RESUMO
The choroid plexus (CP) is part of the blood-cerebrospinal fluid barrier (BCSFB) and was recently described as an important component of the circadian clock system. It is the principal source of cerebrospinal fluid (CSF) and responsible for the synthesis and secretion of various neuroprotective peptides including those involved in amyloid-ß (Aß) transport/degradation, contributing to Aß homeostasis. Inadequate Aß metabolic clearance and transport across the BCSFB have been associated with circadian dysfunctions in Alzheimer's disease (AD) patients. To investigate whether AD pathology influences Aß scavengers circadian expression, we collected CP at different time points from an AD mouse model (APP/PS1) (female and male animals, aged 6- and 12-months-old) and analyzed their mRNA expression by Real-time RT-PCR. Only angiotensin-converting enzyme (Ace) expression in 6-month-old female wild-type mice and transthyretin (Ttr) expression in 12-month-old female wild-type mice presented significant rhythmicity. The circadian rhythmicity of Ace and Ttr, prompt us to analyze the involvement of circadian rhythm in Aß uptake. A human CP papilloma (HIBCPP) cell line was incubated with Aß-488 and uptake was evaluated at different time points using flow cytometry. Aß uptake displayed circadian rhythmicity. Our results suggest that AD might affect Aß scavengers rhythmicity and that Aß clearance is a rhythmic process possibly regulated by the rhythmic expression of Aß scavengers.
Assuntos
Doença de Alzheimer , Humanos , Masculino , Feminino , Camundongos , Animais , Lactente , Doença de Alzheimer/metabolismo , Plexo Corióideo/metabolismo , Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Ritmo Circadiano , Camundongos Transgênicos , Precursor de Proteína beta-Amiloide/genética , Modelos Animais de DoençasRESUMO
Endothelial cells play a major part in the regulation of vascular permeability and angiogenesis. According to their duty to fit the needs of the underlying tissue, endothelial cells developed different subtypes with specific endothelial microdomains as caveolae, fenestrae and transendothelial channels which regulate nutrient exchange, leukocyte migration, and permeability. These microdomains can exhibit diaphragms that are formed by the endothelial cell-specific protein plasmalemma vesicle-associated protein (PLVAP), the only known protein component of these diaphragms. Several studies displayed an involvement of PLVAP in diseases as cancer, traumatic spinal cord injury, acute ischemic brain disease, transplant glomerulopathy, Norrie disease and diabetic retinopathy. Besides an upregulation of PLVAP expression within these diseases, pro-angiogenic or pro-inflammatory responses were observed. On the other hand, loss of PLVAP in knockout mice leads to premature mortality due to disrupted homeostasis. Generally, PLVAP is considered as a major factor influencing the permeability of endothelial cells and, finally, to be involved in the regulation of vascular permeability. Following these observations, PLVAP is debated as a novel therapeutic target with respect to the different vascular beds and tissues. In this review, we highlight the structure and functions of PLVAP in different endothelial types in health and disease.
Assuntos
Retinopatia Diabética , Células Endoteliais , Animais , Encéfalo/metabolismo , Permeabilidade Capilar/fisiologia , Proteínas de Transporte/metabolismo , Células Endoteliais/metabolismo , Proteínas de Membrana/metabolismo , HumanosRESUMO
The objectives of these studies were twofold: 1) to characterize the human choroid plexus papilloma (HIBCPP) cell line as a model of the blood-cerebrospinal fluid barrier (BCSFB) via morphology, tightness, and polarization of transporters in choroid plexus epithelia (CPe), and 2) to utilize Ussing-style electrophysiology to elucidate signaling pathways associated with the activation of the transient receptor potential vanilloid 4 (TRPV4) channel involved in cerebrospinal fluid (CSF) secretion. RT-PCR was implemented to determine gene expression of cell fate markers, junctional complex proteins, and transporters of interest. Scanning electron microscopy and confocal three-dimensional renderings of cultures grown on permeable supports were utilized to delineate the morphology of the brush border, junctional complexes, and polarization of key transporters. Electrophysiology was used to understand and explore TRPV4-mediated signaling in the HIBCPP cell line, considering both short-circuit current (Isc) and conductance responses. HIBCPP cells grown under optimized culture conditions exhibited minimal multilayering, developed an intermediate resistance monolayer, retained differentiation properties, and expressed, and correctly localized, junctional proteins and native transporters. We found that activation of TRPV4 resulted in a robust, multiphasic change in electrogenic ion flux and increase in conductance accompanied by substantial fluid secretion. This response appears to be modulated by a number of different effectors, implicating phospholipase C (PLC), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K) in TRPV4-mediated ion flux. The HIBCPP cell line is a representative model of the human BCSFB, which can be utilized for studies of transporter function, intracellular signaling, and regulation of CSF production.
