Chemokine-dependent mechanisms of leukocyte trafficking across a model of the blood-brain barrier

Leukocyte transmigration across the blood-brain barrier (BBB) is a multistep process that can be mediated by chemokines. These low-molecular-weight chemoattractant proteins are secreted by cells within the central nervous system (CNS) in response to injury or on activation. Leukocytes transmigrate toward this chemokine gradient, crossing the BBB and gaining access to the CNS parenchyma. Depending on the chemokine, the nature of the insult, and the type of cell that transmigrates, the BBB integrity may be disrupted, leading to its increased permeability. Both the inflammation resulting from leukocyte transmigration and BBB perturbations contribute to CNS pathology. The mechanisms that mediate leukocyte transmigration and BBB disruption, as well as tissue culture models that are used to study leukocyte trafficking, are the focus of this review.     

Mycobacteria employ two different mechanisms to cross the blood–brain barrier

Central nervous system (CNS) infection by Mycobacterium tuberculosis is one of the most devastating complications of tuberculosis, in particular in early childhood. In order to induce CNS infection, M. tuberculosis needs to cross specialised barriers protecting the brain. How M. tuberculosis crosses the blood–brain barrier (BBB) and enters the CNS is not well understood. Here, we use transparent zebrafish larvae and the closely related pathogen Mycobacterium marinum to answer this question. We show that in the early stages of development, mycobacteria rapidly infect brain tissue, either as free mycobacteria or within circulating macrophages. After the formation of a functionally intact BBB, the infiltration of brain tissue by infected macrophages is delayed, but not blocked, suggesting that crossing the BBB via phagocytic cells is one of the mechanisms used by mycobacteria to invade the CNS. Interestingly, depletion of phagocytic cells did not prevent M. marinum from infecting the brain tissue, indicating that free mycobacteria can independently cause brain infection. Detailed analysis showed that mycobacteria are able to cause vasculitis by extracellular outgrowth in the smaller blood vessels and by infecting endothelial cells. Importantly, we could show that this second mechanism is an active process that depends on an intact ESX‐1 secretion system, which extends the role of ESX‐1 secretion beyond the macrophage infection cycle.     

Immune response and blood–brain barrier dysfunction during viral neuroinvasion

The blood-brain barrier (BBB), which protects the CNS from pathogens, is composed of specialized brain microvascular endothelial cells (BMECs) joined by tight junctions and ensheathed by pericytes and astrocyte endfeet. The stability of the BBB structure and function is of great significance for the maintenance of brain homeostasis. When a neurotropic virus invades the CNS via a hematogenous or non-hematogenous route, it may cause structural and functional disorders of the BBB, and also activate the BBB anti-inflammatory or pro-inflammatory innate immune response. This article focuses on the structural and functional changes that occur in the three main components of the BBB (endothelial cells, astrocytes, and pericytes) in response to infection with neurotropic viruses transmitted by hematogenous routes, and also briefly describes the supportive effect of three cells on the BBB under normal physiological conditions. For example, all three types of cells express several PRRs, which can quickly sense the virus and make corresponding immune responses. The pro-inflammatory immune response will exacerbate the destruction of the BBB, while the anti-inflammatory immune response, based on type I IFN, consolidates the stability of the BBB. Exploring the details of the interaction between the host and the pathogen at the BBB during neurotropic virus infection will help to propose new treatments for viral encephalitis. Enhancing the defense function of the BBB, maintaining the integrity of the BBB, and suppressing the pro-inflammatory immune response of the BBB provide more ideas for limiting the neuroinvasion of neurotropic viruses. In the future, these new treatments are expected to cooperate with traditional antiviral methods to improve the therapeutic effect of viral encephalitis.     

Characteristics of glycine transport across the inner blood–retinal barrier

Although glycine plays a pivotal role in neurotransmission and neuromodulation in the retina and is present in high concentration in the retina, the source of retinal glycine is still unclear. The purpose of the present study was to investigate glycine transport across the inner blood–retinal barrier (inner BRB). [¹⁴C]Glycine transport at the inner BRB was characterized using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells) as an in vitro model of the inner BRB and in vivo vascular injection techniques. [¹⁴C]Glycine uptake by TR-iBRB2 cells was Na⁺- and Cl⁻-dependent, and concentration-dependent with Michaelis–Menten constants of 55.4 μM and 8.02 mM, and inhibited by glycine transporter 1 (GlyT1) and system A inhibitors. These uptake studies suggest that GlyT1 and system A are involved in [¹⁴C]glycine uptake by TR-iBRB2 cells. RT-PCR analysis demonstrated that GlyT1 and system A (encoding ATA 1 and ATA2) mRNA are expressed in TR-iBRB2 cells. An in vivo study suggested that [¹⁴C]glycine is transported from blood to the retina whereas [¹⁴C]α-methylaminoisobutyric acid, a selective substrate for system A, is not. In conclusion, GlyT1 most likely mediates glycine transport at the inner BRB and is expected to play an important role in regulating the glycine concentration in the neural retina.     

