IL-1R and TLR-5 mediate corneal epithelial defense against Pseudomonas aeruginosa colonization and traversal respectively

Abstract

 

Purpose

 

We previously reported that corneal epithelial defense against P. aeruginosa traversal is MyD88-dependent. MyD88 is an adaptor molecule required for many of the signaling events mediated by Toll-like receptors (TLRs) and the Interleukin-1 receptor (IL-1R). To decipher the molecular mechanisms involved in MyD88-dependent protective activity, we tested the hypothesis that one or more of these receptors is critical for host defense against bacterial traversal.

 

Methods

 

Ex vivo whole eyeballs of C57BL/6 wild-type (control) and single gene (IL-1R, TLR-5 or TLR-7) knockout mice were rinsed with PBS, blotted with tissue paper on the corneal surface to enable susceptibility to bacterial adhesion (or were not blotted), followed by 6 h incubation at 35 °C in 10¹¹ cfu/ml GFP-expressing P. aeruginosa PAO1 then imaged by confocal microscopy. Corneal cells of the unprocessed whole eyeballs and bacteria were visualized using reflection of 633 nm and 488 nm confocal lasers respectively. Z stacks (entire corneal epithelial thickness, 1.0 µm steps) were collected from ≥3 random fields/eye. 3-D image reconstruction was performed by Image-J.

 

Results

 

Bacteria did not adhere to wild-type or TLR-7 knockout mouse corneas unless they were blotted prior to inoculation. In contrast, bacteria bound to non-blotted IL-1R knockout corneas, with partial penetration into the cornea also occurring if corneas were blotted. Blotted TLR-5 knockout mouse corneas showed deep bacterial traversal through to the basal lamina. Non-blotted TLR-5 knockout mouse corneas showed little or no bacterial adherence.

 

Conclusions

 

The data suggest that the IL-1R is involved in preventing P. aeruginosa adhesion to the intact corneal epithelium, while TLR-5 primarily contributes to host defenses against bacterial traversal after adhesion. In contrast, TLR-7, an intracellular receptor known to recognize single stranded viral RNA, is not required for preventing bacterial colonization or traversal. The effectors downstream of the IL-1R and TLR-5 involved in protecting the healthy mouse corneal epithelium against P. aeruginosa are to be determined.

 

 

P. aeruginosa (green) traverses the corneal epithelium of TLR-5 knockout, but not wild-type, mice.

 

Keywords: 482 cornea: epithelium • 664 pseudomonas • 594 microbial pathogenesis: experimental studies