Neutrophils mediate blood-spinal cord barrier disruption in demyelinating neuroinflammatory diseases

Disruption of the blood-brain and blood-spinal cord barriers (BBB and BSCB, respectively) and immune cell infiltration are early pathophysiological hallmarks of multiple sclerosis (MS), its animal model experimental autoimmune encephalomyelitis (EAE), and neuromyelitis optica (NMO). However, their contribution to disease initiation and development remains unclear. In this study, we induced EAE in lys-eGFP-ki mice and performed single, nonterminal intravital imaging to investigate BSCB permeability simultaneously with the kinetics of GFP(+) myeloid cell infiltration. We observed a loss in BSCB integrity within a day of disease onset, which paralleled the infiltration of GFP(+) cells into the CNS and lasted for ∼4 d. Neutrophils accounted for a significant proportion of the circulating and CNS-infiltrating myeloid cells during the preclinical phase of EAE, and their depletion delayed the onset and reduced the severity of EAE while maintaining BSCB integrity. We also show that neutrophils collected from the blood or bone marrow of EAE mice transmigrate more efficiently than do neutrophils of naive animals in a BBB cell culture model. Moreover, using intravital videomicroscopy, we demonstrate that the IL-1R type 1 governs the firm adhesion of neutrophils to the inflamed spinal cord vasculature. Finally, immunostaining of postmortem CNS material obtained from an acutely ill multiple sclerosis patient and two neuromyelitis optica patients revealed instances of infiltrated neutrophils associated with regions of BBB or BSCB leakage. Taken together, our data provide evidence that neutrophils are involved in the initial events that take place during EAE and that they are intimately linked with the status of the BBB/BSCB.

A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells

The human blood brain barrier (BBB) is a selective barrier formed by human brain endothelial cells (hBECs), which is important to ensure adequate neuronal function and protect the central nervous system (CNS) from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs) express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.

DARC shuttles inflammatory chemokines across the blood-brain barrier during autoimmune central nervous system inflammation

The Duffy antigen/receptor for chemokines, DARC, belongs to the family of atypical heptahelical chemokine receptors that do not couple to G proteins and therefore fail to transmit conventional intracellular signals. Here we show that during experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, the expression of DARC is upregulated at the blood-brain barrier. These findings are corroborated by the presence of a significantly increased number of subcortical white matter microvessels staining positive for DARC in human multiple sclerosis brains as compared to control tissue. Using an in vitro blood-brain barrier model we demonstrated that endothelial DARC mediates the abluminal to luminal transport of inflammatory chemokines across the blood-brain barrier. An involvement of DARC in experimental autoimmune encephalomyelitis pathogenesis was confirmed by the observed ameliorated experimental autoimmune encephalomyelitis in Darc(-/-) C57BL/6 and SJL mice, as compared to wild-type control littermates. Experimental autoimmune encephalomyelitis studies in bone marrow chimeric Darc(-/-) and wild-type mice revealed that increased plasma levels of inflammatory chemokines in experimental autoimmune encephalomyelitis depended on the presence of erythrocyte DARC. However, fully developed experimental autoimmune encephalomyelitis required the expression of endothelial DARC. Taken together, our data show a role for erythrocyte DARC as a chemokine reservoir and that endothelial DARC contributes to the pathogenesis of experimental autoimmune encephalomyelitis by shuttling chemokines across the blood-brain barrier.

