PM10 and Pseudomonas aeruginosa: effects on corneal epithelium

Abstract

Purpose: In vivo data indicate that mouse corneas exposed to PM₁₀ showed early perforation and thinning after infection with Pseudomonas aeruginosa. To understand the mechanisms underlying this finding, we tested the effects of PM₁₀ and the mitochondria targeted anti-oxidant SKQ1 in immortalized human corneal epithelial cells (HCET) that were challenged with Pseudomonas aeruginosa strain 19660.

Methods: Mouse corneas were infected with strain 19660 after a 2 week whole-body exposure to PM₁₀ or control air and assessed by clinical scores, slit lamp photography and western blot. HCET were exposed to 100μg/ml PM₁₀ for 24h before challenge with strain 19660 (MOI 20). A subset of cells were pre-treated with 50nM SKQ1 for 1h before PM₁₀ exposure. Phase contrast microscopy was used to study cell morphology, cell viability was measured by an MTT assay, and ROS by DCFH-DA. Levels of pro-inflammatory markers and anti-oxidant enzymes were evaluated by RT-PCR, western blot and ELISA. Reduced glutathione (GSH) and malondialdehyde (MDA) levels were evaluated by assay kits.

Results: In vivo, whole body exposure to PM₁₀ vs. control air exposed mouse corneas showed early perforation and/or corneal thinning at 3 days post infection, accompanied by increased TNF-α and decreased SOD2 protein levels. In vitro, PM₁₀ induced a dose dependent reduction in cell viability of HCET and significantly increased mRNA levels of pro-inflammatory molecules compared to control. Exposure to PM₁₀ before bacterial challenge further amplified the reduction in cell viability and GSH levels. Furthermore, PM₁₀ exposure also exacerbated the increase in MDA and ROS levels and phase contrast microscopy revealed more rounded cells after strain 19660 challenge. PM₁₀ exposure also further increased the mRNA and protein levels of pro-inflammatory molecules, while anti-inflammatory IL-10 was decreased. SKQ1 reversed the rounded cell morphology observed by phase contrast microscopy, increased levels of MDA, ROS and pro-inflammatory molecules, and restored IL-10.

Conclusions: PM₁₀ induces decreased cell viability, oxidative stress and inflammation in HCET and has an additive effect upon bacterial challenge. SKQ1 protects against oxidative stress and inflammation induced by PM₁₀ after bacterial challenge by reversing these effects. The findings provide insight into mechanisms underlying early perforation and thinning observed in infected corneas of PM₁₀ exposed mice.

Keywords: P. aeruginosa; PM10; cornea; epithelium; human.