RESUMEN

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.

Asunto(s)

Barrera Hematoencefálica , Esclerosis Múltiple , Canales Catiónicos TRPV , Humanos , Barrera Hematoencefálica/metabolismo , Sistema Nervioso Central/metabolismo , Inflamación/metabolismo , Esclerosis Múltiple/patología , Canales Catiónicos TRPV/metabolismo

RESUMEN

Considering its intolerance to ischemia, it is of critical importance for the brain to efficiently process microvascular occlusions and maintain tissue perfusion. In addition to collateral microvascular flow and enzymatic degradation of emboli, the endothelium has the potential to engulf microparticles and thereby recanalize the vessel, through a process called angiophagy. Here, we set out to study the dynamics of angiophagy in relation to cytoskeletal remodeling in vitro and reperfusion in vivo. We show that polystyrene microspheres and fibrin clots are actively taken up by (brain) endothelial cells in vitro, and chart the dynamics of the actin cytoskeleton during this process using live cell imaging. Whereas microspheres were taken up through the formation of a cup structure by the apical endothelial membrane, fibrin clots were completely engulfed by the cells, marked by dense F-actin accumulation surrounding the clot. Both microspheres and fibrin clots were retained in the endothelial cells. Notably, fibrin clots were not degraded intracellularly. Using an in vivo microembolization rat model, in which microparticles are injected into the common carotid artery, we found that microspheres are transported by the endothelium from the microvasculature into the brain parenchyma. Microembolization with microspheres caused temporal opening of the blood-brain barrier and vascular nonperfusion, followed by microsphere extravasation and restoration of vessel perfusion over time. Taken together, angiophagy is accompanied by active cytoskeletal remodeling of the endothelium, and is an effective mechanism to restore perfusion of the occluded microvasculature in vivo.

Asunto(s)

Circulación Cerebrovascular , Células Endoteliales/fisiología , Endotelio Vascular/fisiología , Embolia Intracraneal/patología , Microesferas , Microvasos/fisiología , Fagocitosis/fisiología , Animales , Encéfalo , Células Endoteliales/patología , Endotelio Vascular/citología , Endotelio Vascular/patología , Femenino , Células Endoteliales de la Vena Umbilical Humana/patología , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Masculino , Microvasos/patología , Ratas , Trombosis

RESUMEN

BACKGROUND: Non-invasive imaging of the activation status of microglia and the ability to identify a pro- or anti-inflammatory environment can provide valuable insights not only into pathogenesis of neuro-inflammatory and neurodegenerative diseases but also the monitoring of the efficacy of immunomodulatory therapies. P2X7R is highly expressed on pro-inflammatory microglia and [11C]SMW139, a specific P2X7R tracer for positron emission tomography imaging, showed good pharmacokinetics, stability, and brain permeability in vivo. Our objective was to evaluate the potential of [11C]SMW139 for PET imaging of neuroinflammation in vivo in the experimental autoimmune encephalomyelitis (EAE) model. METHODS: We induced EAE in Lewis rats by immunization with MBP 69-88 in complete Freund's adjuvant (CFA). We determined the affinity of [11C]SMW139 to human and rat P2X7R using saturation binding assay. Using this tracer, PET imaging was performed at the peak of disease and in the recovery phase. In vivo blocking experiments were conducted to validate the specific brain uptake of the tracer. Immunohistochemistry staining and autoradiography were performed to evaluate the level of neuroinflammation and validate the specific binding of [11C]SMW139. RESULTS: [11C]SMW139 showed good affinity for the rat P2X7R with a Kd of 20.6 ± 1.7 nM. The uptake of [11C]SMW139 was significantly higher in EAE animals at the peak of disease compared to the recovery phase but not in CFA control animals. The amplitude of increase of [11C]SMW139 uptake showed significant positive correlation with clinical scores mainly in the spinal cord (Pearson = 0.75, Spearman = 0.76; p < 0.0001). Treating EAE animals with P2X7R antagonist JNJ-47965567 blocked the uptake of [11C]SMW139 in the spinal cord, cerebellum, and brain stem, demonstrating specific accumulation of the tracer. P-glycoprotein blocking with tariquidar (30 mg/kg) did not affect tracer penetration in the brain showing that [11C]SMW139 is not a Pgp substrate. CONCLUSION: Our data shows that [11C]SMW139 is a promising PET tracer for imaging neuroinflammation and evaluating the dynamics of pro-inflammatory microglia in the brain. This can provide crucial insights into the role of microglia in disease progression and enables the development of novel treatment strategies aimed at modulating the immune response in order to promote neuroprotection.

