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Rachel Redfern, Kendall Stout, Karen Dionne; Toll-like Receptor Agonists Stimulate Matrix Metalloproteinase-9 Production in Ocular Surface Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5999.
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Dry eye syndrome (DES) is a chronic multifactorial inflammatory condition that can lead to corneal ulceration and chronic ocular discomfort. The pathogenesis of DES is not fully understood and toll-like receptors (TLRs) may be in part responsible for the production of dry eye associated proinflammatory cytokines and matrix metalloproteinases (MMPs) that lead to ocular surface pathology. Previously we have shown that topical application of a TLR agonist cocktail to the ocular surface in mice with experimental dry eye leads to corneal ulceration, yet the mechanism is unknown. This study examined the hypothesis that TLR agonists stimulate the production of MMP-9 in human ocular surface cells.
Primary human corneal epithelial cells (HCEC) and SV40 HCEC were treated with either IL-1β, TLR6/2 (FSL), TLR3 (POLYI:C), TLR4 (LPS), TLR5 (Flagellin) or TLR9 (ODN2006) agonists or the vehicle control for 48 hrs. Following treatment, the cell culture media was collected to detect MMP-9 protein secretion. Total RNA was extracted from cell culture lysates. Real-time PCR and ELISA were used to quantitate MMP-9 mRNA and protein respectively. A statistically significant change (p<0.05) in MMP-9 expression compared to the vehicle control was determined by ANOVA and Student's t-test.
In SV40 HCEC, MMP-9 mRNA was significantly upregulated by 3.2±0.4 fold in response to IL-1β, 3.6±0.3 fold by TLR6/2 agonist and 6.4±0.8 fold in response to TLR5 agonist. MMP-9 protein was significantly increased from 1.2±1.3ng/ml in vehicle treated samples to 4.5±0.8 ng/ml by IL-1β, 3.7±1.1ng/ml by TLR6/2 agonist and 2.5±1.1ng/ml by TLR5 agonist. There was no significant change in MMP-9 mRNA or protein in response to TLR4 agonist in SV40 HCEC. In primary HCEC, MMP-9 protein was significantly increased from 7.5±1.3ng/ml in vehicle treated samples to 10.5±4.5 ng/ml by IL-1β, 13.5±0.7ng/ml by TLR3 agonist and 10.2±3.8ng/ml by TLR5 agonist and 13.1±0.2ng/ml by TLR9 agonist. There was no significant change in response to TLR6/2 agonist in primary HCEC.
These novel results are the first to show that TLR agonists stimulate the production of MMP-9 in various ocular surface cells. Given that MMP-9 production in dry eye patients is thought to be predictive of dry eye associated corneal ulceration, TLR antagonists may serve as a novel therapeutic option in the treatment of dry eye.
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