Airborne conidia (spores) represent infectious propagules that are responsible for transmission of major human mycoses. Humans inhale ubiquitous Aspergillus fumigatus conidia on a daily basis. Conidial germination into tissue-invasive hyphae leads to invasive aspergillosis (IA), a devastating cause of infectious morbidity and mortality in patients with impaired respiratory innate immune function. Although conidial engulfment and killing are hallmarks of alveolar macrophage (AM) function during respiratory infection, antifungal effector mechanisms of these cells remain poorly defined, since assays that measure alveolar macrophage conidiacidial activity in the lung have been difficult to achieve. To dissect AM conidiacidal activity, we developed a fluorescent Aspergillus reporter (FLARE) strain that, upon conidial uptake, tags AMs with a fluorescent signature. Conidial killing induces a change in the fluorescence signature, enabling us to observe and quantify cell type-specific conidial killing in the lung and test tube. We harness the FLARE strain to demonstrate that AMs employ distinct conidiacidal mechanisms and implicate matrix metalloprotease 12 (MMP12) in this process. With this approach, we examine a model of AM function that integrates matrix metalloprotease 12 as a significant cytotoxic effector mechanism to achieve fungicidal activity. The hypothesis that underlies this proposal is that AM-specific factors - that include MMP12 - control A. fumigatus conidial germination at the earliest stages of respiratory fungal infection.
The aims will (1) investigate MMP12 as a major effector of AM conidiacidal activity and (2) integrate alternate AM conidiacidal mechanisms using a functional RNAi-based approach. The experimental design incorporates both a candidate gene (MMP12) and a systematic discovery approach to dissect AM antifungal activity. These studies will define matrix metalloproteolytic activity as a potential novel antifungal effector mechanism and will identify candidate genes that regulate the full spectrum of AM conidiacidal activity.

Public Health Relevance

These studies utilize a novel fluorescence-based method to visualize how inhaled fungal conidia (spores) are eliminated by macrophages, a resident infection-fighting cell in the lung. Our studies will investigate the role of matrix metalloproteas (MMP) activity as a novel macrophage antifungal effector mechanism and conduct a functional discovery approach to account for full macrophage conidial killing activity. These studies will advance our knowledge of lung defense mechanisms against environmental fungi and identify targets for potential therapeutic gain.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI105617-01A1
Application #
8584085
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Duncan, Rory A
Project Start
2013-08-07
Project End
2015-07-31
Budget Start
2013-08-07
Budget End
2014-07-31
Support Year
1
Fiscal Year
2013
Total Cost
$207,740
Indirect Cost
$90,240
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Espinosa, Vanessa; Jhingran, Anupam; Dutta, Orchi et al. (2014) Inflammatory monocytes orchestrate innate antifungal immunity in the lung. PLoS Pathog 10:e1003940
Hohl, Tobias M (2014) Overview of vertebrate animal models of fungal infection. J Immunol Methods 410:100-12