Asunto(s)

Encéfalo/metabolismo , Radioisótopos de Carbono/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Esclerosis Múltiple/metabolismo , Tomografía de Emisión de Positrones/métodos , Receptores Purinérgicos P2X7/metabolismo , Animales , Encéfalo/diagnóstico por imagen , Encefalomielitis Autoinmune Experimental/diagnóstico por imagen , Femenino , Células HEK293 , Humanos , Masculino , Esclerosis Múltiple/inducido químicamente , Esclerosis Múltiple/diagnóstico por imagen , Agonistas del Receptor Purinérgico P2X/química , Agonistas del Receptor Purinérgico P2X/metabolismo , Ratas , Ratas Endogámicas Lew , Ratas Wistar

RESUMEN

The choroid plexus (CP) is a key regulator of the central nervous system (CNS) homeostasis through its secretory, immunological and barrier properties. Accumulating evidence suggests that the CP plays a pivotal role in the pathogenesis of multiple sclerosis (MS), but the underlying mechanisms remain largely elusive. To get a comprehensive view on the role of the CP in MS, we studied transcriptomic alterations of the human CP in progressive MS and non-neurological disease controls using RNA sequencing. We identified 17 genes with significantly higher expression in progressive MS patients relative to that in controls. Among them is the newly described long non-coding RNA HIF1A-AS3. Next to that, we uncovered disease-affected pathways related to hypoxia, secretion and neuroprotection, while only subtle immunological and no barrier alterations were observed. In an ex vivo CP explant model, a subset of the upregulated genes responded in a similar way to hypoxic conditions. Our results suggest a deregulation of the Hypoxia-Inducible Factor (HIF)-1 pathway in progressive MS CP. Importantly, cerebrospinal fluid levels of the hypoxia-responsive secreted peptide PAI-1 were higher in MS patients with high disability relative to those with low disability. These findings provide for the first time a complete overview of the CP transcriptome in health and disease, and suggest that the CP environment becomes hypoxic in progressive MS patients, highlighting the altered secretory and neuroprotective properties of the CP under neuropathological conditions. Together, these findings provide novel insights to target the CP and promote the secretion of neuroprotective factors into the CNS of progressive MS patients.

Asunto(s)

Plexo Coroideo/metabolismo , Hipoxia/genética , Esclerosis Múltiple Crónica Progresiva/genética , Esclerosis Múltiple Recurrente-Remitente/genética , Neuroprotección/genética , Neurosecreción/genética , Adrenomedulina/líquido cefalorraquídeo , Adrenomedulina/genética , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica , Ontología de Genes , Glicoproteínas/líquido cefalorraquídeo , Glicoproteínas/genética , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Péptidos y Proteínas de Señalización Intercelular/líquido cefalorraquídeo , Péptidos y Proteínas de Señalización Intercelular/genética , Ventrículos Laterales , Masculino , Metalotioneína/genética , Persona de Mediana Edad , Esclerosis Múltiple Crónica Progresiva/líquido cefalorraquídeo , Esclerosis Múltiple Recurrente-Remitente/líquido cefalorraquídeo , Inhibidor 1 de Activador Plasminogénico/líquido cefalorraquídeo , Inhibidor 1 de Activador Plasminogénico/genética , ARN sin Sentido/genética , ARN Largo no Codificante , RNA-Seq

