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Sixto M. Leal, Jr., Brian Cobb, Kyung Kwon-Chung, Michelle Momany, Nancy Keller, Eric Pearlman; Neutrophil Nox2, but not iNOS Or Fungal-Derived Gliotoxin Mediate Hyphal Killing During Aspergillus And Fusarium Corneal Infection. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5862.
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© ARVO (1962-2015); The Authors (2016-present)
Aspergillus and Fusarium species are the major cause of fungal keratitis worldwide, often requiring surgical intervention in the form of corneal transplantation. Utilizing a murine model in which conidia are injected into the corneal stroma, we examined the role of neutrophils, pattern recognition receptors (PRRs) , and reactive oxygen and nitrogen species (ROS/RNS), as well as fungal-derived toxins in mediating fungal killing during corneal infection.
10^5 A. fumigatus (Af293, B-5233, delta gliP, gliZ, laeA), A.flavus, or F. oxysporum conidia were injected into the corneal stroma of C57BL/6 mice, neutrophil-depleted C57BL/6 mice, NADPH oxidase (NOX2) deficient mice (CybB-/- ), or iNOS-/- mice, and corneal opacity, histology, and colony forming units (CFU) were examined post-infection.
Neutrophil-depleted C57BL/6 mice infected with A.fumigatus, A.flavus, or F. oxysporum conidia exhibited increased corneal opacity, fungal growth, and CFU. Additionally, Dectin-1 and TLR4 were detected on the surface of infiltrating neutrophils via flow cytometry. Since both these PRRs can induce an oxidative burst, we infected mice lacking functional NADPH Oxidase. Increased corneal opacity, atypical abscess formation, minimal in situ ROS production and elevated CFU was observed in CybB-/- mice, despite similar kinetics of neutrophil recruitment as immunocompetent mice. In contrast, iNOS-/- and 1400W-treated C57BL/6 mice (1400W-irreversible iNOS inhibitor) exhibited no phenotypic differences compared to C57BL/6 mice, demonstrating that ROS but not RNS production is critical for fungal killing. In vitro studies with purified peritoneal neutrophils confirmed these findings. Despite evidence in the literature for inhibition of NOX2 by Aspergillus-derived secondary metabolites, no role for these toxins in ROS inhibition or fungal survival was observed during corneal infection.
Our data demonstrate that infiltrating neutrophils expressing surface Dectin-1 and TLR4 mediate fungal killing through NADPH-oxidase-dependent reactive oxygen species production, which is unimpeded by fungal production of gliotoxin during cornea infection, and is not contingent upon iNOS-dependent reactive nitrogen species formation.
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