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

Abstract

Background and ObjectivesMultiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease characterized by infiltration of immune cells in multifocal areas of the CNS. The specific molecular processes allowing autoreactive immune cells to enter the CNS compartment through the blood-brain barrier remain elusive.MethodsUsing endothelial cell (EC) enrichment and single-cell RNA sequencing, we characterized the cells implicated in the neuroinflammatory processes in experimental autoimmune encephalomyelitis, an animal model of MS. Validations on human MS brain sections of the most differentially expressed genes in venous ECs were performed using immunohistochemistry and confocal microscopy.ResultsWe found an upregulation of genes associated with antigen presentation and interferon in most populations of CNS-resident cells, including ECs. Interestingly, instead of transcriptionally distinct profiles, a continuous gradient of gene expression separated the arteriovenous zonation of the brain vasculature. However, differential gene expression analysis presented more transcriptomic alterations on the venous side of the axis, suggesting a prominent role of venous ECs in neuroinflammation. Furthermore, analysis of ligand-receptor interactions identified important potential molecular communications between venous ECs and infiltrated immune populations. To confirm the relevance of our observation in the context of human disease, we validated the protein expression of the most upregulated genes (Ackr1andLcn2) in MS lesions.DiscussionIn this study, we provide a landscape of the cellular heterogeneity associated with neuroinflammation. We also present important molecular insights for further exploration of specific cell processes that promote infiltration of immune cells inside the brain of experimental autoimmune encephalomyelitis mice.

MCAM+ brain endothelial cells contribute to neuroinflammation by recruiting pathogenic CD4+ T lymphocytes

“…To comprehensively characterize the profile of Mcam + BBB ECs, we analysed a dataset of single-cell RNA profiles of ECs isolated from the brains of naïve and MOG 35-55 -immunized EAE animals. 21 Compared to naïve mice, computational analyses first confirmed the increase in Mcam mRNA average expression in brain ECs in EAE mice and further revealed an increase in the frequency of Mcam + brain ECs in EAE ( Fig. 1D ).…”

“…( D – J ) Analysis of single-cell RNA sequencing dataset of brain ECs in naïve and EAE mice. 21 ( D ) Left and right bar charts show Mcam mRNA expression (mean read per cell ± SEM) in ECs and frequency of Mcam + ECs (mean of total ECs ± SEM), respectively, in brains of naïve and EAE mice. ( E ) Violin plot (log 2 fold change) and ( F ) GSEA of the DEGs in Mcam + ECs in EAE; P < 0.05.…”

“…Having previously shown that mouse brain ECs can be transcriptionally organized on the basis of gradients of gene expression rather than distinct clustering, 21 we next sought to further characterize the expression of Mcam in brain ECs along an arteriovenous axis reflective of the cerebrovascular tree. To generate this axis, we used a pseudotime analysis to unbiasedly rank the sequenced ECs along a transcriptomic trajectory and confirmed its arteriovenous architecture using previously published markers.…”

“… 20 The single-cell RNA sequencing results were determined by analysing the transcriptomes of brain ECs and brain-infiltrating leucocytes from naïve mice and mice with active EAE at peak of disease. 21 Differential gene expression analysis was performed using the FindMarkers function from Seurat (R package, v.4.0.1) and was relative between the compared conditions or to remaining cells within the condition. The Gene Set Enrichment Analysis (GSEA) was achieved using the Gene Ontology Molecular Function and Biological Processes databases, obtained through mSigDb’s collections ( http://www.gsea-msigdb.org/gsea/msigdb/genesets.jsp?collection=GO:MF ).…”

Blood–brain barrier endothelial cells in neurodegenerative diseases: Signals from the “barrier”

“…Molecular MRI targeting at P-selectin is used for the detection of BEC activation, which is promising for clinical translation to monitor or even predict the activity and severity of MS, indicating BBB disruption and BEC activation in the early 10.3389/fnins.2023.1047778 stage of MS (Gauberti et al, 2018). Moreover, recent single-cell transcriptomics identified that enhanced adhesive properties and immune transendothelial migration in MS were mainly localized in venous BECs (Fournier et al, 2023).…”