Treatment of secondary injury after ischemic stroke through targeting microglia

描述（由申请人提供）：虽然已知活化的小胶质细胞/巨噬细胞（M β）有助于卒中后继发性脑损伤，但M β也有助于修复过程。为了解释这种截然不同的作用，提出了两种M β表型，“有害”/M1和“愈合”/M2。重要的是，M1的M2可以被极化为M2，反之亦然。虽然衍生自中风损伤细胞的各种分子具有向M1型迁移的倾向（导致促炎细胞因子和促氧化剂的产生），但其它分子（例如由T细胞产生的），如IL-4、IL 10或TGF β，向M2型迁移。M2巨噬细胞控制清除过程（吞噬作用介导的毒性和促炎细胞碎片的清除）和营养因子的产生。因此，毒性的来源以及阻碍神经重组的生物和物理障碍被去除，同时提供管理神经元发芽（修复过程）的营养支持。如果没有M2 M3，就不可能治愈。虽然中风后不久M1和M2 M2表型共存，但M2表型在2-3天内减弱，使M1型占主导地位。这对大脑的清理、修复和恢复产生不利影响，同时增加细胞毒性。探索这些过程的机制和旨在纠正M2极化损失的治疗方法是本提案的主题。我们的新发现表明，在响应缺血，神经元合成和释放IL-4（典型的M2极化细胞因子）在中风的前3天，随后在IL-4和M2 M β水平下降。IL-4诱导的M2 M β极化需要激活RXR-PPARγ通路。从卒中后第1天开始，给予IL-4或PPARγ和RXR激动剂（整流因子）5天，可改善恢复和神经元重新连接。M细胞中PPARγ的缺失阻碍了恢复。目标1.阐明体外神经元在M β极化和卒中后修复中的作用：IL-4介导的PPARγ：RXR执行的过程涉及吞噬（清除）和营养刺激。假设：神经元充当“损伤”的传感器并产生IL-4/IL 13，其向小胶质细胞发出信号以诱导M2极化，同时抑制与细胞毒性相关的M1表型。目标二。阐明体内IL-4信号传导至PPARγ：RXR的基础，并将其应用于靶向吞噬细胞介导的脑清除、重塑和卒中后恢复的临床相关方法。假设：由于中风后由神经元产生IL 4/IL 13驱动的朝向“愈合”M2表型的初始极化在中风后几天内丧失，因此旨在恢复IL-4介导的信号传导的方法，例如通过重组IL-4和PPARγ：RXR活化（M2中IL-4的分子靶点），以纠正M2极化丧失将有助于中风后恢复。

DESCRIPTION (provided by applicant): Though the activated microglia/macrophages (Mɸ) are known to contribute to secondary brain injury after stroke, Mɸ are also instrumental in the repair process. To explain such contrasting roles, two Mɸ phenotypes, "harmful"/M1 and "healing"/M2, have been proposed. Importantly, the M1 Mɸ can be polarized to M2 and vice versa. While various molecules derived from stroke-injured cells have the propensity to polarize Mɸ toward M1 type (causing the generation of pro-inflammatory cytokines and pro-oxidants), other molecules (e.g. generated by T cells), such as IL-4, IL10 or TGFß, polarize Mɸ toward the M2 type. The M2 Mɸ control the cleanup processes (phagocytosis- mediated removal of toxic and pro-inflammatory cellular debris), and the production of trophic factors. Thus, the source of toxicity and the biological and physical barriers hampering neural reorganization are removed, while providing trophic support governing neuronal sprouting (the repair process). Without the M2 Mɸ, healing would not be possible. While both M1 and M2 Mɸ phenotypes co-exist shortly after stroke, M2 phenotype weakens within 2-3 days allowing M1 type to dominate. This adversely affects the brain cleanup, repair, and recovery, while boosting cytotoxicity. Exploring the mechanisms underlying such processes and therapeutic approaches aiming at rectifying the M2 polarization loss are the subjects of this proposal. Our novel findings indicate that in response to ischemia, neurons synthesize and release IL-4 (canonical M2 polarizing cytokine) during the first 3 days of stroke, followed by a decline in both IL-4 and M2 Mɸ levels. IL-4-induced M2 Mɸ polarization requires the activation of RXR-PPARγ pathway. The administration of IL-4 or agonists of PPARγ and RXR (rectifying factors) for 5 days, starting at 1d post stroke, improves recovery and neuronal re-wiring. Loss of PPARγ in Mɸ impedes recovery. Aim 1. Elucidate, in vitro, the role of neurons in Mɸ polarization and post stroke repair: IL-4- medaited PPARγ:RXR-executed process involving phagocytosis (cleanup) and trophic stimulation. Hypothesis: Neurons act as sensors of "injury" and generate IL-4/IL13 that signals to microglia to induce M2 polarization, while suppressing M1 phenotype associated with cytotoxicity. Aim 2. Elucidate, in vivo, the basis of IL-4 signaling to PPARγ:RXR, and apply them into clinically relevant approaches targeting the phagocyte-mediated brain cleanup, remodeling, and post- stroke recovery. Hypothesis: Because the initial polarization toward the "healing" Mɸ phenotype after stroke, driven by neuronal production of IL4/IL13, is lost within few days after stroke, approaches aiming at restoring IL-4-medaited signaling, such as via recombinant IL-4 and PPARγ:RXR activation (molecular targets of IL-4 in Mɸ), to rectify M2 polarization loss will assist in post stroke recovery.