ROLE OF THE CD14 LPS RECEPTOR IN CORNEAL INFLAMMATION

革兰氏阴性菌感染的角膜与生物体，如假单胞菌可以有深远的后果，包括患者的细菌性角膜炎，可导致视力丧失。对假单胞菌的炎性反应的很大一部分是由LPS介导的。我们最近报道，角膜表达的主要LPS受体之一，CDI 4。该项目的长期目标是确定角膜本身在介导宿主对细菌感染的先天免疫反应中的作用。本申请将检验角膜细胞表达功能性CD 14受体的假设，当被LPS激活时，该受体能够触发促炎肽的表达，促炎肽促进革兰氏阴性角膜感染的宿主消退。为了通过实验检验这一假设，我们将进行以下具体目的：具体目的#1：检查角膜中CD 14受体的表达和调节;具体目的#2：评估角膜CD 14受体的功能能力;具体目的#3：评估CD 14活化诱导角膜中先天性炎症反应的能力;以及具体目的#4：在假单胞菌属细菌性角膜炎小鼠实验模型中，确定角膜CD 14受体在介导角膜炎症反应中的体内作用。为了进行这些研究，将使用人和鼠角膜细胞和角膜组织以及其中CD 14表达缺失、减少或过表达的遗传改变的小鼠品系。角膜CD 14表达将在组成型和暴露于细菌试剂（假单胞菌属、LPS、LPS/LBP）和细胞因子（IL-1和TNF α）后进行测量。将通过细胞内钙应答、酪氨酸激酶活性和NF-κ B活性来测量CD 14对细菌试剂的功能活性。通过测量角膜细胞因子（IL-1、IL-6和TNF α）、趋化因子（IL-8）和细胞粘附分子（ICAM-1）的表达来确定角膜CD 14诱导的对细菌试剂的先天性炎症反应。使用CD 14基因改变动物的鼠模型将用于评估CDI 4在实验假单胞菌属细菌性角膜炎中的体内作用。角膜CD 14的激活可能具有有益和有害的炎症反应。了解CD 14在介导角膜先天性免疫中的作用可能会导致角膜感染性疾病管理的新方法。

Gram negative infections of the cornea with organisms such as Pseudomonas can have profound consequences for patients including bacterial keratitis which can lead to visual loss. A significant portion of the inflammatory response to Pseudomonas is mediated by LPS. We have recently reported that the cornea expresses one of the principal LPS receptors, CDI4. The long-term goal of this project is to determine the role of the cornea itself in mediating host innate immune responses to bacterial infections. This application will test the hypothesis that corneal cells express functional CD14 receptors that, when activated by LPS, are capable of triggering the expression of proinflammatory peptides which facilitate the host resolution of gram negative corneal infections. To experimentally test this hypothesis, we will undertake the following Specific Aims: SPECIFIC AIM #1: To examine the expression and regulation of the CD14 receptor in the cornea; SPECIFIC AIM #2: To assess the functional competence of corneal CD14 receptors; SPECIFIC AIM #3: To assess the ability of CD14 activation to induce innate inflammatory responses in the cornea; and SPECIFIC AIM #4: To determine the in vivo role of the corneal CD14 receptor in mediating corneal inflammatory responses in a murine experimental model of Pseudomonas bacterial keratitis. To carry out these studies, human and murine corneal cells and corneal tissue will be used as well as genetically altered strains of mice in which CD14 expression is absent, diminished, or overexpressed. Cornea CD14 expression will be measured constitutively and after exposure to bacterial reagents (Pseudomonas, LPS, LPS/LBP) and cytokines (IL-1 and TNFa). CD14 functional activity to bacterial reagents will be measured by intracellular calcium responses, tyrosine kinase activity, and NF-kappaB activity. Corneal CD14 induced innate inflammatory responses to bacterial reagents will be determined by measuring the expression of corneal cytoidnes (IL-I, IL-6, and TNFa), chemokines (IL-8), and cell adhesion molecules (ICAM-1). A murine model using CD14 genetically altered animals will be utilized to assess the in vivo role of CDI4 in experimental Pseudomonas bacterial keratitis. The activation of corneal CD14 may have both beneficial and detrimental inflammatory responses. Understanding the role of CD14 in mediating corneal innate immunity may result in novel approaches to the management of corneal infectious diseases.