Notch signaling is impaired during inflammation in a Lunatic Fringe-dependent manner.

The blood-brain barrier (BBB) assures brain homeostasis through the specialized function of brain endothelial cells (BECs). Dysfunction of the BBB due to inflammatory processes is associated with several neurological disorders, including multiple sclerosis (MS). Understanding the mechanisms that underlie these processes may ultimately lead to new therapeutic strategies to restore BBB function, thereby fighting disease progression. In this study, we demonstrate for the first time a critical role of the Notch signaling pathway in the function of the BBB under resting and inflammatory conditions. Inhibition of the Notch signaling, either by a γ-secretase inhibitor or by genetic ablation of endothelial NOTCH, led to BBB dysfunction in vitro as evidenced by reduced transendothelial electrical resistance (TEER), altered localization and loss of endothelial junction molecules and enhanced macromolecular permeability. Inflamed BECs showed impaired Notch signaling as indicated by reduced level of the downstream targets HES-1 and HES-5. Notably, barrier function was further reduced when the Notch signaling was inhibited under inflammatory conditions, suggesting an additive effect of the Notch signaling and inflammation in BECs. In contrast, inducible overexpression of Notch-intracellular domain 1 (NICD1) rescued the detrimental effect caused by inflammation. Furthermore, we provide evidence that inflammation reduced the expression of the glycosyltransferase Lunatic Fringe (LFNG), a known positive regulator of Notch glycosylation and signaling, thereby leading to disrupted barrier function of BECs. Together, our data demonstrate the functional importance of the conserved Notch signaling pathway in control of the brain endothelial barrier and shed light on the role of LFNG in the regulation of Notch glycosylation and signaling in the adult brain vasculature in both health and disease.

Internalization and presentation of myelin antigens by the brain endothelium guides antigen-specific T cell migration.

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.

Acid Sphingomyelinase-Derived Ceramide Regulates ICAM-1 Function during T Cell Transmigration across Brain Endothelial Cells.

Multiple sclerosis (MS) is a chronic demyelinating disorder of the CNS characterized by immune cell infiltration across the brain vasculature into the brain, a process not yet fully understood. We previously demonstrated that the sphingolipid metabolism is altered in MS lesions. In particular, acid sphingomyelinase (ASM), a critical enzyme in the production of the bioactive lipid ceramide, is involved in the pathogenesis of MS; however, its role in the brain vasculature remains unknown. Transmigration of T lymphocytes is highly dependent on adhesion molecules in the vasculature such as intercellular adhesion molecule-1 (ICAM-1). In this article, we hypothesize that ASM controls T cell migration by regulating ICAM-1 function. To study the role of endothelial ASM in transmigration, we generated brain endothelial cells lacking ASM activity using a lentiviral shRNA approach. Interestingly, although ICAM-1 expression was increased in cells lacking ASM activity, we measured a significant decrease in T lymphocyte adhesion and consequently transmigration both in static and under flow conditions. As an underlying mechanism, we revealed that upon lack of endothelial ASM activity, the phosphorylation of ezrin was perturbed as well as the interaction between filamin and ICAM-1 upon ICAM-1 clustering. Functionally this resulted in reduced microvilli formation and impaired transendothelial migration of T cells. In conclusion, in this article, we show that ASM coordinates ICAM-1 function in brain endothelial cells by regulating its interaction with filamin and phosphorylation of ezrin. The understanding of these underlying mechanisms of T lymphocyte transmigration is of great value to develop new strategies against MS lesion formation.

Immune cell trafficking across the barriers of the central nervous system in multiple sclerosis and stroke.

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 migration across the barriers of the CNS and the role of activated astrocytes in this process. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.

Fingolimod attenuates ceramide-induced blood-brain barrier dysfunction in multiple sclerosis by targeting reactive astrocytes.

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.

Sphingosine 1-phosphate receptor 5 mediates the immune quiescence of the human brain endothelial barrier.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Our findings demonstrate that S1P5 in brain ECs contributes to optimal barrier formation and maintenance of immune quiescence of the barrier endothelium.

Pediatric HIV-related oral manifestations: a five-year retrospective study.

The purpose of this study was to carry out a five-year retrospective descriptive follow-up of the oral manifestation frequency, systemic condition and type of medication used in HIV-infected children and adolescents after the introduction of combined antiretroviral therapy. Fifty-eight patients were examined in 2001/2002, and their previous medical and dental records (1997 to 2000) were researched from files. There was an occurrence of 7 new cases of AIDS in a sample of 19 children, while 46.5% of the entire sample (n = 58) progressed as to classification of HIV infection. No difference was noted among the frequencies of oral manifestations, categories of the immunosuppression and viral load categories. The oral manifestations in the group of children and adolescents followed up in this study remained stable, even after treatment with combined antiretroviral therapy. However, a downward trend in the frequency of oral candidiasis and parotid enlargement was noted.

Pediatric HIV-related oral manifestations: a five-year retrospective study

O objetivo deste estudo foi realizar um acompanhamento descritivo retrospectivo da freqüência de manifestações bucais, da condição sistêmica e do tipo de medicação utilizada em um grupo de crianças e adolescentes infectados pelo HIV após a introdução da terapia anti-retroviral combinada. Cinqüenta e oito pacientes foram examinados em 2001/2002, enquanto seus exames médicos e odontológicos retrospectivos (1997 a 2000) foram pesquisados em prontuário. Foram observados 7 novos casos de AIDS em uma amostra de 19 pacientes, enquanto 46,5% da amostra total (n = 58) progrediram quanto à classificação da infecção pelo HIV. Não foram observadas diferenças entre as freqüências de manifestações bucais, das categorias de imunossupressão e da carga viral. O quadro de manifestações bucais no grupo de crianças e adolescentes acompanhados neste estudo manteve-se estável, mesmo após a introdução da terapia anti-retroviral combinada. Contudo, foi observada uma tendência de diminuição da freqüência de candidíase bucal e hipertrofia de parótidas.