miR-27a-3p regulates expression of intercellular junctions at the brain endothelium and controls the endothelial barrier permeability

Background The brain endothelial barrier permeability is governed by tight and adherens junction protein complexes that restrict paracellular permeability at the blood-brain barrier (BBB). Dysfunction of the inter-endothelial junctions has been implicated in neurological disorders such as multiple sclerosis, stroke and Alzheimer’s disease. The molecular mechanisms underlying junctional dysfunction during BBB impairment remain elusive. MicroRNAs (miRNAs) have emerged as versatile regulators of the BBB function under physiological and pathological conditions, and altered levels of BBB-associated microRNAs were demonstrated in a number of brain pathologies including neurodegeneration and neuroinflammatory diseases. Among the altered micro-RNAs, miR-27a-3p was found to be downregulated in a number of neurological diseases characterized by loss of inter-endothelial junctions and disruption of the barrier integrity. However, the relationship between miR-27a-3p and tight and adherens junctions at the brain endothelium remains unexplored. Whether miR-27a-3p is involved in regulation of the junctions at the brain endothelium remains to be determined. Methods Using a gain-and-loss of function approach, we modulated levels of miR-27a-3p in an in-vitro model of the brain endothelium, key component of the BBB, and examined the resultant effect on the barrier paracellular permeability and on the expression of essential tight and adherens junctions. The mechanisms governing the regulation of junctional proteins by miR-27a-3p were also explored. Results Our results showed that miR-27a-3p inhibitor increases the barrier permeability and causes reduction of claudin-5 and occludin, two proteins highly enriched at the tight junction, while miR-27a-3p mimic reduced the paracellular leakage and increased claudin-5 and occludin protein levels. Interestingly, we found that miR-27-3p induces expression of claudin-5 and occludin by downregulating Glycogen Synthase Kinase 3 beta (GSK3ß) and activating Wnt/ß-catenin signaling, a key pathway required for the BBB maintenance. Conclusion For the first time, we showed that miR-27a-3p is a positive regulator of key tight junction proteins, claudin-5 and occludin, at the brain endothelium through targeting GSK3ß gene and activating Wnt/ß-catenin signaling. Thus, miR-27a-3p may constitute a novel therapeutic target that could be exploited to prevent BBB dysfunction and preserves its integrity in neurological disorders characterized by impairment of the barrier’s function.

Transcriptome analysis of Plasmodium falciparum isolates from Benin reveals specific gene expression associated with cerebral malaria

The host and parasitic factors leading to cerebral malaria (CM) are not yet fully elucidated and CM Plasmodium falciparum isolates transcriptome profile remains largely unknown. Based on RNA-seq data from 15 CM and 15 uncomplicated malaria (UM) children from Benin, we identified an increased ring stage signature in CM parasites. Reduced circulating time may result from a higher adherence ability of CM isolates and consistent with this hypothesis, we measured an overexpression of var genes in CM. var genes domains expression was more restricted in CM isolates compared to UM, reflecting the specific binding to receptors in host brain endothelium capillaries. However, ICAM-1 binding motif was found expressed in both CM and UM, questioning its role in PfEMP1 adhesion to ICAM-1 receptor. UM isolates increased circulation time may also be modulated by a more efficient immune response against infected erythrocytes surface proteins, which we could not demonstrate on our cohort. Identification of deregulated genes involved in adhesion, excluding variant surface antigens, also supports the hypothesis of an increased CM adhesion capacity. Finally, numerous upregulated genes involved in entry into host pathway were found, reflecting a greater erythrocytes invasion capacity of CM parasites.

Pyruvate oxidase as a key determinant of pneumococcal viability during transcytosis across brain endothelium.

