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Single-Cell Transcriptomics Identifies Brain Endothelium Inflammatory Networks in Experimental Autoimmune Encephalomyelitis

ABSTRACT: Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease characterized by infiltration of immune cells in multifocal areas of the central nervous system (CNS). The specific molecular processes allowing autoreactive immune cells to enter the CNS compartment through the blood-brain barrier (BBB) remain elusive. Using endothelial cells (ECs) enrichment and single-cell RNA sequencing (scRNA-Seq), we characterized the cells implicated in the neuroinflammatory processes in experimental auto-immune encephalomyelitis, an animal model of MS. We found an upregulation of genes associated with antigen presentation and interferon in most populations of CNS-resident cells, including ECs. Interestingly, a gradient of gene expression rather than distinct transcriptional profiles separated the arteriovenous zonation of the brain vasculature. However, differential gene expression analysis showed more transcriptomic alterations in venous ECs, suggesting their prominent role in neuroinflammation. Furthermore, analysis of ligand-receptor interactions identified important crosstalk between venous ECs and infiltrating immune subsets. To confirm the relevance of our observation in the context of human disease, we validated the protein expression of the most differentially expressed genes in MS lesions. Here, we demonstrate a landscape of the cellular heterogeneity involved in neuroinflammation and provide important molecular insights in unraveling specific cell processes that lead to the infiltration of the brain by immune cells in EAE.

ORGANISM(S): Mus musculus

PROVIDER: GSE199460 | GEO | 2022/12/01

REPOSITORIES: GEO

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Project description:The blood-brain barrier (BBB) dynamically controls and maintains a precisely balanced brain microenvironment necessary for reliable functioning. It is made up of highly specialized endothelial cells (ECs) in the lining of the vascular wall. These ECs are surrounded by the basal lamina, astrocytic perivascular endfeet, pericytes, microglia and neuronal processes that have been shown to contribute to barrier function1. In essence, the brain endothelium limits both transcellular and paracellular passage of cells and molecules into the central nervous system (CNS). Transcellular passage of hydrophilic molecules is limited due to a low rate of transcytotic vesicles, an extremely low pinocytotic activity, expression of active efflux transporters, and high metabolic activity. Paracellular diffusion of hydrophilic molecules and trafficking of immune cells is restricted by a network of tight junctions (TJs)2-5. Due to these characteristics, the BBB is able to protect the CNS from sudden changes in blood composition and uncontrolled influx of immune cells. In a large number of neuro-inflammatory diseases, an impaired function and an opening of the BBB are observed. In diseases of the CNS like multiple sclerosis or stroke or after brain trauma, the BBB becomes inflamed (mediated by for example reactive oxygen species (ROS) and hypoxia) and its function severely impaired which gives rise to enhanced cellular infiltration, thus contributing to tissue damage and neurological deficits. Due to the specialized nature of brain ECs, transmigration of leukocytes through cerebrovascular endothelium is likely to differ from that in other vascular beds. Generally, the transmigration process is closely controlled by the interaction of leukocytes with the endothelial surface followed by low velocity rolling, arrest, firm adhesion, and finally transmigration. For the diapedesis of immune cells across the cerebral vasculature a re-arrangement of the cytoskeleton and opening of the TJ complexes is required to allow cells to pass into the sub endothelial space. In the initial step of the adhesive cascade leukocytes adhere to the endothelium with low affinity. This adhesion is mediated by several members of the selectin family and their corresponding ligands. Despite the low affinity of these interactions, resulting contact of leukocytes with the endothelium leads to further activation of both cell types and finally transmigration of the leukocytes through the BBB6,7. The glycosylation of endothelial cells of different vascular bed origins To date it is unknown which specific carbohydrate structures on the BBB endothelium mediate leukocyte capture and rolling, and whether these structures are differentially expressed onto inflamed brain EC compared to normal brain ECs and vascular beds of other organs. Initial studies using qPCR and FACS analysis within our group reveal that brain endothelial cells have a different profile in their glycosylation-related genes compared to microvascular ECs upon inflammation, which may result in a different glycosylation profile of adhesive structures and may underlie rolling, adhesion and diapedesis of leukocytes. In this project we wish to identify specific, glycosylated structures on brain endothelial cells that mediate capture, rolling and diapedesis of leukocytes in the brain. To investigate the expression of glycosyltransferases in dendritic cells and the changes in expression associated with maturation. RNA preparations from stimulated and non-stimulated hcMEC/D3 (human brain endothelial cells line) and FMVEC (human promary microvascular endothelial cells isolated from foreskins) were sent to both Microarray Core (E) and Core(C). The RNA was put on an RNeasy Column, amplified, labeled, and hybridized to the Glycov3 microarrays. Data was sent to Dr. van Kooyk's lab for analysis.

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Single-Cell Transcriptomics Identifies Brain Endothelium Inflammatory Networks in Experimental Autoimmune Encephalomyelitis

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