Microfluidic blood–brain barrier model provides in vivo‐like barrier properties for drug permeability screening

Efficient delivery of therapeutics across the neuroprotective blood–brain barrier (BBB) remains a formidable challenge for central nervous system drug development. High‐fidelity in vitro models of the BBB could facilitate effective early screening of drug candidates targeting the brain. In this study, we developed a microfluidic BBB model that is capable of mimicking in vivo BBB characteristics for a prolonged period and allows for reliable in vitro drug permeability studies under recirculating perfusion. We derived brain microvascular endothelial cells (BMECs) from human induced pluripotent stem cells (hiPSCs) and cocultured them with rat primary astrocytes on the two sides of a porous membrane on a pumpless microfluidic platform for up to 10 days. The microfluidic system was designed based on the blood residence time in human brain tissues, allowing for medium recirculation at physiologically relevant perfusion rates with no pumps or external tubing, meanwhile minimizing wall shear stress to test whether shear stress is required for in vivo‐like barrier properties in a microfluidic BBB model. This BBB‐on‐a‐chip model achieved significant barrier integrity as evident by continuous tight junction formation and in vivo‐like values of trans‐endothelial electrical resistance (TEER). The TEER levels peaked above 4000 Ω · cm² on day 3 on chip and were sustained above 2000 Ω · cm² up to 10 days, which are the highest sustained TEER values reported in a microfluidic model. We evaluated the capacity of our microfluidic BBB model to be used for drug permeability studies using large molecules (FITC‐dextrans) and model drugs (caffeine, cimetidine, and doxorubicin). Our analyses demonstrated that the permeability coefficients measured using our model were comparable to in vivo values. Our BBB‐on‐a‐chip model closely mimics physiological BBB barrier functions and will be a valuable tool for screening of drug candidates. The residence time‐based design of a microfluidic platform will enable integration with other organ modules to simulate multi‐organ interactions on drug response. Biotechnol. Bioeng. 2017;114: 184–194. © 2016 Wiley Periodicals, Inc.     

Control of the blood–brain barrier function in cancer cell metastasis

Cerebral metastases are the most common brain neoplasms seen clinically in the adults and comprise more than half of all brain tumours. Actual treatment options for brain metastases that include surgical resection, radiotherapy and chemotherapy are rarely curative, although palliative treatment improves survival and life quality of patients carrying brain‐metastatic tumours. Chemotherapy in particular has also shown limited or no activity in brain metastasis of most tumour types. Many chemotherapeutic agents used systemically do not cross the blood–brain barrier (BBB), whereas others may transiently weaken the BBB and allow extravasation of tumour cells from the circulation into the brain parenchyma. Increasing evidence points out that the interaction between the BBB and tumour cells plays a key role for implantation and growth of brain metastases in the central nervous system. The BBB, as the tightest endothelial barrier, prevents both early detection and treatment by creating a privileged microenvironment. Therefore, as observed in several in vivo studies, precise targetting the BBB by a specific transient opening of the structure making it permeable for therapeutic compounds, might potentially help to overcome this difficult clinical problem. Moreover, a better understanding of the molecular features of the BBB, its interrelation with metastatic tumour cells and the elucidation of cellular mechanisms responsible for establishing cerebral metastasis must be clearly outlined in order to promote treatment modalities that particularly involve chemotherapy. This in turn would substantially expand the survival and quality of life of patients with brain metastasis, and potentially increase the remission rate. Therefore, the focus of this review is to summarise the current knowledge on the role and function of the BBB in cancer metastasis.     

Strategies used by bacterial pathogens to cross the blood–brain barrier

The skull, spine, meninges, and cellular barriers at the blood–brain and the blood–cerebrospinal fluid interfaces well protect the brain and meningeal spaces against microbial invasion. However, once in the bloodstream, a range of pathogenic bacteria is able to reach the brain and cause meningitis. Despite advances in antibacterial therapy, bacterial meningitis remains one of the most important infectious diseases worldwide. The most common causative bacteria in children and adults are Streptococcus pneumoniae and Neisseria meningitidis associated with high morbidity and mortality, while among neonates, most cases of bacterial meningitis are due to group B Streptococcus and Escherichia coli. Here we summarise our current knowledge on the strategies used by these bacterial pathogens to survive in the bloodstream, to colonise the brain vasculature and to cross the blood–brain barrier.     