Assuntos
Plexo Corióideo , Fosfatidilinositol 3-Quinases , Humanos , Plexo Corióideo/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Linhagem Celular , Barreira Hematoencefálica/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transdução de Sinais , Células Epiteliais/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismoRESUMO
The choroid plexus epithelium (CPe) forms a barrier between the cerebral blood supply and the cerebrospinal fluid (CSF), establishing the blood-CSF barrier (BCSFB). CSF is actively secreted by the CPe via tightly controlled processes involving multiple channels, transporters, and pumps. The importance of controlling CSF production and composition has been accentuated recently with an appreciation of CSF dysfunction in many pathologies. For mechanistic studies of CSF production, isolated CPe cell lines are valuable for the testing of hypotheses and potential drug targets. Although several continuous CPe cell lines have been described, none appear to have all the characteristics of the native epithelium and each must be used judiciously. The porcine choroid plexus-Riems (PCP-R) cell line forms a high-resistance monolayer characteristic of a barrier epithelium. Conservation of this phenotype is unusual among CPe cell lines, making this model useful for studies of the effects of infection, injury, and drugs on permeability. We have recently discovered that, although this line expresses many of the transporters expressed in the native tissue, some are mispolarized. As a result, inferences regarding fluid/electrolyte flux and the resultant CSF production should be pursued with caution. Furthermore, extended culture periods and changes in media composition result in significant morphological and functional variability. These studies provide a more detailed characterization of the PCP-R cell line concerning transporter expression, polarization, and functionality, as well as plasticity in culture, with the goal to provide the scientific community with information necessary to optimize future experiments with this model.
Assuntos
Proteínas de Transporte , Plexo Corióideo , Animais , Barreira Hematoencefálica/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Líquido Cefalorraquidiano/metabolismo , Plexo Corióideo/metabolismo , Epitélio/metabolismo , SuínosRESUMO
The human central nervous system (CNS) is separated from the blood by distinct cellular barriers, including the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CFS) barrier (BCSFB). Whereas at the center of the BBB are the endothelial cells of the brain capillaries, the BCSFB is formed by the epithelium of the choroid plexus. Invasion of cells of either the BBB or the BCSFB is a potential first step during CNS entry by the Gram-positive bacterium Listeria monocytogenes (Lm). Lm possesses several virulence factors mediating host cell entry, such as the internalin protein family-including internalin (InlA), which binds E-cadherin (Ecad) on the surface of target cells, and internalin B (InlB)-interacting with the host cell receptor tyrosine kinase Met. A further family member is internalin (InlF), which targets the intermediate filament protein vimentin. Whereas InlF has been shown to play a role during brain invasion at the BBB, its function during infection at the BCSFB is not known. We use human brain microvascular endothelial cells (HBMEC) and human choroid plexus epithelial papilloma (HIBCPP) cells to investigate the roles of InlF and vimentin during CNS invasion by Lm. Whereas HBMEC present intracellular and surface vimentin (besides Met), HIBCPP cells do not express vimentin (except Met and Ecad). Treatment with the surface vimentin modulator withaferin A (WitA) inhibited invasion of Lm into HBMEC, but not HIBCPP cells. Invasion of Lm into HBMEC and HIBCPP cells is, however, independent of InlF, since a deletion mutant of Lm lacking InlF did not display reduced invasion rates.