Streptococcus pneumoniae (SPN/pneumococcus), invades myriad of host tissues following efficient breaching of cellular barriers. However, strategies adopted by pneumococcus for evasion of host intracellular defences governing successful transcytosis across host cellular barriers remain elusive. In this study, using brain endothelium as a model host barrier, we observed that pneumococcus containing endocytic vacuoles (PCVs) formed following SPN internalization into brain microvascular endothelial cells (BMECs), undergo early maturation and acidification, with a major subset acquiring lysosome-like characteristics. Exploration of measures that would preserve pneumococcal viability in the lethal acidic pH of these lysosome-like vacuoles revealed a critical role of the two-component system response regulator, CiaR, which has been previously implicated in induction of acid tolerance response. Pyruvate oxidase (SpxB), a key sugar metabolizing enzyme that catalyses oxidative decarboxylation of pyruvate to acetyl phosphate, was found to contribute to acid stress tolerance, presumably via acetyl phosphate-mediated phosphorylation and activation of CiaR, independent of its cognate kinase CiaH. Hydrogen peroxide, the by-product of SpxB catalysed reaction, was also found to improve pneumococcal intracellular survival by oxidative inactivation of lysosomal cysteine cathepsins, thus compromising the degradative capacity of the host lysosomes. Expectedly, a Δ spxB mutant was found to be significantly attenuated in its ability to survive inside the BMEC endocytic vacuoles, reflecting in its reduced transcytosis ability. Collectively, our studies establish SpxB as an important virulence determinant facilitating pneumococcal survival inside host cells, ensuring successful trafficking across host cellular barriers. IMPORTANCE Host cellular barriers have innate immune defences to restrict microbial passage into sterile compartments. Here, by focussing on the blood-brain barrier endothelium, we investigated mechanisms which enable Streptococcus pneumoniae to traverse through host barriers. Pyruvate oxidase, a pneumococcal sugar metabolizing enzyme was found to play a crucial role in this, via generation of acetyl phosphate and hydrogen peroxide. A two-pronged approach consisting of acetyl phosphate-mediated activation of acid tolerance response and hydrogen peroxide-mediated inactivation of lysosomal enzymes enabled pneumococci to maintain viability inside the degradative vacuoles of the brain endothelium, for successful transcytosis across the barrier. Thus, pyruvate oxidase is a key virulence determinant and can potentially serve as a viable candidate for therapeutic interventions for better management of invasive pneumococcal diseases.

Vimentin regulates chemokine expression and NOD2 activation in brain endothelium during Group B streptococcal infection.

Streptococcus agalactiae (Group B Streptococcus , GBS), is an opportunistic pathogen capable of causing invasive disease in susceptible individuals including the newborn. Currently GBS is the leading cause of meningitis in the neonatal period. We have recently shown that GBS interacts directly with host type III intermediate filament vimentin to gain access to the central nervous system. This results in characteristic meningeal inflammation and disease progression; however, the specific role of vimentin in the inflammatory process is unknown. Here we investigate the contribution of vimentin to the pathogenesis of GBS meningitis. We show that a CRISPR targeted deletion of vimentin in human cerebral microvascular endothelial cells (hCMEC) reduced GBS induction of neutrophil attractants IL-8 and CXCL-1, as well as NFκB activation. We further show that inhibition of vimentin localization also prevented similar chemokine activation by GBS. One known chemokine regulator is the nucleotide-binding oligomerization domain containing protein 2 (NOD2), which is known to interact directly with vimentin. Thus, we hypothesized that NOD2 would also promote GBS chemokine induction. We show that GBS infection induced NOD2 transcription in hCMEC comparable to the muramyl dipeptide (MDP) NOD2 agonist, and the chemokine induction was reduced in the presence of a NOD2 inhibitor. Using a mouse model of GBS meningitis we also observed increased NOD2 transcript and NOD2 activation in brain tissue of infected mice. Lastly, we show that NOD2 mediated IL8 and CXCL1 induction required vimentin, further indicating the importance of vimentin in mediating inflammatory responses in brain endothelium.

