Date Published: August 2, 2018
Publisher: Public Library of Science
Author(s): Emily E. Rosowski, Nicholas Raffa, Benjamin P. Knox, Netta Golenberg, Nancy P. Keller, Anna Huttenlocher, Amariliz Rivera.
In immunocompromised individuals, Aspergillus fumigatus causes invasive fungal disease that is often difficult to treat. Exactly how immune mechanisms control A. fumigatus in immunocompetent individuals remains unclear. Here, we use transparent zebrafish larvae to visualize and quantify neutrophil and macrophage behaviors in response to different A. fumigatus strains. We find that macrophages form dense clusters around spores, establishing a protective niche for fungal survival. Macrophages exert these protective effects by inhibiting fungal germination, thereby inhibiting subsequent neutrophil recruitment and neutrophil-mediated killing. Germination directly drives fungal clearance as faster-growing CEA10-derived strains are killed better in vivo than slower-growing Af293-derived strains. Additionally, a CEA10 pyrG-deficient strain with impaired germination is cleared less effectively by neutrophils. Host inflammatory activation through Myd88 is required for killing of a CEA10-derived strain but not sufficient for killing of an Af293-derived strain, further demonstrating the role of fungal-intrinsic differences in the ability of a host to clear an infection. Altogether, we describe a new role for macrophages in the persistence of A. fumigatus and highlight the ability of different A. fumigatus strains to adopt diverse modes of virulence.
Humans inhale hundreds of Aspergillus fumigatus spores from the environment every day and yet almost all immunocompetent individuals successfully contend with the fungal infection. Immunocompromised patients, however, especially acute leukemia patients, hematopoietic cell transplant recipients, and solid-organ transplant recipients, are at risk of developing invasive aspergillosis . In invasive disease, Aspergillus spores germinate into filamentous hyphae and invade and destroy tissues and organs, with mortality rates as high as 50 to 60% in patient populations . Limited antifungal treatments exist and there is growing resistance among fungi to these drugs . The development of successful immunotherapy-based treatments to this infection requires a more comprehensive understanding of the interplay of immune mechanisms that control Aspergillus in vivo.
Here we have used larval zebrafish to image Aspergillus fumigatus infection in live, intact hosts, over the course of a multi-day infection to visualize and quantify both immune cell behavior and fungal development and growth. Using a method to visually quantify spore killing, we find that faster-growing CEA10-derived strains of A. fumigatus are cleared significantly better than slower-growing Af293-derived strains. We report that clearance of CEA10-derived strains is primarily due to neutrophil function and is driven by fungal germination, whereas slower-growing Af293-derived strains do not recruit as many neutrophils and therefore can persist in the host for >5 days. Many fungal pathogens can survive within macrophages , but the role of macrophages in vivo in A. fumigatus pathogenesis has remained ambiguous, and we find that macrophages form tight clusters around injected spores. Macrophages inhibit spore germination, and without spore germination, neutrophil recruitment and neutrophil-mediated killing are diminished, thus macrophages act as a protective niche against neutrophil-mediated killing (Fig 10).