Abstract
Perforin-mediated cytotoxicity plays a crucial role in microbial defense, tumor surveillance, and primary autoimmune disorders. However, the contribution of the cytolytic protein perforin to ischemia-induced secondary tissue damage in the brain has not been fully investigated. Here, we examined the kinetics and subpopulations of perforin-positive cells and then evaluated the direct effects of perforin-mediated cytotoxicity on outcomes after ischemic stroke. Using flow cytometry, we showed that perforin+CD45+ immune cells could be detected at 12 h and that the percentage of these cells increased largely until on day 3 and then significantly declined on day 7. Surprisingly, the percentage of Perforin+CD45+ cells also unexpectedly increased from day 7 to day 14 after ischemic stroke in Perforin1-EGFP transgenic mice. Our results suggested that Perforin+CD45+ cells play vital roles in the ischemic brain at early and late stages and further suggested that Perforin+CD45+ cells are a heterogeneous population. Surprisingly, in addition to CD8+ T cells, NK cells, and NKT cells, central nervous system (CNS)-resident immune microglia, which are first triggered and activated within minutes after ischemic stroke in mice, also secreted perforin during ischemic brain injury. In our study, the percentage of perforin+ microglia increased from 12 h after ischemic stroke, increased largely until on day 3 after ischemic stroke, and then moderately declined from days 3 to 7. Intriguingly, the percentage of perforin+ microglia also dramatically increased from days 7 to 14 after ischemic stroke. Furthermore, compared with wild-type littermates, Perforin 1–/– mice exhibited significant increases in the cerebral infarct volume, neurological deficits, and neurogenesis and inhibition of neurotoxic astrogliosis. Interestingly, the number of CD45+CD3+ T cells was significantly decreased in Perforin 1–/– mice compared with their wild-type littermates, especially the number of γδ T cells. In addition, Perforin 1–/– mice had lower levels of IL-17 than their wild-type littermates. Our results identified a critical function of perforin-mediated neurotoxicity in the ischemic brain, suggesting that targeting perforin-mediated neurotoxicity in brain-resident microglia and invading perforin+CD45+ immune cells may be a potential strategy for the treatment of ischemic stroke.
Highlights
Ischemic stroke is the leading cause of death and disability in adults worldwide (Voskoboinik et al, 2015; Yan et al, 2015; Hankey, 2017; Stinear et al, 2020)
Current evidence suggests that IL-10 and transforming growth factor-β (TGF-β) are anti-inflammatory cytokines mainly produced by microglia, regulatory T cells, and astrocytes, which are required for sensory motor recovery after ischemic stroke (Lu et al, 2005; Kleinschnitz et al, 2013; Mao et al, 2017)
To investigate the kinetics of perforin-positive immune cells, i.e., Perforin+CD45+ cells, we induced distal middle cerebral artery occlusion (dMCAO) in Prf1-EGFP transgenic mice, in which a green fluorescent protein (GFP) reporter gene was integrated into the prf1 allele
Summary
Ischemic stroke is the leading cause of death and disability in adults worldwide (Voskoboinik et al, 2015; Yan et al, 2015; Hankey, 2017; Stinear et al, 2020). Recent studies have suggested that blood-borne innate and adaptive immune cells may determine the outcome of stroke (Kleinschnitz et al, 2013; Ito et al, 2019; Kang et al, 2020). Current evidence suggests that IL-10 and transforming growth factor-β (TGF-β) are anti-inflammatory cytokines mainly produced by microglia, regulatory T cells, and astrocytes, which are required for sensory motor recovery after ischemic stroke (Lu et al, 2005; Kleinschnitz et al, 2013; Mao et al, 2017). Whether microglia contribute to functional recovery after ischemic stroke through direct cytotoxic effects and their underlying signaling mechanisms have not been clearly defined
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