Dengue virus infection of blood-brain barrier cells: consequences of severe disease

More than 500 million people worldwide are infected each year by any of the four-dengue virus (DENV) serotypes. The clinical spectrum caused during these infections is wide and some patients may develop neurological alterations during or after the infection, which could be explained by the cryptic neurotropic and neurovirulent features of flaviviruses like DENV. Using in vivo and in vitro models, researchers have demonstrated that DENV can affect the cells from the blood–brain barrier (BBB) in several ways, which could result in brain tissue damage, neuronal loss, glial activation, tissue inflammation and hemorrhages. The latter suggests that BBB may be compromised during infection; however, it is not clear whether the damage is due to the infection per se or to the local and/or systemic inflammatory response established or activated by the BBB cells. Similarly, the kinetics and cascade of events that trigger tissue damage, and the cells that initiate it, are unknown. This review presents evidence of the BBB cell infection with DENV and the response established toward it by these cells; it also describes the consequences of this response on the nervous tissue, compares these evidence with the one reported with neurotropic viruses of the Flaviviridae family, and shows the complexity and unpredictability of dengue and the neurological alterations induced by it. Clinical evidence and in vitro and in vivo models suggest that this virus uses the bloodstream to enter nerve tissue where it infects the different cells of the neurovascular unit. Each of the cell populations respond individually and collectively and control infection and inflammation, in other cases this response exacerbates the damage leaving irreversible sequelae or causing death. This information will allow us to understand more about the complex disease known as dengue, and its impact on a specialized and delicate tissue like is the nervous tissue

Magnetoliposomas multifuncionales como vehículos de administración de fármacos para el tratamiento potencial de la enfermedad de Parkinson

La enfermedad de Parkinson (EP) es el segundo trastorno neurodegenerativo más frecuente después de la enfermedad de Alzheimer. Por ello, el desarrollo de nuevas tecnologías y estrategias para tratarla es una prioridad sanitaria mundial. Los tratamientos actuales incluyen la administración de levodopa, inhibidores de la monoaminooxidasa, inhibidores de la catecol-O-metiltransferasa y fármacos anticolinérgicos. Sin embargo, la liberación efectiva de estas moléculas, debido a la limitada biodisponibilidad, es un reto importante para el tratamiento de la EP. Como estrategia para resolver este desafío, en este estudio desarrollamos un novedoso sistema de liberación de fármacos multifuncional magnético y sensible a estímulos redox, basado en nanopartículas de magnetita funcionalizadas con la proteína translocadora de alto rendimiento OmpA y encapsuladas en liposomas de lecitina de soja. Los magnetoliposomas multifuncionales (MLP) obtenidos se ensayaron en neuroblastoma, glioblastoma, astrocitos primarios humanos y de rata, células endoteliales de rata de barrera hematoencefálica, células endoteliales microvasculares primarias de ratón y en un modelo celular inducido por EP. Los MLP demostraron un excelente rendimiento en ensayos de biocompatibilidad, incluyendo hemocompatibilidad (porcentajes de hemólisis por debajo del 1%), agregación plaquetaria, citocompatibilidad (viabilidad celular por encima del 80% en todas las líneas celulares probadas), potencial de membrana mitocondrial (alteraciones no observadas) y producción intracelular de ROS (impacto insignificante en comparación con los controles). Además, las nanovehículas mostraron una aceptable internalización celular (área cubierta cercana al 100% a los 30 min y a las 4 h) y capacidad de escape endosomal (disminución significativa de la colocalización lisosomal tras 4 h de exposición). Además, se emplearon simulaciones de dinámica molecular para comprender mejor el mecanismo de translocación subyacente de la proteína OmpA, mostrando hallazgos clave relativos a interacciones específicas con fosfolípidos. En general, la versatilidad y el notable rendimiento in vitro de este novedoso nanovehículo lo convierten en una tecnología de administración de fármacos adecuada y prometedora para el tratamiento potencial de la EP.Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease. Therefore, development of novel technologies and strategies to treat PD is a global health priority. Current treatments include administration of Levodopa, monoamine oxidase inhibitors, catechol-O-methyltransferase inhibitors, and anticholinergic drugs. However, the effective release of these molecules, due to the limited bioavailability, is a major challenge for the treatment of PD. As a strategy to solve this challenge, in this study we developed a novel multifunctional magnetic and redox-stimuli responsive drug delivery system, based on the magnetite nanoparticles functionalized with the high-performance translocating protein OmpA and encapsulated into soy lecithin liposomes. The obtained multifunctional magnetoliposomes (MLPs) were tested in neuroblastoma, glioblastoma, primary human and rat astrocytes, blood brain barrier rat endothelial cells, primary mouse microvascular endothelial cells, and in a PD-induced cellular model. MLPs demonstrated excellent performance in biocompatibility assays, including hemocompatibility (hemolysis percentages below 1%), platelet aggregation, cytocompatibility (cell viability above 80% in all tested cell lines), mitochondrial membrane potential (non-observed alterations) and intracellular ROS production (negligible impact compared to controls). Additionally, the nanovehicles showed acceptable cell internalization (covered area close to 100% at 30 min and 4 h) and endosomal escape abilities (significant decrease in lysosomal colocalization after 4 h of exposure). Moreover, molecular dynamics simulations were employed to better understand the underlying translocating mechanism of the OmpA protein, showing key findings regarding specific interactions with phospholipids. Overall, the versatility and the notable in vitro performance of this novel nanovehicle make it a suitable and promising drug delivery technology for the potential treatment of PD