Assuntos
Listeria monocytogenes , Humanos , Barreira Hematoencefálica/metabolismo , Vimentina/metabolismo , Filamentos Intermediários/metabolismo , Células Endoteliais/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Quercetin-3-glucuronide (Q3GA), the main phase II metabolite of quercetin (Q) in human plasma, is considered to be a more stable form of Q for transport with the bloodstream to tissues, where it can be potentially deconjugated by ß-glucuronidase (ß-Gluc) to Q aglycone, which easily enters the brain. This study evaluates the effect of lipopolysaccharide (LPS)-induced acute inflammation on ß-Gluc gene expression in the choroid plexus (ChP) and its activity in blood plasma, ChP and cerebrospinal fluid (CSF), and the concentration of Q and its phase II metabolites in blood plasma and CSF. Studies were performed on saline- and LPS-treated adult ewes (n = 40) receiving Q3GA intravenously (n = 16) and on primary rat ChP epithelial cells and human ChP epithelial papilloma cells. We observed that acute inflammation stimulated ß-Gluc activity in the ChP and blood plasma, but not in ChP epithelial cells and CSF, and did not affect Q and its phase II metabolite concentrations in plasma and CSF, except Q3GA, for which the plasma concentration was higher 30 min after administration (p < 0.05) in LPS- compared to saline-treated ewes. The lack of Q3GA deconjugation in the ChP observed under physiological and acute inflammatory conditions, however, does not exclude its possible role in the course of neurodegenerative diseases.
Assuntos
Plexo Corióideo/metabolismo , Glucuronidase/metabolismo , Quercetina/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Plexo Corióideo/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Glucuronidase/sangue , Glucuronidase/líquido cefalorraquidiano , Humanos , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Cultura Primária de Células , Quercetina/análogos & derivados , Quercetina/sangue , Quercetina/líquido cefalorraquidiano , Ratos , Ratos Wistar , OvinosRESUMO
The Gram-negative diplococcus Neisseria meningitidis, also called meningococcus, exclusively infects humans and can cause meningitis, a severe disease that can lead to the death of the afflicted individuals. To cause meningitis, the bacteria have to enter the central nervous system (CNS) by crossing one of the barriers protecting the CNS from entry by pathogens. These barriers are represented by the blood-brain barrier separating the blood from the brain parenchyma and the blood-cerebrospinal fluid (CSF) barriers at the choroid plexus and the meninges. During the course of meningococcal disease resulting in meningitis, the bacteria undergo several interactions with host cells, including the pharyngeal epithelium and the cells constituting the barriers between the blood and the CSF. These interactions are required to initiate signal transduction pathways that are involved during the crossing of the meningococci into the blood stream and CNS entry, as well as in the host cell response to infection. In this review we summarize the interactions and pathways involved in these processes, whose understanding could help to better understand the pathogenesis of meningococcal meningitis.
Assuntos
Barreira Hematoencefálica , Interações Hospedeiro-Patógeno , Meningite Meningocócica/microbiologia , Neisseria meningitidis/fisiologia , Transdução de Sinais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/microbiologia , Plexo Corióideo/metabolismo , Plexo Corióideo/microbiologia , Humanos , Meninges/metabolismo , Meninges/microbiologiaRESUMO
Bacteria evolved many strategies to survive and persist within host cells. Secretion of bacterial effectors enables bacteria not only to enter the host cell but also to manipulate host gene expression to circumvent clearance by the host immune response. Some effectors were also shown to evade the nucleus to manipulate epigenetic processes as well as transcription and mRNA procession and are therefore classified as nucleomodulins. Others were shown to interfere downstream with gene expression at the level of mRNA stability, favoring either mRNA stabilization or mRNA degradation, translation or protein stability, including mechanisms of protein activation and degradation. Finally, manipulation of innate immune signaling and nutrient supply creates a replicative niche that enables bacterial intracellular persistence and survival. In this review, we want to highlight the divergent strategies applied by intracellular bacteria to evade host immune responses through subversion of host gene expression via bacterial effectors. Since these virulence proteins mimic host cell enzymes or own novel enzymatic functions, characterizing their properties could help to understand the complex interactions between host and pathogen during infections. Additionally, these insights could propose potential targets for medical therapy.