Characterisation of the interaction between circulating and in vitro cultivated endothelial progenitor cells and the endothelial barrier

In vitro cultured endothelial progenitor cells (cEPC) are used for intracoronary cell therapy in cardiac regeneration. The aim of this study was to investigate whether cEPC and circulating mononuclear cells (MNC), which include a small number of in vivo circulating EPC, are able to transmigrate through the endothelial barrier into the cardiac tissue. MNC and EPC were isolated from the peripheral blood from healthy male volunteers (n = 13, 25+/-6 years) and stained with a fluorescent marker. The cells were perfused in vitro through organs with endothelial layers of different phenotypes (rat aorta, human umbilical vein, isolated mouse heart). The endothelium and the basal lamina were then stained by immunofluorescence and the cryo-sections analysed using a confocal laser scanning microscope. After perfusion through the rat aorta, an adhesion/integration of MNC was observed at the endothelial layer and the basal lamina beneath endothelial cells. However, no migration of MNC over the endothelial barrier was found. This remained true even when the cell numbers were increased (from 0.5 to 10 million cells/h), when the time of perfusion was prolonged (1.5-4 h) and when the aorta was cultivated for 24 h. In the Langendorff-perfused mouse heart with intact endothelium, no migration of MNC (1 x 10(7)) or cEPC (1 x 10(6)) was observed after 0.5 and 2 h. In conclusion, MNC and cEPC do not possess any capacity to transmigrate the endothelial barrier. In the context of stem cell therapy, these cells may therefore serve as endothelial regenerators but not as cardiomyocyte substitutes.     

Hydrogen sulfide protects blood–brain barrier integrity following cerebral ischemia

By using two structurally unrelated hydrogen sulfide (H₂S) donors 5‐(4‐methoxyphenyl) ‐3H‐1, 2‐dithiole‐3‐thione (ADT) and sodium hydrosulfide (NaHS), this study investigated if H₂S protected blood–brain barrier (BBB) integrity following middle cerebral artery occlusion (MCAO). ICR mice underwent MCAO and received H₂S donors at 3 h after reperfusion. Infarction, neurological scores, brain edema, Evans blue (EB) extravasation, and tight junction protein expression were examined at 48 h after MCAO. We also investigated if ADT protected BBB integrity by suppressing post‐ischemic inflammation‐induced Matrix Metalloproteimase‐9 (MMP9) and Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). ADT increased blood H₂S concentrations, decreased infarction, and improved neurological deficits. Particularly, ADT reduced EB extravasation, brain edema and preserved expression of tight junction proteins in the ischemic brain. NaHS also increased blood H₂S levels and reduced EB extravasation following MCAO. Moreover, ADT inhibited expression of pro‐inflammatory markers induced Nitric Oxide Synthase (iNOS) and IL‐1β while enhanced expression of anti‐inflammatory markers arginase 1 and IL‐10 in the ischemic brain. Accordingly, ADT attenuated ischemia‐induced expression and activity of MMP9. Moreover, ADT reduced NOX‐4 mRNA expression, NOX activity, and inhibited nuclear translocation of Nuclear Factor Kappa‐B (NF‐κB) in the ischemic brain. In conclusion, H₂S donors protected BBB integrity following experimental stroke possibly by acting through NF‐κB inhibition to suppress neuroinflammation induction of MMP9 and NOX4‐derived free radicals.

     

Identification and quantification of blood–brain barrier transporters in isolated rat brain microvessels

The blood–brain barrier (BBB ) maintains brain homeostasis by tightly regulating the exchange of molecules with systemic circulation. It consists primarily of microvascular endothelial cells surrounded by astrocytic endfeet, pericytes, and microglia. Understanding the make‐up of transporters in rat BBB is essential to the translation of pharmacological and toxicological observations into humans. In this study, experimental workflows are presented in which the optimization of (a) isolation of rat brain microvessels (b) enrichment of endothelial cells, and (c) extraction and digestion of proteins were evaluated, followed by identification and quantification of BBB proteins. Optimization of microvessel isolation was indicated by 15‐fold enrichment of endothelial cell marker Glut1 mRNA , whereas markers for other cell types were not enriched. Filter‐aided sample preparation was shown to be superior to in‐solution sample preparation (10251 peptides vs. 7533 peptides). Label‐free proteomics was used to identify nearly 2000 proteins and quantify 1276 proteins in isolated microvessels. A combination of targeted and global proteomics was adopted to measure protein abundance of 6 ATP‐binding cassette and 27 solute carrier transporters. Data analysis using proprietary Progenesis and open access MaxQuant software showed overall agreement; however, Abcb9 and Slc22a8 were quantified only by MaxQuant, whereas Abcc9 and Abcd3 were quantified only by Progenesis. Agreement between targeted and untargeted quantification was demonstrated for Abcb1 (19.7 ± 1.4 vs. 17.8 ± 2.3) and Abcc4 (2.2 ± 0.7 vs. 2.1 ± 0.4), respectively. Rigorous quantification of BBB proteins, as reported in this study, should assist with translational modeling efforts involving brain disposition of xenobiotics.

