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Ellen J. Lee, James T. Rosenbaum, Stephen R. Planck; Investigation into Interactions of P. aeruginosa with PMNs and DCs During Murine Corneal Infection. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5809.
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Pseudomonas aeruginosa infection of the cornea is accompanied by an exuberant inflammatory response. Our previous imaging studies of P. aeruginosa keratitis documented some bacterial strain-dependent differences in polymorphonuclear leukocyte (PMN) and dendritic cell (DC) recruitment to and migration in the infected cornea. To further investigate immune cell behavior in response to bacteria, we utilized recently-generated fluorescent bacteria to visualize their relationships and interactions with host PMNs and DCs at the single cell level.
Mice (susceptible C57BL/6 background) expressing eGFP in the lysozyme M locus, or mice expressing eYFP under the control of the CD11c promoter were used to visualize PMNs or DCs, respectively. Scarified central corneas of anesthetized animals were inoculated with PA14 (cytotoxic, expresses ExoU) or PAO1 (invasive, lacks ExoU expression) that constitutively express red fluorescent protein (RFP) (5x105-5x106 cfu in 5 µl). In vivo real-time and time-lapse epifluorescence videomicroscopy (up to 1 h equivalent) were performed ~16-20 h post-inoculation (n=5/strain). Corneas were also whole mounted for ex vivo examination with wide-field and confocal fluorescence microscopy (n=7/strain). Corneal areas that were populated with bacteria as well as distant from (>250 µm) visible bacteria were examined.
Bacteria first invaded the central cornea along the length of the scratch injury, from which they disseminated peripherally. PMNs migrated in from the limbus in directed fashion toward the centrally-located bacteria (speeds similar for PA14 vs. PAO1; 7.2±1.4 vs. 6.1±0.9 µm/min, respectively) until the leading edges of bacteria were reached, after which migration became more random or stopped. At this interface, few bacteria of either strain were visualized within PMNs such that differences in internalization were not detectable. In contrast, DCs exhibited random restricted local migration regardless of the presence of bacteria or bacterial strain. Subepithelial DCs frequently possessed membrane blebs. Those cells exhibiting blebs were not necessarily located in bacteria-laden areas, and none of the blebs appeared to contain internalized bacteria (based on 210 cells in 14 eyes).
The scarcity of phagocytosed bacteria visualized within PMNs and DCs during this early stage of infection should be further investigated, since P. aeruginosa has been shown in vitro to possess factors that affect internalization into mammalian cells.
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