RESUMEN

Chronic inflammation is a key pathological hallmark of multiple sclerosis (MS) and suggests that resolution of inflammation, orchestrated by specialized pro-resolving lipid mediators (LM), is impaired. Here, through targeted-metabololipidomics in peripheral blood of patients with MS, we revealed that each disease form was associated with distinct LM profiles that significantly correlated with disease severity. In particular, relapsing and progressive MS patients were associated with high eicosanoids levels, whereas the majority of pro-resolving LM were significantly reduced or below limits of detection and correlated with disease progression. Furthermore, we found impaired expression of several pro-resolving LM biosynthetic enzymes and receptors in blood-derived leukocytes of MS patients. Mechanistically, differentially expressed mediators like LXA4, LXB4, RvD1 and PD1 reduced MS-derived monocyte activation and cytokine production, and inhibited inflammation-induced blood-brain barrier dysfunction and monocyte transendothelial migration. Altogether, these findings reveal peripheral defects in the resolution pathway in MS, suggesting pro-resolving LM as novel diagnostic biomarkers and potentially safe therapeutics.

Asunto(s)

Monocitos , Esclerosis Múltiple , Barrera Hematoencefálica , Eicosanoides , Humanos , Inflamación , Mediadores de Inflamación , Esclerosis Múltiple/tratamiento farmacológico

RESUMEN

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.

Asunto(s)

Antiinflamatorios/farmacología , Astrocitos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Receptor de Melanocortina Tipo 4/agonistas , alfa-MSH/análogos & derivados , Adulto , Anciano , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Femenino , Humanos , Interleucina-11/biosíntesis , Interleucina-6/biosíntesis , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/tratamiento farmacológico , Fenotipo , Fosforilación , Receptor de Melanocortina Tipo 4/efectos de los fármacos , Receptor de Melanocortina Tipo 4/genética , alfa-MSH/farmacología

RESUMEN

Dysfunction of the blood-brain barrier (BBB) contributes significantly to the pathogenesis of several neuroinflammatory diseases, including multiple sclerosis (MS). Potential players that regulate BBB function are the liver X receptors (LXRs), which are ligand activated transcription factors comprising two isoforms, LXRα, and LXRß. However, the role of LXRα and LXRß in regulating BBB (dys)function during neuroinflammation remains unclear, as well as their individual involvement. Therefore, the goal of the present study is to unravel whether LXR isoforms have different roles in regulating BBB function under neuroinflammatory conditions. We demonstrate that LXRα, and not LXRß, is essential to maintain barrier integrity in vitro. Specific knockout of LXRα in brain endothelial cells resulted in a more permeable barrier with reduced expression of tight junctions. Additionally, the observed dysfunction was accompanied by increased endothelial inflammation, as detected by enhanced expression of vascular cell adhesion molecule (VCAM-1) and increased transendothelial migration of monocytes toward inflammatory stimuli. To unravel the importance of LXRα in BBB function in vivo, we made use of the experimental autoimmune encephalomyelitis (EAE) MS mouse model. Induction of EAE in a constitutive LXRα knockout mouse and in an endothelial specific LXRα knockout mouse resulted in a more severe disease score in these animals. This was accompanied by higher numbers of infiltrating leukocytes, increased endothelial VCAM-1 expression, and decreased expression of the tight junction molecule claudin-5. Together, this study reveals that LXRα is indispensable for maintaining BBB integrity and its immune quiescence. Targeting the LXRα isoform may help in the development of novel therapeutic strategies to prevent BBB dysfunction, and thereby neuroinflammatory disorders.

Asunto(s)

Barrera Hematoencefálica/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Células Endoteliales/inmunología , Receptores X del Hígado/inmunología , Animales , Barrera Hematoencefálica/patología , Línea Celular , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Células Endoteliales/patología , Técnicas de Silenciamiento del Gen , Humanos , Receptores X del Hígado/genética , Ratones , Ratones Noqueados , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/inmunología

