Abstract
Purpose:
Corneal Toll-like receptor 4 (TLR4) expression is higher in the infected cornea of C57BL/6 (B6) over BALB/c mice. It is critical in Pseudomonas aeruginosa keratitis and participates in: PMN infiltration, pro-inflammatory cytokine production, bacterial killing, and neovascularization. The purpose of this study was to determine its role in apoptosis.
Methods:
Primary cultured corneal epithelial cells derived from B6 and BALB/c mice (confluent, 2nd passage) were stimulated with ultrapure LPS (1 μg/ml for 18h) and analyzed using a Mouse Apoptosis RT2 Profiler™ PCR array. RT-PCR confirmed selected genes from the array, and tested genes commonly associated with apoptosis. Immunostaining localized selected apoptotic genes identified by array and RT-PCR in the infected cornea of B6 vs BALB/c mice. BALB/c vs TLR4 functionally deficient, C.C3-Tlr4lps-d (Tlr4lps-d) mice also were infected, corneas harvested (6 h-3 days p.i.), analyzed by PCR array (described above) and selected genes confirmed by RT-PCR.
Results:
PCR array comparing LPS stimulated corneal epithelial cells from B6 vs BALB/c mice identified 19 apoptosis-related genes with 2 fold or greater change. RT-PCR comparing LPS stimulated with control B6 derived corneal epithelial cells showed increased expression of Bcl2, caspase 12, and FADD. In BALB/c derived epithelium, caspases 1, 3, 8, 12, Fas, and FADD were increased in LPS stimulated over controls, while caspase 9 levels were decreased. PCR array comparing infected corneas of BALB/c with Tlr4lps-d mice identified 8 genes with a 2 fold or greater difference between groups. In Tlr4lps-d vs wildtype mice, RT-PCR showed that Fas and FasL levels were decreased early, but unchanged later in disease; caspase 3 and FADD were inititally down-regulated and then up-regulated, while caspase 8, Bcl2 and Bok were up-regulated at all times after infection.
Conclusions:
The data provide evidence that TLR4 contributes to regulation/modulation of apoptosis in the cornea following P. aeruginosa infection.
Keywords: 426 apoptosis/cell death •
664 pseudomonas •
573 keratitis