Single-cell analysis of cerebrospinal fluid reveals common features of neuroinflammation
Single-Cell Analysis Reveals Common Features of Neuroinflammation in Cerebrospinal Fluid
Neuroinflammation is a core pathological feature of neurological diseases, involving complex interactions between the cerebrospinal fluid (CSF) and the peripheral immune system. Although previous studies have revealed the clonal expansion of lymphocytes in the CSF of patients with multiple sclerosis (MS) and other neuroinflammatory diseases, significant scientific questions remain regarding the detailed characteristics of these immune cells, their dynamic changes, and their distinctions in health and disease. To dissect the unique features of the CSF immune microenvironment and clarify its biological significance in disease states, Benjamin M. Jacobs and colleagues conducted a comprehensive study.
This article, collaboratively completed by researchers from the University of Cambridge, Queen Mary University of London, and the Technical University of Munich, was published on January 21, 2025, in the top-tier journal Cell Reports Medicine. The study was funded by organizations such as the UK MS Society and the European Union Horizon 2020 research program. Combining single-cell RNA sequencing with lymphocyte receptor sequencing, the researchers analyzed cells and their transcriptional programs within CSF, uncovering cross-disease common immune characteristics in MS and other neuroinflammatory disorders.
Research Background and Objectives
The researchers highlight that while the CSF has long been regarded as an “immune-privileged” site, it has been found to contain abundant immune cells in various neurological disease contexts. Chronic inflammatory diseases such as MS are especially remarkable, with the clonal expansion of specific lymphocytes leading to immune abnormalities, including the formation of oligoclonal bands. However, it remains unclear how B cells and T cells access the CSF during inflammation, what drives this clonal expansion, and whether these factors are unique to MS or shared across other neuroinflammatory disorders.
Therefore, the primary objective of this study was to systematically analyze the immune microenvironment of CSF at single-cell resolution during both inflammatory and non-inflammatory states. The goal was to uncover shared and distinct features of MS and other neuroinflammatory diseases to provide insight for new therapeutic targets and a deeper understanding of disease mechanisms.
Study Design and Methodology
Research Subjects and Samples
The study analyzed 354,055 CSF cells and 422,809 peripheral immune cells (PBMCs) collected from a total of 123 participants, divided into four groups: - Multiple Sclerosis (MS): 76 peripheral blood samples, 203,220 CSF cells. - Other Inflammatory Neurological Disorders (OIND): 19 patients, 30,796 CSF cells. - Infectious Neurological Disorders (ID): 23 patients, 83,339 CSF cells. - Non-Inflammatory Neurological Disorders (NIND): 36 control samples, 36,700 CSF cells.
The research utilized single-cell RNA sequencing (scRNA-seq) integrated with immune receptor sequencing to perform an exhaustive analysis of these cell populations, supplemented with differential gene expression (DGE) calculations and bioinformatics approaches.
Study Workflow and Experimental Methods
(1) Single-Cell Transcriptomics and Cellular Composition Analysis
Using Uniform Manifold Approximation and Projection (UMAP), the researchers found significant differences in cell type distribution between CSF and PBMC: 1. Under non-inflammatory conditions, the CSF was enriched with dendritic cells (DCs), CD8+ T cells, regulatory T cells (Tregs), and specialized macrophages, whereas PBMC was dominated by B cells and monocytes. 2. Under inflammatory conditions, CSF showed marked enrichment of antibody-secreting cells (ASCs). This phenomenon was particularly pronounced in MS and infectious neurological disorders.
(2) Lymphocyte Clonality and Immune Receptor Repertoire Analysis
The study detected 602 B cell clonal groups and 11,541 T cell clonotypes: - B Cells: Clonal expansion was more pronounced in MS patients (53.4%), dominated by IgG1-positive antibody-secreting cells. - T Cells: Expanded T cell clones consisted mainly of effector memory CD8+ cells, with numerous virus-specific TCR (T-cell receptor) clones reactive to Epstein-Barr Virus (EBV) and Cytomegalovirus (CMV).
(3) Differential Gene Expression and Functional Enrichment
- Immune cells in the CSF universally exhibited upregulation of markers related to tissue residency, cytotoxicity, and antigen presentation.
- Gene set enrichment analysis (GSEA) revealed widespread activation of cholesterol homeostasis genes in CSF cells, driven by the transcription factors SREBF1 and SREBF2.
- Among MS-specific genes, CCL22 was significantly upregulated in antibody-secreting cells, while genes like CD99 and CRIP2 were prominently expressed in plasmacytoid dendritic cells and CD4+ T cells, respectively.
(4) Single-Cell eQTL Analysis
The study uncovered several CSF-specific expression quantitative trait loci (eQTLs), including MS risk-associated genes like EAF2 and ZC2HC1A. Reduced EAF2 expression may play a role in B cell clonal expansion and overproliferation, while ZC2HC1A expression was predominantly restricted to CSF cells, suggesting a regulatory role in MS pathogenesis.
Key Findings and Results
Enrichment of Antibody-Secreting Cells (ASCs) in CSF Marks Neuroinflammation
Inflammatory conditions, including MS, OIND, and infectious neurological disorders, exhibited a significant increase in ASCs. However, this phenomenon is a general hallmark of neuroinflammation and not unique to MS.Distinct Transcriptional Programs and Clonal Expansion in CSF Immune Cells
Antibody-secreting cells and memory B cells in the CSF displayed unique transcriptional characteristics that were significantly distinct from PBMCs. Clonal expansion in T cells predominantly involved an effector memory phenotype combined with virus-specific responses.Limited Disease-Specific Transcriptional Changes in MS
While the study identified some MS-specific genes (e.g., CCL22), the majority of transcriptional features and immune cell behaviors in the CSF were highly consistent across different neuroinflammatory diseases.CSF-Specific eQTL Mechanisms Linked to MS Risk
The study highlighted key genes regulating cholesterol metabolism, immune cell migration, and clonal expansion. Findings emphasize the regulatory effects of MS risk loci on immune responses.
Significance and Highlights
This study represents a groundbreaking application of single-cell technology, providing an unprecedented high-resolution view of immune cells in the CSF under both healthy and pathological conditions. Its significance includes: 1. Revealing shared immune features across MS and other neuroinflammatory diseases, offering vital clues for precision medicine. 2. Enhancing the understanding of MS pathogenesis, particularly in the context of immune microenvironments and clonal immune cell expansion. 3. Identifying key genes that regulate CSF immune cell behavior and are linked to MS risk, advancing therapeutic drug targeting and development.
The study is not only methodologically and scale-wise innovative but also offers new perspectives for investigating the immune pathology of neurological diseases. It provides a solid theoretical foundation for future in-depth research on neuroinflammatory mechanisms.