IFN-γ treatment restores functional metabolic defect in CGD monocytes. (A-B) Calculated glycolytic metabolic parameters on the Glyco Stress Test (A) and calculated mitochondrial parameters on Mito Stress Test (B) of monocytes from HC (n = 5 for Glyco Stress Test, n = 7 for Mito Stress Test) and patients with CGD (n = 7 for Glyco Stress Test, n = 8 for Mito Stress Test) after 12 hours treatment with medium (RPMI) or IFN-γ (50 ng/mL) measured by Seahorse XF technology. (C) Mean normalized expression of genes associated with NAD⁺ salvage pathway differentially expressed in CD14⁺ monocytes on 4 hours ex vivo whole blood stimulations with IFN-γ vs unstimulated within the group of HC (n = 4) and patients with CGD (n = 4), respectively. Dots are colored by donor identity, and dot size is scaled to the percentage of cells expressing the respective gene. (D-E) IL-6 (D) and IL-1β (E) production in CGD monocytes (n = 6) and HC (n = 5) preincubated for 4 hours with or without IFN-γ (50 ng/mL) and then stimulated for 24 hours with either medium (RPMI), live A fumigatus (1 × 10⁷ conidia per mL), heat-killed C albicans UC820 (1 × 10⁶ yeast per mL), Pam₃Cys (10 μg/mL), or LPS (10 ng/mL). (F) A fumigatus outgrowth in monocytes preincubated for 4 hours with or without IFN-γ (50 ng/mL) after the in vitro killing assay, expressed as percentage killing in respect to the total inoculum (CGD n = 2, HC n = 2). (G) Monocytes from HC (n = 1) and patients with CGD (n = 2) were preincubated with IFN-γ (50 ng/mL) and then trained for 24 hours by incubation with 5 μg/mL BCG in the presence of 10% HPS. Thereafter, the stimulus was removed, and cells were kept in RPMI with 10% HPS (regular medium) or in a medium containing 10% human serum and IFN-γ (50 ng/mL). On day 6, a second stimulation with LPS (10 ng/mL) was performed for an additional 24 hours. IL-6 levels were measured in cell culture supernatants after the second stimulation. (H) Mean normalized expression of selected differentially expressed genes related to glutamine metabolism in CD14⁺ monocytes on 4 hours of ex vivo whole blood stimulation with IFN-γ vs unstimulated within the group of patients with CGD (n = 4) and HC (n = 4), respectively. Dots are colored by donor identity, and dot size is scaled to the percentage of cells expressing the respective gene. (I) Mean normalized gene expression of CXCL8 in neutrophils on 4 hours of in vitro whole blood stimulation with IFN-γ vs unstimulated demonstrated differential expression in each group (CGD n = 4, HC n = 4) comparing in vitro IFN-γ treated vs untreated cells after 4 hours of incubation. Dots are colored by donor identity, and dot size is scaled to the percentage of cells expressing the respective gene. (J) IL-8 production of HC (n = 3) and CGD neutrophils (n = 2) preincubated for 30 minutes with or without IFN-γ (50 ng/mL) and then stimulated with either medium (RPMI) or PMA (50 ng/mL) for 4 hours. (A, B, D-G, J) Data are illustrated as mean ± SEM, and statistical analysis was performed using the Wilcoxon signed-rank test comparing within the patient group (or within the control group) the IFN-γ–treated condition with the untreated condition.