RESUMEN

Tumors that lack T cell infiltration are less likely to respond to immune checkpoint inhibition and could benefit from cancer vaccination for the initiation of anti-tumor T cell responses. An attractive vaccine strategy is in vivo targeting of dendritic cells (DCs), key initiators of antigen-specific T cell responses. In this study we generated tumor-derived apoptotic extracellular vesicles (ApoEVs), which are potentially an abundant source of tumor-specific neo-antigens and other tumor-associated antigens (TAAs), and which can be manipulated to express DC-targeting ligands for efficient antigen delivery. Our data demonstrates that by specifically modifying the glycocalyx of tumor cells, high-mannose glycans can be expressed on their cell surface and on extracellular vesicles derived after the induction of apoptosis. High-mannose glycans are the natural ligands of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), a dendritic cell associated C-type lectin receptor (CLR), which has the ability to efficiently internalize its cargo and direct it to both major histocompatibility complex (MHC)-I and MHC-II pathways for the induction of CD8+ and CD4+ T cell responses, respectively. Compared to unmodified ApoEVs, ApoEVs carrying DC-SIGN ligands are internalized to a higher extent, resulting in enhanced priming of tumor-specific CD8+ T cells. This approach thus presents a promising vaccination strategy in support of T cell-based immunotherapy of cancer.

RESUMEN

The blood-brain barrier (BBB) has a major role in maintaining brain homeostasis through the specialized function of brain endothelial cells (BECs). Inflammation of the BECs and loss of their neuroprotective properties is associated with several neurological disorders, including the chronic neuro-inflammatory disorder multiple sclerosis (MS). Yet, the underlying mechanisms of a defective BBB in MS remain largely unknown. Endothelial to mesenchymal transition (EndoMT) is a pathophysiological process in which endothelial cells lose their specialized function and de-differentiate into mesenchymal cells. This transition is characterized by an increase in EndoMT-related transcription factors (TFs), a downregulation of brain endothelial markers, and an upregulation of mesenchymal markers accompanied by morphological changes associated with cytoskeleton reorganization. Here, we postulate that EndoMT drives BEC de-differentiation, mediates inflammation-induced human BECs dysfunction, and may play a role in MS pathophysiology. We provide evidence that stimulation of human BECs with transforming growth factor (TGF)-ß1 and interleukin (IL)-1ß promotes EndoMT, a process in which the TF SNAI1, a master regulator of EndoMT, plays a crucial role. We demonstrate the involvement of TGF-ß activated kinase 1 (TAK1) in EndoMT induction in BECs. Finally, immunohistochemical analysis revealed EndoMT-associated alterations in the brain vasculature of human post-mortem MS brain tissues. Taken together, our novel findings provide a better understanding of the molecular mechanisms underlying BECs dysfunction during MS pathology and can be used to develop new potential therapeutic strategies to restore BBB function.

Asunto(s)

Encéfalo/fisiopatología , Inflamación/complicaciones , Esclerosis Múltiple/genética , Esclerosis Múltiple/fisiopatología , Células Cultivadas , Células Endoteliales/metabolismo , Transición Epitelial-Mesenquimal , Humanos

RESUMEN

Increasing evidence suggests that vascular dysfunction in the brain is associated with early stages of Alzheimer's disease. Amyloid-ß deposition in the microvasculature of the brain, a process referred to as capillary cerebral amyloid angiopathy (capillary CAA), propagates vascular remodelling, which results in impaired function of the blood-brain barrier, reduced cerebral perfusion and increased hypoxia. While improving vascular function may be an attractive new way to fight capillary CAA, the underlying factors that mediate vascular alterations in Alzheimer's disease and capillary CAA pathogenesis remain largely unknown. Here we provide first evidence that angiopoietin like-4 (ANGPTL4), a hypoxia-induced factor, is highly expressed by reactive astrocytes in well characterized post-mortem tissues of patients with capillary CAA. Our in vitro studies reveal that ANGPTL4 is upregulated and secreted by human cortical astrocytes under hypoxic conditions and in turn stimulates endothelial cell migration and sprouting in a 3D spheroid model of human brain endothelial cells. Interestingly, plasma levels of ANGPTL4 are significantly increased in patients with vascular dementia compared to patients with subjective memory complaints. Overall, our data suggest that ANGPTL4 contributes to pathological vascular remodelling in capillary CAA and that detection of ANGPTL4 levels may improve current diagnostics. Ways of counteracting the detrimental effects of ANGPTL4 and thus promoting cerebral vascular function may provide novel treatment regimens to halt the progression of Alzheimer's disease.

