Shamila Khan

Experimental Pseudomonas aeruginosa keratitis in interleukin-10 gene knockout mice.

ABSTRACT Pseudomonas aeruginosa keratitis is one of the most destructive diseases of the cornea. The host response to this infection is critical to the outcome. The cytokine interleukin-10 (IL-10) is thought to play an important role in modulating excessive inflammation and antimicrobial defenses. We have found that in IL-10−/− mice there is a significant decrease in bacterial load in corneas at 7 days postchallenge with P. aeruginosa. This decrease was accompanied by a reduction in neutrophil numbers in the cornea and changes in cytokine levels compared to those of wild-type mice. A characteristic increase in neovascularization in the cornea was found in the IL-10−/− mice. This increased angiogenesis correlated with an increased expression of KC, whereas the kinetics of macrophage inflammatory peptide 2 expression correlated with neutrophil numbers. This finding suggests that KC may play a role in corneal angiogenesis. The source of IL-10 in mouse corneas was identified as a subpopulation of infiltrating cells and keratocytes. This study demonstrates that IL-10 plays an important role in regulating the balance of inflammatory mediators during P. aeruginosa infection of the cornea.

The role of CXC chemokine receptor 2 in Pseudomonas aeruginosa corneal infection

Pseudomonas is one of the leading causes of contact lens‐related microbial keratitis. Despite the use of antibiotics, the host inflammatory response continues to cause damage to the cornea, which may lead to blindness. CXCR2‐binding chemokines have been implicated in the pathogenesis of Pseudomonas keratitis, and the exact role of this receptor remains to be elucidated. Corneas of CXCR2 knockout and wild‐type mice (Cmkar 2−/− and Cmkar 2+/+) were scratched, and 2 × 106 CFU/mL Pseudomonas 6294 or 6206 was added to corneas. Twenty‐four hours postinfection, mice were killed, and eyes were harvested for enumeration of bacteria, myeloperoxidase (MPO) levels, and inflammatory mediators. Cmkar 2−/− had 20‐ to 100‐fold more bacteria than Cmkar 2+/+ mice. There were no differences in MPO levels between gene knockout and Cmkar 2+/+ mice. Histology revealed PMN were restricted to the limbal area. Levels of CXCR2 chemokines (keratinocyte‐derived chemokine and MIP‐2) were elevated significantly in gene knockout mice. A lack of CXCR2 leads to an inability to control bacterial numbers as a result of the inability of PMN to reach the site of infection in the avascular cornea. These results imply that CXCR2 is critical to the extravasation of neutrophils into the avascular cornea.

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‐γ 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‐γ 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.

The corneal response to infection with Staphylococcus aureus in the absence of interleukin-4.

Interleukin‐4 (IL‐4) has previously been implicated in a protective response to Staphylococcus aureus corneal infection. Consequently, the specific role of IL‐4 during S. aureus corneal infection was investigated using IL‐4 gene knockout mice. The eyes of IL‐4−/− mice and wild‐type mice were challenged topically with S. aureus and examined at 24 h post‐infection. Keratitis was examined clinically and histologically. Bacterial and polymorphonuclear leucocytes (PMN) numbers were enumerated and cytokine and chemokine levels determined by enzyme‐linked immunosorbent assay. Exogenous IL‐4 was administered to both IL‐4−/− and wild‐type mice and clinical parameters were determined. A lack of IL‐4 resulted in a significant increase in clinical scores, pathology, bacterial load and neutrophil numbers. The absence of IL‐4 also resulted in an upregulation of interferon (IFN)‐γ and a downregulation of IL‐6, IL‐10 and the chemokines KC and macrophage inflammatory protein‐2. Administration of exogenous IL‐4 to IL‐4−/− mice was protective but time‐dependent. This study highlights the protective role of IL‐4 during S. aureus infection and emphasizes the balance between IL‐4 and IFN‐γ in achieving bacterial control and maintaining the integrity of the cornea. This information may lead to the development of novel therapeutic strategies potentially improving the prognosis for infection of this unique avascular site.

Interleukin-4 is not Critical to Pathogenesis in a Mouse Model of Pseudomonas aeruginosa Corneal Infection

Purpose: To determine the contribution of interleukin-4 (IL-4) to the initial host response during corneal infection with Pseudomonas aeruginosa in a mouse model. Methods: Corneas of 6− to 8-week-old IL-4−/− and wild-type mice were topically challenged with P. aeruginosa. Ocular tissue was collected 24 hr and 7 days postchallenge. Viable bacterial counts, myeloperoxidase assays, cytokine levels, and clinical and histological examinations were performed. Results: During challenge with P. aeruginosa, no differences were observed clinically, histologically, or in bacterial load between IL-4−/− and wild-type mice at either time point. However, differences in cytokine levels of IL-6, KC, and IL-10 were observed. Conclusions: The data presented indicate that IL-4, a central Th2 cytokine, may not be critical to the pathogenesis or bacterial clearance in this model of P. aeruginosa bacterial keratitis during the early stages of the infectious process.