Single-Cell RNA Sequencing of Peripheral Blood Reveals That Monocytes With High Cathepsin S Expression Aggravate Cerebral Ischemia-Reperfusion Injury

Background: Stroke persists as a major cause of morbidity and mortality worldwide. After a stroke, peripheral immune cells are rapidly activated and then infiltrate the central nervous system to cause inflammation in the brain. However, it is not clear when and how these peripheral immune cells affect the central inflammatory response and whether there are intervention targets that can alleviate ischemia-reperfusion injury. In this study, we collected mouse peripheral blood samples at different time points after stroke for single-cell sequencing to reveal the dynamic changes in peripheral immune cells. Methods: We performed single-cell sequencing on peripheral blood of mice at 1, 3, 7, and 14 days after ischemia-reperfusion to analyze the changes of subpopulations after cerebral ischemia-reperfusion; Real-time PCR, western blot and enzyme‑linked immunoabsorbent assay were used to perform mRNA and protein levels verification; Immunoprecipitation verifies the interaction of proteins and between junctional adhesion molecule (JAM-A) and Cathepsin S (CTSS) protein, in vitro enzyme digestion and silver staining method to detect the protease digestion effect of CTSS.Results: Peripheral monocyte subpopulations increased significantly after ischemia-reperfusion. Pseudo-time trajectory analysis and gene function analysis further suggested that CTSS may play an important role in regulating monocyte activation and leading to proteolysis. Next, we found that the expression of CTSS was significantly increased in monocytes after I/R in mice. Then, we used CTSS inhibitors and knockout mouse experiments to prove that inhibiting its expression can significantly reduce infarct volume and reduce blood–brain barrier (BBB) leakage. In addition, we found that CTSS destroys BBB by binding to JAM family proteins to cause them degradation. Conclusion: Inhibition of Cathepsin S attenuated cerebral ischemia reperfusion injury and Cathepsin S can be used as a novel target for drug intervention after stroke.