Distinct innate immune phagocyte responses to Aspergillus fumigatus conidia and hyphae in zebrafish larvae

Abstract

Aspergillus fumigatus is the most common filamentous fungal pathogen of immunocompromised hosts, resulting in invasive aspergillosis (IA) and high mortality rates. Innate immunity is known to be the predominant host defense against A. fumigatus; however, innate phagocyte responses to A. fumigatus in an intact host and their contributions to host survival remain unclear. Here, we describe a larval zebrafish A. fumigatus infection model amenable to real-time imaging of host-fungal interactions in live animals. Following infection with A. fumigatus, innate phagocyte populations exhibit clear preferences for different fungal morphologies: macrophages rapidly phagocytose conidia and form aggregates around hyphae, while the neutrophil response is dependent upon the presence of hyphae. Depletion of macrophages rendered host larvae susceptible to invasive disease. Moreover, a zebrafish model of human leukocyte adhesion deficiency with impaired neutrophil function also resulted in invasive disease and impaired host survival. In contrast, macrophage-deficient but not neutrophil-deficient larvae exhibited attenuated disease following challenge with a less virulent (ΔlaeA) strain of A. fumigatus, which has defects in secondary metabolite production. Taking these results together, we have established a new vertebrate model for studying innate immune responses to A. fumigatus that reveals distinct roles for neutrophils and macrophages in mediating host defense against IA.

FIG 1

Aspergillus fumigatus causes invasive disease in immunodeficient larvae. (A) Cartoon of zebrafish larvae at ∼32 hpf undergoing microinjection of conidia into the hindbrain ventricle (shaded red) through the otic vesicle. (B) Immunocompetent larvae (control MO) are not susceptible to infection following challenge with A. fumigatus (gpdA::GFP::his2A). Treatment with the pu.1 morpholino (pu.1 MO) depletes morphant larvae of macrophages and neutrophils and renders them highly susceptible (P < 0.0001) to infection following challenge with A. fumigatus. pu.1 morphant larvae showed no mortality following injection of PBS, heat-killed A. fumigatus (HK), or the classically avirulent ΔsidA strain of A. fumigatus deficient in siderophore biosynthesis (P > 0.05). Survival data are pooled from three independent experimental replicates, and P values were calculated by Cox proportional-hazard regression analysis. (C) Immunocompetent larvae significantly reduce viable conidia by days 5 and 7 postinfection. *, P < 0.05 as determined by repeated-measures ANOVA and Tukey's HSD method for multiple-comparison test. (D) At 2 days postinfection, control larvae harbor ungerminated conidia (gray arrows), while macrophage- and neutrophil-deficient pu.1 morphants succumb to invasive A. fumigatus (gpdA::YFP) growth. Scale bar, 100 μm.

FIG 2

Macrophages phagocytose A. fumigatus conidia, colocalize with hyphae, and mediate host survival. (A and B) Immunocompetent larvae with fluorescent macrophages [Tg(mpeg1:dendra2)] injected with A. fumigatus (gpdA::mCherry::his2A). (A) Time lapse imaging immediately following injection shows rapid phagocytosis of conidia by macrophages by 6 hpi (gray arrows) (see also Movie S2 in the supplemental material). Scale bar, 100 μm. (B) Macrophages closely associate with hyphae in immunocompetent larvae (see also Movie S3 in the supplemental material). Scale bar, 50 μm. (C) Macrophage-deficient irf8 morphant larvae exhibit significant (P < 0.0001) mortality following challenge with A. fumigatus (gpdA::GFP::his2A) and not with a mock PBS infection. Survival data are pooled from five independent experimental replicates, and the P value was calculated by Cox proportional-hazard regression analysis.

FIG 3

Neutrophils do not phagocytose A. fumigatus conidia. Immunocompetent larvae with fluorescent neutrophils [Tg(mpx:dendra2)] were injected with A. fumigatus (gpdA::mCherry::his2A). (A) Time lapse imaging immediately following infection shows a lack of conidial phagocytosis by neutrophils (see also Movie S4 in the supplemental material). Gray arrows indicate conidial clumping resulting from macrophage phagocytosis. (B) Removal of macrophages with the irf8 morpholino (irf8 MO) leaves conidia in the extracellular space in the absence of macrophage phagocytosis (see gray arrows in panel A), and neutrophils remain nonphagocytic toward conidia (see also Movie S5 in the supplemental material). (C) Stimulated neutrophil recruitment with P. aeruginosa in a coinjection with A. fumigatus shows a continued lack of neutrophil phagocytosis of conidia. All images are representative of more than three experimental replicates. Scale bar, 100 μm.

FIG 4

Neutrophils respond to A. fumigatus hyphae and mediate host survival. (A and B) Immunocompetent larvae with fluorescent neutrophils [Tg(mpx:dendra2)] injected with A. fumigatus (gpdA::mCherry::his2A) and screened at 1 day postinfection (dpi) for the presence of hyphae. (A) Time lapse imaging reveals a persistent neutrophil response around A. fumigatus hyphae characterized by both tight colocalization with the mycelium and migration toward and adherence to hyphal filaments (gray arrow) by individual neutrophils (see also Movie S6 in the supplemental material). (B) Representative images from daily imaging of hypha-containing fish demonstrate host clearance of hyphae at 2 dpi. (C) Larvae expressing the inhibitory Rac2D57N mutation specifically in neutrophils [Tg(mpx:mCherry-2A-Rac2D57N)] are highly susceptible (P < 0.001) to A. fumigatus (gpdA::GFP::his2A) infection. Survival data were pooled from five independent experimental replicates, and the P value was calculated by Cox proportional-hazard regression analysis. Scale bar, 100 μm.

FIG 5

Virulence of A. fumigatus ΔlaeA is attenuated only in a macrophage-deficient host. (A) In macrophage-deficient irf8 morphant larvae, the ΔlaeA strain of A. fumigatus (pyrG−; ΔlaeA::A. parasiticus pyrG) is significantly less virulent (P = 0.00027) than a complement control (pyrG−; A. parasiticus pyrG). (B) Virulence attenuation is not observed (P = 0.772) with A. fumigatus ΔlaeA in larvae with impaired neutrophil function [Tg(mpx:mCherry-2A-Rac2D57N)]. Survival data were pooled from three independent experimental replicates except for the PBS condition in Rac2^D57N, which was included in two of the three replicates, and P values were calculated by Cox proportional-hazard regression analysis.