Assuntos
Infecções Bacterianas/genética , Interações Hospedeiro-Patógeno , Fatores de Virulência/metabolismo , Animais , Bactérias/patogenicidade , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Epigênese Genética , Humanos , Estabilidade Proteica , Estabilidade de RNARESUMO
Echovirus-30 (E-30) is responsible for the extensive global outbreaks of meningitis in children. To gain access to the central nervous system, E-30 first has to cross the epithelial blood-cerebrospinal fluid barrier. Several meningitis causing bacteria preferentially infect human choroid plexus papilloma (HIBCPP) cells in a polar fashion from the basolateral cell side. Here, we investigated the polar infection of HIBCPP cells with E-30. Both apical and basolateral infections caused a significant decrease in the transepithelial electrical resistance of HIBCPP cells. However, to reach the same impact on the barrier properties, the multiplicity of infection of the apical side had to be higher than that of the basolateral infection. Furthermore, the number of infected cells at respective time-points after basolateral infection was significantly higher compared to apical infection. Cytotoxic effects of E-30 on HIBCPP cells during basolateral infection were observed following prolonged infection and appeared more drastically compared to the apical infection. Gene expression profiles determined by massive analysis of cDNA ends revealed distinct regulation of specific genes depending on the side of HIBCPP cells' infection. Altogether, our data highlights the polar effects of E-30 infection in a human in vitro model of the blood-cerebrospinal fluid barrier leading to central nervous system inflammation.
Assuntos
Barreira Hematoencefálica/virologia , Plexo Corióideo/virologia , Enterovirus Humano B/patogenicidade , Redes Reguladoras de Genes , Adulto , Barreira Hematoencefálica/metabolismo , Polaridade Celular , Sobrevivência Celular , Plexo Corióideo/citologia , Plexo Corióideo/metabolismo , Plexo Corióideo/patologia , Impedância Elétrica , Feminino , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Biológicos , Células Tumorais CultivadasRESUMO
Non-typeable Haemophilus influenzae (NTHI) is a pathogen of the human respiratory tract causing the majority of invasive H. influenzae infections. Severe invasive infections such as septicemia and meningitis occur rarely, but the lack of a protecting vaccine and the increasing antibiotic resistance of NTHI impede treatment and emphasize its relevance as a potential meningitis causing pathogen. Meningitis results from pathogens crossing blood-brain barriers and invading the immune privileged central nervous system (CNS). In this study, we addressed the potential of NTHI to enter the brain by invading cells of the choroid plexus (CP) prior to meningeal inflammation to enlighten NTHI pathophysiological mechanisms. A cell culture model of human CP epithelial cells, which form the blood-cerebrospinal fluid barrier (BCSFB) in vivo, was used to analyze adhesion and invasion by immunofluorescence and electron microscopy. NTHI invade CP cells in vitro in a polar fashion from the blood-facing side. Furthermore, NTHI invasion rates are increased compared to encapsulated HiB and HiF strains. Fimbriae occurrence attenuated adhesion and invasion. Thus, our findings underline the role of the BCSFB as a potential entry port for NTHI into the brain and provide strong evidence for a function of the CP during NTHI invasion into the CNS during the course of meningitis.
Assuntos
Plexo Corióideo/citologia , Plexo Corióideo/microbiologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Infecções por Haemophilus/metabolismo , Haemophilus influenzae/patogenicidade , Interações Hospedeiro-Patógeno , Aderência Bacteriana , Barreira Hematoencefálica , Linhagem Celular Tumoral , Polaridade Celular , Sobrevivência Celular , DNA Bacteriano/genética , Fímbrias Bacterianas , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/genética , Haemophilus influenzae/isolamento & purificação , Humanos , Meningite/líquido cefalorraquidiano , Meningite/microbiologia , Virulência , Fatores de VirulênciaRESUMO
Streptococcus suis is a zoonotic agent causing meningitis in pigs and humans. Neutrophils, as the first line of defense against S. suis infections, release neutrophil extracellular traps (NETs) to entrap pathogens. In this study, we investigated the role of the secreted nuclease A of S. suis (SsnA) as a NET-evasion factor in vivo and in vitro. Piglets were intranasally infected with S. suis strain 10 or an isogenic ssnA mutant. DNase and NET-formation were analyzed in cerebrospinal fluid (CSF) and brain tissue. Animals infected with S. suis strain 10 or S. suis 10ΔssnA showed the presence of NETs in CSF and developed similar clinical signs. Therefore, SsnA does not seem to be a crucial virulence factor that contributes to the development of meningitis in pigs. Importantly, DNase activity was detectable in the CSF of both infection groups, indicating that host nucleases, in contrast to bacterial nuclease SsnA, may play a major role during the onset of meningitis. The effect of DNase 1 on neutrophil functions was further analyzed in a 3D-cell culture model of the porcine blood-CSF barrier. We found that DNase 1 partially contributes to enhanced killing of S. suis by neutrophils, especially when plasma is present. In summary, host nucleases may partially contribute to efficient innate immune response in the CSF.
