The miR-183/96/182 Cluster in Pseudomonas aeruginosa-induced Keratitis

铜绿假单胞菌（PA）是一种条件致病菌，可引起快速发展和破坏性的 角膜疾病，是全球视力障碍和失明的原因。它也是最常见的 在发达国家发现了隐形眼镜相关疾病的致病微生物。最令人担忧的是， 抗药性细菌菌株的不断出现对有效控制疾病构成了严重挑战， 需要进行管理和预防性治疗。因此，研究的长期目标 提出的是测试微RNA（miRNAs）的调节作用，这是一个新认识的重要基因水平， 表达调控，在细菌性角膜炎，确定新的治疗靶点，并提供替代治疗 战略布局为了支持这一目标，初步发表的研究表明，miR-183/96/182簇， 产生miR-183、miR-96和miR-182，在角膜和先天免疫细胞中表达，包括 巨噬细胞（Mφ）和多形核中性粒细胞（PMN）。灭活本 小鼠中的簇导致促炎趋化细胞因子减少（例如，MCP 1、MIP 2和IL-1β）在 在miR-183/96/182簇敲除（ko）小鼠中，PA诱导的角膜炎的严重程度降低。 与ko小鼠中趋化性细胞因子减少一致，PMN数量在早期（给药后1天）减少， 感染，DPI），并且细菌负荷增加。然而，在疾病后期（5 dpi）， 两组，但细菌负荷显着下降，在KO动物。其他初步数据显示， KO小鼠的PMN具有增强的吞噬和杀伤能力，与体内数据一致， 尽管PMN数量相似，但角膜中的细菌负荷减少。关于潜在的治疗策略， 在PA感染的野生型中预防性结膜下和局部应用抗miR的初步实验， 型小鼠，提供了成功下调miR-183/96/182簇功能的信息 降低PA角膜炎的严重程度。因此，为达致长远目标，我们的目标如下： 提出：目的1将测试角膜驻留Mφ（髓样细胞）中miR-183/96/182簇的失活 减少促炎趋化细胞因子的产生，特别是MCP 1、IL-1β和 MIP 2的表达减少了PMN和Mφ向感染角膜的浸润。目标2将测试这一点 通过将ko小鼠的骨髓细胞繁殖到骨髓细胞中来恢复miR-183/96/182簇表达 特异性Cre转基因动物，足以在体内逆转角膜对PA感染的反应，以及 吞噬作用和浸润细胞的细胞内杀伤。目的3将测试DAP 12是miR-12的直接靶点。 183/96/182在PMN和Mφ中聚集，并介导其对这些细胞的调节。Aim 4将测试本地 角膜中miR-183/96/182簇功能的敲低对于PA诱导的角膜炎是治疗性的。 预计这些研究将揭示miR-183/96/182簇调控的新分子机制 为细菌性角膜炎的治疗提供了新的靶点。

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that induces a rapidly developing and destructive disease of the cornea and is a global cause of visual impairment and blindness. It is also the most commonly recovered causative organism in contact lens-related disease in developed countries. Of most concern, continued emergence of antibiotic-resistant bacterial strains poses a serious challenge for effective disease management and adjunctive treatments are required. Therefore, the long-term objective of the studies proposed is to test the regulatory role of microRNAs (miRNAs), a newly recognized, important level of gene- expression regulation, in bacterial keratitis, identify new therapeutic targets and provide alternative treatment strategies. To support this goal, preliminary published studies showed that the miR-183/96/182 cluster, which produces miR-183, miR-96 and miR-182, is expressed in the cornea and in innate immune cells, including macrophages (Mφ) and polymorphonuclear neutrophils (PMN), in both mouse and human. Inactivation of this cluster in mice led to decreased pro-inflammatory chemotactic cytokines (e.g., MCP1, MIP2 and IL-1β) in the cornea and a decreased severity of PA-induced keratitis in miR-183/96/182 cluster knockout (ko) mice. Consistent with reduced chemotactic cytokines in ko mice, PMN number was decreased early (1 day post infection, dpi) in disease, and bacterial load increased. Yet later in disease (5 dpi) PMN number was similar in both groups, but bacterial load was significantly decreased in the ko animals. Other preliminary data showed that PMN from ko mice had enhanced phagocytic and killing abilities, consistent with in vivo data showing reduction of bacterial load in the cornea, despite similar PMN number. Regarding potential treatment strategies, a pilot experiment with prophylactic subconjunctival and topical application of anti-miRs in PA-infected wild- type mice, provided information that down-regulation of miR-183/96/182 cluster function successfully decreased the severity of PA keratitis. Therefore, to achieve our long-term objectives, the following aims are proposed: Aim 1 will test that inactivation of the miR-183/96/182 cluster in corneal resident Mφ (myeloid cells) decreases production of pro-inflammatory chemotactic cytokines, specifically, MCP1, IL-1β and MIP2, contributing to a decreased infiltration of PMN and Mφ to the infected cornea. Aim 2 will test that restoration of miR-183/96/182 cluster expression in myeloid cells of ko mice by breeding them to myeloid specific Cre transgenic animals, is sufficient to reverse the corneal response to PA infection in vivo, as well as phagocytosis and intracellular killing by infiltrating cells. Aim 3 will test that DAP12 is a direct target of the miR- 183/96/182 cluster in PMN and Mφ, and mediates its regulation of these cells. Aim 4 will test that local knockdown of miR-183/96/182 cluster function in the cornea is therapeutic for PA-induced keratitis. It is anticipated that these studies will reveal novel molecular mechanisms of miR-183/96/182 cluster regulation of innate immune responses in bacterial keratitis and provide a new target for its treatment.