Epithelial Barrier During Corneal Infection

角膜感染期间的上皮屏障

• 批准号: 6525371
• 负责人: Fu-Shin X Yu
• 金额: $ 28.7万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6525371
• 关键词: CD14 molecule; Pseudomonas aeruginosa; bacteria infection mechanism; biological signal transduction; cell component structure /function; confocal scanning microscopy; cornea; corneal epithelium; enzyme activity; enzyme inhibitors; eye infections; flow cytometry; gel mobility shift assay; human tissue; immunofluorescence technique; immunoprecipitation; keratitis; lipopolysaccharides; mitogen activated protein kinase; neutralizing antibody; nuclear factor kappa beta; organ culture; phosphorylation; swine; tight junctions; western blottings

DESCRIPTION (provided by applicant): The long-term goal of this application is to understand the mechanisms underlying the induction of the inflammatory reaction and breakdown of the epithelial barrier in the cornea upon infection. Recent data has shown that challenging human corneal epithelial (HCE) cells with lipopolysaccharide (LPS) isolated from Pseudomonas aeruginosa (PA), a major cause of bacterial keratitis, increases monolayer permeability and alteration of tight junction (TJ) status. LPS and PA challenges also lead to activation of NF-kappaB, a transcription factor activated by Toll-like receptor 4 (TLR4, the predominant LPS receptor in mammals). Furthermore, PA infection resulted in the loss of epithelial barrier function in cultured pig corneas. The current application will test the hypothesis that in the cornea TLRs confer responsiveness of HCE cells to pathogens, and PA challenge-induced TLR signaling, through activation of NF-kappaB and/or mitogen-activated protein kinase (MAPK), contributes to infection-induced epithelial barrier breakdown. The following studies will be carried out. (1) Alterations of barrier properties in corneal epithelial cells upon PA infection will be assessed using HCE cells cultured on Transwell filters as a model epitheliuin. Parameters to be measured include transepithelial electrical resistance, and paracellular flux, alterations in TJ proteins ZO-1 and ZO-2. (2) Signal transduction pathway(s) that couples PA challenge to alteration of epithelial barrier function in HCE cells will be characterized using antibodies to detect cell surface expression of TLRs and co-receptor CD 14, and pharmacological reagents to inhibit activation of NF-kappaB and MAPK upon HCE cell infection. (3) Whether infection-induced epithelial responses, including barrier breakdown, can be inhibited by modification of TLR-mediated signaling will be assessed. A combination of transfection of TLRs and their mutants and the neutralizing antibodies will be used to assess epithelial response to PA challenge in Transwell model epithelium. TLR neutralizing antibodies, along with other biological and pharmacological reagents, will also be applied to corneal organ culture to determine if infection-induced epithelial responses and barrier breakdown can be inhibited. An understanding of how TLRs transmit signals that lead to epithelial responses, including modulation of barrier function, may allow the development of therapeutic agents that prevent breakdown or enhance recovery of barrier function during infection and, as an adjuvant therapy, eliminate the corneal scarring and vision loss associated with bacterial keratitis.

描述（由申请人提供）：本申请的长期目标是 为了了解炎症诱导的潜在机制， 感染后角膜上皮屏障的反应和破坏。 最近的数据表明，具有挑战性的人角膜上皮（HCE）细胞 与从铜绿假单胞菌（PA）分离的脂多糖（LPS），a 细菌性角膜炎的主要原因，增加单层通透性， 紧密连接（TJ）状态的改变。LPS和PA挑战还导致 Toll样受体激活的转录因子NF-κ B的激活 4（TLR 4，哺乳动物中的主要LPS受体）。此外，PA感染 导致培养的猪角膜上皮屏障功能丧失。 本申请将测试在角膜中TLR赋予角膜细胞增殖的假设。 HCE细胞对病原体的反应性和PA激发诱导的TLR 通过NF-κ B和/或促分裂原活化蛋白的活化进行信号传导 丝裂原活化蛋白激酶（MAPK）参与感染诱导的上皮屏障破坏。 将开展以下研究。(1)屏障改建 PA感染后角膜上皮细胞的特性将使用 HCE细胞在Transwell滤膜上培养作为模型上皮细胞。参数以 包括跨上皮电阻和细胞旁电阻。 流量，TJ蛋白ZO-1和ZO-2的改变。(2)信号转导 将PA激发与上皮屏障改变偶联的途径 HCE细胞中的功能将使用抗体来检测细胞 TLR和共受体CD 14的表面表达，以及药理学试剂 抑制HCE细胞感染后NF-κ B和MAPK的活化。（三） 感染诱导的上皮反应，包括屏障破坏， 可以通过TLR介导的信号传导的修饰来抑制。一 TLR及其突变体的转染和中和的组合 抗体将用于评估上皮细胞对PA激发的反应， Transwell模型上皮。TLR中和抗体，沿着其他 生物和药理试剂也将应用于角膜器官 培养以确定感染诱导的上皮反应和屏障 可以抑制击穿。了解TLR如何传递信号， 导致上皮反应，包括调节屏障功能，可以 允许开发防止分解或增强的治疗剂， 在感染期间恢复屏障功能，作为辅助治疗， 消除与细菌性角膜炎相关的角膜瘢痕和视力丧失 角膜炎。