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IL-10 is important in the resistance response of BALB/c mice to experimental Pseudomonas aeruginosa corneal infection. However, the cellular mechanisms by which this anti-inflammatory cytokine is regulated remain unknown. Because the mammalian target of rapamycin (mTOR) regulates IL-10 in other disease models, the present study tested its role in bacterial keratitis. After infection, corneas of rapamycin versus control-treated BALB/c mice showed worsened disease, and real-time RT-PCR confirmed that mTOR mRNA levels were significantly decreased. Rapamycin treatment also increased clinical score, polymorphonuclear neutrophil (PMN) infiltration (determined by myeloperoxidase assay), and bacterial load, but it diminished PMN bactericidal activity. Inhibition of mTOR also led to elevated mRNA and protein levels of IL-12p40, matrix metalloproteinase 9, and inducible NO synthase, whereas mRNA and protein levels of IL-10, its regulator/effector STAT-3, and suppressor of cytokine signaling 3 (a proinflammatory cytokine regulator) were decreased. Furthermore, mTOR inhibition reduced levels of proapoptotic caspase-3 and increased levels of B cell lymphoma-2 (antiapoptotic), indicative of delayed apoptosis. mTOR inhibition also altered genes related to TLR signaling, including elevation of TLR4, TLR5, and IL-1R1, with decreases in IL-1R-associated kinase 1 and an inhibitor of NF-κB, NF-κB inhibitor–like 1. Rapamycin treatment also increased levels of IFN-γ and CCAAT/enhancer binding protein, β, a gene that regulates expression of preprotachykinin-A (the precursor of substance P). Collectively, these data, as well as a rescue experiment using rIL-10 together with rapamycin, which decreased PMN in cornea, provide concrete evidence that mTOR regulates IL-10 in P. aeruginosa–induced bacterial keratitis and is critical to balancing pro- and anti-inflammatory events, resulting in better disease outcome.

Purpose To determine whether rapamycin altered corneal growth factor levels to impact severity of Pseudomonas aeruginosa keratitis. Methods BALB/c mice were injected intraperitoneally with rapamycin or PBS and infected with P. aeruginosa. Corneas were harvested and mRNA levels of growth factors (EGF, HGF, FGF-7/KGF), receptors (EGFR, c-met, FGFR-2), and signaling molecules (PI3K, Akt, S6K1, and IGF-1R) tested. ELISA determined HGF/c-met, IGF-1, and Substance P (SP) protein levels. Corneal application of recombinant (r)HGF was assessed by clinical score, photography with a slit lamp, real-time RT-PCR (mRNA for mT0R, IL-10, IL-12, IL-18, PI3KCα, Akt), and ELISA (total and phosphorylated [p]c-met); rIGF-1 effects also were tested by ELISA. In vitro, RAW cells and peritoneal macrophages were stimulated with LPS ± rHGF ± c-met inhibitor (CI) and mTOR mRNA levels tested. Results Rapamycin disparately regulated infected corneal mRNA levels of EGF/EGFR and FGF-7/FGFR-2, but HGF/c-met mRNA levels both increased. ELISA confirmed elevated HGF protein. Rapamycin did not change PI3KCα or Akt signaling molecule expression, downregulated S6K1, but upregulated IGF-1R mRNA levels; IGF-1 and SP proteins also were upregulated. After infection, topical rHGF versus PBS increased mRNA levels of IL-12p40, IL-18, PI3KCα, and Akt; mTOR and IL-10 mRNA were downregulated; rIGF-1 increased HGF protein. In vitro, rHGF and LPS lowered RAW cell and macrophage mTOR levels; CI addition restored them. Conclusions Collectively, these data provide evidence that enhanced corneal HGF levels increase signaling through the c-met receptor, decrease mTOR levels, and enhance proinflammatory cytokines, while decreasing anti-inflammatory cytokines, and that HGF signaling is central to disease outcome.

This study analyzed the influence of substance P (SP) on growth factors related to wound healing in mice in the presence of infectious keratitis.Naturally resistant mice were injected intraperitoneally with SP or phosphate-buffered saline and infected with Pseudomonas aeruginosa, and corneal messenger RNA (mRNA) levels of growth factors and apoptosis genes were tested. Enzyme-linked immunosorbent assay determined the protein levels, whereas immunohistochemistry tested the distribution, macrophage phenotype, and cell quantitation. In vitro, macrophages were stimulated with lipopolysaccharide (LPS; with or without SP) and mRNA levels of proinflammatory and antiinflammatory cytokines and apoptosis genes were tested.After SP, epidermal growth factor mRNA and protein levels were disparately regulated early, with no differences later in the disease. Hepatocyte growth factor and fibroblast growth factor-7 mRNA and protein levels were increased after SP treatment. Enumerating dual-labeled stromal cells revealed no difference between SP-treated versus phosphate-buffered saline-treated groups in the percentage of epidermal growth factor-labeled fibroblasts or macrophages, but there were significant increases in both hepatocyte growth factor- and fibroblast growth factor-7-labeled cells. Type 2 (M2) macrophages and caspase-3 mRNA levels were decreased, whereas B-cell lymphoma-2 mRNA expression was increased after SP treatment. In vitro, mRNA levels of several proinflammatory cytokines and B-cell lymphoma-2 were elevated, whereas transforming growth factor β was decreased after macrophage stimulation with SP (with LPS) over LPS alone. (Mice: n = 105 control; 105 experimental.)These data show that treatment with SP in infectious keratitis elevates growth factors but also adversely affects the disease by enhancing the inflammatory response and its sequelae.

Exogenous vasoactive intestinal peptide (VIP) down-regulates pro-inflammatory but up-regulates anti-inflammatory cytokines, growth factors (GFs) and Toll-like receptors promoting healing in experimental Pseudomonas aeruginosa (P. aeruginosa) keratitis. Whether VIP is required for GF or GF receptor (R) expression in normal and infected corneas is unknown and is the purpose of this study.VIP knockout ((-/-)) and wild-type (WT) C57BL/6 (B6) mice were infected and tested using PCR array, real-time RT-PCR, ELISA, and immunostaining. VIP antagonist treatment studies also were done using B6 and BALB/c mice.Infected corneas of VIP(-/-) versus WT B6 mice perforated earlier (2 vs. 5 days postinfection [p.i.]), and array data showed that GFs were differentially changed between groups. RT-PCR revealed that the infected cornea of VIP(-/-) versus WT mice expressed higher mRNA levels of epidermal growth factor (EGF) and hepatocyte growth factor (HGF), reduced FGF, EGFR, and HGFR, with no difference in FGFR; differences between groups were not seen in normal cornea. Immunostaining for GF and GFR in the normal cornea of VIP(-/-) versus WT mice was similar. However, at 1 day p.i., VIP(-/-) versus WT mice had more intense EGF and HGF, similar FGFR, and reduced FGF, EGFR, and HGFR staining. VIP antagonist treatment decreased protein levels for GFR at 5 days p.i. in both B6 and BALB/c mice, with no significant changes in normal cornea.The data showed that endogenous VIP is not requisite for GF or GFR expression in the normal cornea but, after infection, its absence or reduction is critical for their regulation.