Methyl-Beta-Cyclodextrin Restores KIR Channel Function in Brain Endothelium of Female Alzheimer’s Disease Mice

Background: As the sixth-leading cause of death in the United States, Alzheimer’s disease (AD) entails deteriorating endothelial control of blood flow throughout the brain. In particular, reduced inward-rectifying K+ (KIR) channel function in animal models of aging and AD compromises endothelial function and optimal perfusion of brain parenchyma. Deficient endothelial KIR channels may result from aberrant interaction with plasma membrane cholesterol as a primary regulator of membrane fluidity and ion channels. Objective: We tested the hypothesis that mild methyl-β-cyclodextrin (MβCD) treatment to reduce membrane cholesterol may restore endothelial KIR channel function in brain endothelium of old AD mice. Methods: Membrane potential was continuously measured in isolated endothelial tubes from posterior cerebral arteries of young (1 to 3 months) and old (16 to 19 months) female 3xTg-AD mice before and after mild treatment with the cholesterol-removing agent MβCD (1 mmol/L). Elevated extracellular potassium ([K+]E; 15 mmol/L) and NS309 (1μmol/L) activated KIR and Ca2+-activated K+ (SKCa/IKCa) channels respectively before and after MβCD treatment. Results: SKCa/IKCa channel function for producing hyperpolarization remained stable regardless of age group and MβCD treatment (ΔVm: ∼–33 mV). However, as deficient during AD, KIR channel function was restored (ΔVm: –9±1 mV) versus pre-MβCD conditions (–5±1 mV); a progressive effect that reached –14±1 mV hyperpolarization at 60 min following MβCD washout. Conclusion: In female animals, MβCD treatment of brain endothelium selectively restores KIR versus SKCa/IKCa channel function during AD. Thus, the endothelial cholesterol-KIR channel interface is a novel target for ameliorating perfusion of the AD brain.

Insights Into the Mechanisms of Brain Endothelial Erythrophagocytosis

The endothelial cells which form the inner cellular lining of the vasculature can act as non-professional phagocytes to ingest and remove emboli and aged/injured red blood cells (RBCs) from circulation. We previously demonstrated an erythrophagocytic phenotype of the brain endothelium for oxidatively stressed RBCs with subsequent migration of iron-rich RBCs and RBC degradation products across the brain endothelium in vivo and in vitro, in the absence of brain endothelium disruption. However, the mechanisms contributing to brain endothelial erythrophagocytosis are not well defined, and herein we elucidate the cellular mechanisms underlying brain endothelial erythrophagocytosis. Murine brain microvascular endothelial cells (bEnd.3 cells) were incubated with tert-butyl hydroperoxide (tBHP, oxidative stressor to induce RBC aging in vitro)- or PBS (control)-treated mouse RBCs. tBHP increased the reactive oxygen species (ROS) formation and phosphatidylserine exposure in RBCs, which were associated with robust brain endothelial erythrophagocytosis. TNFα treatment potentiated the brain endothelial erythrophagocytosis of tBHP-RBCs in vitro. Brain endothelial erythrophagocytosis was significantly reduced by RBC phosphatidylserine cloaking with annexin-V and with RBC-ROS and phosphatidylserine reduction with vitamin C. Brain endothelial erythrophagocytosis did not alter the bEnd.3 viability, and tBHP-RBCs were localized with early and late endosomes. Brain endothelial erythrophagocytosis increased the bEnd.3 total iron pool, abluminal iron levels without causing brain endothelial monolayer disruption, and ferroportin levels. In vivo, intravenous tBHP-RBC injection in aged (17–18 months old) male C57BL/6 mice significantly increased the Prussian blue-positive iron-rich lesion load compared with PBS-RBC-injected mice. In conclusion, RBC phosphatidylserine exposure and ROS are key mediators of brain endothelial erythrophagocytosis, a process which is associated with increased abluminal iron in vitro. tBHP-RBCs result in Prussian blue-positive iron-rich lesions in vivo. Brain endothelial erythrophagocytosis may provide a new route for RBC/RBC degradation product entry into the brain to produce iron-rich cerebral microhemorrhage-like lesions.

