Infection of Zebrafish Larvae with &lt;em&gt;Aspergillus&lt;/em&gt; Spores for Analysis of Host-Pathogen Interactions

Thrikawala, Savini; Rosowski, Emily E.

doi:10.3791/61165

“…Larvae were injected with spores as described previously [ 16 ]. Prepared spore suspensions at 1.5 X 10 8 / mL were mixed at 2:1 with filter-sterilized 1% phenol red to achieve a final spore concentration of 1 X 10 8 / mL.…”

Section: Methodsmentioning

confidence: 99%

“…The zebrafish larva- Aspergillus infection model overcomes many of these challenges, as larvae are transparent and allow for direct visualization of phagocyte- Aspergillus interactions through high-resolution repetitive imaging of the same larvae over the course of a multi-day infection [ 15 ]. Multiple steps in pathogenesis such as phagocyte recruitment, phagocytosis, spore killing, germination, and hyphal growth or clearance can be quantified using this live imaging technique [ 16 ]. Zebrafish have a well-conserved immune system with humans, but depend solely on their innate immune system for the first few weeks of their life, providing a window to study innate immune mechanisms with no interference from the adaptive system [ 17 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

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

Thrikawala

¹

,

Niu

²

,

Keller

³

et al. 2022

Self Cite

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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.

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“…Zebrafish offer many advantages for in vivo imaging [ 171 , 172 , 174 , 175 ]. Due to their transparent nature, zebrafish larvae provide an infection model that allows for non-invasive cellular imaging in the same live hosts over multiple days of infection (longitudinal studies).…”

Section: How Does Germination Alter the Interactions Of A F...mentioning

confidence: 99%

Novel Insights into Aspergillus fumigatus Pathogenesis and Host Response from State-of-the-Art Imaging of Host–Pathogen Interactions during Infection

Ortiz

¹

,

Pennington

²

,

Thomson

³

et al. 2022

JoF

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Aspergillus fumigatus spores initiate more than 3,000,000 chronic and 300,000 invasive diseases annually, worldwide. Depending on the immune status of the host, inhalation of these spores can lead to a broad spectrum of disease, including invasive aspergillosis, which carries a 50% mortality rate overall; however, this mortality rate increases substantially if the infection is caused by azole-resistant strains or diagnosis is delayed or missed. Increasing resistance to existing antifungal treatments is becoming a major concern; for example, resistance to azoles (the first-line available oral drug against Aspergillus species) has risen by 40% since 2006. Despite high morbidity and mortality, the lack of an in-depth understanding of A. fumigatus pathogenesis and host response has hampered the development of novel therapeutic strategies for the clinical management of fungal infections. Recent advances in sample preparation, infection models and imaging techniques applied in vivo have addressed important gaps in fungal research, whilst questioning existing paradigms. This review highlights the successes and further potential of these recent technologies in understanding the host–pathogen interactions that lead to aspergillosis.

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“…A Plan-Apochromat 20x objective (0.8 NA) and ZEN software were used to acquire Z-stack images of the hindbrain area with 5 μm distance between slices. After imaging, larvae were rinsed with 200 µM PTU in E3 and put back in the same well (16). For AlexaFluor labeled live-dead staining, infected larvae at 2 dpi were mounted in 1% low-melting point agarose (Fisher BioReagents) in a 35mm glass-bottom dish (Greiner) and oriented laterally.…”

Section: Live Imagingmentioning

confidence: 99%

“…The zebrafish larva-Aspergillus infection model overcomes many of these challenges, as larvae are transparent and allow for direct visualization of phagocyte-Aspergillus interactions through high-resolution repetitive imaging of the same larvae over the course of a multi-day infection (15). Multiple steps in pathogenesis such as phagocyte recruitment, phagocytosis, spore killing, germination, and hyphal growth or clearance can be quantified using this live imaging technique (16). Zebrafish have a well-conserved immune system with humans, but depend solely on their innate immune system for the first few weeks of their life, providing a window to study innate immune mechanisms with no interference from the adaptive system (17,18).…”

Section: Introductionmentioning

confidence: 99%

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

Thrikawala

¹

,

Niu

²

,

Keller

³

et al. 2021

Preprint

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0

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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. 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.Author SummaryInvasive aspergillosis causes mortality in >50% of infected patients. It is caused by a free-living fungus Aspergillus fumigatus which releases thousands of airborne spores. While healthy individuals clear inhaled spores efficiently, in immunocompromised individuals these spores grow into filamentous hyphae and destroy lungs and other tissues causing invasive aspergillosis. The immune mechanisms that control this fungal growth in healthy people are still largely unknown. Here, we used a larval zebrafish model of A. fumigatus infection to determine that cyclooxygenase enzymes, which are the target of non-steroidal anti-inflammatory drugs such as aspirin and ibuprofen, are important to control the fungus. Innate immune cells use cyclooxygenase signaling to prevent hyphal growth and tissue destruction. Our study provides new insights into the mechanisms that immune cells deploy to stop invasive growth of A. fumigatus and inform development of future strategies to combat invasive aspergillosis.

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Infection of Zebrafish Larvae with <em>Aspergillus</em> Spores for Analysis of Host-Pathogen Interactions

Cited by 8 publications

References 28 publications

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

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

Novel Insights into Aspergillus fumigatus Pathogenesis and Host Response from State-of-the-Art Imaging of Host–Pathogen Interactions during Infection

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

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