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Published on: May 12, 2020
Silencing Toll-like receptor-9 in Pseudomonas aeruginosa keratitis
Xi Huang1, Ronald P Barrett, Sharon A McClellan
1Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA. xhuang@med.wayne.edu
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.
Insights
Silencing Toll-like receptor (TLR) 9 signaling in Pseudomonas aeruginosa keratitis reduces inflammation but impairs bacterial clearance. This suggests TLR9 plays a dual role in the immune response to this infection.
Area of Science:
- Ophthalmology
- Immunology
- Microbiology
Background:
- Pseudomonas aeruginosa keratitis is a severe eye infection.
- Toll-like receptor 9 (TLR9) is involved in innate immunity.
- The role of TLR9 in P. aeruginosa keratitis is not fully understood.
Purpose of the Study:
- To investigate the effect of silencing TLR9 signaling on P. aeruginosa keratitis.
- To determine the impact on corneal inflammation and bacterial clearance.
Main Methods:
- Corneal TLR9 mRNA levels were assessed in susceptible (B6) and resistant (BALB/c) mice.
- B6 mice received CpG DNA (TLR9 agonist) or control injections.
- Langerhans cells (LCs) were stimulated and treated with TLR9 siRNA.
- Mice were treated with TLR9 siRNA before P. aeruginosa infection.
- Corneal opacity, leukocyte infiltration, cytokine levels, and bacterial load were measured.
Main Results:
- TLR9 mRNA was higher in susceptible B6 mice.
- CpG DNA increased corneal inflammatory markers in B6 mice.
- TLR9 siRNA reduced inflammatory markers in stimulated LCs and infected corneas.
- TLR9 silencing decreased corneal opacity and inflammation but increased bacterial load and perforation risk.
Conclusions:
- TLR9 signaling is crucial in P. aeruginosa keratitis.
- Silencing TLR9 reduces corneal inflammation.
- However, TLR9 silencing may impair bacterial killing, potentially increasing infection severity.
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