A novel inflammatory cell with neuroprotective and neuroregenerative properties

轴索病变是许多中枢神经系统(CNS)疾病的早期和突出的病理特征， 包括脑和脊髓损伤，视神经病变，多发性硬化症，早期阿尔茨海默病， 和皮质下缺血。所有这些神经疾病的不良临床结果在很大程度上是由于 包括视网膜神经节细胞(RGC)在内的成年中枢神经系统神经元再生能力有限 产生视神经)。迫切需要开发新的治疗干预措施，以克服 修复成年中枢神经系统的障碍，促进轴突再生。这里提出的研究是基于我们的 以细胞表面表型为特征的促再生中性粒细胞新亚群的发现 Ly6GlowCD14+，局部积聚在眼后房或腹膜腔内 给药酵母细胞壁抽提物酵母多糖。这些中性粒细胞带有一个环状的核，并表达 高水平的模式识别受体Dectin-1，以及精氨酸酶-1和CD206的转录本。在……里面 初步研究证实酵母多糖过继转移可诱导Ly6GlowCD14+中性粒细胞 直接进入视神经挤压(ONC)损伤小鼠的玻璃体足以将RGC从细胞死亡中拯救出来 刺激切断的RGC轴突再生。此外，从培养物中收获的条件培养液 Ly6GlowCD14+中性粒细胞诱导分离的RGC和背根神经节神经元突起生长 体外培养。目前提案的总体目标是阐明构成差异的途径， 这些非常规修复性中性粒细胞的存活和作用机制，并利用 在开发减轻甚至逆转损害的免疫调节疗法方面获得的知识 中枢神经系统神经元和轴突。在目标1中，我们将检验我们的假设，即转化生长因子-驱动 酵母多糖对体内Ly6GlowCD14+中性粒细胞的分化作用在《目标2》中我们将 确定缺氧诱导因子-1在细胞稳定、存活和生物学功能中的作用 Ly6GlowCD14+中性粒细胞。在目标3中，我们将优化生成促再生的协议 骨髓前体细胞体外培养的中性粒细胞。选定的中性粒细胞株将被注入患有ONC的小鼠 以评估其作为一种自体细胞疗法的疗效。此外，我们将使用蛋白质组学和遗传学 表征Ly6Glow中性粒细胞条件培养液中存在的可溶性因子的方法 对增强的轴突生长负责。未来的方向将是对候选人进行管理 神经再生素作为疾病改良剂对轴索病小鼠的影响。我们希望这些数据 由我们的研究产生的结果将最终导致基于细胞的创新疗法和/或 具有神经保护/再生特性的免疫调节药物，可恢复丧失的神经功能 在中枢神经系统创伤或其他以轴索病变为特征的情况下。

Axonopathy is an early and prominent pathological feature of many central nervous system (CNS) disorders, including brain and spinal cord trauma, optic neuropathy, Multiple Sclerosis, early stage Alzheimer’s disease, and subcortical ischemia. Poor clinical outcomes in all of these neurological conditions are due, in large part, to the limited regenerative capacity of adult CNS neurons, including retinal ganglion cells (RGC, the neurons that give rise to the optic nerve). There is a dire need to develop novel therapeutic interventions that overcome barriers to repair in the adult CNS and promote axonal regrowth. The studies proposed here are based on our discovery of a novel subset of pro-regenerative neutrophils, characterized by the cell surface phenotype Ly6GlowCD14+, that accumulate in the posterior chamber of the eye or the peritoneal cavity following local administration of the yeast cell wall extract, zymosan. These neutrophils bear a ring-form nucleus and express high levels of pattern recognition receptor, dectin-1, as well as transcripts for arginase-1 and CD206. In preliminary studies we demonstrated that adoptive transfer of zymosan-elicited Ly6GlowCD14+ neutrophils directly into the vitreous of mice with optic nerve crush (ONC) injury is sufficient to rescue RGC from cell death and to stimulate the regrowth of severed RGC axons. Furthermore, conditioned media harvested from cultures of Ly6GlowCD14+neutrophils induce neurite outgrowth of dissociated RGC and dorsal root ganglion neurons in vitro. The overall goal of the current proposal is to elucidate the pathways that underlie the differentiation, survival and mechanism of action of these unconventional reparative neutrophils, and to leverage the knowledge gained for the development of immunomodulatory therapies that mitigate, or even reverse, damage to CNS neurons and axons. In Aim 1 we will test our hypothesis that transforming growth factor (TGF)- drives the differentiation of Ly6GlowCD14+ neutrophils in vivo following the administration of zymosan. In Aim 2 we will determine the role of hypoxia induced factor (HIF)-1 in the stabilization, survival and biological functions of Ly6GlowCD14+ neutrophils. In Aim 3 we will optimize protocols for the generation of pro-regenerative neutrophils from bone marrow precursors ex vivo. Selected neutrophil lines will be infused into mice with ONC injury to assess their efficacy as an autologous cellular therapy. In addition, we will use proteomic and genetic approaches to characterize the soluble factors present in Ly6Glow neutrophil-conditioned media that are responsible for enhanced neurite outgrowth. A future direction will be to administer candidate neuroregenerative factors to mice with axonopathy as disease modifying agents. We are hopeful that the data generated by our study will ultimately lead to the development of innovative cell based therapies and/ or immunomodulatory drugs with neuroprotective/ regenerative properties that restore lost neurological functions in patients with CNS trauma or other conditions characterized by axonopathy.