Harnessing the Anti-infective Activity of the Ocular Surface to Prevent Infection

Bacterial keratitis, a common infection in contact lens wearers, can lead to blindness caused by pathogenic and host factors. Seventy percent of bacterial keratitis cases are caused by the Pseudomonas aeruginosa, a gram-negative bacterium. Fortunately, a healthy cornea provides a defense system against bacterial invasion. Understanding the mechanism of how a healthy maintains its health would be beneficial in providing therapies for corneal infections and more importantly, in providing preventative measures. My thesis project comprises of two parts: 1) To understand the role of surfactant protein-D (SP-D) in providing protection against P. aeruginosa colonization on the healthy eye in vivo; 2) To study the effect of tear fluid on corneal epithelial cells and to identify tear fluid-modulated corneal cell defenses. We showed that healthy murine eyes clear both cytotoxic and invasive P. aeruginosa strains efficiently and that SP-D, present in human tear fluid, aided in this clearance. Our studies also revealed that protease mutants were more efficiently cleared from the healthy ocular surface as opposed to its parent strain. Experiments to study mechanisms for these differences revealed that purified elastase could degrade tear fluid SP-D in vivo. Together, these data showed that SP-D can contribute to the clearance of P. aeruginosa from the healthy ocular surface and that proteases can compromise that clearance. The data also suggested that SP-D degradation in vivo is a mechanism by which P. aeruginosa proteases could contribute to virulence. Human tear fluid is known to increase transepithelial resistance of corneal epithelia in vitro and protects them against P. aeruginosa invasion, cytotoxicity and translocation but only retards the growth of some P. aeruginosa strains. However, we have also found that if a murine eye is removed and suspended in bacteria without tear fluid, the corneal epithelium remains resistant to P. aeruginosa translocation. This suggested the importance of tear fluid and corneal epithelial cells' interaction for protection. Through my thesis project, we discovered that pre-exposing the corneal epithelial cells to tear fluid is sufficient to induce protective activity against P. aeruginosa invasion and cytotoxicity. Our bacteriostatic study involving corneal cell lysates fractionated by size, showed that fraction < 3 kDa is bactericidal and that Histatin 5 (< 3 kDa), a potent antimicrobial whose expression is induced by tear fluid, may be a contributing bactericide. Furthermore, our microarray data revealed that RNase7, another potent antimicrobial heavily studied in human skin, and ST2, a member of the IL1R family, are upregulated with tear fluid, suggesting the protective role of tear fluid by modulating corneal cell defenses. This thesis demonstrates using a novel "null-infection" model (in which the cornea is not damaged prior to inoculation with P. aeruginosa) that very large inoculation of P. aeruginosa is rapidly cleared from the healthy ocular surface in vivo and that SP-D plays a signification role in this protection from bacterial colonization. Furthermore, we demonstrate here that tear fluid provides protection by modulating corneal cell defenses. Understanding the roles of corneal cell defenses that provide protection against microbes in a healthy eye environment is essential in developing potential strategies for bacterial keratitis due to contact lens wear in the future