Research Overview Britta Engelhardt

The key achievements of our research include:

• Defining the mechanisms of immune cell trafficking across the BBB, including the discovery of distinct pathways for transcellular and paracellular T cell migration.
• Elucidating the role of tricellular junctions in the BBB as critical sites for T cell entry during neuroinflammation.
• Development of cutting-edge microfluidic in vitro BBB models and in vivo live-cell imaging techniques to study CNS immune interactions in real time.
• Discovering expression of VE-cadherin in adherens junctions of leptomeningeal fibroblasts and identifying VE-cadherin as suitable landmark for in vivo imaging of the leptomeninges
• Unravling that antigen-presentation at the BBB prohibits CD8 T cell entry into the CNS and triggers CD8 T cell mediated BBB breakdown
• Contributions to the understanding of multiple sclerosis (MS) pathogenesis, particularly how therapies like natalizumab influence T cell behavior at the BBB and how dysfunction of the BBB may contribute to MS pathogenesis.
• Investigations into how brain barriers coordinate CNS immune privilege through anatomical and molecular “zonation” concepts.
• Leadership in exploring how SARS-CoV-2, stroke, Alzheimer’s disease and other insults impact BBB integrity and immune cell dynamics.
• Coordination of major interdisciplinary research networks (e.g., BtRAIN, JUSTBRAIN)

Imaging the brain barriers in vivo

Two-photon intravital microscopy (2P-IVM) has advanced our understanding of CNS immune surveillance, but a limitation of current IVM technologies is that they lack simultaneous visualization of the brain barriers and immune cells. We have therefore developed novel fluorescent reporter mice allowing for simultaneous imaging of the different CNS barriers. We employ a leptomeningeal -arachnoid and pia mater- mouse reporter as well as a glia limitans mouse reporter.  Making use of cervical spinal cord, skull thinning and cranial window preparations and our fluorescent barrier and border reporter mouse models, we currently investigate by 2P-IVM, in collaboration with Benoit Zuber (Institute for Anatomy, University of Bern) and Christer Betsholtz (Uppsala, Sweden), the role of these different barriers in controlling the migration of fluorescently labelled T cells the CNS, in healthy conditions and during neuroinflammation.

Role of the choroid plexus in immune cell entry into the CNS during brain disorders

Following up on the role of the chemokine receptor CCR6 and its ligand CCL20 for Th17 cell entry into the CNS, we have investigated the migration of Th17 cell across in vitro models of the BCSFB and have found that CCR6 is not required for Th17 cell migration across the BCSFB (to the Article).

We are now exploring other potential anatomical pathways of encephalitogenic Th17 cells entry from the choroid plexus stroma into the brain in EAE. To visualize Th17 cell migration from the choroid plexus stroma into the brain, we developed a fluorescent brain barrier reporter mouse that enables for simultaneous imaging of the blood-brain barrier by expression of GFP and the BCSFB of the choroid plexus by expression of tdTomato. In collaboration with Maria Lehtinen (Harvard Medical School, Boston, USA), we successfully established a surgical window preparation that allows for two-photon intravital imaging (2P-IVM) of the lateral ventricle choroid plexus, enabling us to investigate whether Th17 cells can reach the ventricular space from the ChP stroma directly crossing the BCSFB in vivo.

As this surgical preparation does not allow to image the base of the choroid plexus, we complement this study with whole-brain imaging to gain a more comprehensive view of other potential Th17 cell brain entry sites. To this end, we have employed both confocal and light-sheet fluorescence microscopy (in collaboration with the Wyss Center in Geneva) with the aim to fully map the distribution of Th17 in the brain during the time course of EAE. By elucidating the anatomical routes through which Th17 cells invade the brain, we aim to contribute to a better understanding of EAE and multiple sclerosis pathogenesis.