Assuntos
Proteínas de Bactérias/metabolismo , Desoxirribonuclease I/metabolismo , Meningites Bacterianas/enzimologia , Neutrófilos/enzimologia , Infecções Estreptocócicas/enzimologia , Streptococcus suis/enzimologia , Doenças dos Suínos/enzimologia , Animais , Meningites Bacterianas/genética , Meningites Bacterianas/veterinária , Mutação , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/veterinária , Streptococcus suis/genética , Suínos , Doenças dos Suínos/genéticaRESUMO
The choroid plexus (CP), composed of capillaries surrounded by a barrier epithelium, is the main producer of cerebrospinal fluid (CSF). The CP epithelium regulates the transport of ions and water between the blood and the ventricles, contributing to CSF production and composition. Several studies suggest a connection between the cation channel transient receptor potential vanilloid-4 (TRPV4) and transepithelial ion movement. TRPV4 is a nonselective, calcium-permeable cation channel present in CP epithelia reported to be activated by cytokines and inflammatory mediators. Utilizing the PCP-R (porcine choroid plexus-Riems) cell line, we investigated the effects of various cytokines and inflammatory mediators on TRPV4-mediated activity. Select proinflammatory cytokines (TNF-α, IL-1ß, TGF-ß1) had inhibitory effects on TRPV4-stimulated transepithelial ion flux and permeability changes, whereas anti-inflammatory cytokines (IL-10, IL-4, and IL-6) had none. Quantitative mRNA analysis showed that these cytokines had no effect on TRPV4 transcription levels. Inhibition of the transcription factor NF-κB, involved in the production and regulation of several inflammatory cytokines, inhibited TRPV4-mediated activity, suggesting a link between TRPV4 and cytokine production. Contrary to published studies, the proinflammatory mediator arachidonic acid (AA) had inhibitory rather than stimulatory effects on TRPV4-mediated responses. However, inhibition of AA metabolism also caused inhibitory effects on TRPV4, suggesting a complex interaction of AA and its metabolites in the regulation of TRPV4 activity. Together these data imply that TRPV4 activity is involved in the inflammatory response; it is negatively affected by proinflammatory mediators. Furthermore, arachidonic acid metabolites, but not arachidonic acid itself, are positive regulators of TRPV4.
Assuntos
Plexo Corióideo/metabolismo , Citocinas/metabolismo , Células Epiteliais/metabolismo , Mediadores da Inflamação/metabolismo , Canais de Cátion TRPV/fisiologia , Animais , Linhagem Celular , Plexo Corióideo/citologia , Plexo Corióideo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Leucina/análogos & derivados , Leucina/farmacologia , Sulfonamidas/farmacologia , Suínos , Canais de Cátion TRPV/agonistasRESUMO
The blood-cerebrospinal fluid barrier (BCSFB) plays important roles during the transport of substances into the brain, the pathogenesis of central nervous system (CNS) diseases, and neuro-immunological processes. Along these lines, transmigration of granulocytes across the blood-cerebrospinal fluid (CSF) barrier (BCSFB) is a hallmark of inflammatory events in the CNS. Choroid plexus (CP) epithelial cells are an important tool to generate in vitro models of the BCSFB. A porcine CP epithelial cell line (PCP-R) has been shown to present properties of the BCSFB, including a strong barrier function, when cultivated on cell culture filter inserts containing a membrane with 0.4 µm pore size. For optimal analysis of pathogen and host immune cell interactions with the basolateral side of the CP epithelium, which presents the physiologically relevant "blood side", the CP epithelial cells need to be grown on the lower face of the filter in an inverted cell culture insert model, with the supporting membrane possessing a pore size of at least 3.0 µm. Here, we demonstrate that PCP-R cells cultivated in the inverted model on filter support membranes with a pore size of 3.0 µm following a "conventional" protocol grow through the pores and cross the membrane, forming a second layer on the upper face. Therefore, we developed a cell cultivation protocol, which strongly reduces crossing of the membrane by the cells. Under these conditions, PCP-R cells retain important properties of a BCSFB model, as was observed by the formation of continuous tight junctions and a strong barrier function demonstrated by a high transepithelial electrical resistance and a low permeability for macromolecules. Importantly, compared with the conventional cultivation conditions, our optimized model allows improved investigations of porcine granulocyte transmigration across the PCP-R cell layer.
