Epithelial Barrier During Corneal Infection

描述(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 感染时角膜上皮屏障的反应和破坏。 最近的数据表明，挑战人类角膜上皮(HCE)细胞 用从铜绿假单胞菌(PA)中分离的脂多糖(LPS)， 细菌性角膜炎的主要原因，增加单层通透性和 更改紧密连接(TJ)状态。LP和PA挑战也会导致 Toll样受体激活转录因子核因子-kappaB的研究 4(TLR4，哺乳动物的主要内毒素受体)。此外，PA感染 导致培养的猪角膜上皮屏障功能丧失。 目前的应用将测试这样的假设：在角膜中，TLRs赋予 HCE细胞对病原体的反应性和PA攻击诱导的TLR 核因子-kappaB和/或丝裂原激活蛋白激活的信号转导 蛋白激酶(MAPK)参与了感染诱导的上皮屏障的破坏。 将开展以下研究。(1)更改障碍物 PA感染后角膜上皮细胞的特性将使用 在Transwell滤膜上培养的HCE细胞作为模型上皮细胞。参数设置为 要测量的包括跨上皮电阻和细胞旁电阻 通量，TJ蛋白ZO-1和ZO-2的变化。(2)信号转导 偶联PA挑战上皮屏障改变的途径(S) 将使用抗体检测细胞来表征HCE细胞的功能 TLRs和共受体CD14的表面表达及其药理试剂 抑制HCE细胞感染时核因子-kappaB和MAPK的激活。(3) 感染诱导的上皮反应，包括屏障破坏， 是否可以通过修饰TLR介导的信号转导来抑制将被评估。一个 TLRs及其突变体的联合转染和中和 抗体将用于评估上皮细胞对PA挑战的反应 Transwell模型上皮。TLR中和抗体，以及其他 生物和药理试剂，也将应用于角膜器官 培养以确定感染是否诱导上皮反应和屏障 击穿可以被抑制。了解TLR如何传输信号 导致上皮反应，包括调节屏障功能，可能 允许开发可防止分解或增强的治疗剂 感染期间屏障功能的恢复，作为一种辅助治疗， 消除与细菌相关的角膜疤痕和视力丧失 角膜炎。

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.