Abstract
Purpose: :
Rapamycin inhibits mTOR, a ubiquitous peptide that signals downstream of the PI3K/Akt cell survival pathway and regulates IL-10 levels. Substance P (SP) is a neuropeptide with a complex role in the infected cornea of BALB/c mice, i.e., exacerbation of inflammatory processes with concurrent up-regulation of growth factor expression. Growth factors (e.g., HGF) signal through the PI3K/Akt/mTOR pathway. This work examines the relationship between SP, mTOR, and the growth factors in a Pseudomonas aeruginosa model of corneal infection.
Methods: :
BALB/c mice were injected intraperitoneally with rapamycin (sterile saline controls) or SP (selected experiments) from the day before infection until 3-5 days post-infection (p.i.). Corneas were harvested at days 1, 3 and 5 p.i. for real-time RT-PCR, ELISA, bacterial plate counts, and MPO. Real-time RT-PCR determined mRNA levels of mTOR, STAT-3, pro- and anti-inflammatory cytokines (IL-12p40, IL-10), pro- and anti-apoptotic genes (caspase-3, Bcl-2), and growth factors (EGF, HGF, FGF-7). A select group of these molecules was tested using ELISA. HGF was applied topically to the cornea (1μg/5μl before and after infection) to determine if mTOR could be modulated by this growth factor, the only one elevated in common between the two treatments.
Results: :
After infection, the cornea of rapamycin- (and SP, as shown before) vs. control-treated BALB/c mice showed worsened disease. In this regard, rapamycin and SP treatment were similar: they increased clinical scores, MPO levels, bacterial load, and IL-12p40 expression. Both treatments similarly decreased levels of mTOR, STAT-3 (an effector of IL-10 expression), and IL-10. Both treatments also delayed apoptosis (decreased mRNA levels of caspase-3 and increased Bcl-2) in the infected cornea. As with SP-treatment, rapamycin increased mRNA and protein levels of HGF, but was not similar to SP with regard to elevating EGF or FGF-7. Unexpectedly, topical HGF treatment increased mRNA levels of mTOR and IL-10, but also raised levels of pro-inflammatory cytokines (IL-12p40 and IL-1β).
Conclusions: :
These data provide evidence that rapamycin and SP treatment similarly downregulate mTOR, STAT-3, and IL-10, while upregulating IL-12p40, and HGF levels after infection. Nonetheless, topical HGF, although able to upregulate mTOR and IL-10 fails to be protective, in that proinflammatory cytokines are upregulated as well.
Keywords: cornea: basic science • growth factors/growth factor receptors • neuropeptides