Assuntos
Barreira Hematoencefálica/fisiologia , Técnicas de Cultura de Células/métodos , Plexo Corióideo/citologia , Células Epiteliais , Granulócitos , Migração Transendotelial e Transepitelial/fisiologia , Animais , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Granulócitos/citologia , Granulócitos/metabolismo , Modelos Biológicos , SuínosRESUMO
BACKGROUND: Echovirus 30 (E-30) is one of the most frequently isolated pathogens in aseptic meningitis worldwide. To gain access to the central nervous system (CNS), E-30 and immune cells have to cross one of the two main barriers of the CNS, the epithelial blood-cerebrospinal fluid barrier (BCSFB) or the endothelial blood-brain barrier (BBB). In an in vitro model of the BCSFB, it has been shown that E-30 can infect human immortalized brain choroid plexus papilloma (HIBCPP) cells. METHODS: In this study we investigated the migration of different T cell subpopulations, naive and effector T cells, through HIBCPP cells during E-30 infection. Effects of E-30 infection and the migration process were evaluated via immunofluorescence and flow cytometry analysis, as well as transepithelial resistance and dextran flux measurement. RESULTS: Th1 effector cells and enterovirus-specific effector T cells migrated through HIBCPP cells more efficiently than naive CD4+ T cells following E-30 infection of HIBCPP cells. Among the different naive T cell populations, CD8+ T cells crossed the E-30-infected HIBCPP cell layer in a significantly higher number than CD4+ T cells. A large amount of effector T cells also remained attached to the basolateral side of the HIBCPP cells compared with naive T cells. Analysis of HIBCPP barrier function showed significant alteration after E-30 infection and trans- as well as paracellular migration of T cells independent of the respective subpopulation. Morphologic analysis of migrating T cells revealed that a polarized phenotype was induced by the chemokine CXCL12, but reversed to a round phenotype after E-30 infection. Further characterization of migrating Th1 effector cells revealed a downregulation of surface adhesion proteins such as LFA-1 PSGL-1, CD44, and CD49d. CONCLUSION: Taken together these results suggest that naive CD8+ and Th1 effector cells are highly efficient to migrate through the BCSFB in an inflammatory environment. The T cell phenotype is modified during the migration process through HIBCPP cells.
Assuntos
Movimento Celular/imunologia , Plexo Corióideo/metabolismo , Plexo Corióideo/virologia , Infecções por Echovirus/imunologia , Linfócitos T/imunologia , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/virologia , Humanos , Linfócitos T/metabolismo , Células Tumorais CultivadasRESUMO
Infections of the central nervous system (CNS) are still a major cause of morbidity and mortality worldwide. Traversal of the barriers protecting the brain by pathogens is a prerequisite for the development of meningitis. Bacteria have developed a variety of different strategies to cross these barriers and reach the CNS. To this end, they use a variety of different virulence factors that enable them to attach to and traverse these barriers. These virulence factors mediate adhesion to and invasion into host cells, intracellular survival, induction of host cell signaling and inflammatory response, and affect barrier function. While some of these mechanisms differ, others are shared by multiple pathogens. Further understanding of these processes, with special emphasis on the difference between the blood-brain barrier and the blood-cerebrospinal fluid barrier, as well as virulence factors used by the pathogens, is still needed.
Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Meningites Bacterianas/metabolismo , Fatores de Virulência/metabolismo , Animais , Sistema Nervoso Central , Líquido Cefalorraquidiano , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Humanos , Meningites Bacterianas/microbiologia , Transdução de Sinais , VirulênciaRESUMO
The choroid plexus (CP) is responsible for the production of a large amount of the cerebrospinal fluid (CSF). As a highly vascularized structure, the CP also presents a significant frontier between the blood and the central nervous system (CNS). To seal this border, the epithelium of the CP forms the blood-CSF barrier, one of the most important barriers separating the CNS from the blood. During the course of infectious disease, cells of the CP can experience interactions with intruding pathogens, especially when the CP is used as gateway for entry into the CNS. In return, the CP answers to these encounters with diverse measures. Here, we will review the distinct responses of the CP during infection of the CNS, which include engaging of signal transduction pathways, the regulation of gene expression in the host cells, inflammatory cell response, alterations of the barrier, and, under certain circumstances, cell death. Many of these actions may contribute to stage an immunological response against the pathogen and subsequently help in the clearance of the infection.
Assuntos
Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar , Infecções do Sistema Nervoso Central/líquido cefalorraquidiano , Plexo Corióideo/irrigação sanguínea , Plexo Corióideo/metabolismo , Animais , Barreira Hematoencefálica/imunologia , Infecções do Sistema Nervoso Central/imunologia , Infecções do Sistema Nervoso Central/patologia , Plexo Corióideo/imunologia , Interações Hospedeiro-Patógeno , Humanos , Prognóstico , Transdução de SinaisRESUMO
The choroid plexus (CP) epithelium plays a major role in the production of cerebrospinal fluid (CSF). A polarized cell line, the porcine CP-Riems (PCP-R) line, which exhibits many of the characteristics of the native epithelium, was used to study the effect of activation of the transient receptor potential vanilloid 4 (TRPV4) cation channel found in the PCP-R cells as well as in the native epithelium. Ussing-style electrophysiological experiments showed that activation of TRPV4 with a specific agonist, GSK1016790A, resulted in an immediate increase in both transepithelial ion flux and conductance. These changes were inhibited by either of two distinct antagonists, HC067047 or RN1734. The change in conductance was reversible and did not involve disruption of epithelial junctional complexes. Activation of TRPV4 results in Ca2+ influx, therefore, we examined whether the electrophysiological changes were the result of secondary activation of Ca2+-sensitive channels. PCP-R cells contain two Ca2+-activated K+ channels, the small conductance 2 (SK2) and the intermediate conductance (IK) channels. Based on inhibitor studies, the former is not involved in the TRPV4-mediated electrophysiological changes whereas one of the three isoforms of the IK channel (KCNN4c) may play a role in the apical secretion of K+. Blocking the activity of this IK isoform with TRAM34 inhibited the TRPV4-mediated change in net transepithelial ion flux and the increased conductance. These studies implicate TRPV4 as a hub protein in the control of CSF production through stimulation by multiple effectors resulting in transepithelial ion and subsequent water movement.
Assuntos
Plexo Corióideo/metabolismo , Células Epiteliais/metabolismo , Potenciais da Membrana/fisiologia , Canais de Cátion TRPV/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Plexo Corióideo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Leucina/análogos & derivados , Leucina/farmacologia , Isoformas de Proteínas/metabolismo , Sulfonamidas/farmacologia , SuínosRESUMO
BACKGROUND: Echovirus (E) 30 (E-30) meningitis is characterized by neuroinflammation involving immune cell pleocytosis at the protective barriers of the central nervous system (CNS). In this context, infection of the blood-cerebrospinal fluid barrier (BCSFB), which has been demonstrated to be involved in enteroviral CNS pathogenesis, may affect the tight junction (TJ) and adherens junction (AJ) function and morphology. METHODS: We used an in vitro human choroid plexus epithelial (HIBCPP) cell model to investigate the effect of three clinical outbreak strains (13-311, 13-759, and 14-397) isolated in Germany in 2013, and compared them to E-30 Bastianni. Conducting transepithelial electrical resistance (TEER), paracellular dextran flux measurement, quantitative real-time polymerase chain reaction (qPCR), western blot, and immunofluorescence analysis, we investigated TJ and AJ function and morphology as well as strain-specific E-30 infection patterns. Additionally, transmission electron and focused ion beam microscopy electron microscopy (FIB-SEM) was used to evaluate the mode of leukocyte transmigration. Genome sequencing and phylogenetic analyses were performed to discriminate potential genetic differences among the outbreak strains. RESULTS: We observed a significant strain-dependent decrease in TEER with strains E-30 Bastianni and 13-311, whereas paracellular dextran flux was only affected by E-30 Bastianni. Despite strong similarities among the outbreak strains in replication characteristics and particle distribution, strain 13-311 was the only outbreak isolate revealing comparable disruptive effects on TJ (Zonula Occludens (ZO) 1 and occludin) and AJ (E-cadherin) morphology to E-30 Bastianni. Notwithstanding significant junctional alterations upon E-30 infection, we observed both para- and transcellular leukocyte migration across HIBCPP cells. Complete genome sequencing revealed differences between the strains analyzed, but no explicit correlation with the observed strain-dependent effects on HIBCPP cells was possible. CONCLUSION: The findings revealed distinct E-30 strain-specific effects on barrier integrity and junctional morphology. Despite E-30-induced barrier alterations leukocyte trafficking did not exclusively occur via the paracellular route.