     

Staphylococcus aureus‐mediated blood–brain barrier injury: an in vitro human brain microvascular endothelial cell model

Blood–brain barrier (BBB) disruption constitutes a hallmark event during pathogen‐mediated neurological disorders such as bacterial meningitis. As a prevalent opportunistic pathogen, Staphylococcus aureus (SA) is of particular interest in this context, although our fundamental understanding of how SA disrupts the BBB is very limited. This paper employs in vitro infection models to address this. Human brain microvascular endothelial cells (HBMvECs) were infected with formaldehyde‐fixed (multiplicity of infection [MOI] 0–250, 0–48 hr) and live (MOI 0–100, 0–3 hr) SA cultures. Both Fixed‐SA and Live‐SA could adhere to HBMvECs with equal efficacy and cause elevated paracellular permeability. In further studies employing Fixed‐SA, infection of HBMvECs caused dose‐dependent release of cytokines/chemokines (TNF‐α, IL‐6, MCP‐1, IP‐10, and thrombomodulin), reduced expression of interendothelial junction proteins (VE‐Cadherin, claudin‐5, and ZO‐1), and activation of both canonical and non‐canonical NF‐κB pathways. Using N‐acetylcysteine, we determined that these events were coupled to the SA‐mediated induction of reactive oxygen species (ROS) within HBMvECs. Finally, treatment of HBMvECs with Fixed‐ΔSpA (MOI 0–250, 48 hr), a gene deletion mutant of Staphylococcal protein A associated with bacterial infectivity, had relatively similar effects to Newman WT Fixed‐SA. In conclusion, these findings provide insight into how SA infection may activate proinflammatory mechanisms within the brain microvascular endothelium to elicit BBB failure.     

Type 1 diabetes exacerbates blood–brain barrier alterations during experimental epileptic seizures in an animal model

The aim of this study was to perform the effects of diabetes on the permeability of the blood–brain barrier (BBB) during pentylenetetrazole (PTZ)‐induced epileptic attacks. For this propose, the animals were divided into four groups. These groups contained were intact, PTZ‐treated, diabetic and PTZ‐treated diabetic individuals, respectively. To evaluate the functioning of the BBB, Evans blue was used as a BBB permeability indicator, and the expressions of zonula occludens‐1 and glial fibrillary acidic protein involving the functioning of the BBB were determined immunohistochemically. Also, the changes in the release of serum tumour necrosis factor‐alpha and interleukin‐10 and interleukin‐12 were studied by using enzyme‐linked immunosorbent assay method. BBB permeability in the seizures under diabetic conditions showed a considerable increase (p < 0·01) in all of the brain we studied. The immunoreactive staining intensity of zonula occludens‐1 and glial fibrillary acidic protein was found reduced in the brain regions of diabetic rats (p < 0·01). However, the serum level of tumour necrosis factor‐alpha increased in diabetes and diabetes + PTZ groups, and the serum level of interleukin‐12 increased significantly in all experimental groups (p < 0·05). In conclusion, diabetes dramatically increases BBB damage during epileptic seizures, and it may be derived from an elevation of paracellular passage. Copyright © 2015 John Wiley & Sons, Ltd.     

A putative nicotine pump at the metabolic blood–brain barrier of the tobacco hornworm

In mammals, P-glycoprotein immunostaining at the blood–brain barrier has implicated the multidrug pump in the restricted movement of many cytotoxic agents into the central nervous system (NCS). Since many insects require as sophisticated blood–brain barrier system to protect their CNS from plant-derived neurotoxins, we have investigated the possibility that a P-glycoprotein homolog constitutes a component of the insect blood–brain barrier. We have used the nicotine-resistant tobacco hornworm (Manduca sexta) to address this issue. Manduca has been previously shown, in physiological studies, to have an alkaloid (nicotine/morphine/atropine) pump at its excretory malpighian tubules. We show (1) that the tubules are P-glycoprotein immunopositive, (2) that Manduca has a metabolic blood–brain barrier for nicotine, (3) that the barrier co-localizes with P-glycoprotein immunostaining, and (4) that detoxifying enzymes as well as the nicotine pump are likely to account for the metabolic blood–brain to nicotine. These findings may provide insights on two major fronts, the troublesome problem of multi-insecticide resistance, a phenomenon that parallels multidrug resistance in tumor cells, and the problem of tolerance to addictive neuroactive drugs like nicotine or morphine. 1994 John Wiley & Sons, Inc.