Single-cell analyses reveal the dynamic functions of Itgb2+ microglia subclusters at different stages of cerebral ischemia-reperfusion injury in transient middle cerebral occlusion mice model

Fanning Zeng; Jun Cao; Zexuan Hong; Yujun Liu; Jie Hao; Zaisheng Qin; Xin Zou; Tao Tao

doi:10.3389/fimmu.2023.1114663

Single-cell analyses reveal the dynamic functions of Itgb2⁺ microglia subclusters at different stages of cerebral ischemia-reperfusion injury in transient middle cerebral occlusion mice model

Front Immunol. 2023 Mar 30:14:1114663. doi: 10.3389/fimmu.2023.1114663. eCollection 2023.

Authors

Fanning Zeng^{1

2}, Jun Cao³, Zexuan Hong³, Yujun Liu⁴, Jie Hao⁵, Zaisheng Qin², Xin Zou^{4

6}, Tao Tao¹

Affiliations

¹ Department of Anesthesiology, Central People's Hospital of Zhanjiang, Zhanjiang, Guangdong, China.
² Department of Anesthesiology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China.
³ Department of Anesthesiology, Affiliated Shenzhen Maternity and Child Healthcare Hospital, Southern Medical University, Shenzhen, Guangdong, China.
⁴ Center for Tumor Diagnosis and Therapy, Jinshan Hospital, Fudan University, Shanghai, China.
⁵ Institute of Clinical Science, Zhongshan Hospital, Fudan University, Shanghai, China.
⁶ Department of Pathology, Jinshan Hospital, Fudan University, Shanghai, China.

Abstract

Introduction: The underlying pathophysiological mechanisms of cerebral ischemia reperfusion injury (CIRI) is intricate, and current studies suggest that neuron, astrocyte, microglia, endothelial cell, and pericyte all have different phenotypic changes of specific cell types after ischemic stroke. And microglia account for the largest proportion after CIRI. Previous transcriptomic studies of ischemic stroke have typically focused on the 24 hours after CIRI, obscuring the dynamics of cellular subclusters throughout the disease process. Therefore, traditional methods for identifying cell types and their subclusters may not be sufficient to fully unveil the complexity of single-cell transcriptional profile dynamics caused by an ischemic stroke.

Methods: In this study, to explore the dynamic transcriptional profile of single cells after CIRI, we used single-cell State Transition Across-samples of RNA-seq data (scSTAR), a new bioinformatics method, to analyze the single-cell transcriptional profile of day 1, 3, and 7 of transient middle cerebral artery occlusion (tMCAO) mice. Combining our bulk RNA sequences and proteomics data, we found the importance of the integrin beta 2 (Itgb2) gene in post-modeling. And microglia of Itgb2+ and Itgb2- were clustered by the scSTAR method. Finally, the functions of the subpopulations were defined by Matescape, and three different time points after tMCAO were found to exhibit specific functions.

Results: Our analysis revealed a dynamic transcriptional profile of single cells in microglia after tMCAO and explored the important role of Itgb2 contributed to microglia by combined transcriptomics and proteomics analysis after modeling. Our further analysis revealed that the Itgb2+ microglia subcluster was mainly involved in energy metabolism, cell cycle, angiogenesis, neuronal myelin formation, and repair at 1, 3, and 7 days after tMCAO, respectively.

Discussion: Our results suggested that Itgb2+ microglia act as a time-specific multifunctional immunomodulatory subcluster during CIRI, and the underlying mechanisms remain to be further investigated.

Keywords: Itgb2; cerebral ischemia-reperfusion injury; microglia; scSTAR; single cell RNA-seq.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Disease Models, Animal
Infarction, Middle Cerebral Artery / genetics
Infarction, Middle Cerebral Artery / metabolism
Ischemic Stroke* / metabolism
Mice
Microglia / metabolism
Reperfusion Injury* / genetics
Reperfusion Injury* / metabolism
Single-Cell Analysis

Grants and funding

This research was funded by the National Natural Science Foundation of China (Grant Number: 81973305), the Discipline Construction Fund of Central People’s Hospital of Zhanjiang (Grant Number: 2020A01 and 2020A02), National Natural Science Foundation of China (82170045 to J.H.), and The Innovative Research Team of High-level Local Universities in Shanghai (SHSMU-ZLCX20212301 to J.H).