Cyclooxygenase production of PGE2 promotes phagocyte control of A. fumigatus hyphal growth in larval zebrafish

Abstract

Invasive aspergillosis is a common opportunistic infection, causing >50% mortality in infected immunocompromised patients. The specific molecular mechanisms of the innate immune system that prevent pathogenesis of invasive aspergillosis in immunocompetent individuals are not fully understood. Here, we used a zebrafish larva-Aspergillus infection model to identify cyclooxygenase (COX) enzyme signaling as one mechanism that promotes host survival. Larvae exposed to the pan-COX inhibitor indomethacin succumb to infection at a significantly higher rate than control larvae. COX signaling is both macrophage- and neutrophil-mediated. However, indomethacin treatment has no effect on phagocyte recruitment. Instead, COX signaling promotes phagocyte-mediated inhibition of germination and invasive hyphal growth. Increased germination and invasive hyphal growth is also observed in infected F0 crispant larvae with mutations in genes encoding for COX enzymes (ptgs2a/b). Protective COX-mediated signaling requires the receptor EP2 and exogenous prostaglandin E2 (PGE2) rescues indomethacin-induced decreased immune control of fungal growth. Collectively, we find that COX signaling activates the PGE2-EP2 pathway to increase control A. fumigatus hyphal growth by phagocytes in zebrafish larvae.

Glucocorticoids suppress NF-κB-mediated neutrophil control ofAspergillus fumigatushyphal growth

“…We enumerated macrophage and neutrophil recruitment to the infection site through daily, live confocal imaging of infected larvae. In line with previous findings [26, 39], macrophages arrive first and form clusters around spores starting at 1 dpi (Fig 2A). A significantly higher number of macrophages arrive at 2 dpi in control larvae compared to dexamethasone-treated larvae, yet ~90 macrophages still arrive at the infection site in dexamethasone-treated larvae (Fig 2B).…”

“…To determine if the increased susceptibility of dexamethasone-treated larvae to A. fumigatus infection was due to signaling through this receptor, and not to off-target effects on either the host or pathogen, we used CRISPR/Cas9 to target the zebrafish glucocorticoid receptor gene . This is likely due to increased fungal growth attracting more macrophages and neutrophils to the infection site, as described previously [39]. Injected A. fumigatus spores can germinate, and after germination, the fungal filamentous growth can branch and form a network of hyphae, which we classified as invasive hyphae.…”

“…The first step in the phagocyte response is macrophage-mediated phagocytosis of spores and spore killing [33, 40]. To test whether dexamethasone treatment decreases macrophage-mediated spore killing, we used an established live-dead staining method in which A. fumigatus spores expressing GFP are coated with AlexaFluor546 via cell wall cross-linking [26, 39]. We injected these spores into larvae expressing BFP in macrophagesm ( Tg(mfap4:BFP) ) and imaged the infection site at 2 dpi.…”

Aspergillus fumigatus transcription factor ZfpA regulates hyphal development and alters susceptibility to antifungals and neutrophil killing during infection

“…The larval zebrafish is an ideal model to directly observe how ZfpA affects fungal development and immune cell behavior. The innate immune response of zebrafish larvae to Aspergillus shares many similarities with that of mammals, including fungal clearance by macrophages and neutrophils, with the unique advantage of repeated live-imaging of larvae over the course of multi-day infections [ 19 – 24 ]. Additionally, A .…”

Aspergillus fumigatustranscription factor ZfpA regulates hyphal development and alters susceptibility to antifungals and neutrophil killing during infection

“…The larval zebrafish is an ideal model to directly observe how ZfpA affects fungal development and immune cell behavior. The innate immune response of zebrafish larvae to Aspergillus shares many similarities with that of mammals, including fungal clearance by macrophages and neutrophils, with the unique advantage of repeated live-imaging of larvae over the course of multi-day infections [19][20][21][22][23][24]. Additionally, A. fumigatus infections in zebrafish can be successfully treated with clinically relevant antifungals [25], allowing us to screen for changes in drug susceptibility of ZfpA mutants in vivo.…”