角膜上皮创伤诱导的炎症级联反应与角膜的神经再生

The focus of this application is on the restoration of the corneal nerves following corneal injury. The general hypothesis is that specific molecular mechanisms within the innate inflammatory process participate directly or indirectly in nerve restoration. Within the unique environment of the cornea, we are defining temporal cascades in the inflammatory process that coordinate sequential healing mechanisms. An initial surface injury to the corneal epithelium and the subbasal nerve plexus results in limbal inflammation with leukocyte migration into the corneal stroma and epithelium. This reaction to injury is critical to host defense against infection, and has direct and indirect effects on tissue repair, for example, factors supporting early nerve growth delivered by the infiltrating leukocytes. Dysregulation of the inflammatory response can be detrimental to nerve repair given the ability of host defense mechanisms to cause tissue injury or the need to deliver cytokines and growth factors at critical stages of healing. The balance and timing of the innate inflammatory response to injury for coordinated tissue and nerve restoration is a potentially rich context for therapeutic considerations. Our evidence is consistent with the interpretation that neutrophils and associated platelets deliver VEGF-A adjacent to the injured nerves at the beginning of neurite sprouting, and this VEGF is important to early nerve regeneration. The neutrophil/VEGF step depends on the production of CCL20 that attracts γδ T cells. The γδ T cells express IL-17A and IL-22 necessary for the efficient recruitment of the neutrophils and platelets to the corneal stroma and the healing of the stratified corneal epithelium. NK cell accumulation follows γδ T cell, neutrophil and platelet accumulation by about 6-12 hours, and is dependent on γδ T cells, CXCL10 and CCL2. The NK cells appear to be important in limiting the extent of neutrophil accumulation, keeping the balance in favor of healing. These steps in the cascade occur within the first 24 hours after injury and the neutrophil/VEGF step is transient, waning within the first 30 hours. These include (1) the ability of NKp46+ CD3ˉ NK cells to not only limit acute inflammation but sustain epithelial CD11c+ MHCII+ dendritic cells, (2) the influence of IL-20+ CD301a+ CD11cˉ myeloid cells on epithelial healing and nerve regeneration, and (3) the influence of a macrophage metalloproteinase, MMP12, on nerve regeneration. The CD301a+ myeloid cells appear to exhibit a macrophage phenotype previously shown to positively promote nerve growth in vitro. Pursuit of the specific aims of this application will provide a cellular and molecular model within which support for nerve regeneration in the wounded cornea can be approached, and dysregulated or destructive inflammation can be managed.

申请者前期的研究工作提示角膜创伤后存在一个非常复杂的炎症性级联反应网络。本项目主要聚焦于角膜创伤后炎症级联反应对角膜神经再生的促进作用及其机制。将以角膜上皮细胞创伤后触发产生的致炎性趋化因子和细胞因子为始动环节，围绕申请者最近发现的在角膜创伤后发挥负性调节作用的NK细胞和巨噬细胞为中心环节，使用高分辨荧光显微镜、基因敲除、细胞和分子生物学技术寻找促进角膜神经再生的分子和细胞的网络。其中包括NK细胞分泌的抗炎性细胞因子、对其他白细胞起杀伤作用的细胞毒受体和对树突状细胞迁移的影响，以及巨噬细胞产生的IL-10家族成员（IL-20和IL-24）的调节作用和金属蛋白酶的负反馈作用。其最终目标是阐明角膜创伤后炎症反应促进神经再生的细胞和分子机制,并对炎症失控背景下的角膜神经再生提供潜在的治疗方案。

角膜的创伤修复和炎症反应是一复杂的级联反应。不良的修复过程常造成视力的严重损害。通过使用模式动物、基因表达、流式细胞分选、特异性白细胞亚群去除等技术，我们在角膜创伤后炎症反应和修复机制领域取得了数项突破性研究成果。主要的发现有：（1）确立了角膜巨噬细胞的两个亚群（CD64+CCR2+和CD64-CCR2-巨噬细胞）及其在创伤修复过程中的平衡作用；CD64+CCR2+巨噬细胞通过产生致炎性细胞因子促进炎症反应，但CD64-CCR2-亚群则通过产生炎症抑制性细胞因子抑制炎症反应；（2）首次证实在角膜存在2型自然淋巴样细胞（ILC2s）并通过IL-33依赖性的产生双调蛋白在促进角膜的创伤修复过程中发挥着独特的作用；（3）发现抗生素诱导的肠道菌群失调通过调节CD64+CCR2-巨噬细胞在角膜的分布及其基因转录的缺陷抑制角膜神经的再生和功能的修复；（４）自主神经对角膜创伤后触发的炎症反应和再上皮化过程发挥着重要的调节作用，交感神经兴奋加强炎症反应、抑制创伤修复；与此相反，副交感神经兴奋则抑制炎症、加速创伤修复；使用肾上腺素能受体阻断剂具有显著加速角膜创伤修复和抑制角膜炎症的作用；（5）进一步的研究显示，CCR2+巨噬细胞亚群优势表达肾上腺素能受体，但CCR2-则优势表达胆碱能受体；（６）发现埃坡霉素B通过稳定微管和增加神经纤维内线粒体数量促进角膜神经的再生和功能的恢复；（７）发现角膜上皮的有丝分裂受昼夜节律的严密控制，时差和药理性干扰分子时钟影响角膜的创伤修复。这些发现和数据均发表在国际权威杂志。在该研究中提出的新概念和新假设对于进一步阐明角膜创伤修复后级联性细胞和分子反应机制以及制定促进角膜创伤修复和控制炎症反应的新措施提供了重要的理论基础，且具有潜在的重要的临床价值。