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

铜绿假单胞菌是一种条件致病菌，可引起迅速发展和破坏性的致病菌。 这是一种角膜疾病，是全球范围内造成视力损害和失明的原因。它也是最常见的 发达国家隐形眼镜相关疾病的病原菌恢复情况。最令人担忧的是， 抗生素耐药细菌菌株的不断出现对有效的疾病构成了严峻的挑战 需要进行管理和辅助治疗。因此，研究的长期目标是 提出的是测试microRNAs(MiRNAs)的调节作用，miRNAs是一种新发现的重要水平的基因- 细菌性角膜炎中的表达调控，确定新的治疗靶点并提供替代治疗 战略。为支持这一目标，已发表的初步研究表明，miR-183/96/182群组 产生miR-183、miR-96和miR-182，在角膜和先天性免疫细胞中表达，包括 巨噬细胞(Mφ)和中性粒细胞(PMN)。停用此功能 小鼠体内的聚集性导致炎性趋化细胞因子(如MCP1、MIP2和IL-1β)减少 在miR-183/96/182簇基因敲除(KO)小鼠中，角膜和PA诱导的角膜炎的严重程度降低。 与KO小鼠趋化细胞因子减少一致，PMN数量在早期(1天后)减少 感染，DPI)，细菌负荷增加。然而，在后来的疾病(5dpi)中，PMN数量在 但KO组动物的细菌负荷显著降低。其他初步数据显示 来自KO小鼠的PMN具有增强的吞噬和杀伤能力，这与体内数据显示的一致 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， 一项预防性结膜下和局部应用抗miRs的初步实验，在感染PA的野生. 型小鼠，提供了miR-183/96/182簇功能下调成功的信息 降低PA角膜炎的严重程度。因此，为了实现我们的长远目标，我们的目标如下 建议：AIM 1将在角膜常驻Mφ(髓系细胞)中测试miR-183/96/182簇的失活 减少促炎趋化细胞因子的产生，特别是MCP1，IL-1β和 MIP2，有助于减少中性粒细胞和M-φ对感染角膜的渗透。目标2将测试这一点 培育成髓系细胞恢复miR-183/96/182在KO小鼠髓系细胞中的表达 特定的Cre转基因动物，足以逆转体内对PA感染的角膜反应，以及 渗透细胞的吞噬作用和细胞内杀伤作用。目标3将测试DAP12是miR的直接目标- 183/96/182聚集在中性粒细胞和M-φ上，并介导其对这些细胞的调节。目标4将测试当地人 抑制角膜miR-183/96/182簇功能对PA诱导的角膜炎有治疗作用。 预计这些研究将揭示miR-183/96/182簇调控的新的分子机制 研究细菌性角膜炎的先天免疫反应，为其治疗提供新的靶点。

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.