Novel insights into the development and maintenance of the blood-brain barrier

The blood-brain barrier (BBB) is essential for maintaining homeostasis within the central nervous system (CNS) and is a prerequisite for proper neuronal function. The BBB is localized to microvascular endothelial cells that strictly control the passage of metabolites into and out of the CNS. Complex and continuous tight junctions and lack of fenestrae combined with low pinocytotic activity make the BBB endothelium a tight barrier for water soluble moleucles. In combination with its expression of specific enzymes and transport molecules, the BBB endothelium is unique and distinguishable from all other endothelial cells in the body. During embryonic development, the CNS is vascularized by angiogenic sprouting from vascular networks originating outside of the CNS in a precise spatio-temporal manner. The particular barrier characteristics of BBB endothelial cells are induced during CNS angiogenesis by cross-talk with cellular and acellular elements within the developing CNS. In this review, we summarize the currently known cellular and molecular mechanisms mediating brain angiogenesis and introduce more recently discovered CNS-specific pathways (Wnt/β-catenin, Norrin/Frizzled4 and hedgehog) and molecules (GPR124) that are crucial in BBB differentiation and maturation. Finally, based on observations that BBB dysfunction is associated with many human diseases such as multiple sclerosis, stroke and brain tumors, we discuss recent insights into the molecular mechanisms involved in maintaining barrier characteristics in the mature BBB endothelium.

β2 integrin-mediated crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed blood-brain barrier

In acute neuroinflammatory states such as meningitis, neutrophils cross the blood-brain barrier (BBB) and contribute to pathological alterations of cerebral function. The mechanisms that govern neutrophil migration across the BBB are ill defined. Using live-cell imaging, we show that LPS-stimulated BBB endothelium supports neutrophil arrest, crawling, and diapedesis under physiological flow in vitro. Investigating the interactions of neutrophils from wild-type, CD11a(-/-), CD11b(-/-), and CD18(null) mice with wild-type, junctional adhesion molecule-A(-/-), ICAM-1(null), ICAM-2(-/-), or ICAM-1(null)/ICAM-2(-/-) primary mouse brain microvascular endothelial cells, we demonstrate that neutrophil arrest, polarization, and crawling required G-protein-coupled receptor-dependent activation of β2 integrins and binding to endothelial ICAM-1. LFA-1 was the prevailing ligand for endothelial ICAM-1 in mediating neutrophil shear resistant arrest, whereas Mac-1 was dominant over LFA-1 in mediating neutrophil polarization on the BBB in vitro. Neutrophil crawling was mediated by endothelial ICAM-1 and ICAM-2 and neutrophil LFA-1 and Mac-1. In the absence of crawling, few neutrophils maintained adhesive interactions with the BBB endothelium by remaining either stationary on endothelial junctions or displaying transient adhesive interactions characterized by a fast displacement on the endothelium along the direction of flow. Diapedesis of stationary neutrophils was unchanged by the lack of endothelial ICAM-1 and ICAM-2 and occurred exclusively via the paracellular pathway. Crawling neutrophils, although preferentially crossing the BBB through the endothelial junctions, could additionally breach the BBB via the transcellular route. Thus, β2 integrin-mediated neutrophil crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed BBB.

Distinctive insular forms of threespine stickleback (Gasterosteus aculeatus) from western Mediterranean islands

Neutral and adaptive variation among populations within a species is a major component of biological diversity and may be pronounced among insular populations due to geographical isolation and island specific evolutionary forces at work. Detecting and preserving potential evolutionary significant units below the species rank has become a crucial task for conservation biology. Combining genetic, phenotypic and ecological data, we investigated evolutionary patterns among the enigmatic threespine stickleback populations from western Mediterranean islands, all of which are threatened by habitat deterioration and climate change. We find indications that these populations derive from different genetic lineages, being genetically highly distinct from the stickleback of mainland Europe and the northern Atlantic as well as from each other. Mediterranean island stickleback populations are also phenotypically distinct from mainland populations but interestingly stickleback from Iceland have converged on a similar phenotype. This distinctive island stickleback phenotype seems to be driven by distinct selective regimes on islands versus continents. Overall, our results reveal the status of western Mediterranean island stickleback as evolutionarily distinct units, important for conservation of biodiversity.

Active eosinophilic esophagitis is characterized by epithelial barrier defects and eosinophil extracellular trap formation.