Assuntos
Barreira Hematoencefálica/virologia , Líquido Cefalorraquidiano/virologia , Plexo Corióideo/virologia , Surtos de Doenças , Enterovirus Humano B/isolamento & purificação , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/ultraestrutura , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Células Cultivadas , Líquido Cefalorraquidiano/metabolismo , Plexo Corióideo/metabolismo , Plexo Corióideo/ultraestrutura , Enterovirus Humano B/metabolismo , Humanos , Filogenia , Especificidade da EspécieRESUMO
The Gram-negative bacterium Haemophilus influenzae (H. influenzae) can commensally colonize the upper respiratory tract, but also cause life threatening disease including epiglottitis, sepsis and meningitis. The H. influenzae capsule protects the bacteria against both phagocytosis and opsonization. Encapsulated H. influenzae strains are classified into serotypes ranging from a to f dependent on their distinct polysaccharide capsule. Due to the implementation of vaccination the incidence of invasive H. influenzae type b (Hib) infections has strongly decreased and infections with other capsulated types, including H. influenzae type f (Hif), are emerging. The pathogenesis of H. influenzae meningitis is not clarified. To enter the central nervous system (CNS) the bacteria generally have to cross either the blood-brain barrier (BBB) or the blood-cerebrospinal fluid barrier (BSCFB). Using a cell culture model of the BCSFB based on human choroid plexus papilloma (HIBCPP) cells and different H. influenzae strains we investigated whether Hib and Hif invade the cells, and if invasion differs between encapsulated vs. capsular-deficient and fimbriated vs. non-fimbriated variants. We find that Hib can adhere to and invade into HIBCPP cells. Invasion occurs in a strongly polar fashion, since the bacteria enter the cells preferentially from the basolateral "blood "side. Fimbriae and capsule attenuate invasion into choroid plexus (CP) epithelial cells, and capsulation can influence the bacterial distribution pattern. Finally, analysis of clinical Hib and Hif isolates confirms the detected invasive properties of H. influenzae. Our data point to roles of capsule and fimbriae during invasion of CP epithelial cells.
Assuntos
Aderência Bacteriana/fisiologia , Cápsulas Bacterianas/patologia , Barreira Hematoencefálica/microbiologia , Fímbrias Bacterianas/patologia , Infecções por Haemophilus/patologia , Haemophilus influenzae/patogenicidade , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Haemophilus influenzae/classificação , Haemophilus influenzae/isolamento & purificação , Interações Hospedeiro-Patógeno/fisiologia , HumanosRESUMO
Porcine infections are currently not the state-of-the-art model to study human diseases. Nevertheless, the course of human and porcine toxoplasmosis is much more comparable than that of human and murine toxoplasmosis. For example, severity of infection, transplacental transmission, and interferon-gamma-induced antiparasitic effector mechanisms are similar in pigs and humans. In addition, the severe immunosuppression during acute infection described in mice does not occur in the experimentally infected ones. Thus, we hypothesise that porcine Toxoplasma gondii infection data are more representative for human toxoplasmosis. We therefore suggest that the animal model chosen must be critically evaluated for its assignability to human diseases.