Fungal keratitis: Immune recognition, neutrophil-hyphae interactions, and fungal anti-oxidative defenses

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Fungal keratitis: Immune recognition, neutrophil-hyphae interactions, and fungal anti-oxidative defenses


Leal, Sixto Manuel, Jr.



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Filamentous fungal infections of the cornea caused by Aspergillus and Fusarium species are an important cause of blindness and visual impairment worldwide accounting for > 65% of corneal ulcers in developing countries and an estimated 80,000 total cases in India alone. Tissue pathology is often severe leading to either extensive scar tissue formation, cornea transplantation (>10% ), or enucleation of the affected eye. The series of studies described herein delineate our multifaceted approach to investigate: 1. the mechanism by which neutrophils are recruited into fungal-infected corneas, 2. the effectors required by neutrophils to kill fungal hyphae, and 3. the microbial factors utilized by hyphae to resist attack.In collaboration with Aravind Eye Hospital in Tamil Nadu, India, we first characterized the human immune response during fungal keratitis using ulcerative material from infected corneas as well as infected whole corneas from patients undergoing corneal transplantation. Analysis of these tissues identified neutrophils as the major cellular infiltrate with smaller populations of macrophages and T cells. In addition, utilizing qPCR and RNA extracted from fungal-infected human corneas we identified elevated expression of: 1. fungal-sensing cell-surface receptors (Dectin-1, TLR2, and TLR4), 2. neutrophil-recruiting cytokines (IL-8, IL-1946;, IL-17), 3. proteins involved in IL-1 946; maturation (NALP3, ASC), and 4. cytokines associated with adaptive immune responses (IFN-947;, IL-17, but not IL-4).Having characterized the human immune response during fungal keratitis, we next developed and utilized a novel mouse model of trauma-induced fungal keratitis and knockout mice to identify that 946;-glucan on A. fumigatus germinating conidia activates Dectin-1 on resident macrophages to produce IL-1946;, and CXCL1/KC, which together with IL-1R1/ MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4 but not MD-2 dependent fungal killing. In the next series of studies we show that hyphae activate neutrophil NADPH oxidase via complement receptor 3 (CD11b/CD18) and utilizing a novel neutrophil adoptive transfer model that neutrophil NADPH oxidase is essential for killing hyphae in vivo. In addition, utilizing mutant A. fumigatus strains, our mouse model, and a novel in vitro neutrophil-hyphae killing assay we show that the ROS-sensing transcription factor Yap1, the ROS detoxifying-enzyme superoxide dismutase, and the Yap1-regulated thioredoxin antioxidant pathway, but not fungal 2° metabolites such as gliotoxin are required for resistance to oxidation by neutrophils.In conclusion, this work has significantly advanced our knowledge of the immune and microbial mediators of disease outcome during fungal keratitis. In this effort we identified: Dectin-1, IL-1R1, CXCR2, CD18, Syk, and MyD88 as potential anti-inflammatory targets to minimize excessive neutrophil recruitment during fungal keratitis. Lastly, we identify fungal superoxide dismutase and thioredoxin as potential anti-fungal targets to enhance fungal sensitivity to oxidative stress and neutrophils.