Asunto(s)

Proteína 4 Similar a la Angiopoyetina/metabolismo , Astrocitos/metabolismo , Angiopatía Amiloide Cerebral/metabolismo , Anciano , Anciano de 80 o más Años , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Encéfalo/patología , Hipoxia de la Célula , Movimiento Celular , Células Endoteliales/metabolismo , Femenino , Humanos , Masculino , Microvasos/patología , Remodelación Vascular

RESUMEN

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.

Asunto(s)

Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Glicosiltransferasas/metabolismo , Inflamación/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Encéfalo/metabolismo , Línea Celular , Supervivencia Celular/fisiología , Glicosilación , Humanos , Permeabilidad

RESUMEN

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.

Asunto(s)

Presentación de Antígeno , Antígenos/inmunología , Encéfalo/patología , Linfocitos T CD4-Positivos/inmunología , Movimiento Celular , Endotelio/inmunología , Vaina de Mielina/inmunología , Linfocitos T CD4-Positivos/fisiología , Células Cultivadas , Endocitosis , Endotelio/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos

RESUMEN

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.

Asunto(s)

Encéfalo/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Linfocitos T/inmunología , Migración Transendotelial y Transepitelial/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Encéfalo/citología , Encéfalo/inmunología , Adhesión Celular/genética , Adhesión Celular/inmunología , Línea Celular , Ceramidas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Femenino , Filaminas/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/inmunología , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/inmunología , Fosforilación/genética , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/inmunología

RESUMEN

BACKGROUND: Reactive oxygen species play a key role in the pathogenesis of multiple sclerosis as they induce blood-brain barrier disruption and enhance transendothelial leukocyte migration. Thus, therapeutic compounds with antioxidant and anti-inflammatory potential could have clinical value in multiple sclerosis. The aim of the current study was to elucidate the therapeutic effects of monomethyl fumarate on inflammatory-mediated changes in blood-brain barrier function and gain insight into the underlying mechanism. METHODS: The effects of monomethyl fumarate on monocyte transendothelial migration across and adhesion to inflamed human brain endothelial cells (hCMEC/D3) were quantified using standardized in vitro migration and adhesion assays. Flow cytometry analysis and qPCR were used to measure the concomitant effects of monomethyl fumarate treatment on protein expression of cell adhesion molecules. Furthermore, the effects of monomethyl fumarate on the expression and nuclear localization of proteins involved in the activation of antioxidant and inflammatory pathways in human brain endothelial cells were elucidated using nuclear fractionation and Western blotting. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni post-hoc test. RESULTS: Our results show that monomethyl fumarate induced nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 and concomitant production of the antioxidant enzymes heme oxygenase-1 and NADPH:quinone oxidoreductase-1 in brain endothelial cells. Importantly, monomethyl fumarate treatment markedly decreased monocyte transendothelial migration across and adhesion to inflamed human brain endothelial cells. Treatment of brain endothelial cells with monomethyl fumarate resulted in a striking reduction of vascular cell adhesion molecule expression. Surprisingly, monomethyl fumarate did not affect nuclear translocation of nuclear factor-кB suggesting that monomethyl fumarate potentially affects activity of nuclear factor-ĸB downstream of nuclear translocation. CONCLUSIONS: Taken together, we show that monomethyl fumarate, the primary metabolite of dimethyl fumarate, which is currently used in the clinics for the treatment of relapsing-remitting multiple sclerosis, demonstrates beneficial therapeutic effects at the inflamed blood-brain barrier.

Asunto(s)

Antiinflamatorios/farmacología , Antioxidantes/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Fumaratos/farmacología , Leucocitos/efectos de los fármacos , Maleatos/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Migración Transendotelial y Transepitelial/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Citoprotección , Células Endoteliales/metabolismo , Células Endoteliales/patología , Hemo-Oxigenasa 1/metabolismo , Humanos , Leucocitos/metabolismo , Leucocitos/patología , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo

RESUMEN

Recent evidence suggests that reactive oxygen species (ROS) produced by inflammatory cells drive axonal degeneration in active multiple sclerosis (MS) lesions by inducing mitochondrial dysfunction. Mitochondria are endowed with a variety of antioxidant enzymes, including peroxiredoxin-3 and thioredoxin-2, which are involved in limiting ROS-induced damage. In this study, we explored the distribution and role of the mitochondrial antioxidants peroxiredoxin-3 and thioredoxin-2 as well as their regulator peroxisome proliferator-activated receptor gamma coactivator1-alpha (PGC-1α) in MS pathogenesis. Immunohistochemical analysis of a large cohort of MS patients revealed a striking upregulation of PGC-1α and downstream mitochondrial antioxidants in active demyelinating MS lesions. Enhanced expression was predominantly observed in reactive astrocytes. To elucidate the functional role of astrocytic PGC-1α in MS pathology, we generated human primary astrocytes that genetically overexpressed PGC-1α. Upon an oxidative insult, these cells were shown to produce less ROS and were found to be more resistant to ROS-induced cell death compared to control cells. Intriguingly, also neuronal cells co-cultured with PGC-1α-overexpressing astrocytes were protected against an exogenous oxidative attack compared to neuronal cells co-cultured with control astrocytes. Finally, enhanced astrocytic PGC-1α levels markedly reduced the production and secretion of the pro-inflammatory mediators interleukin-6 and chemokine (C-C motif) ligand 2. Our findings suggest that increased astrocytic PGC-1α in active MS lesions might initially function as an endogenous protective mechanism to dampen oxidative damage and inflammation thereby reducing neurodegeneration. Activation of PGC-1α therefore represents a promising therapeutic strategy to improve mitochondrial function and repress inflammation.

Asunto(s)

Astrocitos/metabolismo , Encéfalo/metabolismo , Encefalitis/etiología , Encefalitis/patología , Esclerosis Múltiple/complicaciones , Factores de Transcripción/metabolismo , Adulto , Anciano , Antioxidantes/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/patología , Proteína Proteolipídica de la Mielina/metabolismo , Estrés Oxidativo/fisiología , Peroxiredoxina III/genética , Peroxiredoxina III/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Especies Reactivas de Oxígeno , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Factores de Transcripción/genética , Sustancia Blanca/metabolismo , Sustancia Blanca/patología

RESUMEN

Multiple sclerosis (MS) lesions are characterized by the presence of activated astrocytes, which are thought to actively take part in propagating lesion progression by secreting pro-inflammatory mediators. Conversely, reactive astrocytes may exert disease-dampening effects through the production of trophic factors and anti-inflammatory mediators. Astrocytic control of the blood-brain barrier (BBB) is crucial for normal brain homeostasis and BBB disruption is a well-established early event in MS lesion development. Here, we set out to unravel potential protective effects of reactive astrocytes on BBB function under neuroinflammatory conditions as seen in MS, where we focus on the role of the brain morphogen retinoic acid (RA). Immunohistochemical analysis revealed that retinaldehyde dehydrogenase 2 (RALDH2), a key enzyme for RA synthesis, is highly expressed by reactive astrocytes throughout white matter lesions compared to control and normal appearing white matter. In vitro modeling of reactive astrocytes resulted in increased expression of RALDH2, enhanced RA synthesis, and a protective role for astrocyte-derived RA on BBB function during inflammation-induced barrier loss. Furthermore, RA induces endothelial immune quiescence and decreases monocyte adhesion under inflammatory conditions. Finally, we demonstrated that RA attenuated oxidative stress in inflamed endothelial cells, through activation of the antioxidant transcription factor nuclear factor E2 related factor 2. In summary, RA synthesis by reactive astrocytes represents an endogenous protective response to neuroinflammation, possibly aimed at protecting the BBB against inflammatory insult. A better understanding of RA signaling in MS pathophysiology may lead to the discovery of novel targets to halt disease progression.

