Single-cell transcriptomic landscape reveals the role of intermediate monocytes in aneurysmal subarachnoid hemorrhage

Abstract

Objective: Neuroinflammation is associated with brain injury and poor outcomes after aneurysmal subarachnoid hemorrhage (SAH). In this study, we performed single-cell RNA sequencing (scRNA-seq) to analyze monocytes and explore the mechanisms of neuroinflammation after SAH.

Methods: We recruited two male patients with SAH and collected paired cerebrospinal fluid (CSF) and peripheral blood (PB) samples from each patient. Mononuclear cells from the CSF and PB samples were sequenced using 10x Genomics scRNA-seq. Additionally, scRNA-seq data for CSF from eight healthy individuals were obtained from the Gene Expression Omnibus database, serving as healthy controls (HC). We employed various R packages to comprehensively study the heterogeneity of transcriptome and phenotype of monocytes, including monocyte subset identification, function pathways, development and differentiation, and communication interaction.

Results: (1) A total of 17,242 cells were obtained in this study, including 7,224 cells from CSF and 10,018 cells from PB, mainly identified as monocytes, T cells, B cells, and NK cells. (2) Monocytes were divided into three subsets based on the expression of CD14 and CD16: classical monocytes (CM), intermediate monocytes (IM), and nonclassical monocytes (NCM). Differentially expressed gene modules regulated the differentiation and biological function in monocyte subsets. (3) Compared with healthy controls, both the toll-like receptor (TLR) and nod-like receptor (NLR) pathways were significantly activated and upregulated in IM from CSF after SAH. The biological processes related to neuroinflammation, such as leukocyte migration and immune response regulation, were also enriched in IM. These findings revealed that IM may play a key role in neuroinflammation by mediating the TLR and NLR pathways after SAH.

Interpretation: In conclusion, we establish a single-cell transcriptomic landscape of immune cells and uncover the heterogeneity of monocyte subsets in SAH. These findings offer new insights into the underlying mechanisms of neuroinflammation and therapeutic targets for SAH.

Keywords: ScRNA-seq; TLR; aneurysmal subarachnoid hemorrhage; monocytes; neuroinflammation.

FIGURE 1

Single-cell transcriptomic landscape of immune cells after aneurysmal subarachnoid hemorrhage (SAH). (A) Overview of the experiment design and data analysis process. ScRNA-seq (10x Genomics) was performed on immune cells from CSF and PB samples corrected from SAH patients. (B) T-SNE visualization of 17,242 cells from CSF and PB. These cells were divided into 19 clusters (0–18). (C) The 19 cell clusters were further identified as 5 cell types: monocytes, T cells, B cells, natural killer cells, and platelets. (D) Violin plot showing the expression of the marker genes in each immune cell type. (E) The feature plot displaying the relative distribution of marker genes in each immune cell type, with low expression in gray and high expression in red. (F) The total number and percentage of each immune cell type from PB and CSF.

FIGURE 2

Identification of monocyte subsets. (A, B) Monocytes were divided into three subsets, including classical monocytes (CM), intermediate monocytes (IM), and nonclassical monocytes (NCM). (C) Violin plot showing the expression of marker genes across monocyte subsets. (D) The total number and percentage of monocyte subsets from PB and CSF.

FIGURE 3

Gene regulation mechanism of monocyte differentiation. (A) Pseudotime trajectory of monocyte differentiation. Monocytes gradually differentiated from right to left, with a minor branch at the bottom right. (B) Monocyte subsets with different colors were mapped onto the trajectory, representing a lineage differentiation from CM to IM and NCM. (C) The heatmap showed that two gene modules, one and two, were significantly differentially expressed along the monocyte differentiation, and their representative GO biological processes were listed on the right. (D) The differentiation trajectory had two branches: the major (red Arrow) and the minor (black Arrow). (E) Gene module one exhibited high expression along the minor branch, whereas Gene module two displayed high expression along the major branch. Their respective representative GO biological processes were shown on the right.

FIGURE 4

IM may play a key role in neuroinflammation after SAH. (A) Compared with PB, many signaling pathways were highly activated in IM from CSF, especially the TLR and NLR (red Arrow). The one above the line represents the monocyte subsets (CM, IM, and NCM), and the one below represents sample types (CSF and PB). (B) Compared to PB, the TLR and NLR pathways showed significant upregulation in IM from CSF (red arrow). The blue bars represent upregulated pathways, and the green bars represent downregulated pathways. (C, D) TLR and NLR pathways were significantly activated and upregulated in IM from CSF after SAH compared with HC (red Arrow). (E) Compared with HC, the activation of transcription factors was also considerably upregulated in IM from SAH. (F) GO biological processes related to neuroinflammation were enriched in IM from CSF after SAH.

FIGURE 5

The communication interaction between different cell types. (A) The number and strength of communication interactions among various immune cell types in CSF. The line color matches the cell type, and the thickness indicates the intensity of communication interaction. (B) The number and strength of communication interactions among immune cell types in PB. (C) Compared with PB, the number and strength of communication interaction among monocyte subsets were significantly increased in CSF. The red line indicates upregulation, while the blue line indicates downregulation. (D, E) The communication probability of ligand-receptor pairs was different between CSF and PB. The MIF- (CD74+CXCR4) and MIF- (CD74+CD44) pairs were highly activated in both CSF and PB (red box).