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

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

Copper-Triggered Aggregation of Ubiquitin

Neurodegenerative disorders share common features comprising aggregation of misfolded proteins, failure of the ubiquitin-proteasome system, and increased levels of metal ions in the brain. Protein aggregates within affected cells often contain ubiquitin, however no report has focused on the aggregation propensity of this protein. Recently it was shown that copper, differently from zinc, nickel, aluminum, or cadmium, compromises ubiquitin stability and binds to the N-terminus with 0.1 micromolar affinity. This paper addresses the role of copper upon ubiquitin aggregation. In water, incubation with Cu(II) leads to formation of spherical particles that can progress from dimers to larger conglomerates. These spherical oligomers are SDS-resistant and are destroyed upon Cu(II) chelation or reduction to Cu(I). In water/trifluoroethanol (80∶20, v/v), a mimic of the local decrease in dielectric constant experienced in proximity to a membrane surface, ubiquitin incubation with Cu(II) causes time-dependent changes in circular dichroism and Fourier-transform infrared spectra, indicative of increasing β-sheet content. Analysis by atomic force and transmission electron microscopy reveals, in the given order, formation of spherical particles consistent with the size of early oligomers detected by gel electrophoresis, clustering of these particles in straight and curved chains, formation of ring structures, growth of trigonal branches from the rings, coalescence of the trigonal branched structures in a network. Notably, none of these ubiquitin aggregates was positive to tests for amyloid and Cu(II) chelation or reduction produced aggregate disassembly. The early formed Cu(II)-stabilized spherical oligomers, when reconstituted in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes and in POPC planar bilayers, form annular and pore-like structures, respectively, which are common to several neurodegenerative disorders including Parkinson's, Alzheimer's, amyotrophic lateral sclerosis, and prion diseases, and have been proposed to be the primary toxic species. Susceptibility to aggregation of ubiquitin, as it emerges from the present study, may represent a potential risk factor for disease onset or progression while cells attempt to tag and process toxic substrates

Micromechanical Properties of Injection-Molded Starch–Wood Particle Composites

The micromechanical properties of injection molded starch–wood particle composites were investigated as a function of particle content and humidity conditions. The composite materials were characterized by scanning electron microscopy and X-ray diffraction methods. The microhardness of the composites was shown to increase notably with the concentration of the wood particles. In addition,creep behavior under the indenter and temperature dependence were evaluated in terms of the independent contribution of the starch matrix and the wood microparticles to the hardness value. The influence of drying time on the density and weight uptake of the injection-molded composites was highlighted. The results revealed the role of the mechanism of water evaporation, showing that the dependence of water uptake and temperature was greater for the starch–wood composites than for the pure starch sample. Experiments performed during the drying process at 70°C indicated that the wood in the starch composites did not prevent water loss from the samples.Peer reviewe

NLRC4 regulates caspase-1 and IL-1beta production in a CD11blowLy6Glow population of cells required for resistance to Pseudomonas aeruginosa keratitis.

Psbetaeudomonas (P.) aeruginosa infection of the cornea in BALB/c mice does not result in perforation and the mice have been classified as resistant. However, regulation of this response via inflammasome activation remained untested. Therefore, BALB/c mice were infected with P. aeruginosa ATCC strain 19660 and NLRP3 and NLRC4 protein tested by ELISA. Since NLRC4 vs NLRP3 protein levels were significantly higher in the corneas of BALB/c at 1 and 5 days postinfection we used silencing to knockdown NLRC4. Silencing NLRC4 vs scrambled siRNA treatment exacerbated disease in BALB/c mice, reduced myeloperoxidase levels and elevated bacterial plate counts at 5 days postinfection. It also increased pro IL-1beta, but reduced total protein for IL-1beta and IL-18 at 5 days postinfection. Flow cytometry to identify cells affected by silencing, showed reduced caspase-1 levels in a CD11blowLy6Glow population of cells, (but not PMN or macrophages) from the infected cornea of siNLRC4 treated mice that produced less mature IL-1beta. These data provide evidence that the NLRC4 inflammasome contributes to resistance through regulation of caspase-1, IL-1beta and IL-18 in a CD11blowLy6Glow population of cells

Testican-1 Promotes Resistance against Pseudomonas aeruginosa–Induced Keratitis through Regulation of MMP-2 Expression and Activation

This study is the first to demonstrate the expression of the proteoglycan testican-1 in the cornea after induction of bacterial keratitis. This molecule is also shown to influence MMP-2 expression and activation, indicating a potential target for disease manipulation and treatment involving ocular tissue destruction

VIP Promotes Resistance in the Pseudomonas aeruginosa–Infected Cornea by Modulating Adhesion Molecule Expression

The neuropeptide VIP immunomodulates corneal expression of adhesion molecules after Pseudomonas aeruginosa–induced ocular infection. Consequently, corneal perforation is precluded and disease outcome is improved