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E. J. Lee, D. J. Evans, S. M. J. Fleiszig, J. T. Rosenbaum, S. R. Planck; Differential Effects of the P. aeruginosa Type III Secretion System (T3SS) on Corneal Dendritic Cells and Neutrophil Recruitment During Infection. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3870.
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© ARVO (1962-2015); The Authors (2016-present)
Intoxication by effectors of the ExsA-regulated type III secretion system (T3SS) of Pseudomonas aeruginosa has been investigated extensively in epithelial cells, which form an important defensive barrier to infection. Less well studied is the effect of the T3SS on the host inflammatory response during infection, except that it is involved in ring infiltration by cytotoxic P. aeruginosa. The goal of this study was to examine the effect of the T3SS on trafficking of polymorphonuclear leukocytes (PMNs) in contrast to dendritic cells (DCs) in P. aeruginosa-infected murine corneas.
Mice (C57BL/6 background) expressing GFP in the lysozyme M locus, or mice expressing YFP under the control of the CD11c promoter, were used to visualize PMNs or DCs, respectively. Scarified corneas of anesthetized animals were inoculated with phosphate-buffered saline (PBS), with wild type (wt) cytotoxic PA103 or invasive PAO1, or with their respective T3SS (exsA-) mutants (PA103exsA::Ω, PAO1exsA::Ω)(5x105 cfu in 5 µl). PA103, but not PAO1, encodes ExoU, a T3SS toxin. At 6 and 24 h post-infection corneas were examined by in vivo microscopy and then whole mounted for quantitative analysis ex vivo (n=3-5/group). At 24 h, in vivo time-lapse videomicroscopy was used to analyze dynamic cell migration (n=4).
As previously reported, PMNs accumulated primarily in the central cornea with PAO1 infection, but formed a dense ring in the mid-periphery with PA103. While T3SS mutation of PA103 resulted in drastic reduction in the number of infiltrating PMNs compared to wt, in PAO1 it increased the area of the central infiltrate (by ~38%) compared to wt. For PA103, PMNs outside the ring exhibited typical amoeboid morphology and migration speed of 8.4±1.2 µm/min, while those in the ring were mostly rounded and migrated at reduced speeds of 2.6±0.9 µm/min (p=0.038), with many cells immobile. Cell fragments were noted within, but not outside, the ring. At 24 h, the number of DC in PA103-infected eyes was lower in the central cornea (p=0.029), but not the periphery (p=0.057) compared to PAO1-infected eyes. T3SS mutation did not affect DC recruitment into the cornea for either strain. In all cases, DC in infected corneas exhibited "blebs" emanating from cell bodies and what appeared to be eventual fragmentation.
The T3SS of P. aeruginosa has differential effects on PMN and DC recruitment. These cell types respond to different cytokines/chemokines. It is possible that the T3SS indirectly modulates PMN and DC responses by altering expression of such mediators.
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