Abstract Body: Background: Ischemic preconditioning (IPC) is a robust protective phenomenon in which a brief ischemic exposure confers tolerance against a subsequent prolonged ischemic challenge. Elucidating the mechanisms of IPC is a critical challenge in the stroke field. Innate immune pathways, particularly type I interferon (IFN) signaling, in microglia are critical in establishing protection in both grey and white matter. Our previous studies demonstrated that type I IFN receptor (IFNAR1) in microglia is required for both IPC-induced axonal protection and interferon stimulated gene (ISG) expression. We also showed that exposure of primary microglia to either IFN-beta or ischemia/reperfusion-like conditions results in phosphorylation of STAT1, a key signaling kinase downstream of IFNAR1. Therefore, we hypothesized that STAT1 is a critical mediator of the microglial response to IPC. Here we report the impact of systemic STAT1 knock-out on microglial responses after IPC.
Methods: We performed a transient (15 min) middle cerebral artery occlusion on 4 wild-type (WT) and 4 Stat1^-/- mice and collected tissue 72 hours later. We processed tissue for single nucleus RNA-sequencing using 10X Genomics kits. We captured ~10,000 nuclei per sample and sequenced at a depth of 25,000 reads. We performed comparative analysis between ipsilateral and contralateral hemispheres to identify focal changes in microglial subpopulations and gene expression due to IPC. We performed immunofluorescent microscopy and quantitative stereology to validate IPC-induced changes in microglial number/morphology and expression of ISG protein products such as IFITM3.
Results: We describe global in vivo transcriptomic changes in microglial subpopulations after IPC and found that STAT1-deficiency alters both the distribution of microglial subpopulations and cluster-specific transcriptional profiles. We identify and define an interferon responsive microglial cluster induced by IPC that is dependent on STAT1. We also provide data demonstrating how ischemia/reperfusion and innate immune signaling affect STAT1 phosphorylation signaling dynamics in microglia in vitro. We show that phosphorylation is dependent on both Toll-like receptor 4 (TLR4) and IFNAR1.
Conclusions: Our results support a central role for STAT1 in mediating microglial phenotype in vivo following IPC. Our findings indicate that STAT1 is an important regulator of microglial type I IFN signaling in the context of ischemia.