In vitro integrity evaluation of the Blood Brain Barrier and its disturbance caused by dengue virus infection

El sistema nervioso (SN), está protegido por una barrera de difusión celular conocida como Barrera Hematoencefálica (BHE), compuesta por diferentes tipos de células que limitan el paso de moléculas desde los capilares hacia el parénquima cerebral, lo cual garantiza la homeostasis del tejido. A pesar de esto, algunas moléculas y agentes infecciosos logran atravesar la BHE e ingresar al tejido nervioso para inducir graves daños en la fisiología de este. Dentro de los patógenos que logran alterar la BHE e infectar el tejido nervioso, se encuentran algunos miembros de la familia flaviviridae típicamente neurotrópicos, sin embargo el virus del dengue (DENV), un virus considerado no neurotrópico, puede infectar y alterar la fisiología del tejido nervioso ocasionando signos como encefalitis, parálisis y alteraciones motoras y cognitivas que pueden ser permanentes. Para comprender los cambios de tropismo y los factores neurológicos e inmunológicos asociados a la neuroinfección por DENV, en nuestro laboratorio se desarrolló un modelo de neuroinfección en ratones lactantes utilizando una cepa de virus neuroadaptado (D4MB-6), que infecta neuronas y otras células del tejido nervioso e induce la alteración de la BHE en ratones lactantes. Por lo tanto, el presente proyecto buscó evaluar en un modelo de BHE in vitro, si la alteración de la permeabilidad de la barrera endotelial cerebrovascular asociada a la infección con el DENV-4 neuroadaptado o no, favorece el paso de virus libre o asociado a células del sistema inmune lo cual, permitiría la infección y dispersión viral en el tejido nervioso. Para esto, se estableció un modelo de BHE in vitro en monocapa o co-cultivo utilizando cultivos primarios de endotelio cerebrovascular con o sin astrocitos obtenidos a partir de cerebros de ratones lactantes, siendo únicamente las células endoteliales susceptibles a la infección con el virus parental o D4MB-6. Los resultados obtenidos mostraron en ambos modelos de BHE que a las 10 horas post-infección (hpi) con cada virus, hubo una disminución en los valores de resistencia transendotelial (TEER), asociada a un aumento en la permeabilidad. Adicionalmente en este mismo tiempo post infección se detectaron partículas virales infecciosas en la cámara inferior de los insertos, lo que sugiere que la infección ocasionó una alteración en la integridad de las células endoteliales, lo que permitió el paso paracelular de las partículas virales. Se encontró que la infección con el D4MB-6 indujo la relocalización -sin afectar la expresión-, de la proteína ZO-1 y un aumento en la expresión de los transcritos para las proteínas VCAM, PECAM, TNFα y MCP-1 comparado con lo observado durante la infección con el virus parental. Esto último está relacionado con un proceso de activación endotelial que al parecer favoreció el proceso de transmigración de monocitos/macrófagos J774 en ambos modelos de BHE estandarizados. Lo anterior convierte el proceso de alteración de la BHE en uno de los posibles mecanismos utilizados por el DENV para ingresar y dispersarse dentro del tejido nervioso.Magíster en Ciencias Básicas BiomédicasMaestríaThe nervous system (NS) is protected by a cellular diffusion barrier named Blood Brain Barrier (BBB). This barrier is formed by different cell types that limit the passage of molecules from the capillaries into the brain parenchyma, nevertheless some molecules and infectious agents are able to cross the BBB and infect the nervous tissue. Among the viruses that manage to alter the BBB and infect nervous tissue, there are some flaviviridae family members known as neurotropic flavivirus, however Dengue virus (DENV), a no-neurotropic flavivirus, can alter the physiology of the nervous tissue, causing clinical signs such as encephalitis, paralysis and motor and cognitive alterations that might be permanent. To understand some of the changes in the virus tropism, and in the neurological and immunological factors associated with DENV infection in the NS, we developed a neuroinfection model in suckling mice using a strain of neuroadapted virus (D4MB-6) than infects neurons and some others cells of the nervous tissue and can alter the BBB permeability in suckling mice. Therefore, the present project evaluated the permeability alteration of the cerebrovascular endothelial barrier, associated with DENV-4 or D4MB-6 infection in an in vitro BBB model. We tried to answer if this alteration can promote the passage of virus into the nervous tissue allowing infection and viral spread. We establish a BBB in vitro model using primary cultures (isolated from suckling mice) of endothelial cells (BBB model in monolayer) or endothelial cells and astrocytes (co-culture BBB model). The results showed that only the endothelial cells were susceptible to the infection with each evaluated virus; also in both BBB models at 10 hours post-infection, there was a decreased in the transendothelial resistance values (TEER), associated with an increase in the permeability of the model. Additionally at this time, we detected infectious viral particles in the supernatants of the lower chamber of the inserts, suggesting that infection results in an alteration in the integrity of the endothelial cells and permits the paracellular passage of viral particles. We also found that the infection with D4MB-6 induced the relocation of the ZO-1 protein, but it did not affect the expression pattern of this protein. Infection also induced the over expression of primary transcripts of VCAM, PECAM, TNFα y MCP-1 proteins, compared with the observations made when the cells were infected with DENV-4. This is related with an endothelial activation process that favored the transmigration of immune cells (J774) in both BBB models. Our results suggest that the alteration process of the BBB is one of the possible mechanisms used by the DENV to enter and spread in to the nervous tissue

