Stimulating the autophagic-lysosomal axis enhances host defense against fungal infection in a zebrafish model of invasive Aspergillosis

ABSTRACT

The increasing prevalence of antifungal-resistant human pathogenic fungi, particularly azole-resistant Aspergillus fumigatus, is a life-threatening challenge to the immunocompromised population. Autophagy-related processes such as LC3-associated phagocytosis have been shown to be activated in the host response against fungal infection, but their overall effect on host resistance remains uncertain. To analyze the relevance of these processes in vivo, we used a zebrafish animal model of invasive Aspergillosis. To confirm the validity of this model to test potential treatments for this disease, we confirmed that immunosuppressive treatments or neutropenia rendered zebrafish embryos more susceptible to A. fumigatus. We used GFP-Lc3 transgenic zebrafish to visualize the autophagy-related processes in innate immune phagocytes shortly after phagocytosis of A. fumigatus conidia, and found that both wild-type and melanin-deficient conidia elicited Lc3 recruitment. In macrophages, we observed GFP-Lc3 accumulation in puncta after phagocytosis, as well as short, rapid events of GFP-Lc3 decoration of single and multiple conidia-containing vesicles, while neutrophils covered single conidia-containing vesicles with bright and long-lasting GFP-Lc3 signal. Next, using genetic and pharmacological stimulation of three independent autophagy-inducing pathways, we showed that the antifungal autophagy response improves the host survival against A. fumigatus infection, but only in the presence of phagocytes. Therefore, we provide proof-of-concept that stimulating the (auto)phagolysosomal pathways is a promising approach to develop host-directed therapies against invasive Aspergillosis, and should be explored further either as adjunctive or stand-alone therapy for drug-resistant Aspergillus infections.

Abbreviations: DMSO: dimethyl sulfoxide; HR: hazard ratio; HDT: host-directed therapy; Hpf: hours post fertilization; IA: invasive Aspergillosis; LAP: LC3-associated phagocytosis; MTZ: metronidazole; PTU: N-phenylthiourea; ROS: reactive oxygen species.

KEYWORDS:

• Aspergillus
• autophagic defense
• fungal infection
• host-pathogen interaction
• immunomodulation
• innate immunity
• phagocytes
• zebrafish

Acknowledgments

We thank Michiel van der Vaart and Monica Varela for confocal microscopy advice and critical proofreading of the manuscript, and all members of the fish facility team for zebrafish care at the Institute of Biology Leiden. The A. fumigatus melanin-deficient strain ∆pksP was a kind gift from Dr. Jean-Paul Latgé (Institut Pasteur, Paris, France). The fluorescent zebrafish lines labelling Lc3, macrophages or neutrophils were kind gifts from Dan Klionsky (University of Michigan, USA), Georges Lutfalla (University of Montpellier, France), and Stephen Renshaw (University of Sheffield). This work and G.F.-C. were supported by a European Marie Curie fellowship (H2020-COFUND-2015-FP707404).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All images presented in this manuscript except Figure 2D-I, and 5F are stills from confocal timelapse imaging experiments. All raw confocal imaging data is freely available for download and reuse via Biostudies under accession number S-BSST698 (https://www.ebi.ac.uk/biostudies/studies/S-BSST698), in which each file is tagged with the figures that belong to it.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2022.2090727

Additional information

Funding

This work was supported by the H2020 Marie Skłodowska-Curie Actions [H2020-COFUND-2015-FP707404].