1. Calcineurin inhibition impairs neutrophil recruitment in a zebrafish model of invasive aspergillosisAt 2 days post‐fertilization (dpf), Lyz:dsRed or mpx:GFP larvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2.
Calcineurin inhibition impairs neutrophil recruitment in a zebrafish model of invasive aspergillosisAt 2 days post‐fertilization (dpf), Lyz:dsRed or mpx:GFP larvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafish larvae were microinjected with AF eGFP conidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed. A, BAbsolute neutrophil numbers in treated and untreated mpx:GFP larvae were quantified by direct counting of fluorescent neutrophils in non‐infected whole fish (n = 11‐13 per group). There were no significant differences. CFor survival analysis, lyz:dsRed larvae were microinjected with ˜10 conidia and the effects of FK506 on the outcome from invasive aspergillosis was determined by Kaplan–Meier analysis for survival over 6 days post‐infection (n = 16 per group). Immunosuppression led to a significant increase in mortality. DTNF‐α mRNA levels were measured in whole non‐infected or infected larvae 48 h p.i. Immunosuppression led to significant and almost complete inhibition of TNF‐α expression during infection. E–GLyz:dsRed larvae were infected with ˜50 eGFP‐expressing conidia of A. fumigatus and were imaged daily from 24 h p.i. (E) The effects of FK506 on neutrophil recruitment were assessed by integrated density analysis of red fluorescent neutrophils at sites of infection in ImageJ. A representative image showing impaired neutrophil recruitment to the site of infection in immunosuppressed larvae is shown (60 h p.i.). The arrows indicate infectious foci. (F) Neutrophil recruitment was minimal throughout the first 60 h p.i. in immunosuppressed larvae (n = 8 per group). (G) Fungal growth was determined by measurement of the length of individual fluorescent hyphal elements (n = 8 per group). Data information: All data represent mean ± SEM and were statistically analysed by Student's t‐test.
Susanne Herbst et al. EMBO Mol Med. 2015;7:
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