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

铜绿假单胞菌诱导的角膜炎中的 miR-183/96/182 簇

• 批准号: 9307421
• 负责人: SHUNBIN XU
• 金额: $ 38.5万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9307421
• 关键词: Adaptor Signaling Protein; Adverse event; Anti-Inflammatory Agents; Anti-inflammatory; Antibiotic Therapy; Antibiotics; Bacterial Antibiotic Resistance; Blindness; Breeding; CCL2 gene; Cells; Contact Lenses; Cornea; Data; Developed Countries; Developing Countries; Disease; Disease Management; Disease Outcome; Down-Regulation; Equilibrium; Gene Expression; Gene Expression Regulation; Genetic Transcription; Goals; Human; Immune; Immune response; In Vitro; Infection; Infiltration; Inflammatory; Inflammatory Response; Injection of therapeutic agent; Innate Immune Response; Innate Immune System; Interleukin-1 beta; Keratitis; Knock-out; Knockout Mice; Left; Lipopolysaccharides; Mediating; MicroRNAs; Microglia; Modeling; Molecular; Mus; Myelogenous; Myeloid Cells; Natural Immunity; Organism; Perforation; Phagocytes; Phagocytosis; Play; Population; Production; Pseudomonas aeruginosa; Publishing; Recruitment Activity; Regimen; Regulation; Regulator Genes; Research; Role; Severities; Severity of illness; TYROBP gene; Testing; Therapeutic; Tissues; Topical application; Transgenic Animals; Untranslated RNA; Virulence Factors; Visual impairment; Wild Type Mouse; alternative treatment; cell type; chemokine; cytokine; experimental study; in vivo; killings; knock-down; knockout animal; macrophage; microbial; mouse model; neutrophil; new therapeutic target; novel; overexpression; pathogen; prophylactic; response; restoration; therapeutic target; treatment strategy

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.

铜绿假单胞菌（PA）是一种机会性病原体，可诱导快速发展和破坏性 角膜疾病是视觉障碍和失明的全球原因。它也是最常见的 发达国家中与晶状体相关疾病中的病原体有机体恢复。最关心的是 持续出现抗生素的细菌菌株对有效疾病具有严重的挑战 需要管理和辅助治疗。因此，研究的长期目标 提出的是测试microRNA（miRNA）的调节作用，这是一种新认识的，重要的基因水平 表达调节，在细菌角膜炎中，识别新的治疗靶标并提供替代治疗 策略。为了支持这一目标，初步发表的研究表明，miR-183/96/182群集，该研究 产生miR-183，miR-96和miR-182，在角膜和先天免疫细胞中表达，包括 小鼠和人类中的巨噬细胞（Mφ）和多形性嗜中性粒细胞（PMN）。失活 小鼠的簇导致促炎性趋化性细胞因子（例如MCP1，MIP2和IL-1β）降低 MiR-183/96/182簇敲除（KO）小鼠的角膜和PA诱导的角膜炎的严重程度降低。 与KO小鼠中趋化细胞因子降低一致，PMN的数量提早提高（1天后1天 感染，疾病中的DPI，细菌负荷增加。后来在疾病（5 DPI）的PMN数字中相似 两组，但是KO动物的细菌负荷显着降低。显示的其他初步数据 来自KO小鼠的PMN具有增强的吞噬性和杀戮能力，与显示的体内数据一致 在角膜中减少细菌负荷，目的地相似的PMN数量。关于潜在的治疗策略， 预防性下结节和抗MIRS局部应用的试验实验 类型小鼠，提供了信息，即Mir-183/96/182群集功能的下调成功 降低了PA角膜炎的严重程度。因此，为了实现我们的长期目标，以下目标是 提议：AIM 1将测试角膜居民Mφ（髓样细胞）中miR-183/96/182群集的灭活 促炎性趋化细胞因子的产生，特别是MCP1，IL-1β和 MIP2，导致PMN和Mφ浸润减少对感染的角膜。 AIM 2将测试 通过将它们育成髓样 特定的CRE转基因动物足以扭转对体内PA感染的角膜反应，以及 吞噬作用和浸润细胞的细胞内杀死。 AIM 3将测试DAP12是miR-的直接目标 183/96/182聚类在PMN和Mφ中，并介导其对这些细胞的调节。 AIM 4将测试本地 miR-183/96/182角膜中的簇功能的敲低是PA诱导的角膜炎的治疗性。 预计这些研究将揭示miR-183/96/182簇调节的新分子机制 细菌性角膜炎中先天免疫反应，并为其治疗提供了新的靶标。