Treatment of secondary injury after ischemic stroke through targeting microglia

描述(申请人提供)：虽然已知激活的小胶质细胞/巨噬细胞(Mɸ)会导致中风后的继发性脑损伤，但Mɸ也在修复过程中起着重要作用。为了解释这种不同的作用，已经提出了两种Mɸ表型，“有害的”/M1和“治愈的”/M2。重要的是，M1 Mɸ可以极化为M2，反之亦然。虽然来自中风损伤细胞的各种分子有将Mɸ极化为M1型的倾向(导致促炎细胞因子和促氧化剂的产生)，但其他分子(如由T细胞产生的)，如IL-4、IL10或转化生长因子？等，使Mɸ极化为M2型。M2 Mɸ控制着清除过程(吞噬作用介导的有毒和促炎细胞碎片的清除)，以及营养因子的产生。这样，毒性的来源以及阻碍神经重组的生物和物理障碍就被消除了，同时提供了管理神经元萌发(修复过程)的营养支持。如果没有M2 Mɸ，治愈是不可能的。虽然M1和M2两种Mɸ表型在卒中后不久共存，但M2表型在2-3天内减弱，M1型占主导地位。这会对大脑的清理、修复和恢复产生不利影响，同时增加细胞毒性。探索这种过程的机制和旨在纠正M2偏振损失的治疗方法是本提案的主题。我们的新发现表明，在卒中的前3天，神经元对缺血的反应是合成和释放IL-4(典型的M2极化细胞因子)，随后IL-4和M2 Mɸ水平都下降。IL-4诱导的M2 Mɸ极化需要激活RXR-PPARγ通路。从中风后1d开始，给予IL-4或PPAR激动剂γ和RXR(整流因子)5天，可促进恢复和神经元重新连接。PPARγ在Mɸ中的丢失阻碍了恢复。目的1.体外研究神经元在Mɸ极化和卒中后修复中的作用：IL-4介导的PPARγ：RXR执行的吞噬(清除)和营养刺激过程。假设：神经元作为“损伤”的感受器，产生IL-4/IL-13，向小胶质细胞发出信号，诱导M2极化，同时抑制与细胞毒性相关的M1表型。目的2.在体内阐明IL-4信号转导PPARγ：RXR的基础，并将其应用于针对吞噬细胞介导的脑清理、重塑和卒中后恢复的临床相关方法。假设：由于卒中后在神经元产生IL-4/IL-13的驱动下，卒中后M-ɸ表型的初始极化在卒中后几天内消失，旨在恢复IL-4介导的信号的方法，如通过重组IL-4和PPARγ：RXR激活(M-ɸ中IL-4的分子靶点)，纠正M2极化丢失将有助于卒中后的恢复。

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.