May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
siRNA Targeting TLR–9: In vitro and in vivo Effects in Models of Bacterial Infection
Author Affiliations & Notes
  • X. Huang
    Anatomy & Cell Biology, Wayne State University, Detroit, MI
  • L.D. Hazlett
    Anatomy & Cell Biology, Wayne State University, Detroit, MI
  • Footnotes
    Commercial Relationships  X. Huang, None; L.D. Hazlett, None.
  • Footnotes
    Support  NIH Grant EY02986; EY04068; MEBTC 25MWE
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 5083. doi:
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      X. Huang, L.D. Hazlett; siRNA Targeting TLR–9: In vitro and in vivo Effects in Models of Bacterial Infection . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5083.

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Abstract

Abstract: : Purpose: Our purpose was threefold: 1) to determine changes in expression for TLR–9, IL–1ß and MIP–2 in Langerhans cells (LC) after stimulation with synthetic CpG–ODN or heat–killed Pseudomonas aeruginosa (P. aeruginosa); 2) to inhibit expression of pro–inflammatory cytokines by TLR–9 specific silencing in LC; and 3) to test the protective effects of silencing TLR–9 in an in vivo model of P. aeruginosa keratitis where excessive up–regulation of IL–1 and MIP–2 occur and where corneal perforation is the outcome. Methods: LC (XS52 cell line) were cultured in complete RPMI (with 10% FCS) and 5% fibroblast (NS line) supernatant supplemented with 0.5ng/ml murine recombinant GM–CSF. Synthetic, unmethylated CpG–ODN, GpC–ODN (control) and CpG–inhibitor were designed and LC treated with these ODN or heat–killed bacteria. siRNA duplexes targeting the coding region of TLR–9 were produced and used for LC transfection. Cell lysates were prepared for total RNA extraction and supernatant was collected for ELISA assay. For real–time RT–PCR, total RNA was isolated using TRIzol, and mRNA expression fold differences calculated after normalizing to ß–actin. For in vivo study, C57BL/6 mice were injected subconjunctivally with siRNA or scrambled siRNA for TLR–9, and infected with P. aeruginosa. Disease was monitored by clinical score, slit lamp, plate counts, myeloperoxidase (MPO) assay for PMN enumeration, RT–PCR and ELISA analysis. Results: Six hours after treatment, when compared with controls, significant up–regulation of mRNA expression for TLR–9 (8 vs. 13 fold), IL–1ß (225 vs. 684 fold) and MIP–2 (237 vs. 825 fold) was detected in LC treated with CpG–ODN vs. heat–killed PA, respectively. Treatment with a CpG–inhibitor completely blocked mRNA up–regulation of each after CpG–ODN, but not after heat–killed bacteria. Silencing of TLR–9 in vitro reduced CpG–ODN induced IL–1ß and MIP–2 mRNA and protein expression levels in LC. In vivo in the infected cornea, TLR–9 silencing reduced these cytokines and infected corneas did not perforate, despite a greater bacterial load and reduced PMN infiltrate. Conclusions: These results suggest that murine LC respond to synthetic CpG–ODN by up–regulating TLR–9, IL–1ß and MIP–2. It also suggests that heat–killed P. aeruginosa not only up–regulates TLR–9, but probably other Toll receptors as well that in turn may contribute to the upregulation of IL–1ß and MIP–2. siRNA induces TLR–9 silencing and appears promising as an effective alternative or adjunctive treatment for P. aeruginosa bacterial keratitis.

Keywords: microbial pathogenesis: experimental studies • inflammation • Pseudomonas 
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