Restoration of anti-Aspergillus defense by neutrophil extracellular traps in human chronic granulomatous disease after gene therapy is calprotectin-dependent. J Allergy Clin Immunol 127(5):1243-1252.e7

Division of Immunology/Hematology/BMT, University Children's Hospital Zurich, Zurich, Switzerland.
The Journal of allergy and clinical immunology (Impact Factor: 11.48). 03/2011; 127(5):1243-52.e7. DOI: 10.1016/j.jaci.2011.01.021
Source: PubMed


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.
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.
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 Zn(2+) or α-S100A9 antibody, and with induced NETs from wild-type or S100A9(-/-) mouse neutrophils.
We identified the host Zn(2+) 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 Zn(2+) 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.
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.


Available from: Constantin Felix Urban, Feb 28, 2014
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    • "demonstrated that a functional NADPH-oxidase is essential for NET release, and subsequent findings from patients with chronic granulomatous disease (CGD), in whom a mutation in the NADPH-oxidase impairs ROS generation, demonstrated that these individuals lacked the ability to produce NETs (Bianchi et al. 2011). However, several publications have demonstrated an alternative NADPH-oxidase–independent pathway leading to NET formation (Pilsczek et al. 2010; Douda et al. 2015). "
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    ABSTRACT: Neutrophil extracellular traps (NETs) represent a novel paradigm in neutrophil-mediated immunity. NETs are believed to constitute a highly conserved antimicrobial strategy comprising decondensed nuclear DNA and associated histones that are extruded into the extracellular space. Associated with the web-like strands of DNA is an array of antimicrobial peptides (AMPs), which facilitate the extracellular destruction of microorganisms that become entrapped within the NETs. NETs can be released by cells that remain viable or following a unique form of programmed cell death known as NETosis, which is dependent on the production of reactive oxygen species (ROS) and the decondensing of the nuclear DNA catalyzed by peptidyl arginine deiminase-4. NETs are produced in response to a range of pathogens, including bacteria, viruses, fungi, and protozoa, as well as host-derived mediators. NET release is, however, not without cost, as the concomitant release of cytotoxic molecules can also cause host tissue damage. This is evidenced by a number of immune-mediated diseases, in which excess or dysfunctional NET production, bacterial NET evasion, and decreased NET removal are associated with disease pathogenesis. Periodontitis is the most prevalent infectious-inflammatory disease of humans, characterized by a dysregulated neutrophilic response to specific bacterial species within the subgingival plaque biofilm. Neutrophils are the predominant inflammatory cell involved in periodontitis and have previously been found to exhibit hyperactivity and hyperreactivity in terms of ROS production in chronic periodontitis patients. However, the contribution of ROS-dependent NET formation to periodontal health or disease remains unclear. In this focused review, we discuss the mechanisms, stimuli, and requirements for NET production; the ability of NET-DNA and NET-associated AMPs to entrap and kill pathogens; and the potential immunogenicity of NETs in disease. We also speculate on the potential role of NETs in the pathogenesis of periodontitis.
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    • "While no precipitate was detectable in cells derived from the noncorrected X-CGD clone, genetically corrected cells revealed blueblack formazan precipitates, although to a lesser extent than in healthy control samples. In order to demonstrate that the amount of produced ROS in the genetically edited cells was sufficient to induce neutrophil extracellular traps (NETs), i.e. networks of extracellular DNA to capture extracellular pathogens [33] [34], we quantified NET formation using Sytox green (Fig. 5C). Our results indicate that the levels of ROS produced in genetically corrected cells were sufficient to induce NET formation and therefore to reverse the cellular disease phenotype. "
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    • "deficient phagocytic microbial killing activity. [1] [2] [3] Life expectancy is compromised despite life-long antimicrobial prophylaxis. [4] [5] Allogeneic hematopoietic stem cell (HSC) transplantation (allo-HSCT) from a human leukocyte antigen (HLA)-identical donor, preferably using reduced intensity conditioning, is currently the only curative treatment, associated with excellent disease-free survival. "
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