BACKGROUND Eosinophilic esophagitis (EoE) exhibits esophageal dysfunction owing to an eosinophil-predominant inflammation. Activated eosinophils generate eosinophil extracellular traps (EETs) able to kill bacteria. There is evidence of an impaired barrier function in EoE that might allow pathogens to invade the esophagus. This study aimed to investigate the presence and distribution of EETs in esophageal tissues from EoE patients and their association with possible epithelial barrier defects. METHODS Anonymized tissue samples from 18 patients with active EoE were analyzed. The presence of DNA nets associated with eosinophil granule proteins forming EETs and the expression of filaggrin, the protease inhibitor lympho-epithelial Kazal-type-related inhibitor (LEKTI), antimicrobial peptides, and cytokines were evaluated by confocal microscopy following immune fluorescence staining techniques. RESULTS Eosinophil extracellular trap formation occurred frequently and was detected in all EoE samples correlating with the numbers of infiltrating eosinophils. While the expression of both filaggrin and LEKTI was reduced, epithelial antimicrobial peptides (human beta-defensin-2, human beta-defensin-3, cathelicidin LL-37, psoriasin) and cytokines (TSLP, IL-25, IL-32, IL-33) were elevated in EoE as compared to normal esophageal tissues. There was a significant correlation between EET formation and TSLP expression (P = 0.02) as well as psoriasin expression (P = 0.016). On the other hand, a significant negative correlation was found between EET formation and LEKTI expression (P = 0.016). CONCLUSION Active EoE exhibits the presence of EETs. Indications of epithelial barrier defects in association with epithelial cytokines are also present which may have contributed to the activation of eosinophils. The formation of EETs could serve as a firewall against the invasion of pathogens.

Accumulation rates and predominant atmospheric sources of natural and anthropogenic Hg and Pb on the Faroe Islands

A monolith representing 5420 14C yr of peat accumulation was collected from a blanket bog at Myrarnar, Faroe Islands. The maximum Hg concentration (498 ng/g at a depth of 4.5 cm) coincides with the maximum concentration of anthropogenic Pb (111 μg/g). Age dating of recent peat accumulation using 210Pb (CRS model) shows that the maxima in Hg and Pb concentrations occur at AD 1954 ± 2. These results, combined with the isotopic composition of Pb in that sample (206Pb/207Pb = 1.1720 ± 0.0017), suggest that coal burning was the dominant source of both elements. From the onset of peat accumulation (ca. 4286 BC) until AD 1385, the ratios Hg/Br and Hg/Se were constant (2.2 ± 0.5 × 10-4 and 8.5 ± 1.8 × 10-3, respectively). Since then, Hg/Br and Hg/Se values have increased, also reaching their maxima in AD 1954. The age date of the maximum concentrations of anthropogenic Hg and Pb in the Faroe Islands is consistent with a previous study of peat cores from Greenland and Denmark (dated using the atmospheric bomb pulse curve of 14C), which showed maximum concentrations in AD 1953. The average rate of atmospheric Hg accumulation from 1520 BC to AD 1385 was 1.27 ± 0.38 μg/m2/yr. The Br and Se concentrations and the background Hg/Br and Hg/Se ratios were used to calculate the average rate of natural Hg accumulation for the same period, 1.32 ± 0.36 μg/m2/yr and 1.34 ± 0.29 μg/m2/yr, respectively. These fluxes are similar to the preanthropogenic rates obtained using peat cores from Switzerland, southern Greenland, southern Ontario, Canada, and the northeastern United States. Episodic volcanic emissions and the continual supply of marine aerosols to the Faroe Islands, therefore, have not contributed significantly to the Hg inventory or the Hg accumulation rates, relative to these other areas. The maximum rate of Hg accumulation was 34 μg/m2/yr. The greatest fluxes of anthropogenic Hg accumulation calculated using Br and Se, respectively, were 26 and 31 μg/m2/yr. The rate of atmospheric Hg accumulation in 1998 (16 μg/m2/yr) is comparable to the values recently obtained by atmospheric transport modeling for Denmark, the Faroe Islands, and Greenland.