In Vitro Infection with Dengue Virus Induces Changes in the Structure and Function of the Mouse Brain Endothelium.

BACKGROUND:The neurological manifestations of dengue disease are occurring with greater frequency, and currently, no information is available regarding the reasons for this phenomenon. Some viruses infect and/or alter the function of endothelial organs, which results in changes in cellular function, including permeability of the blood-brain barrier (BBB), which allows the entry of infected cells or free viral particles into the nervous system. METHODS:In the present study, we standardized two in vitro models, a polarized monolayer of mouse brain endothelial cells (MBECs) and an organized co-culture containing MBECs and astrocytes. Using these cell models, we assessed whether DENV-4 or the neuro-adapted dengue virus (D4MB-6) variant infects cells or induces changes in the structure or function of the endothelial barrier. RESULTS:The results showed that MBECs, but not astrocytes, were susceptible to infection with both viruses, although the percentage of infected cells was higher when the neuro-adapted virus variant was used. In both culture systems, DENV infection changed the localization of the tight junction proteins Zonula occludens (ZO-1) and Claudin-1 (Cln1), and this process was associated with a decrease in transendothelial resistance, an increase in macromolecule permeability and an increase in the paracellular passing of free virus particles. MBEC infection led to transcriptional up-regulation of adhesion molecules (VCAM-1 and PECAM) and immune mediators (MCP-1 and TNF- α) that are associated with immune cell transmigration, mainly in D4MB-6-infected cells. CONCLUSION:These results indicate that DENV infection in MBECs altered the structure and function of the BBB and activated the endothelium, affecting its transcellular and paracellular permeability and favoring the passage of viruses and the transmigration of immune cells. This phenomenon can be harnessed for neurotropic and neurovirulent strains to infect and induce alterations in the CNS

Endothelial Dysfunction, HMGB1, and Dengue: An Enigma to Solve

Dengue is a viral infection caused by dengue virus (DENV), which has a significant impact on public health worldwide. Although most infections are asymptomatic, a series of severe clinical manifestations such as hemorrhage and plasma leakage can occur during the severe presentation of the disease. This suggests that the virus or host immune response may affect the protective function of endothelial barriers, ultimately being considered the most relevant event in severe and fatal dengue pathogenesis. The mechanisms that induce these alterations are diverse. It has been suggested that the high mobility group box 1 protein (HMGB1) may be involved in endothelial dysfunction. This non-histone nuclear protein has different immunomodulatory activities and belongs to the alarmin group. High concentrations of HMGB1 have been detected in patients with several infectious diseases, including dengue, and it could be considered as a biomarker for the early diagnosis of dengue and a predictor of complications of the disease. This review summarizes the main features of dengue infection and describes the known causes associated with endothelial dysfunction, highlighting the involvement and possible relationship between HMGB1 and DENV

MBEC susceptibility to DENV infection.

