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

Abstract

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

Sevoflurane blocks KLF5‐mediated transcriptional activation of ITGB2 to inhibit macrophage infiltration in hepatic ischemia/reperfusion injury

“…Consistently, ITGB2 has been shown as a target of KLF5 in breast cancer cells 10 . ITGB2‐positive microglia subcluster has been recently related to energy metabolism, cell cycle, angiogenesis, neuronal myelin formation, and repair in response to cerebral I/R injury 11 . Because myocardin‐related transcription factor A has been identified to regulate the transcriptional level of ITGB2 to expedite macrophage infiltration and cardiac hypertrophy in mice 12 and macrophage‐mediated excessive inflammatory response is of great importance to hepatic I/R injury, 13 we hypothesized that Sevo elicited protection to the livers with I/R injury by controlling macrophage infiltration via the KLF5/ITGB2 axis.…”

Perspective from single‐cell sequencing: Is inflammation in acute ischemic stroke beneficial or detrimental?

“…Cluster 1 is involved in inflammation, cluster 3 is associated with metabolic processes, and clusters 6 and 7 are involved in glial cell differentiation, gliogenesis, neurogenesis, and synapses. 21 Zheng et al also found that microglial cells exhibit cellular diversity in five different subtypes after stroke and divided these into five clusters. Cluster MG 0 primarily originates from the sham-operated group and is characterized by high expression of genes such as P2ry12, Selplg, Tmem119, Gpr34, Siglech, Olfm13, P2ry13, Csf1r, and Hexb.…”

“…The activation of microglial cells is the initial step in the central nervous system inflammatory response, followed by the infiltration of immune cells, such as neutrophils, macrophages/monocytes, natural killer cells, and T cells, and the activation of neurons 26 . In a study by Zeng et al., the percentage of activated microglial cells was significantly higher than that in the sham‐operated group on the first, third, and seventh day after AIS 21 . Moreover, activated microglial cells produce various mediators, including inducible nitric oxide synthase (iNOS), nitric oxide (NO), 27,28 pro‐inflammatory cytokines (such as TNF‐a), and anti‐inflammatory cytokines (such as TGF‐a), 29 collectively leading to complex immune reactions in the brains of patients with stroke, 16,30–32 including reactions with contradictory effects 33–35 .…”

“…Lastly, Cluster 7 exhibits high expression of Hist1h2ap and Lag3 . Cluster 1 is involved in inflammation, cluster 3 is associated with metabolic processes, and clusters 6 and 7 are involved in glial cell differentiation, gliogenesis, neurogenesis, and synapses 21 . Zheng et al.…”

“…Following an ischemic brain injury, dramatic changes in the internal environment or ischemic neuronal death can activate microglial cells within minutes. 20 As the predominant resident immune cells in the brain, microglial cells comprise the largest proportion of cells after cerebral I/R injury 21 and are closely associated with the early excessive ROS response during I/R. 2 , 22 , 23 , 24 , 25 The activation of microglial cells is the initial step in the central nervous system inflammatory response, followed by the infiltration of immune cells, such as neutrophils, macrophages/monocytes, natural killer cells, and T cells, and the activation of neurons.…”