FIGURE

Fig. 3

ID
ZDB-FIG-150421-10
Publication
Herbst et al., 2015 - Phagocytosis-dependent activation of a TLR9-BTK-calcineurin-NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus
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Fig. 3

Calcineurin inhibition impairs neutrophil recruitment in a zebrafish model of invasive aspergillosis

At 2 days postfertilization (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., B. Absolute neutrophil numbers in treated and untreated mpx:GFP larvae were quantified by direct counting of fluorescent neutrophils in noninfected whole fish (n = 1113 per group). There were no significant differences.

C. For 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 postinfection (n = 16 per group). Immunosuppression led to a significant increase in mortality.

D. TNFα mRNA levels were measured in whole noninfected or infected larvae 48 h p.i. Immunosuppression led to significant and almost complete inhibition of TNFα expression during infection.

E–G. Lyz:dsRed larvae were infected with ~50 eGFPexpressing 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 ttest.

Expression Data
Genes:
Fish:
Conditions:
Anatomical Terms:
Stage: Days 7-13

Expression Detail
Antibody Labeling
Phenotype Data
Fish:
Conditions:
Observed In:
Stage: Days 7-13

Phenotype Detail
Acknowledgments
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