The role of T cells in the pathogenesis of Alzheimer’s Disease

Alzheimer’s Disease (AD) is a neurodegenerative disease, and the most common and severe type of dementia worldwide. The pathological phenomena are well described – extracellular accumulation of amyloid-β peptides as amyloid plaques, and aggregation of abnormally-phosphorylated tau protein into intraneuronal neurofibrillary tangles in the cortex. Data from us and other suggest a potential role of the adaptive immune system in AD pathogenesis. We make use of the ArcAβ mouse model crossed into our fluorescent border and barrier reporter mice to explore the anatomical pathways and molecular mechanisms of T cell infiltration into the CNS during the time course of AD by advanced ex vivo and in vivo imaging.

Role of the arachnoid barrier in immune cell entry into the CNS during neuroinflammation

The dura mater has been proposed as an important meningeal tissue layer for CNS immune surveillance. The dura mater harbors lymphatic vessels and a high number of immune cells along the dural venous sinuses. However the dura is separated from the brain by the arachnoid barrier. The arachnoid barrier is formed by a specialized fibroblasts within the arachnoid mater that are connected by tight and tricellular junctions and express specific transporters and enzymes as other brain barrier forming cells. The arachnoid barrier establishes a blood–cerebrospinal fluid barrier (BCSFB) between the dura mater with blood vessels lacking barrier properties and the cerebrospinal fluid (CSF)-filled subarachnoid space. Making use of fluorescent barrier and border reporter mice we investigate if immune mediators and immune cells from the dura mater and the CNS can communicate across the arachnoid barrier making use of advanced in vivo imaging.

Osseous channels connecting the skull and vertebral bone marrow with the dura mater (Herisson et al., 2018) have been identified as potential shortcut pathways for immune cells to reach the dura without entering the bloodstream. This finding led to the suggestion of a skull-meninges-brain axis and bidirectional communication between the skull and the CNS, questioning the concept of CNS immune privilege and the role of the brain barriers along this axis. By using brain barrier reporter mice and considering the meningeal anatomy, we investigate the existence of this axis in CNS immunity, with a particular focus on immune cell trafficking routes and the diffusion of immune mediators across the arachnoid mater.

Imaging cerebrospinal fluid drainage in vivo

Cerebrospinal fluid (CSF) outflow plays a crucial role in maintaining brain homeostasis by clearing metabolic waste and regulating intracranial pressure. Understanding CSF outflow mechanisms is essential for developing targeted therapies and improving clinical management. To explore the complexity of cerebrospinal fluid (CSF) outflow pathways in vivo and how these pathways change with aging and disease, we have established a deep tissue imaging technique employing Gradient Refractive Index (GRIN) lenses allowing to directly visualize CSF drainage from the brain across the cribriform plate to the nasal mucosa.

Furthermore in the context of an SNSF funded Sinergia project entitled “Fluid Dynamics of the Central Nervous System: 3D Functional Anatomy & Pathophysiology in Mouse Models” we are working in collaboration with the research groups of Dr. Steven Proulx in house; Professor Bert Müller (University of Basel) and Professor Vartan Kurtcuoglu (University of Zurich). We have developed ex vivo and in vivo synchrotron radiation micro-computed tomography (SRµCT) imaging allowing to study CSF outflow in living mice. This advanced technique allows for high-resolution, real-time visualization of CSF contrast agent distribution across the entire CSF compartment in the mouse brain in vivo.

Studying the behavior of neutrophils and CSF clearance from the brain after ischemic stroke using intravital imaging modalities

Ischemic stroke is one of the leading causes of mortality worldwide. Despite extensive research, therapeutic options for stroke remain limited in effectiveness, underscoring the need to elucidate further mechanisms driving stroke pathology. Specifically, it has not been considered in sufficient detail that in addition to dysfunction of the blood-brain barrier, breakdown of other brain barrier such as the arachnoid barrier or the glia limitans may contribute to inflammation and edema after ischemic stroke. In the framework of the ERANET Neuron Project entitled “Deciphering Cellular and Acellular Barrier Dysfunction in Cerebrovascular Diseases” we aim to understand the roles of different brain barriers after stroke.

Employing novel fluorescent reporter mice for the brain borders and for neutrophils and two-photon imaging of the brain we investigate neutrophil behavior within different vascular and meningeal compartments in the brain after ischemic stroke.

