| Summary: | Microbial keratitis (MK) is a leading cause of blindness in the developing world. Pseudomonas aeruginosa (PA) is the most common pathogen isolated from contact lens related MK, and is usually associated with significant visual complications. The intact corneal epithelium forms a strong barrier to bacterial penetration into deeper tissue, and only when bacteria reach the stroma, through a breach in the epithelium, does a typical ulcer develop, with stromal suppuration and necrosis. Since corneal fibroblasts are one of the most prominent cellular components in the stroma, they probably have an important role in the pathogenesis of corneal ulceration. The aim of this study was to establish an in vitro model of PA microbial keratitis in order to examine the microbiological, biochemical and ultrastructural changes associated with live infection of primary human corneal fibroblasts (hCF) with PA, and to assess the role of these cells in the initiation and progression of the pathogenesis of the disease. Human corneal fibroblast monolayers were infected with bacteria, and bacterial association was assessed at 3, 6 and 9h, bacterial invasion was assessed at 9h and cytokine profiles were assessed at 9 and 24 h post challenge. Host cell death was assessed at 9h using a lactate dehydrogenase assay (LDH). Gentamicin was added at 3h to eliminate all extra cellular bacteria and limit pathogen induced effects, and bacterial replication and survival and its effect on host cell viability were assessed. Induction of cytokine and matrix metalloproteinase (MMP) production by hCF in response to PA infection was assessed by a sandwich immunoassay and a gelatine zymography. To examine the interactions of PA with the cornea as an organ, donor corneal buttons remaining after DSEK were incubated in a medium inoculated with PA for 3-9h, then processed for scanning and transmission electron microscopy. Scanning and Transmission Electron Micrographs (SEM/TEM) of bacterial association and invasion of corneal cells were acquired and showed that bacteria associated to superficial epithelial cells but only invaded and colonized the stroma in the presence of a mechanical breach to the epithelial basement membrane. PAO1 bacteria caused monolayer disruption within 24h with all infective doses (101-108 CFU/mL). PAO1 associated to hCF in a dose- and time-dependant manner utilizing type IV pilus and flagella. Bacterial internalization was detected by the gentamicin assay, which also demonstrated the ability of PAO1 to survive and replicate within the fibroblasts, and was dependant on the SRC tyrosine kinase and the actin microfilament system, where internalisation was diminished when these systems were inhibited. Cytotoxicity was observed by 9h post challenge, and was reliant on bacterial type III secretion system and flagella, where mutant bacterial strains induced less cytotoxicity. The more virulent PA14 bacteria induced more cytotoxicity than PAO1. The presence of intracellular bacteria did not affect cell viability in the presence of gentamicin. In response to PAO1 infection, hCF produced the pro-inflammatory IL-1β and GM-CSF, although GM-CSF seems to be released later than IL-1β as shown by protecting the cells with gentamicin 3h post challenge. Infection with PA14 caused an earlier and more potent cytokine production than PAO1, reflecting the pattern of cytotoxicity. Bacterial components did not have a significant effect on cytokine production by corneal fibroblasts. Cytokine production was partly reliant on bacterial type III secretion system and flagella, and wild type PA14 strains induced an earlier and more potent IL-1β production by infected corneal fibroblasts. Gelatine zymography showed that hCF produce specific MMPs in response to live PA challenge for 24h, and these MMPs were not released in response to treatment with individual bacterial components and virulence factors. Bacteria alone produced alkaline protease, a bacterial MMP. Sandwich immunoassays showed that MMP-1, MMP-2, MMP-3 and MMP-9 were produced by infected fibroblasts to a level similar to uninfected cells. Gentamicin protected cells produced more MMPs than unprotected cells.
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