Asunto(s)

Astrocitos/efectos de los fármacos , Barrera Hematoencefálica/fisiopatología , Encéfalo/patología , Esclerosis Múltiple/patología , Tretinoina/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Astrocitos/metabolismo , Autopsia , Células Cultivadas , Citocinas/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Células HEK293 , Humanos , Masculino , Persona de Mediana Edad , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Retinal-Deshidrogenasa/genética , Retinal-Deshidrogenasa/metabolismo , Factores de Tiempo

RESUMEN

To ensure efficient energy supply to the high demanding brain, nutrients are transported into brain cells via specific glucose (GLUT) and monocarboxylate transporters (MCT). Mitochondrial dysfunction and altered glucose metabolism are thought to play an important role in the progression of neurodegenerative diseases, including multiple sclerosis (MS). Here, we investigated the cellular localization of key GLUT and MCT proteins in human brain tissue of non-neurological controls and MS patients. We show that in control brain tissue GLUT and MCT proteins were abundantly expressed in a variety of central nervous system cells, particularly in microglia and endothelial cells. In active MS lesions, GLUTs and MCTs were highly expressed in infiltrating leukocytes and reactive astrocytes. Astrocytes manifest increased MCT1 staining and maintain GLUT expression in inactive lesions, whereas demyelinated axons exhibit significantly reduced GLUT3 and MCT2 immunoreactivity in inactive lesions. Finally, we demonstrated that the co-transcription factor peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α), an important protein involved in energy metabolism, is highly expressed in reactive astrocytes in active MS lesions. Overexpression of PGC-1α in astrocyte-like cells resulted in increased production of several GLUT and MCT proteins. In conclusion, we provide for the first time a comprehensive overview of key nutrient transporters in white matter brain samples. Moreover, our data demonstrate an altered expression of these nutrient transporters in MS brain tissue, including a marked reduction of axonal GLUT3 and MCT2 expression in chronic lesions, which may impede efficient nutrient supply to the hypoxic demyelinated axons thereby contributing to the ongoing neurodegeneration in MS.

Asunto(s)

Encéfalo/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Esclerosis Múltiple/metabolismo , Sustancia Blanca/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Astrocitos/metabolismo , Astrocitos/patología , Axones/metabolismo , Axones/patología , Encéfalo/irrigación sanguínea , Encéfalo/patología , Línea Celular , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Transportador de Glucosa de Tipo 3/metabolismo , Humanos , Leucocitos/metabolismo , Leucocitos/patología , Masculino , Microglía/metabolismo , Microglía/patología , Persona de Mediana Edad , Esclerosis Múltiple/patología , Esclerosis Múltiple Crónica Progresiva/metabolismo , Esclerosis Múltiple Crónica Progresiva/patología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Factores de Transcripción/metabolismo , Sustancia Blanca/irrigación sanguínea , Sustancia Blanca/patología

RESUMEN

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.

Asunto(s)

Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Encéfalo/inmunología , Linfocitos T CD8-positivos/fisiología , Encefalomielitis Autoinmune Experimental/inmunología , Esclerosis Múltiple/inmunología , Migración Transendotelial y Transepitelial/fisiología , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Barrera Hematoencefálica/fisiología , Encéfalo/irrigación sanguínea , Encéfalo/patología , Linfocitos T CD4-Positivos/fisiología , Línea Celular , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Microvasos/patología , Microvasos/fisiopatología , Esclerosis Múltiple/patología , Miembro 4 de la Subfamilia B de Casete de Unión a ATP

RESUMEN

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.

Asunto(s)

Plexo Coroideo/fisiopatología , Claudina-3/metabolismo , Encefalomielitis Autoinmune Experimental/fisiopatología , Esclerosis Múltiple/fisiopatología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Encéfalo/irrigación sanguínea , Encéfalo/patología , Encéfalo/fisiopatología , Plexo Coroideo/patología , Claudina-3/genética , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Femenino , Humanos , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Microvasos/patología , Microvasos/fisiopatología , Persona de Mediana Edad , Esclerosis Múltiple/patología , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos , Índice de Severidad de la Enfermedad

RESUMEN

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.

Asunto(s)

Barrera Hematoencefálica/fisiopatología , Encéfalo/patología , Células Endoteliales/fisiología , Inflamación/patología , MicroARNs/metabolismo , Esclerosis Múltiple/patología , Barrera Hematoencefálica/efectos de los fármacos , Línea Celular Transformada , Citocinas/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/fisiología , Humanos , MicroARNs/genética , ARN Interferente Pequeño/farmacología , Migración Transendotelial y Transepitelial/efectos de los fármacos , Transfección