequent progression of neuronal deficits in a number of brain disorders and has been well-documented for the auto-immune disease multiple sclerosis. Extravasation of cells happens by paracellular transport across the endothelial junctions, transcellularly across the endothelial cells, or both, and requires the active participation of endothelial cells. We and others have shown that this process requires the activity of proteases, including tissue-type plasminogen activator. We here describe a novel role for NMDA receptor, a potential cellular target of tissue-type plasminogen activator, in human brain endothelial cells. Our results show that the NMDA receptor subunit 1 (NR1) is expressed in brain endothelial cells, regulates tissue-type plasminogen activator-induced signal transduction and controls the passage of monocytes through the brain endothelial cell barrier. Together, our results hold significant promise for the treatment of chronic inflammation in the brain.
Journal of Neurochemistry 04/2010; 113(2):447-53. · 3.97 Impact Factor