Silencing Toll-like receptor-9 in Pseudomonas aeruginosa keratitis.
OBJECTIVE
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.
Huang
Xi
X
Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA. xhuang@med.wayne.edu
Barrett
Ronald P
RP
McClellan
Sharon A
SA
Hazlett
Linda D
LD
D000818:Animals; D015169:Colony Count, Microbial; D003315:Cornea / Q000378:metabolism / Q000382:microbiology; D016207:Cytokines / Q000378:metabolism; D004195:Disease Models, Animal; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D015818:Eye Infections, Bacterial / Q000378:metabolism* / Q000382:microbiology; D005260:Female; D020868:Gene Silencing / Q000502:physiology*; D007124:Immunoenzyme Techniques; D007634:Keratitis / Q000378:metabolism* / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009504:Neutrophils / Q000502:physiology; D011552:Pseudomonas Infections / Q000378:metabolism* / Q000382:microbiology; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D051217:Toll-Like Receptor 9 / Q000235:genetics* / Q000378:metabolism
D016207:Cytokines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051217:Toll-Like Receptor 9