Immune mediated regeneration of retinal ganglion cell axons following optic nerve trauma

轴索病变是青光眼、视神经炎和外伤性视神经病变的早期和显著的病理特征。 神经损伤。在所有这些情况下，永久性视力丧失在很大程度上是视网膜衰竭的次要原因。 神经节细胞(RGC)是视神经的输出神经元，用于生存和再生轴突。有一个 迫切需要开发新的治疗干预措施，克服眼睛修复障碍，促进RGC 存活和RGC轴突再生，从而减轻甚至逆转视力丧失。在这份提案中，我们 研究眼内注射(I.O.)后在玻璃体内积聚的一组新的中性粒细胞。 给小鼠注射真菌胞壁提取物、酵母多糖，并与再生有关的断端 RGC轴突。在初步研究中，我们已经证明了酵母多糖诱导的过继转移 中性粒细胞直接进入视神经挤压(ONC)损伤小鼠的玻璃体，足以挽救RGC和 刺激RGC轴突再生。这些中性粒细胞以环状核和细胞表面为特征。 表型CD14+Ly6Glow。它们表达高水平的精氨酸酶-1和CD206的转录本。我们的主要目标 是阐明推动修复性中性粒细胞分化的因素，并制定方案 在体外产生它们，用于视神经疾病患者的治疗。在目标1中，我们将测试我们的 假设IL-4和粒细胞集落刺激因子(G-CSF)协同作用推动 IO致小鼠体内促再生中性粒细胞的分化。酵母多糖注射剂与ONC 受伤。我们发现CD14+Ly6Glow中性粒细胞表达高水平的IL-4，提示了IL-4的作用 信号分子，而IL-4蛋白在IO后的玻璃体中产生。酵母多糖的给药。G- CSF也在玻璃体中上调，最近发现它可以诱导玻璃体内IL-4受体的表达。 中性粒细胞。我们将确定IL-4、IL-4受体链、G-CSF和G-CSF的动力学和细胞来源 酵母多糖注射眼内的受体。功能的得失实验，使用一组条件敲击- 将进行小鼠和骨髓嵌合体的实验，以评估IL-4和G-CSF信号在 CD14+Ly6Glow中性粒细胞的发育、RGC存活和轴突再生酵母多糖 注射和ONC。这项研究最终可能导致小说的发展，或者改变小说的用途 已建立的促进神经再生中性粒细胞分化和扩增的免疫调节剂 继发于青光眼、视神经炎或外伤的视神经病变患者。我们的探索性实验 已发现小鼠骨髓中性粒细胞具有酵母多糖诱导的CD14+Ly6Glow的特征 中性粒细胞，包括驱动轴突再生的能力，在体外用IL-4和G- 脑脊液。目标2的总体目标是优化从 小鼠骨髓前体细胞体外培养。目标3的目标是评估IL-4的神经再生潜力 经调节的人类中性粒细胞，可能会重新注入视神经疾病患者体内，作为一种 自体细胞疗法。

Axonopathy is an early and prominent pathological feature of glaucoma, optic neuritis and traumatic optic nerve injury. Permanent loss of vision in all of these conditions is secondary, in large part, to a failure of retinal ganglion cells (RGC), the output neurons of the optic nerve, to survive and regenerate their axons. There is a dire need to develop novel therapeutic interventions that overcome barriers to repair in the eye, promote RGC survival and RGC axonal regrowth, thereby mitigating, or even reversing, visual loss. In this proposal we investigate a novel subset of neutrophils that accumulate in the vitreous body following intraocular (i.o.) injection of mice with the fungal cell wall extract, zymosan, and are associated with the regrowth of severed RGC axons. In preliminary studies we have demonstrated that adoptive transfer of zymosan-elicited neutrophils directly into the vitreous of mice with optic nerve crush (ONC) injury is sufficient to rescue RGC and stimulate RGC axon regrowth. These neutrophils are characterized by ring-form nuclei and the cell surface phenotype CD14+Ly6Glow. They express high levels of transcripts for arginase-1 and CD206. Our major goals are to elucidate the factors that drive the differentiation of reparative neutrophils and develop protocols to generate them in vitro for therapeutic application in individuals with optic neuropathy. In Aim 1 we will test our hypothesis that IL-4 and granulocyte-colony stimulating factor (G-CSF) act synergistically to drive the differentiation of pro-regenerative neutrophils in vivo in mice subjected to i.o. zymosan injection and ONC injury. A role of IL-4 was suggested by our finding that CD14+Ly6Glow neutrophils express high levels of IL-4 signaling molecules, and IL-4 protein is produced in the vitreous following i.o. administration of zymosan. G- CSF is also upregulated in the vitreous and it was recently shown to induce IL-4 receptor expression on neutrophils. We will determine the kinetics and cellular source of IL-4, IL-4 receptor chains, G-CSF and G-CSF receptor in zymosan injected eyes. Loss and gain of function experiments, using a panel of conditional knock- out mice and bone marrow chimeras, will be performed to assess the roles of IL-4 and G-CSF signaling in the development of CD14+Ly6Glow neutrophils, RGC survival and axonal regeneration following i.o. zymosan injection and ONC. This research could ultimately lead to the development of novel, or the repurposing of established, immunomodulators to promote the differentiation and expansion of neuroregenerative neutrophils in patients with optic neuropathy secondary to glaucoma, optic neuritis or trauma. Our exploratory experiments have shown that murine bone marrow neutrophils acquire characteristics of zymosan-elicited CD14+Ly6Glow neutrophils, including the ability to drive axonal regeneration, following in vitro polarization with IL-4 and G- CSF. The overall goal of Aim 2 is to optimize protocols for the generation of pro-regenerative neutrophils from murine bone marrow precursors ex vivo. The goal of Aim 3 is to assess the neuroregenerative potential of IL-4 modulated human neutrophils that could, potentially, be re-infused into patients with optic neuropathy, as an autologous cell therapy.