Epithelial Barrier During Corneal Infection

角膜感染期间的上皮屏障

基本信息

批准号：
6487010
负责人：
Fu-Shin X Yu
金额：
$ 32.29万
依托单位：
AUGUSTA UNIVERSITY
依托单位国家：
美国
项目类别：
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

项目摘要

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了解诱发炎症的机制感染后角膜上皮屏障的反应和破坏。最近的数据表明，挑战人角膜上皮（HCE）细胞含有从铜绿假单胞菌 (PA) 中分离出的脂多糖 (LPS)，细菌性角膜炎的主要原因，增加单层渗透性和紧密连接（TJ）状态的改变。 LPS 和 PA 挑战也会导致激活 NF-kappaB（一种由 Toll 样受体激活的转录因子） 4（TLR4，哺乳动物中主要的 LPS 受体）。此外，PA感染导致培养猪角膜上皮屏障功能丧失。当前的应用程序将测试以下假设：在角膜中 TLR 赋予 HCE 细胞对病原体的反应性以及 PA 攻击诱导的 TLR 通过激活 NF-κB 和/或丝裂原激活蛋白来传递信号激酶 (MAPK) 有助于感染引起的上皮屏障破坏。将进行以下研究。 (1) 屏障变更 PA 感染后角膜上皮细胞的特性将使用以下方法进行评估在 Transwell 过滤器上培养的 HCE 细胞作为上皮蛋白模型。参数为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 的改变。 (2)信号转导将 PA 挑战与上皮屏障改变相结合的途径 HCE 细胞中的功能将使用抗体来检测细胞来表征 TLR 和辅助受体 CD 14 的表面表达以及药理学试剂抑制 HCE 细胞感染后 NF-kappaB 和 MAPK 的激活。 (3) 感染是否诱导上皮反应，包括屏障破坏，将评估是否可以通过修改 TLR 介导的信号传导来抑制。一个 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 挑战的反应 Transwell 模型上皮。 TLR 中和抗体以及其他生物药理试剂，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可以抑制击穿。了解 TLR 如何传输信号导致上皮反应，包括屏障功能的调节，可能允许开发防止分解或增强的治疗剂感染期间屏障功能的恢复，作为辅助治疗，消除与细菌相关的角膜疤痕和视力丧失角膜炎。