The blood-brain barrier (BBB) is a complex vascular structure consisting of microvascular endothelial cells that line the vessel wall, astrocyte end-feet, pericytes, as well as the basal lamina. BBB cells act in concert to maintain the characteristic impermeable and low paracellular flux of the brain vascular network, thus ensuring a homeostatic neuronal environment. Alterations in BBB stability that occur during injury have dire consequences on disease progression and it is clear that BBB cell-specific responses, positive or negative, must make a significant contribution to injury outcome. Reduced oxygenation, or hypoxia, is a characteristic of many brain diseases that significantly increases barrier permeability. Recent data suggest that hypoxia-inducible factor (HIF-1), the master regulator of the hypoxic response, probably mediates many hypoxic effects either directly or indirectly via its target genes. This review discusses current knowledge of physiological cell-specific regulation of barrier function, their responses to hypoxia as well as consequences of hypoxic-and HIF-1-mediated mechanisms on barrier integrity during select brain diseases. In the final sections, the potential of current advances in targeting HIF-1 as a therapeutic strategy will be overviewed. AbbreviationsBBB, blood-brain barrier; HIF-1, hypoxia-inducible factor-1; TJ, tight junction; ECs, endothelial cells; TEER, transendothelial electrical resistanceThe maintenance of CNS homeostasis is performed largely by the blood-brain barrier (BBB), which together with neurons and microglia form an organization referred to as the neurovascular unit (NVU). The BBB is dynamic performing both passive and active features of the brain endothelium essentially acting as a vascular gatekeeper that controls movement of substances from the circulating blood into the brain parenchyma -a role crucial for neuronal, and therefore CNS, homeostasis. Accumulating experimental evidence supports the hypothesis that opening of the BBB triggers a chain of events leading to neuronal dysfunction and damage resulting in neurological disease, and when coupled with previous insults BBB disruption could have serious detrimental consequences for patient outcome. Despite this knowledge, our understanding of physiological barrier function, as well as during disease, is very limited. In addition, the contribution of the perivascular cells that modulate barrier characteristics and their individual responses to injury is poorly characterized. This review will discuss the mechanisms through which hypoxia, a characteristic state of many brain diseases, disrupts barrier function and the importance of BBB cell-specific responses to barrier integrity. Additionally, consequences of hypoxia-mediated barrier modulation during brain disease and future therapeutic use of hypoxia-inducible factor-1 (HIF-1) modulators in the clinics will be reviewed. Physiology BBB organization and cell-specific functionThe BBB is a complex structure consisting of microvascular endothelial cells (ECs) that ...

The blood-brain barrier (BBB) constitutes a critical barrier for the maintenance of central nervous system homeostasis. Brain microvascular endothelial cells line the vessel walls and express tight junction (TJ) complexes that restrict paracellular passage across the BBB, thereby fulfilling a crucial role in ensuring brain function. Hypoxia, an impaired O(2) delivery, is known to cause BBB dysfunction but the mechanisms that drive this disruption remain unclear. This study discloses the relevance of the master regulator of the hypoxic response, hypoxia-inducible factor-1 (HIF-1), in hypoxia-induced barrier disruption using the rat brain endothelial cell line RBE4. Hypoxic exposure rapidly induced stabilization of the HIF-1 oxygen-dependent alpha subunit (HIF-1α) concomitantly with BBB impairment and TJ disruption mainly through delocalization and increased tyrosine phosphorylation of TJ proteins. Similar observations were obtained by normoxic stabilization of HIF-1α using CoCl(2), deferoxamine, and dimethyloxalylglycine underlining the involvement of HIF-1 in barrier dysfunction particularly via TJ alterations. In agreement inhibition of HIF-1 stabilization by 2-methoxyestradiol and YC-1 improved barrier function in hypoxic cells. Overall our data suggests that activation of HIF-1-mediated signaling disrupts TJ resulting in increased BBB permeability.