Differential ubiquitination as an effective strategy employed by Blood-Brain Barrier for prevention of bacterial transcytosis

The protective mechanisms of blood-brain barrier (BBB) prohibiting entry of blood borne pathogens and toxins into the central nervous system (CNS) is critical for maintenance of homeostasis in the brain. These include various forms of intracellular defence mechanisms which are vital to block bacterial transcytosis, the major route of trafficking adopted by meningeal pathogens to transit into the CNS. However, mechanistic details of the defence mechanisms and their exploitation to prevent bacterial meningitis remain unexplored. In this study, we established that brain endothelium driven ubiquitination acts as a major intracellular defence mechanism for clearance of S. pneumoniae, a critical neurotropic pathogen, during its transit through BBB. Our findings suggest that brain endothelium employs differential ubiquitination with either K48 or K63-Ub chain topologies as an effective strategy to target SPN towards diverse killing pathways. While K63-Ub decoration triggers autophagic killing, K48-Ub directs pneumococcus exclusively to the proteasome machinery. Indeed, time lapse fluorescence imaging involving proteasomal marker LMP2 revealed that in BBB, majority of the ubiquitinated SPN were cleared by proteasome. Fittingly, pharmacological inhibition of proteasome and autophagy pathway not only led to exclusive accumulation of K48-Ub and K63-Ub marked SPN, respectively, but also triggered significant increment in intracellular SPN burden. Moreover, genetic impairment of formation of either K48 or K63-Ub chain topology demonstrated that though both chain types play important roles in disposal of intracellular SPN, K48-Ub chains and subsequent proteasomal degradation has more pronounced contribution towards ubiquitinated SPN killing in brain endothelium. Collectively, these observations for the first time illustrated a pivotal role of differential ubiquitination in orchestrating a symphony of intracellular defence mechanisms blocking pathogen trafficking into the brain which could be further exploited to prevent bacterial CNS infections.

A type VII secretion system in Group B Streptococcus mediates cytotoxicity and virulence

Type VII secretion systems (T7SS) have been identified in Actinobacteria and Firmicutes and have been shown to secrete effector proteins with functions in virulence, host toxicity, or interbacterial killing in a few genera. Bioinformatic analysis indicates that Group B streptococcal (GBS) isolates encode four distinct subtypes of T7SS machinery, three of which encode adjacent putative T7SS effectors with WXG and LXG motifs. However, the function of T7SS in GBS pathogenesis is not known. Here we show that the most abundant GBS T7SS subtype is important for virulence and cytotoxicity in brain endothelium and that these phenotypes are dependent on the WXG100 effector EsxA. We further show that the WXG motif is required for cytotoxicity in brain endothelium and that EsxA is a pore-forming protein. This work reveals the importance of a T7SS in host-GBS interactions and has implications for the functions of T7SS effectors in other Gram-positive bacteria.

Comparison of polypeptides that bind the transferrin receptor for targeting gold nanocarriers

The ability to target therapeutic agents to specific tissues is an important element in the development of new disease treatments. The transferrin receptor (TfR) is one potential target for drug delivery, as it expressed on many dividing cells and on brain endothelium, the key cellular component of the blood-brain barrier. The aim of this study was to compare a set of new and previously-described polypeptides for their ability to bind to brain endothelium, and investigate their potential for targeting therapeutic agents to the CNS. Six polypeptides were ranked for their rate of endocytosis by the human brain endothelial cell line hCMEC/D3 and the murine line bEnd.3. One linear polypeptide and two cyclic polypeptides showed high rates of uptake. These peptides were investigated to determine whether serum components, including transferrin itself affected uptake by the endothelium. One of the cyclic peptides was strongly inhibited by transferrin and the other cyclic peptide weakly inhibited. As proof of principle the linear peptide was attached to 2nm glucose coated gold-nanoparticles, and the rate of uptake of the nanoparticles measured in a hydrogel model of the blood-brain barrier. Attachment of the TfR-targeting polypeptide significantly increased the rates of endocytosis by brain endothelium and increased movement of nanoparticles across the cells.