Restoration of anti-Aspergillus defense by neutrophil extracellular traps in human chronic granulomatous disease after gene therapy is calprotectin-dependent

Background: Aspergillus spp infection is a potentially lethal disease in patients with neutropenia or impaired neutrophil function. We showed previously that Aspergillus hyphae, too large for neutrophil phagocytosis, are inhibited by reactive oxygen species-dependent neutrophil extracellular trap (NET) formation. This process is defective in chronic granulomatous disease (CGD) because of impaired phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function. Objective: To determine the antifungal agent and mechanism responsible for reconstitution of Aspergillus growth inhibition within NETs after complementation of NADPH oxidase function by gene therapy (GT) for CGD. Methods: Antifungal activity of free and NET-released calprotectin was assessed by incubation of Aspergillus nidulans with purified calprotectin, induced NETs from human controls, and CGD neutrophils after GT in the presence or absence of Zn2+ or alpha-S100A9 antibody, and with induced NETs from wildtype or S100A9(-/-) mouse neutrophils. Results: We identified the host Zn2+ chelator calprotectin as a neutrophil-associated antifungal agent expressed within NETs, reversibly preventing A nidulans growth at low concentrations, and leading to irreversible fungal starvation at higher concentrations. Specific antibody-blocking and Zn2+ addition abolished calprotectin-mediated inhibition of A nidulans proliferation in vitro. The role of calprotectin in anti-Aspergillus defense was confirmed in calprotectin knockout mice. Conclusion: Reconstituted NET formation by GT for human CGD was associated with rapid cure of pre-existing therapy-refractory invasive pulmonary aspergillosis in vivo, underlining the role of functional NADPH oxidase in NET formation and calprotectin release for antifungal activity. These results demonstrate the critical role of calprotectin in human innate immune defense against Aspergillus infection. (J Allergy Clin Immunol 2011;127:1243-52.)