<p><b>(A)</b>. MBEC cultured on glass coverslips were inoculated for 48 h with mock suspension or infected with DENV-4 or D4MB-6 at an MOI:1 and stained for detecting viral envelope protein (red) and ZO-1 protein (green). Both viruses infected the MBECs and showed perinuclear localization of viral E protein (arrow). On the other hand, the typical plasma membrane localization pattern of ZO-1 was detected in the mock and DENV-4 infected cultures, while in the D4MB-6 infected cells was located mainly in the cytoplasm, in clusters or in a discontinuous pattern in the perimembrane region. Bar = 20 μm. (<b>B).</b> Infection percentages of MBEC after 24 or 48 h p.i. Infection proportion was significantly higher with D4MB-6 at 48 h p.i. (49%), with regard to 11% of the cells infected with DENV-4. Data are shown as the mean +/- SD of 3 independent cultures performed by duplicate.</p

Scheme barrier models and evaluation of TEER and permeability assay in each models.

<p><b>(A).</b> Endothelial barrier model scheme. Transwell inserts were used to establish the two barrier models. The first one (Monolayer model) consisted in MBEC cultured on the luminal side of the membrane (upper side) for four days until monolayer reaches TEER values between 1 to 1,5 kΩ. For establishing the second one barrier model (co-culture model), the glial cells were seed on the abluminal side inverting the insert for three days, then it was flipped to the right position before seeding the MBEC in the luminal side. (<b>B).</b> Transendothelial electrical resistance. MBEC in each barrier model were infected or treated with mock inoculum. Since 10 h p.i. there was a significant reduction in TEER in DENV-4 and D4MB-6 infected compared with mock-inoculated barrier models. This TEER loss was sustained up to 48 h p.i. (p<0.05, Kruskal-Wallis and Bonferroni tests), however, TEER changes were less drastic in MBEC-astrocytes co-culture with regard to monolayer barrier model. (<b>C)</b>. Permeability assay. Using the same culture and infection protocols described above, dextran blue (DB) permeability assays were performed. DB was added in the insert’s upper chamber and at each time point; the lower chamber medium culture was collected to quantify the DB pass through by spectrophotometry. Since 10 to 48 h p.i. the infection with both virus strains induced a significant increase in lower chamber DB concentration coinciding with TEER loss. Barrier damage and DB quantified in the lower medium of MBEC-astrocytes co-culture were too low (between 0,2 to 1,2%) indicating a protective role of astrocytes. Data shown are mean of TEER or DB percentage from triplicates of two independent cultures and SD.</p

Virus titration in the upper and lower chambers in the co-culture model.

<p>Virus titration in the upper and lower chambers in the co-culture model.</p

RNA quantitation of viruses, cell proteins, inflammatory mediators and adhesion molecules.

<p>MBECs were infected with D4MB-6 and then processed to amplify a segment of dengue M protein gene, and cellular transcripts to ZO-1, TNF-α, MCP-1, PECAM, and VCAM using a quantitative RT-PCR. There were no significant changes in the amount of viral RNA and ZO-1 expression in the evaluated time points. TNF-α transcription was increased at 24 h p.i., while MCP-1 expression was increased at 10 h p.i. PECAM transcription increased at 2 and 24 h p.i. but not at 10 h p.i., while, VCAM expression was increased early (2 h p.i) and then gradually decreased over time. The data are shown as the relative expression obtained from duplicates of two independent experiments. The data were analyzed using the methods described in Pffafl et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157786#pone.0157786.ref023" target="_blank">23</a>] or Schefe et al, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157786#pone.0157786.ref024" target="_blank">24</a>].</p

DENV transmigration model of passage through the BBB.

<p><b>(A).</b> After the virus inoculation, MBECs are activated and/or infected, inducing early TJP relocalization and the expression of MCP-1 and adhesion molecules (10 h p.i.). These changes allowed viral paracellular transport by which the particles passed through to abluminal side. Adhesion molecule expression also changed, bringing immune cells closer and favor rolling. (<b>B).</b> After viral replication in MBEC, transcellular virus transport occurs, and the integrity of the barrier is damaged, resulting in an increase in virus paracellular transport to the abluminal side. MBEC activation and the production of inflammatory mediators promote leukocyte adhesion and transmigration, which carries the virus through via a Trojan horse mechanism.</p