Modulating microglia and macrophage functions to promote stroke recovery

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Emerging evidence implicates a pivotal role of cerebral inflammation in the pathophysiology of ischemic stroke. Microglia and macrophages (Mi/MΦ) are important mediators of post-stroke neuroinflammation and assume diverse functional states in response to specific microenvironmental signals, thereby regulating inflammation, injury progression, and brain repair. The key molecular switches and networks that determine the overall functional state of Mi/MΦ after stroke are poorly understood. Identification of these signaling mechanisms may reveal novel therapeutic targets to improve long-term stroke outcomes by boosting beneficial Mi/MΦ functions and harnessing the power of restorative neuroinflammation. Signal transducer and activator of transcription 1 (STAT1) is a transcription factor that is potently activated in canonical interferon signaling and defined as an important mediator of macrophage M1 polarization. Despite clear implication in regulating immune responses, the role of STAT1 in ischemic stroke has been studied solely for its contribution to acute neuronal death during the first 24 hours. To date, how STAT1 controls Mi/MΦ function under the temporally evolving long-term recovery period, the primary endpoint of clinical stroke, is unknown. Our pilot studies show for the first time that: 1) STAT1 is activated (phosphorylated) primarily in Mi/MΦ at the subacute stage (1-5 days) after transient focal cerebral ischemia (tFCI). 2) Mi/MΦ-specific knockout (mKO) of STAT1 does not reduce acute brain infarct yet improves long-term outcomes after tFCI in mice of both sexes. 3) Uniquely, STAT1 mKO not only downregulates proinflammatory genes in post-tFCI Mi/MΦ, but also elevates a panel of pro-repair genes, including Arginase 1, a key protective and pro-repair factor. 4) Intraperitoneal administration of fludarabine, a brain-penetrant and selective STAT1 inhibitor, upregulates Arginase 1 in Mi/MΦ, alleviates the brain invasion of peripheral immune cells, and improves both short-term and long-term outcomes after tFCI. Given these observations, we propose three specific aims to test the novel central hypothesis that genetic deletion or pharmacological inhibition of STAT1 improves long-term outcomes after ischemic stroke by promoting inflammation-resolving and pro-repair microglial/macrophage responses. Aim 1: Test if inducible deletion of STAT1 selectively in Mi/MΦ improves long-term stroke outcomes. Mice with tamoxifen-inducible STAT1 knockout in Mi/MΦ will be subjected to tFCI induced by 1-h middle cerebral artery occlusion (MCAO). Outcomes will be assessed for 5 weeks after tFCI. Aim 2: Test if STAT1 knockout alleviates proinflammatory Mi/MΦ responses but promotes the beneficial pro-resolving/pro-repair activities of Mi/MΦ via Arginase 1 signaling, using the in vivo tFCI model and in vitro primary cultures and co-cultures. We will also test if Mi/MΦ-specific overexpression of Arginase 1 boosts pro-repair Mi/MΦ responses and neurological recovery after tFCI. Aim 3: Test the therapeutic potential of the selective STAT1 inhibitor fludarabine in long-term (5 weeks) stroke outcomes in young adult and aged mice (20 months old) of both sexes following STAIR guidelines.