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A. Yamada, S. R. Heimer, M. S. Gilmore; Toxigenic Staphylococcus aureus Infection Induces CCL20 Secretion in the Corneal Epithelium. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2482.
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CCL20 (Chemokine ligand 20) is a potent chemoattractant for CCR6-positive immature dendritic cells and memory T cells in addition to having antimicrobial activities, and plays a role on mucosal surfaces in inflammation. Little is known regarding the CCL20 secretion in the cornea following exposure to bacteria. We have previously demonstrated in corneal epithelial cell culture model that Staphylococcus aureus infection causes a large increase in CCL20 gene expression by microarray study. Some S. aureus lytic toxins which are regulated by global regulators Agr and Sar can modulate cellular behavior by altering receptor processing and signaling. We investigated whether CCL20 secretion is affected by toxins by using isogenic Agr/Sar mutant ALC135 and wild type RN6390. Furthermore, we measured the level of cytokines which are known to stimulate CCL20 secretion in other cell types. We also investigated whether internalization of S. aureus is necessary for increased CCL20 secretion, and whether CCL20 secretion increased through protein synthesis.
S. aureus RN6390 and ALC135 were incubated with confluent monolayers of human corneal epithelial cells (primary and Araki-Sasaki) in serum-free defined keratinocyte medium (DK-SFM) for 1 to 8 hrs. Initial MOIs were ≤ 20. Co-cultures were assessed periodically for cytokine and chemokine assay. Concentrations of CCL20, IL-1 α, IL-1β, and IL-17 were measured. CytochalasinD blocks S. aureus internalization in corneal epithelial cells. Actinomycin D is a transcriptional inhibitor which blocks protein synthesis. Monolayers were incubated for 1 h in DK-SFM containing the cytochalasin D 0.5µg/ml or actinomycin D 1 or 5µg/ml prior to adding S. aureus.
CCL20 protein secretion increased in both primary and Araki-Sasaki cells in time dependent manner. Throughout all time points, RN6390 infected group had significantly higher level of CCL20 secretion than ALC135 infected group. IL-1 α and IL-1β secretion was significantly higher in RN6390 infected group compared to ALC135 infected group in both cell types. CytochalasinD treatment did not change the level of CCL20 secretion in RN6390 or ALC135 infected cells. Secretion of CCL20 in response to S. aureus infection was inhibited by actinomycin D in dose dependent manner.
Agr/Sar regulated virulence factors are responsible for increased CCL20 secretion associated with increased IL-1 α and IL-1β secretion. S. aureus infection caused increased synthesis of CCL20 protein. Internalization of S. aureus is not necessary for increased CCL20 secretion.
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