Fig. 1: Eosinophils affect C. albicans growth in a Candida-induced eosinophil death mechanism.
A Eosinophil peroxidase (EPX) release in supernatants of eosinophils co-cultured with C. albicans (Ca) for 3 h at the indicated multiplicity of infection (MOI). EPX levels were assessed by a colorimetric assay and quantified using purified human EPX. Data is representative of 1 out of 4 experiments performed with eosinophils obtained from 4 independent donors. Each experiment was performed in technical triplicates. B, C Viable eosinophil numbers (B) and LDH levels (C) following incubation of eosinophils with C. albicans (MOI 0.1 and 1) for 0.5, 3, and 20 h. Viable cells were assessed by trypan blue exclusion test, and LDH was evaluated in supernatants by colorimetric assay; n = 3 biological replicates. D Colony forming unit (CFU) counts of C. albicans incubated with eosinophils (MOI 1) or in medium for 20 h; n = 3 biological replicates. Each experiment was performed in technical duplicates. E CFU counts of C. albicans incubated with supernatants of eosinophils following overnight incubation with medium alone (Eos sup) or with C. albicans (MOI 1; Eos+ Ca sup); n = 3 biological replicates. Each experiment was performed in technical triplicates. F CFU counts of C. albicans incubated with eosinophils (MOI 1) or with purified eosinophil granule proteins or with their combination for 20 h. Eosinophil granule proteins were added at a concentration corresponding to their content in 105 eosinophils; n = 3 biological replicates. Each experiment was performed in technical triplicates (G) Fluorescent microscopy image of eosinophils incubated with FITC-labeled C. albicans (MOI 1) for 30 min. Eosinophil nuclei were stained with DAPI. White arrows show engulfment of C. albicans by eosinophils (×400 magnification). H Scanning electron microscopy (SEM) image of eosinophils incubated with C. albicans (MOI 1) for 30 min. White arrows show the engulfment of C. albicans by eosinophils and red arrows show eosinophil extracellular traps (×10,000 magnification). I EPX release in supernatants of naive or cytochalasin D-pretreated (5 µg/ml) eosinophils incubated for 3 h with C. albicans (MOI 1); n = 3 biological replicates. J CFU counts of C. albicans incubated for 20 h with naive or cytochalasin D-pretreated (5 µg/ml) eosinophils (MOI 1); n = 3 biological replicates. Each experiment was performed in technical triplicates. K, L EPX (K) and LDH (L) release in supernatants of eosinophils co-cultured respectively for 3 and 20 h with WT C. albicans (WT), re-integrant strain (ece1Δ/Δ + ECE1), candidalysin mutant strains (ece1Δ/Δ, ece1Δ/Δ + ECE1Δ184–279; all at MOI 1) or with synthetic candidalysin at the indicated concentrations. Data is representative of 1 out of 3 experiments with eosinophils obtained from 3 independent donors. Each experiment was performed in technical triplicates. Data show the mean ± SD. Significance was analyzed by one-tail Anova (A, B, F, I–L) or two-tailed student t test (C–E) as described in “Materials and Method” section; ns not-significant, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005; ****P ≤ 0.001.
