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.
