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Breakdown of the blood-brain barrier (BBB) and increased immune cell trafficking into the central nervous system (CNS) are hallmarks of the pathogenesis of multiple sclerosis (MS). Platelet endothelial cell adhesion molecule-1 (PECAM-1; CD31) is expressed on cells of the vascular compartment and regulates vascular integrity and immune cell trafficking. Involvement of PECAM-1 in MS pathogenesis has been suggested by the detection of increased levels of soluble PECAM-1 (sPECAM-1) in the serum and CSF of MS patients. Here, we report profound upregulation of cell-bound PECAM-1 in initial (pre-phagocytic) white matter as well as active cortical gray matter MS lesions. Using a human in vitro BBB model we observed that PECAM-1 is not essential for the transmigration of human CD4 + T-cell subsets (Th1, Th1 * , Th2, and Th17) across the BBB. Employing an additional in vitro BBB model based on primary mouse brain microvascular endothelial cells (pMBMECs) we show that the lack of endothelial PECAM-1 impairs BBB properties as shown by reduced transendothelial electrical resistance (TEER) and increases permeability for small molecular tracers. Investigating T-cell migration across the BBB under physiological flow by in vitro live cell imaging revealed that absence of PECAM-1 in pMBMECs did not influence arrest, polarization, and crawling of effector/memory CD4 + T cells on the pMBMECs. Absence of endothelial PECAM-1 also did not affect the number of T cells able to cross the pMBMEC monolayer under flow, but surprisingly favored transcellular over paracellular T-cell diapedesis. Taken together, our data demonstrate that PECAM-1 is critically involved in regulating BBB permeability and although not required for T-cell diapedesis itself, its presence or absence influences the cellular route of T-cell diapedesis across the BBB. Upregulated expression of cell-bound PECAM-1 in human MS lesions may thus reflect vascular repair mechanisms aiming to restore BBB integrity and paracellular T-cell migration across the BBB as it occurs during CNS immune surveillance.

Here we report on the development of a breakthrough microfluidic human in vitro cerebrovascular barrier (CVB) model featuring stem cell-derived brain-like endothelial cells (BLECs) and nanoporous silicon nitride (NPN) membranes (µSiM-CVB). The nanoscale thinness of NPN membranes combined with their high permeability and optical transparency makes them an ideal scaffold for the assembly of an in vitro microfluidic model of the blood–brain barrier (BBB) featuring cellular elements of the neurovascular unit (NVU). Dual-chamber devices divided by NPN membranes yield tight barrier properties in BLECs and allow an abluminal pericyte-co-culture to be replaced with pericyte-conditioned media. With the benefit of physiological flow and superior imaging quality, the µSiM-CVB platform captures each phase of the multi-step T-cell migration across the BBB in live cell imaging. The small volume of <100 µL of the µSiM-CVB will enable in vitro investigations of rare patient-derived immune cells with the human BBB. The µSiM-CVB is a breakthrough in vitro human BBB model to enable live and high-quality imaging of human immune cell interactions with the BBB under physiological flow. We expect it to become a valuable new tool for the study of cerebrovascular pathologies ranging from neuroinflammation to metastatic cancer.

Background: The brain barriers establish compartments in the central nervous system (CNS) that significantly differ in their communication with the peripheral immune system. In this function they strictly control T-cell entry into the CNS. T cells can reach the CNS by either crossing the endothelial blood-brain barrier (BBB) or the epithelial bloodcerebrospinal fluid barrier (BCSFB) of the choroid plexus (ChP). Objective: Analysis of the cellular and molecular mechanisms involved in the migration of different human CD4 + T-cell subsets across the BBB versus the BCSFB. Methods: Human in vitro models of the BBB and BCSFB were employed to study the migration of circulating and CNS-entry experienced CD4 + T helper cell subsets (Th1, Th1*, Th2, Th17) across the BBB and BCSFB under inflammatory and non-inflammatory conditions in vitro. Results: While under non-inflammatory conditions Th1* and Th1 cells preferentially crossed the BBB, under inflammatory conditions the migration rate of all Th subsets across the BBB was comparable. The migration of all Th subsets across the BCSFB from the same donor was 10-to 20-fold lower when compared to their migration across the BBB. Interestingly, Th17 cells preferentially crossed the BCSFB under both, non-inflamed and inflamed conditions. Barriercrossing experienced Th cells sorted from CSF of MS patients showed migratory characteristics indistinguishable from those of circulating Th cells of healthy donors. All Th cell subsets could additionally cross the BCSFB from the CSF to ChP stroma side. T-cell migration across the BCSFB involved epithelial ICAM-1 irrespective of the direction of migration. Conclusions: Our observations underscore that different Th subsets may use different anatomical routes to enter the CNS during immune surveillance versus neuroinflammation with the BCSFB establishing a tighter barrier for T-cell entry into the CNS compared to the BBB. In addition, CNS-entry experienced Th cell subsets isolated from the CSF of MS patients do not show an increased ability to cross the brain barriers when compared to circulating Th cell subsets from healthy donors underscoring the active role of the brain barriers in controlling T-cell entry into the CNS. Also we identify ICAM-1 to mediate T cell migration across the BCSFB.

The blood-brain barrier (BBB) is the brain-specific endothelial cell barrier that is important for maintaining brain homeostasis and preventing the entry of toxic substances. Pathological BBB dysfunction is a critical step of the disease process in several immuno-mediated neurological diseases, including multiple sclerosis (MS), neuromyelitis optica (NMO), neuropsychiatric systemic lupus erythematosus (NPSLE) and neuro-Behçet diseases. The pathological findings from patients with secondary progressive (SP) MS, NMO and NPSLE showed leaky BBB in the active lesions. NMO is a disease with strong evidence of disease-specific and pathogenic autoantibodies (aquaporin 4 [AQP4] autoantibodies). In the development of NMO, circulating AQP4 autoantibodies need to pass through the BBB in order to reach AQP4 on the astrocyte endfeet. Strong evidence suggests that NPSLE is associated with the disruption of the BBB and NPSLE patients frequently have antibodies bound to endothelial cells in their sera. We recently identified two BBB-reactive autoantibodies in immuno-mediated neurological diseases: galectin-3 autoantibodies in SPMS and GRP78 autoantibodies in NMO. In the present review article, we describe the basic structure and cellular biology of the BBB, discuss recent insights regarding the pathophysiology of the BBB breakdown in the setting of immuno-mediated neurological diseases, and describe our recent findings of autoantibody-mediated BBB breakdown.