Immunology and Microbiology Thrombomodulin Protects Against Bacterial Keratitis, Is Anti-Inflammatory, but Not Angiogenic

PURPOSE. Thrombomodulin (TM) is a multidomain, transmembrane protein with antiinflammatory properties. Thrombomodulin domain (D) 1 is lectin-like, interacting with Lewis Y antigen on lipopolysaccharide, and with HMGB1, while TMD23 is associated with angiogenic and anti-inflammatory functions. Thus, we tested if TM is protective against Pseudomonas aeruginosa keratitis and whether it enhanced corneal vascularity. METHODS. Eyes of C57BL/6 (B6) mice were injected with recombinant TM (rTM), rTMD1, or PBS subconjunctivally before and intraperitoneally after infection with P. aeruginosa. Clinical scores, photography with a slit lamp, RT-PCR, ELISA, myeloperoxidase (MPO) assay, viable bacterial plate counts, and India ink perfusion were used to assess the disease response and corneal vascularity (rTM only). RESULTS. Recombinant TM versus PBS treatment reduced clinical scores and corneal opacity. Corneal mRNA levels for HMGB1 were unchanged, but proinflammatory molecules IL-1b, CXCL2, NF-jB, TLR4, and RAGE were decreased; anti-inflammatory molecules SIGIRR and ST2 were increased. ELISA confirmed the mRNA data for HMGB1, IL-1b, and CXCL2 proteins. Both neutrophil influx and viable bacterial plate counts also were decreased after rTM treatment. Protein levels for angiogenic molecules VEGF, VEGFR-1, and VEGFR-2 were measured at 5 days post infection and were not different or reduced significantly after rTM treatment. Further, perfusion with India ink revealed similar vessel ingrowth between the two groups. Similar studies were performed with rTMD1, but disease severity, mRNA, proteins, MPO, and plate counts were not changed from controls. CONCLUSIONS. These data provide evidence that rTM treatment is protective against bacterial keratitis, does not reduce HMGB1, and is not angiogenic

Matrix Metalloproteinase-9 Amplifies the Immune Response to \u3ci\u3ePseudomonas aeruginosa\u3c/i\u3e Corneal Infection

PURPOSE. The purpose of this study was to determine the role of matrix metalloproteinases (MMP) in Pseudomonas aeruginosa keratitis. METHODS. Gene array and selective real-time PCR examined MMP expression in the cornea of susceptible (C57BL/6, B6) versus resistant (BALB/c) mice before and after infection; zymography tested enzyme activity for MMP-2 and -9. Clinical score, Langerhans cell (LC), and Neutrophil (PMN) quantitation were done in recombinant (r) MMP-9, antibody neutralized, and MMP-9−/− mice. The chemotactic potential of MMP-9 was tested in a Boyden chamber assay; light and transmission microscopy and immunostaining for collagen IV and MMP-9 were used to examine the effects and the source of MMP-9 after infection. ELISA was used to assess IL-1β and MIP-2 levels. RESULTS. Gene array (confirmed by PCR) revealed sixfold more MMP-9, and zymography showed greater enzyme activity in the infected cornea of B6 over BALB/c mice. rMMP-9 injection of BALB/c mice enhanced, whereas MMP-9 antibody neutralization in B6 mice and its absence in MMP-9−/− mice decreased corneal disease. MMP-9−/− and antibody neutralized mice had fewer LCs in cornea; rMMP-9-treated mice had more. A myeloperoxidase (MPO) assay showed a similar pattern for PMN. MMP-9 was not chemotactic for LC or PMN. The basement membrane was more intact in MMP-9−/− over wild-type infected mice and correlated with staining for collagen IV; PMN was a source of MMP-9. IL-1β and MIP-2 were increased in rMMP-9 but decreased in MMP-9 antibody neutralized and MMP-9−/− over control groups. CONCLUSIONS. MMP-9 regulates immune function in cornea by proteolysis, potentiating P. aeruginosa keratitis by degrading collagen IV and upregulating chemotactic cytokines/chemokines IL-1β and MIP-2

PM10 and Pseudomonas aeruginosa: effects on corneal epithelium

PurposeIn 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.MethodsMouse 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.ResultsIn 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.ConclusionsPM10 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

Pseudomonas aeruginosa Binds to Extracellular Matrix Deposited by Human Corneal Epithelial Cells

PURPOSE. To measure the effect of extracellular matrix substrate, pH, and O 2 on Pseudomonas aeruginosa binding. METHODS. Extracellular matrix substrates were prepared from human corneal epithelial cells cultured in 2% or 20% O 2 . P. aeruginosa strains ATCC 19660 or PAO1 (suspended in pH 7.0 or 7.5 buffer) were cultured on extracellular matrix substrates in 2% or 20% O 2 . The mean number of adherent bacteria per counted per field Ϯ SEM (n ϭ 15) was determined for combinations of bacteria, extracellular matrix substrate, pH, and O 2 . Binding in the presence of antibodies directed against laminin-5 was also measured. RESULTS. Extracellular matrix substrates produced by cells cultured in 20% O 2 , combined with an environment of pH 7.0, provided the least favorable conditions for binding of strain 19660. In contrast, extracellular matrix substrates produced by cells cultured in 2% O 2 , combined with an environment of pH 7.0, provided the most favorable conditions for binding of strain 19660. Binding of PAO1, however, as a function of extracellular matrix substrate and pH, did not similarly compare with binding of strain 19660. Antibodies against laminin-5 chains served to increase the number of strain 19660 bacteria bound to extracellular matrix substrates compared with the control. CONCLUSIONS. The extracellular matrix secreted by hypoxic corneal epithelial cells is a substrate for binding of P. aeruginosa. Results in previous studies have shown that hypoxic extracellular matrix contains less laminin-5 protein than normoxic matrix. The antibody studies in this report suggest that the decrease in laminin-5 content in hypoxic matrix, relative to matrix secreted by normoxic corneal epithelium, may be responsible for increased bacterial adhesion. (Invest Ophthalmol Vis Sci. 2002;43:3654 -3659) A n intact layer of epithelium firmly attached to the underlying basement membrane creates a barrier that is essential for the cornea to maintain proper vision. The epithelial barrier, along with elements provided by the tear film, plays a role in preventing bacterial binding, colonization, and infectivity of the corneal surface and stromal spread of the bacteria. Microbial infections that disrupt the integrity of corneal epithelial cell-extracellular matrix, if left untreated, will result in cellular loss, inflammatory reactions, and subsequent decreased visual acuity. Pseudomonas aeruginosa is a virulent opportunistic pathogen responsible for a particularly destructive corneal disease