Pathogenic Fusarium and Aspergillus molds are an important cause of vision loss in the USA and worldwide. Preliminary data from an unbiased gene array analyses (Nanostring and RNA sequencing) showed an anticipated increase in expression of multiple genes in neutrophils isolated from Fusarium and Aspergillus infected mouse corneas compared with bone marrow neutrophils from the same mice. However, we found an unexpected significant increase in expression of the pro-inflammatory cytokine IL-1 alpha, and of Spp1/osteopontin (OPN), which has known pro-inflammatory activity, and which exists as a secreted (sOPN) and intracellular (iOPN). Elevated IL-1 alpha and OPN was confirmed by Q-PCR, ELISA and intracellular flow cytometry.
Aim 1 will characterize the membrane, secreted and intracellular activities the pro- and cleaved forms of IL-1 alpha in murine and human neutrophils, and in Fusarium and Aspergillus keratitis.
Aim 2 will identify regulators of OPN expression, and use OPN-/- and iOPN knock-in mice to examine the role of the intracellular versus secreted forms of this protein in human neutrophils and in fungal keratitis.
Aim 3 will characterize the role of neutrophil extracellular traps (NETs), which can limit hyphal growth, but also have the potential to contribute to tissue damage. Proposed experiments will identify the molecular pathways leading to NET formation in infected corneas, and using mice that lack the enzyme required for histone citrullinatation and decondensation (PAD4), we will examine the role of NETs in Fusarium and Aspergillus keratitis. Collectively, the results of these proposed studies will increase our understanding of the pathogenesis of this blinding disease, and will potentially identify novel targets for immune intervention to prevent neutrophil mediated tissue damage and loss of corneal clarity.

Public Health Relevance

Aspergillus and Fusarium are molds that are ubiquitous in the environment, with high airborne spores in hot, humid regions of the USA, and in the developing world. Corneal infection with these organisms, either as a complication of contact lens wear or as a result of ocular trauma results in a painful and sight threatening disease that is extremely difficult to treat. Experiments described in this proposal will target the inflammatory neutrophils that cause corneal damage, visual impairment and blindness, and identify novel targets for interventional therapy for this disease.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY018612-13
Application #
10104502
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mckie, George Ann
Project Start
2008-02-01
Project End
2023-01-31
Budget Start
2021-02-01
Budget End
2022-01-31
Support Year
13
Fiscal Year
2021
Total Cost
Indirect Cost
Name
University of California Irvine
Department
Physiology
Type
Schools of Medicine
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92617
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Clark, Heather L; Minns, Martin S; Sun, Yan et al. (2018) Atovaquone Impairs Growth of Aspergillus and Fusarium Keratitis Isolates by Modulating Mitochondrial Function and Zinc Homeostasis. Invest Ophthalmol Vis Sci 59:1589-1598
Clark, Heather L; Abbondante, Serena; Minns, Martin S et al. (2018) Protein Deiminase 4 and CR3 Regulate Aspergillus fumigatus and ?-Glucan-Induced Neutrophil Extracellular Trap Formation, but Hyphal Killing Is Dependent Only on CR3. Front Immunol 9:1182
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Deng, Zihou; Ma, Shixin; Zhou, Hao et al. (2015) Tyrosine phosphatase SHP-2 mediates C-type lectin receptor-induced activation of the kinase Syk and anti-fungal TH17 responses. Nat Immunol 16:642-52
Underhill, David M; Pearlman, Eric (2015) Immune Interactions with Pathogenic and Commensal Fungi: A Two-Way Street. Immunity 43:845-58
Karthikeyan, Rajapandian Sivaganesa; Vareechon, Chairut; Prajna, Namperumalsamy Venkatesh et al. (2015) Interleukin 17 expression in peripheral blood neutrophils from fungal keratitis patients and healthy cohorts in southern India. J Infect Dis 211:130-4
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