The Immune Regulatory Mechanism Of CD95 On Monocytes And Microglia In Ischemic Stroke

Stroke is one of the most severe cerebrovascular diseases that endanger human health with high incidence,mortality and morbidity.The cascading inflammatory response injury followed by ischemic stroke is one of the leading causes of neuronal death.The reactive oxygen radicals and damage-associated molecular patterns released after tissue injury can stimulate microglia to release pro-inflammatory and chemokines and activate endothelial cells to upregulate adhesion molecules,thereby recruiting a large number of peripheral immune cells to exacerbate the inflammatory response.There is a part of the hypoperfused area around the ischemic core,called the ischemic penumbra.Rescuing tissues within the ischemic penumbra is critical in clinical treatment,and there are differences in the immune microenvironment between ischemic core and ischemic penumbra.Therefore,studying the immune regulatory mechanisms in the ischemic core and ischemic penumbra separately contributes to understanding the spatial heterogeneity of immune responses after stroke injury and is essential for alleviating the inflammatory response within the ischemic penumbra and promoting neural repair after injury.In this study,we will use the myeloid cell-specific CD95 deletion mouse,LysMcre CD95fl/fl,to explore the inflammatory regulation of CD95 signalling pathways by focusing on microglial and monocytes involved in the inflammatory response at the early stage of ischemic stroke injury.This study,based on the LysMcre CD95^fl/fl,a myeloid cell-specific CD95 knockout mouse,focuses on microglia and monocytes after ischemic injury and targets the CD95signalling pathway to reveal the regulatory mechanism of the early inflammatory response to cerebral ischemic injury.Firstly,analyzing the extent of cerebral infarction in mice after the specific deletion of CD95 in myeloid cells.Then analyzing the immunophenotypic and transcriptomic differences in the ischemic core and penumbra,respectively,by flow cytometry and single-cell RNA sequencing to search for potential immunoregulatory mechanisms.Our data shows that the myeloid cell-specific CD95 deletion increased the brain infarct volume in mice three days post cerebral ischemia.Besides,the results of flow cytometric analysis and single-cell RNA sequencing showed that in the ischemic core,myeloid-specific CD95 deletion induced M1-type polarization of monocytes and microglia,which exacerbated the inflammatory response in the ischemic core.In the penumbra,myeloid-specific CD95 deletion inhibited the expression of microglial pro-inflammatory genes,which alleviated the inflammatory response in the ischemic penumbra.This study revealed the differences in the immunoregulatory mechanisms of the CD95 signalling pathway in myeloid cells between the infarct core and penumbra tissues in the early stage of ischemic injury,which laid the foundation for further investigation of the downstream molecular mechanisms of the CD95 signalling pathway.