May 2006
Volume 47, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2006
SIGIRR Promotes Resistance Against P. Aeruginosa Infection by Down–Regulating TLR4 Signaling and IFN– Production
Author Affiliations & Notes
  • X. Huang
    Anatomy & Cell Biology, Wayne State Univ Sch of Med, Detroit, MI
  • L.D. Hazlett
    Anatomy & Cell Biology, Wayne State Univ Sch of Med, Detroit, MI
  • Y. Zhang
    Anatomy & Cell Biology, Wayne State Univ Sch of Med, Detroit, MI
  • S.A. McClellan
    Anatomy & Cell Biology, Wayne State Univ Sch of Med, Detroit, MI
  • R.P. Barrett
    Anatomy & Cell Biology, Wayne State Univ Sch of Med, Detroit, MI
  • Footnotes
    Commercial Relationships  X. Huang, None; L.D. Hazlett, None; Y. Zhang, None; S.A. McClellan, None; R.P. Barrett, None.
  • Footnotes
    Support  R01 EY016058 HIGHWIRE EXLINK_ID="47:5:1906:1" VALUE="EY016058" TYPEGUESS="GEN" /HIGHWIRE and P30 EY04068.
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 1906. doi:
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      X. Huang, L.D. Hazlett, Y. Zhang, S.A. McClellan, R.P. Barrett; SIGIRR Promotes Resistance Against P. Aeruginosa Infection by Down–Regulating TLR4 Signaling and IFN– Production . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1906.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : This study elucidated the role of SIGIRR (single immunoglobulin interleukin–1 receptor–related molecules) in resistance to Pseudomonas aeruginosa (P. aeruginosa).

Methods: : Real–time RT–PCR and western–blot tested corneal SIGIRR mRNA and protein levels in C57BL/6 (B6, susceptible) vs. BALB/c (resistant) mice before and after P. aeruginosa (5 µl of 106 CFU/µl, ATCC stain 19660) challenge. Infected BALB/c mice also were tested after injection with anti–SIGIRR Ab or IgG (control). The corneal disease response was monitored in the two groups by slit lamp, clinical score, histopathology, plate counts, myeloperoxidase (MPO) activity, real–time PCR and ELISA. In vitro, SIGIRR was overexpressed in RAW264.7 macrophage–like cells. Toll–like receptors (TLRs) and cytokine/chemokine expression in SIGIRR vs. vector transfected cells were measured by real–time PCR and/or ELISA. The role of SIGIRR on IL–1, TLR4 (LPS) and TLR3 [poly(I:C)] signaling was tested in various cell lines by a NF–ΚB–dependent luciferase reporter assay.

Results: : SIGIRR was constitutively expressed in the cornea of both B6 and BALB/c mice. SIGIRR mRNA levels in BALB/c cornea were initially (12 h) down–regulated, then elevated significantly at 1–5 days p.i., while levels in B6 cornea were not changed significantly. Protein levels in normal and infected corneas of BALB/c and B6 mice showed a similar trend. BALB/c mice treated with anti–SIGIRR Ab over rat–IgG controls showed increased corneal opacity, stromal damage, and bacterial load. In addition, corneal mRNA levels for IL–1ß, MIP–2, IL–1R1, TLR4, IL–18 and IFN–γ and protein levels for IL–1ß and MIP–2 were significantly upregulated in SIGIRR Ab over controls, while no changes in PMN number, IL–4 or IL–10 mRNA expression was seen. RAW264.7 cells transiently transfected with SIGIRR and stimulated with heat–killed P. aeruginosa or LPS showed significantly decreased mRNA levels for IL–1R1, TLR4, and type–1 cytokines (IL–12, IL–18 and IFN–γ) as well as IL–1ß and MIP–2 protein over those transfected with empty–vector. Finally, overexpression of SIGIRR reduced IL–1 and LPS mediated NF–ΚB activation in a dose–dependent manner in HEK293 and 293–hTLR4–CD14–MD2 cell lines, respectively, but did not inhibit poly(I:C) induced NF–ΚB activation in 293–hTLR3 cells.

Conclusions: : SIGIRR is critical in resistance to P. aeruginosa keratitis, functioning to reduce corneal infection and inflammation by negatively regulating IL–1 and TLR4 (LPS) signaling, inhibiting type–1 immunity, and decreasing bacterial load.

Keywords: microbial pathogenesis: experimental studies • inflammation • cytokines/chemokines 
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