Factors impacting corneal epithelial barrier function against Pseudomonas aeruginosa traversal.

After corneal injury or during contact lens wear, Pseudomonas aeruginosa can cause sight-threatening corneal disease.1–3 However, the healthy human cornea is similar to our other exposed surfaces in its remarkable resistance to infection by this, and other, opportunistic bacterial pathogens. Indeed, we have shown that inoculation of large numbers of P. aeruginosa onto healthy animal corneas in vivo results in rapid bacterial clearance without inflammation or infection.4 Because of this resistance, researchers who study corneal infection commonly use “invasive” methods to enable disease, including scarification before inoculation or intrastromal injection of the inoculum.5–8 It is assumed that the corneal epithelium provides the major barrier to microbes in vivo and that the scarification and intrastromal injection methods work because they enable bacteria to bypass it. Indeed, in vitro studies have clearly shown the potential for corneal epithelial cells to act as a barrier to microbes—for example, through their tight junctions.9,10 However, corneal epithelial cells grown in vitro still permit traversal by P. aeruginosa, even if grown as multilayers,11,12 illustrating the importance of other in vivo factors in defense against infection. Related to this, we have shown that tear fluid13 at the surface of the corneal epithelium plays roles in defense through mechanisms independent of bacteriostatic activity.14 We have also shown that the basal lamina, the basement membrane below the epithelium, prevents bacteria from entering the corneal stroma.12 The role of the corneal epithelium itself in defense against bacterial penetration, and infection, of the cornea in vivo has not been well studied. Paradoxically, this is probably because existing infection models deliberately bypass it to enable disease to be studied. Results of in vitro studies suggest that epithelial tight junctions, present at the corneal surface and within the epithelium15 and critical for cell polarity, are of significance in protecting against P. aeruginosa.16,17 In vitro studies also suggest that factors secreted by corneal epithelial cells (including mucins, defensins, surfactant proteins, and extracellular matrix proteins) can modulate bacterial adhesion, invasion, and traversal of these and other cells found in vivo.12,18–23 Although these in vitro-derived insights will likely be important in vivo, their actual in vivo relevance cannot be explored using existing animal models that bypass the epithelium. The aim of this study was to develop an infection model that does not require bypassing the epithelium to determine the minimal parameters that enable susceptibility to infection, with a goal of better understanding the defenses that protect us against infection during health. Thus, we developed a method for studying epithelial barrier function against bacteria in vivo using tissue paper blotting of the epithelial surface to enable bacterial adhesion. We then used this method to demonstrate roles for both ethylene glycol tetraacetic acid (EGTA)-sensitive factors and SP-D in epithelial defense against P. aeruginosa traversal. That information will form the foundation for further studies focused on the mechanisms of disease and developing methods to prevent it.