BackgroundUndisturbed functioning of the blood–brain barrier (BBB) crucially depends on paracellular signaling between its associated cells; particularly endothelial cells, pericytes and astrocytes. Hypoxic and ischemic injuries are closely associated with disturbed BBB function and the contribution of perivascular cells to hypoxic/ischemic barrier regulation has gained increased attention. Regardless, detailed information on the basal hypoxic/ischemic responses of the barrier-associated cells is rare and the outcome of such cell-specific responses on BBB modulation is not well understood. This study investigated crucial parameters of hypoxic/ischemic adaptation in order to characterize individual perivascular cell responses to stress conditions.MethodsThe brain microvascular endothelial cell line RBE4 (EC cell line) as well as primary rat brain endothelial cells (ECs), pericytes (PCs) and astrocytes (ACs) were exposed to 24 and 48 hours of oxygen deprivation at 1% and 0.2% O2. All primary cells were additionally subjected to combined oxygen and glucose deprivation mimicking ischemia. Central parameters of cellular adaptation and state, such as HIF-1α and HIF-1 target gene induction, actin cytoskeletal architecture, proliferation and cell viability, were compared between the cell types.ResultsWe show that endothelial cells exhibit greater responsiveness and sensitivity to oxygen deprivation than ACs and PCs. This higher sensitivity coincided with rapid and significant stabilization of HIF-1α and its downstream targets (VEGF, GLUT-1, MMP-9 and PHD2), early disruption of the actin cytoskeleton and metabolic impairment in conditions where the perivascular cells remain largely unaffected. Additional adaptation (suppression) of proliferation also likely contributes to astrocytic and pericytic tolerance during severe injury conditions. Moreover, unlike the perivascular cells, ECs were incapable of inducing autophagy (monitored via LC3-II and Beclin-1 expression) - a putative protective mechanism. Notably, both ACs and PCs were significantly more susceptible to glucose than oxygen deprivation with ACs proving to be most resistant overall.ConclusionIn summary this work highlights considerable differences in sensitivity to hypoxic/ischemic injury between microvascular endothelial cells and the perivascular cells. This can have marked impact on barrier stability. Such fundamental knowledge provides an important foundation to better understand the complex cellular interactions at the BBB both physiologically and in injury-related contexts in vivo.

The analysis of cell types involved in cross-priming of particulate Ag is essential to understand and improve immunotherapies using microparticles. In this study, we show that murine splenic dendritic cells (DCs) as well as macrophages (MΦs) are able to efficiently endocytose poly(D,L-lactate-co-glycolate) acid (PLGA) microspheres (MS) and to cross-present encapsulated Ags in the context of MHC class I molecules in vitro. A comparison of purified CD8+ and CD8− DCs indicated that both DC subtypes are able to present OVA-derived epitopes on MHC class I and II in vitro. To determine the contribution of DCs and MΦs to cross-priming of PLGA MS in vivo, DCs were depleted in transgenic CD11c-DTR mice, and MΦs were depleted by clodronate liposomes in wild-type mice before immunizing mice with OVA-encapsulated MS. Our results show that the depletion of DCs or MΦs alone only led to minor differences in the OVA-specific immune responses. However, simultaneous depletion of DCs and MΦs caused a strong reduction of primed effector cells, indicating a redundancy of both cell populations for the priming of PLGA MS-encapsulated Ag. Finally, we analyzed PLGA MS trafficking to draining lymph nodes after s.c. injection. It was evident that fluorescent particles accumulated within draining lymph nodes over time. Further analysis of PLGA MS-positive lymphatic cells revealed that mainly CD8− DCs and MΦs contained MS. Moreover, immune responses in BATF3 knockout mice lacking CD8+ DCs were normal. The results presented in this work strongly suggest that in vivo cross-priming of PLGA MS-encapsulated Ag is performed by CD8− DCs and MΦs.

High-grade gliomas (HGG) exert systemic immunosuppression, which is of particular importance as immunotherapeutic strategies such as therapeutic vaccines are increasingly used to treat HGGs. In a first cohort of 61 HGG patients we evaluated a panel of 30 hematological and 34 plasma biomarkers. Then, we investigated in a second cohort of 11 relapsed HGG patients receiving immunomodulation with metronomic cyclophosphamide upfront to a DC-based vaccine whether immune abnormalities persisted and whether they hampered induction of IFNγ+ T-cell responses. HGG patients from the first cohort showed increased numbers of leukocytes, neutrophils and MDSCs and in parallel reduced numbers of CD4+/CD8+ T-cells, plasmacytoid and conventional DC2s. MDSCs and T-cell alterations were more profound in WHO IV° glioma patients. Moreover, levels of MDSCs and epidermal growth factor were negatively associated with survival. Serum levels of IL-2, IL-4, IL-5 and IL-10 were altered in HGG patients, however, without any impact on clinical outcome. In the immunotherapy cohort, 6-month overall survival was 100%. Metronomic cyclophosphamide led to > 40% reduction of regulatory T cells (Treg). In parallel to Treg-depletion, MDSCs and DC subsets became indistinguishable from healthy controls, whereas T-lymphopenia persisted. Despite low T-cells, IFNγ-responses could be induced in 9/10 analyzed cases. Importantly, frequency of CD8+VLA-4+ T-cells with CNS-homing properties, but not of CD4+ VLA-4+ T-cells, increased during vaccination. Our study identifies several features of systemic immunosuppression in HGGs. Metronomic cyclophosphamide in combination with an active immunization alleviates the latter and the combined treatment allows induction of a high rate of anti-glioma immune responses.Electronic supplementary materialThe online version of this article (10.1007/s00262-018-2214-0) contains supplementary material, which is available to authorized users.

Background: Legionella governs pathogen-host interactions by translocating ϳ300 "effector" proteins through a type IV secretion system. Results: The hitherto unrecognized effector LppA is a phytase that counteracts intracellular bacterial growth restriction by phytate. Conclusion:The chelator phytate is a bacteriostatic component of cell-autonomous immunity, which is degraded by a bacterial effector. Significance: Legionella LppA represents the first translocated phytase and a potential therapeutic target.

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