Killing of C albicans by CARD9-deficient neutrophils. (A) Killing efficacy of patient and control neutrophils of unopsonized and opsonized C albicans conidia was assessed by standard colony-forming unit assay. Patient and control neutrophils were incubated with C albicans conidia for 2 hours, and the colonies were counted after overnight incubation of the remaining Candida conidia. Results are means ± SEM of at least 3 different assays; *P < .05. (B) Neutrophils from healthy controls, from the CARD9-deficient patient and from CGD patients (n = 9), were cocultured overnight with C albicans–GFP and assessed microscopically. Massive outgrowth of the hyphenated form of C albicans–GFP was observed, when cocultured overnight with CARD9-deficient cells. Bright-green fluorescence of hyphae indicates viable Candida. Such outgrowth was not detected when C albicans was cocultured with control neutrophils. (C) Clusters of hyphae were quantified after overnight incubation of 1 × 105 neutrophils with different numbers of Candida conidia. Data are representative for at least 3 experiments. Original magnification, ×20; scale bar, 200 μm. (D) The capacity of patient and control neutrophils to kill Candida hyphae was determined by MTT assay. Neutrophils in different concentrations were incubated with monolayer of Candida hyphae for 2 hours. The viability of hyphae was assessed by the MTT assay. Results are means ± SEM of at least 3 different assays.
