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David J Evans, Matteo Maria Emiliano Metruccio, Connie Tam, Suzanne M J Fleiszig; Role of Dendritic Cells in Corneal Epithelial Defense against Pseudomonas aeruginosa Colonization and Traversal in Vivo. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6256.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported that MyD88 plays a critical role in defending the intact murine corneal epithelium against P. aeruginosa colonization and traversal in vivo. However, relative role(s) of epithelial cell- or immune cell-expressed MyD88 in that defense were unknown. Here, we hypothesized that depletion of dendritic cells in the murine cornea in vivo would compromise epithelial defense against P. aeruginosa.
Dendritic cell (DC) depletion in the murine cornea was achieved using CD11c-DTR/GFP C57BL/6 mice and intraperitoneal injection with diphtheria toxin (DT) (4ng/g of body weight) versus a sterile control. After 24 h to allow DC depletion, one cornea of each anesthetized mouse was rinsed with PBS to wash away tear fluid, then blotted with Kimwipe™ tissue paper (to enable bacterial adhesion) and inoculated with 5 µl of P. aeruginosa strain PAO1-GFP suspension (~1011 cfu/ml). After 4 h, animals were euthanized, eyes carefully enucleated, rinsed with PBS, and corneas imaged using reflection confocal microscopy (633 nm, corneal cells) and 488 nm, GFP). Z stacks (1.0 µm) were collected from ≥3 random fields/sample, and Image J used for 3-D image reconstruction, and to quantify bacterial adherence. Untreated contralateral eyes, i.e. not tissue-paper blotted, uninoculated, were also imaged as controls.
Blotted corneas of DT treated mice showed a significant increase in P. aeruginosa adherence compared to blotted corneas of saline-injected controls (p < 0.05, Mann-Whitney test). Blotted corneas of DT treated mice also showed partial bacterial traversal of the epithelium. Unblotted and uninoculated corneas of DC depleted mice looked healthy and similar to controls.
These data suggest that dendritic cells contribute to corneal epithelial defense against P. aeruginosa colonization and traversal in the tissue-paper blotting murine model, and thus contribute to MyD88-mediated epithelial defense against this bacterium. Further studies will elucidate if these data reflect a direct effect of DCs or crosstalk with the corneal epithelial cells.
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