Modulating microglia and macrophage functions to promote stroke recovery

调节小胶质细胞和巨噬细胞功能促进中风恢复

• 批准号: 10393657
• 负责人: Yejie Shi
• 金额: $ 39.65万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10393657
• 关键词: Acute; Affect; Brain; Brain Infarction; Brain Injuries; Cells; Cerebral Ischemia; Cerebrum; Clinical; Clinical Treatment; Coculture Techniques; Cognitive deficits; Cues; Equilibrium; Foundations; Functional disorder; Future; Genes; Genetic; Guidelines; Homeostasis; Hour; Immune; Immune response; In Vitro; Inflammation; Injury; Interferons; Ischemia; Ischemic Stroke; Knock-out; Lentivirus; Mediator of activation protein; Methods; Microglia; Middle Cerebral Artery Occlusion; Modeling; Molecular; Mus; Nature; Nervous System Physiology; Neurons; Outcome; Peripheral; Pharmacology; Phenotype; Pilot Projects; Quality of life; Recovery; Recovery of Function; Rehabilitation therapy; Resolution; Role; STAT1 protein; Signal Transduction; Solid; Stroke; Tamoxifen; Testing; Therapeutic; Therapeutic Effect; Time; Transgenic Organisms; Treatment Protocols; Work; aged; arginase; brain remodeling; brain repair; brain tissue; clinical translation; clinically relevant; effective therapy; fludarabine; gene repair; gray matter; immune function; improved; in vitro testing; in vivo; in vivo evaluation; inhibitor; intraperitoneal; knock-down; macrophage; monocyte; neuroinflammation; neurological recovery; neuron loss; neurorestoration; new therapeutic target; novel; overexpression; post stroke; preservation; prevent; primary endpoint; repaired; response; sex; small hairpin RNA; stroke outcome; stroke recovery; stroke therapy; stroke victims; therapeutic evaluation; transcription factor; white matter; young adult

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.

新的证据表明，脑炎症在缺血性卒中的病理生理学中起着关键作用。 小胶质细胞和巨噬细胞(Mi/MΦ)是卒中后神经炎症的重要介质，并假定 不同的功能状态对特定的微环境信号做出反应，从而调节炎症， 损伤进展和大脑修复。决定整体性能的关键分子开关和网络 卒中后Mi/MΦ的功能状态尚不清楚。识别这些信令机制可以 揭示通过增强有益的Mi/MΦ功能来改善长期中风预后的新治疗靶点 以及利用恢复性神经炎症的力量。 信号转导和转录激活因子1(STAT1)是一种转录因子，在 典型的干扰素信号，并被定义为巨噬细胞M1极化的重要中介。尽管 STAT1在调节免疫反应中的明确意义已被单独研究过在缺血性中风中的作用 因为它在最初的24小时内导致了急性神经元死亡。到目前为止，STAT1如何控制Mi/MΦ函数 在时间演变的长期恢复期下，临床卒中的主要终点尚不清楚。 我们的初步研究首次表明：1)STAT1主要在Mi/MΦ中被激活(磷酸化) 短暂性局灶性脑缺血后亚急性期(1~5天)。2)Mi/MΦ特异性基因敲除 STAT1不能减少急性脑梗塞，但改善了tFCI后的长期结果，无论性别小鼠。3) 独特的是，STAT1MKO不仅下调了tFCI Mi/MΦ后的促炎基因，而且还上调了a 一组促修复基因，包括精氨酸酶1，一个关键的保护和促修复因子。4)腹膜内 氟达拉滨是一种脑穿透性和选择性的STAT1抑制剂，它能上调Mi/MΦ中的精氨酸酶1， 减轻外周免疫细胞对大脑的侵袭，改善短期和长期结果 在tFCI之后。鉴于这些观察，我们提出了三个具体目标来检验新的中心假设 STAT1基因缺失或药物抑制可改善缺血性卒中后的长期预后 通过促进炎症消退和促进修复的小胶质/巨噬细胞反应。 目的1：检测Mi/MΦ中可诱导的STAT1基因选择性缺失是否能改善卒中的长期预后。小鼠带有 他莫昔芬诱导的Mi/MΦ中STAT1基因敲除将受到大脑中动脉1h诱导的tFCI的影响 闭塞(MCAO)。结果将在tFCI后5周内进行评估。目标2：测试STAT1基因敲除是否缓解 促炎症的Mi/MΦ反应，但通过以下途径促进Mi/MΦ的有益分解/修复活性 精氨酸酶1信号，使用体内tFCI模型和体外原代培养和共培养。我们还将测试 如果Mi/MΦ特异性过表达精氨酸酶1促进修复前期Mi/MΦ反应和神经功能恢复 在tFCI之后。目的3：测试选择性STAT1抑制剂氟达拉滨的长期治疗潜力(5周) 遵循阶梯指南的成年和老年小鼠(20个月大)的卒中结果。