Using intravital dynamic near-infrared imaging we explore the effects of ischemic stroke on the clearance of CSF from the brain into peripheral lymph nodes. To this end we follow the clearance of cisterna magna injected tracers in lymphatic reporter mice after ischemic stroke.  

Discovering the molecular underpinnings of blood-brain barrier dysfunction in multiple sclerosis

Blood-brain barrier (BBB) breakdown is an early hallmark of multiple sclerosis (MS) pathogenesis. The mechanisms leading to BBB breakdown are incompletely understood and are generally thought to be a consequence of the autoimmune neuroinflammatory process in MS.  In this project we challenge this view and propose that intrinsic alterations in BBB endothelial cells manifested at the genetic or epigenetic, transcriptional and ultimately phenotypic level cause or contribute to BBB dysfunction, which in combination with additional risk factors are crucial for the development of clinical MS.

This project makes use of our recently developed hiPSC-derived in vitro model of the BBB where we differentiate hiPSC from healthy individuals as controls (HC) and persons with MS (pwMS) into endothelial precursor cells and by the extended endothelial cell culture method (EECM) into brain microvascular like endothelial cells (EECM-BMEC-like cells). In collaboration with the laboratories of Renaud Du Pasquier (Lausanne, CH) and Eric Shusta (Madison, Wisconsin, USA) we have shown that EECM-BMEC-like cells from pwMS show impaired barrier properties and an inflammatory phenotype when compared to their HC counterparts (Nishihara et al., Brain, 2022; doi: 10.1093/brain/awac019).  To this end we have increased the number of hiPSC HC and pwMS donors including persons with RIS (Radiologically isolated Syndrome), which is a preclinical stage of MS. We have differentiated the hiPSCs into EECM-BMEC-like cells and performed transcriptional profiling by RNAsequencing. We have identified target genes differentially regulated between HC and MS- and RIS-EECM-BMEC-like cells and are currently analyzing their role in BBB dysfunction in MS. Identification of the molecular underpinnings of BBB dysfunction in MS will set the stage for the development of an entirely novel generation of therapeutic approaches aiming for BBB stabilization.

Modeling the human BBB in neuroinflammation

BBB characteristics in brain microvascular endothelial cells rely on the continuous crosstalk with pericytes and astrocytes. Alterations in BBB properties lead to impaired barrier characteristics and increased immune cell infiltration into the CNS. The study of the human BBB is limited by the lack of in vitro tools that accurately model all functional aspects of it. In collaboration with James McGrath (University of Rochester, USA) we have proposed to address this need by building an in vitro human BBB on a μSiM (microphysiological system enabled by a silicon membrane) platform. Our “human BBB-on-a-chip” model features ultrathin, transparent, and permeable silicone membranes that mimic the interface between blood and brain. The membranes harbor ~1 billion 60 nm pores, which allow unhindered solute exchange between ‘blood’ and ‘brain’ compartments. The luminal side of the membrane is lined with human induced pluripotent stem cell (hiPSC)-derived BMECs, which mimic the “blood compartment”. The "brain compartment" on the abluminal side of the membrane constitutes of hiPSC-derived pericytes and astrocytes. The BMECs, pericytes and astrocytes -all differentiated from the same hiPSC source- will be incorporated into the chip to build a fully isogenic human neurovascular unit (hNVU). After completing the establishment of the μSiM-hNVU, we will include physiological flow to investigate immune cell trafficking in healthy control-derived and MS patient-derived BBB models.

Studying the effect of vitamin D on T cell interaction with the BBB

In collaboration with the lab of Anne Astier (Toulouse, France) we investigate the effect of vitamin D on the cellular and molecular mechanisms involved in the multi-step T-cell extravasation across the BBB. To this end, we employ different in vitro models of the human BBB such as for example CD34 cord blood stem cell derived brain like endothelial cells (in collaboration with Fabien Gosselet, Lens, France) and hiPSC- derived brain microvascular endothelial cells (in collaboration with Renaud Du Pasquier, Lausanne, CH). These in vitro models permit us to study how vitamin D modulates human CD4 and CD8 T cell interaction with the BBB under both static and physiological flow conditions and provide further insights on the molecular mechanisms of action of these therapeutical strategies in MS.