Silencing Toll-like receptor-9 in Pseudomonas aeruginosa keratitis

Abstract

Purpose: To determine the effects of silencing Toll-like receptor (TLR) 9 signaling in Pseudomonas aeruginosa keratitis.

Methods: Corneal TLR9 mRNA levels were tested by RT-PCR in C57BL/6 (B6, susceptible) and BALB/c (resistant) mice and compared. The response of B6 mice to CpG DNA, which binds TLR9, was tested after subconjunctival injection of mice with control or CpG DNA; TLR9, IL-1beta, macrophage inflammatory protein (MIP)-2, IL-4, IL-10, IL-12, IL-18, and IFN-gamma levels were measured by RT-PCR. Langerhans cells (LCs) were stimulated with CpG DNA and treated with TLR9 or control siRNA, and mRNA levels of TLR9, IL-1beta, and MIP-2 were detected by RT-PCR. In addition, IL-1beta levels were tested by ELISA. Then B6 mice were injected subconjunctivally with control or TLR9 siRNA before infection and treated topically afterward. Slit lamp, clinical score, RT-PCR, ELISA, myeloperoxidase assay, and plate counts were performed.

Results: TLR9 mRNA levels were sixfold higher in B6 than in BALB/c corneas the day after injection. B6 mice injected with CpG DNA exhibited an increase in corneal mRNA for TLR9, IL-1beta, MIP-2, IL-12, and IFN-gamma over controls. LCs stimulated with CpG DNA and treated with TLR9 siRNA exhibited reduced TLR9, IL-1beta, and MIP-2 levels compared with controls. Finally, B6 mice treated with TLR9 siRNA showed decreases in corneal opacity, polymorphonuclear leukocyte number, IL-12 and IFN-gamma mRNA, IL-1beta, and MIP-2 protein compared with those treated with control siRNA. Fewer corneas perforated in these mice, but bacterial loads were higher than in controls.

Conclusions: Signaling through TLR9 appears important in P. aeruginosa keratitis, and silencing TLR9 signaling reduces inflammation but likely contributes to decreased bacterial killing in the cornea.