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Megan E. Foldenauer, Sharon A. McClellan, Ronald P. Barrett, Linda D. Hazlett; mTOR Inhibition has Similar Effects to Treatment with Substance P in the Cornea of Pseudomonas aeruginosa-Infected BALB/c Mice. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6131.
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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.
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.
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).
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.
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