Leptomeningeal contrast enhancement and blood-CSF barrier dysfunction in aseptic meningitis

OBJECTIVE To investigate the blood-CSF barrier (BCSFB) dysfunction in aseptic meningitis. METHODS In our case series of 14 patients with acute aseptic meningitis, we compared MRI characteristics with CSF findings. RESULTS Contrast enhancement in the sulcal space in a leptomeningeal pattern was visualized in 7 patients with BCSFB dysfunction categorized as moderate to severe as evidenced by the CSF/serum albumin ratio (Qalb) but was not present in those with mild or no barrier disturbance (p = 0.001). The Qalb as a marker for the leakiness of the BCSFB and, more indirectly, of the blood-brain barrier (BBB) was positively correlated with the incidence of leptomeningeal contrast enhancement seen on postcontrast fluid-attenuated inversion recovery (FLAIR) MRI (p = 0.003). Patients with a more pronounced brain barrier dysfunction recovered more slowly and stayed longer in the hospital. CONCLUSIONS The severity of meningeal BBB disturbance can be estimated on postcontrast FLAIR MRI, which may be of diagnostic value in patients with aseptic meningitis.

Engineering an in vitro air-blood barrier by 3D bioprinting

Intensive efforts in recent years to develop and commercialize in vitro alternatives in the field of risk assessment have yielded new promising two- and three dimensional (3D) cell culture models. Nevertheless, a realistic 3D in vitro alveolar model is not available yet. Here we report on the biofabrication of the human air-blood tissue barrier analogue composed of an endothelial cell, basement membrane and epithelial cell layer by using a bioprinting technology. In contrary to the manual method, we demonstrate that this technique enables automatized and reproducible creation of thinner and more homogeneous cell layers, which is required for an optimal air-blood tissue barrier. This bioprinting platform will offer an excellent tool to engineer an advanced 3D lung model for high-throughput screening for safety assessment and drug efficacy testing.

Modeling immune functions of the mouse blood-cerebrospinal fluid barrier in vitro: primary rather than immortalized mouse choroid plexus epithelial cells are suited to study immune cell migration across this brain barrier.

BACKGROUND The blood-cerebrospinal fluid barrier (BCSFB) established by the choroid plexus (CP) epithelium has been recognized as a potential entry site of immune cells into the central nervous system during immunosurveillance and neuroinflammation. The location of the choroid plexus impedes in vivo analysis of immune cell trafficking across the BCSFB. Thus, research on cellular and molecular mechanisms of immune cell migration across the BCSFB is largely limited to in vitro models. In addition to forming contact-inhibited epithelial monolayers that express adhesion molecules, the optimal in vitro model must establish a tight permeability barrier as this influences immune cell diapedesis. METHODS We compared cell line models of the mouse BCSFB derived from the Immortomouse(®) and the ECPC4 line to primary mouse choroid plexus epithelial cell (pmCPEC) cultures for their ability to establish differentiated and tight in vitro models of the BCSFB. RESULTS We found that inducible cell line models established from the Immortomouse(®) or the ECPC4 tumor cell line did not express characteristic epithelial proteins such as cytokeratin and E-cadherin and failed to reproducibly establish contact-inhibited epithelial monolayers that formed a tight permeability barrier. In contrast, cultures of highly-purified pmCPECs expressed cytokeratin and displayed mature BCSFB characteristic junctional complexes as visualized by the junctional localization of E-cadherin, β-catenin and claudins-1, -2, -3 and -11. pmCPECs formed a tight barrier with low permeability and high electrical resistance. When grown in inverted filter cultures, pmCPECs were suitable to study T cell migration from the basolateral to the apical side of the BCSFB, thus correctly modelling in vivo migration of immune cells from the blood to the CSF. CONCLUSIONS Our study excludes inducible and tumor cell line mouse models as suitable to study immune functions of the BCSFB in vitro. Rather, we introduce here an in vitro inverted filter model of the primary mouse BCSFB suited to study the cellular and molecular mechanisms mediating immune cell migration across the BCSFB during immunosurveillance and neuroinflammation.