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

The increasing prevalence of antifungal-resistant human pathogenic fungi, particularly azole-resistant <i>Aspergillus fumigatus</i>, 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 <i>in vivo</i>, 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 <i>A. fumigatus</i>. We used GFP-Lc3 transgenic zebrafish to visualize the autophagy-related processes in innate immune phagocytes shortly after phagocytosis of <i>A. fumigatus</i> 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 <i>A. fumigatus</i> 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 <i>Aspergillus</i> infections. <b>Abbreviations:</b> 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.