ROLE OF THE CD14 LPS RECEPTOR IN CORNEAL INFLAMMATION

像假单胞菌这样的生物引起的角膜革兰氏阴性感染可能会对患者产生深远的后果，包括细菌性角膜炎，这可能会导致视力丧失。对假单胞菌的炎症反应有很大一部分是由内毒素介导的。我们最近报道，角膜表达主要的内毒素受体之一CDI4。该项目的长期目标是确定角膜本身在调节宿主对细菌感染的先天免疫反应中的作用。这一应用将验证角膜细胞表达功能性CD14受体的假设，当被内毒素激活时，能够触发促炎多肽的表达，从而促进革兰氏阴性角膜感染的宿主分解。为了从实验上验证这一假说，我们将承担以下特定目标：特定目的#1：检测CD14受体在角膜中的表达和调控；特定目的#2：评估角膜CD14受体的功能活性；特定目的#3：评估CD14激活在诱导角膜固有炎症反应中的能力；以及特定目的#4：在小鼠细菌性角膜炎小鼠模型中确定角膜CD14受体在介导角膜炎症反应中的作用。为了进行这些研究，将使用人类和小鼠的角膜细胞和角膜组织，以及CD14表达缺失、减少或过度表达的转基因小鼠品系。在接触细菌试剂(假单胞菌、内毒素、脂多糖/LBP)和细胞因子(IL-1和TNFa)后，将对角膜CD14的表达进行结构性测定。CD14对细菌试剂的功能活性将通过细胞内钙反应、酪氨酸激酶活性和核因子-kappaB活性来衡量。通过检测角膜细胞因子(IL-I、IL-6和TNFa)、趋化因子(IL-8)和细胞黏附分子(ICAM-1)的表达来确定CD14诱导的对细菌试剂的先天炎症反应。使用CD14转基因动物的小鼠模型将被用来评估CDI4在实验性假单胞菌细菌性角膜炎中的体内作用。角膜CD14的激活可能既有有益的炎症反应，也有有害的炎症反应。了解CD14在调节角膜天然免疫中的作用可能会导致新的角膜感染性疾病的治疗方法。

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.