Abstract
Purpose:
We have previously shown that conditional Notch1-/- mice develop progressive barrier impairment. We examined the inflammatory changes involved in this process and the possible role of TLR4 signaling and MAPKs as potential downstream pathways.
Methods:
Notch1 was conditionally deleted in 3 month old Notch1 flox/flox, K14-Cre-ERT +/- mice using intra-peritoneal injection of 1 mg/day 4-hydroxy-tamoxifen (4-OHT) for 5 days. A 2 mm central corneal epithelial mechanical debridement was performed by a blunt scraper. Immunofluorescence staining was used to detect TLR4 expression in the epithelium. Neutrophils and dendritic cells were detected using anti-Ly-6G and anti-CD11-b antibodies. MAPKs were examined by immunohistochemistry. RT-PCR was used to evaluate transcription of inflammatory cytokines.
Results:
In wild type mice. TLR4 expression was prominently noted at the leading edge as early as 2 hours after wounding. The expression of TLR4 returned to baseline by 72-96 hr in WT mice whereas in Notch1-/- mice its expression remained persistently elevated at 72 and 96 hours after wounding which correlated with their delayed barrier recovery. Examination of Notch1-/- corneas similarly demonstrated progressive increase in TLR4 expression starting at 2-3 weeks after Notch1 deletion. Notch1-/- epithelium at 3 weeks after Notch1 deletion demonstrated p-JNK staining throughout the epithelium in contrast to no expression in wild type. Expression of phospho-p38 became more prominent in the later stages of the disease concurrent with the development of keratinization. Stromal inflammatory cells including neutrophils and dendritic cells were noted as early as 2 weeks after Notch1 deletion. Expression of IL-1β was increased by 2.5 fold in whole corneas of Notch1-/- mice by RT-PCR.
Conclusions:
These results highlight the inflammatory changes that develop from recurrent epithelial trauma in the barrier impaired corneas of Notch1-/- mice. It further implicates TLR4 (and MAPKs) as a potential mediator of the secondary changes that ensue. TLR signaling may be one of signaling mechanisms that responds to epithelial injuries and contributes to the wound healing process during which endogenous TLR ligands likely play an important role.
Keywords: 482 cornea: epithelium •
714 signal transduction