The role of staphopain a in Staphylococcus aureus keratitis.

Staphylococcus aureus is a common bacterial isolate from cases of microbial keratitis. The virulence factors that contribute to its pathogenicity during this disease have not been fully resolved. The aim of the current study was to examine the effects of the extracellular protease Staphopain A on corneal virulence. Two strains were used, one Staph 38 that gives a high pathology score during keratitis and a less virulent strain ATCC 8325-4. The effect of inhibition of Staphopain by general or specific protease inhibitors on adhesion of strains to fibronectin-coated glass or PMMA was determined. This was followed by an analysis of the effect of Staphopain A on the ability of the bacteria to adhere to and invade corneal epithelial cells. Finally, the effect of inhibiting Staphopain A on pathogenesis in a mouse model of keratitis was studied. Staphopain A increased the adhesion of strains to fibronectin-coated substrata and inhibition of Staphopain A reduced adhesion. The inhibition of Staphopain A by staphostatin A significantly decreased both association with and invasion into human corneal epithelial cells by 15-fold for strain Saur38. Inhibition of Staphopain A significantly reduced the pathology associated with S. aureus keratitis, reducing the infecting numbers of bacteria from 1.8x105 to <1x104 cells/cornea (p ≤ 0.001), significantly reducing the corneal pathology score (p ≤ 0.038) and reducing the numbers of infiltrating PMNs. This study shows that Staphopain increases adhesion and invasion of corneal cells due to increasing fibronectin binding and its inhibition has a significant impact on pathogenicity of S. aureus during keratitis.

A protective role for IL-6 in staphylococcal microbial keratitis.

PURPOSE: To determine whether interleukin-6 (IL-6) plays a protective role in Staphylococcus aureus keratitis in a gene knockout (gko) mouse model and to determine whether IL-6 may be used as a therapy to modulate host responses and control bacterial infection, thereby reducing scarring. METHODS: The eyes of IL-6 gko mice and wild-type mice were challenged topically with S. aureus and examined at 24 hours after infection. Keratitis was examined clinically and histologically. Bacterial and polymorphonuclear leukocytes (PMNs) were enumerated, and cytokine and chemokine levels were determined by ELISA. Exogenous IL-6 was administered to both IL-6 gko and wild-type mice, and clinical parameters were determined. RESULTS: IL-6 gko mice showed more severe disease, with increased bacterial counts and PMNs, than did wild-type mice. Changes in levels of chemokines and cytokines were also observed. Administration of exogenous IL-6 resulted in an improved outcome in IL-6 gko mice, with a threefold reduction in bacterial load. CONCLUSIONS: The data suggest an important regulatory role for IL-6 in modulating excessive inflammatory responses and in controlling bacterial proliferation. IL-6 may play a role in the priming and activation of neutrophils. It could represent a broad-spectrum therapy to improve outcomes in patients who have these potentially blinding infections.

A Staphylococcus aureus mouse keratitis topical infection model: cytokine balance in different strains of mice.

Staphylococcus is a leading cause of the potentially blinding disease microbial keratitis. Even with the use of antibiotic therapy, the host inflammatory response continues to damage the cornea, which may lead to blindness. Manipulation of the host response may help improve patient outcome from this devastating disease. We aim to understand the contribution of the host response to Staphylococcus aureus infection. A S. aureus keratitis mouse model was developed in both C57BL/6 and BALB/c mice using two different strains of S. aureus (8325-4 and Staph 38). Twenty-four hours postinfection, mice were killed and eyes were harvested for enumeration of bacteria, polymorphonuclear leucocytes, chemokines and cytokines. The laboratory strain 8325-4 was not as virulent as the clinical isolate Staph 38. In vitro data showed a 250-fold increase in invasion of human corneal epithelial cells by Staph 38 compared to 8325-4. BALB/c mice were susceptible to S. aureus infection whereas C57BL/6 mice were resistant. The resistant C57BL/6 mice were polarized towards a Th2 response, which may be protective for these mice. IL-4, IL-10 and IL-6 were elevated significantly in C57BL/6 mice infected with Staph 38 (P < 0.05). Macrophage inflammatory peptide (MIP)-2 was also significantly elevated in C57BL/6 mice (P < 0.001). The susceptible BALB/c mice had a muted cytokine response, which suggests that S. aureus might be 'walled off' during infection and might avoid host defences. IL-4, IL-10 and IL-6 cytokines may be protective during Gram-positive corneal infection and therefore may be useful for adjunct therapies in the treatment of this disease.

Contribution of the cornea to cytokine levels in the whole eye induced during the early phase of Pseudomonas aeruginosa challenge.

Pseudomonas aeruginosa keratitis is one of the most destructive diseases of the cornea. The host response to this infection is critical to the outcome, and is regulated by cytokines produced in the ocular tissue. In this study, we assessed the relative contribution of the cytokines produced in the cornea to the inflammatory response of the whole eye to gain a better understanding of the inflammatory and regulatory processes in the ocular environment during localized corneal infection. C57BL/6 mice were challenged by topical application of P. aeruginosa to wounded corneas. Corneas and whole eyes were harvested 24 h post-challenge and bacterial numbers, myeloperoxidase levels and the levels of cytokines known to be important in keratitis were determined. The site of production of IL-6 and KC in the retina was determined by in situ hybridization. Before infection, 90% of macrophage inflammatory protein (MIP)-2 and approximately 80% of all IFN-gamma and IL-10 produced constitutively in the eye was found outside the cornea. Twenty-four hours after infection, bacterial numbers, levels of myeloperoxidase, and levels of MIP-2 and IL-1 were not different, whether measured in cornea or whole eye. However, expression of IL-6, KC, IFN-gamma and IL-10 was significantly greater in whole eyes than in the corneas of infected eyes. The cells expressing IL-6 and KC in the retina were identified by in situ hybridization. This study indicates that during corneal inflammation, the response of the whole eye as well as the cornea needs to be considered.