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: 11.25). 03/2011; 127(5):1243-52.e7. DOI: 10.1016/j.jaci.2011.01.021
Source: PubMed

ABSTRACT 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.

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Available from: Constantin Felix Urban, Feb 28, 2014
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    • ". During NETosis the crucial steps are chromatin decondensation, disintegration of intracellular membranes and release of chromatin threads with the associated proteins [13]. NET formation has an unambiguously beneficial role during infection, since deficiency in NET production, e.g. in chronic granulomatosus disease [14] [15], or degrading the scaffold by bacterial DNases may lead to severe infections [16] [17] [18]. However, the excessive formation of NETs might harm the healthy tissue in their vicinity, as it has been described in acute lung injury [12] [19] [20], cystic fibrosis [21] [22], asthma [23], psoriasis [24], thrombosis [25] [26] [27], preeclampsia [28], appendicitis [10], sepsis [29], Crohn's disease [30], systemic lupus erythematosus (SLE) [31] [32] [33] [34] and small vessel vasculitis [35]. "
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    ABSTRACT: To study if neutrophil extracellular traps (NETs) are present in the peritoneal fluid of endometriosis patients. NETs play a crucial role in fighting against microorganisms. However, exaggerated NET production may lead to tissue damage in their vicinity in pathological conditions. Our study evaluates the presence of NETs in endometriosis peritoneal fluid.Study designPeritoneal fluid (PF) was collected in a case-control study from 52 women, who underwent either diagnostic or operative laparoscopy. The control group consisted of 17 women with infertility, chronic pelvic pain, simple or functional cysts or irregular bleeding. The endometriosis group, altogether 35 patients, comprised 19 patients with stage I and II and 16 patients with stage III and IV endometriosis. First we tested whether the PF is able to stimulate NET production. Neutrophils from healthy volunteers were treated with the PF of endometriosis patients and controls and NETs were detected with Sytox orange extracellular DNA dye and immunofluorescence microscopy. Then we evaluated if NETs were already present in the collected PF using the specific myeloperoxidase (MPO)-DNA capture ELISA method, based on the MPO associated with the NET scaffold.ResultsThe PF of endometriosis patients did not stimulate NET release from healthy granulocytes. However, pre-existent NETs could be detected in 17 endometriosis patients out of 35 (49%). In contrary, in the control group NETs were present in only 3 patients out of 17 (18%), (p = 0.03, OR: 4.4). Moreover, the quantification of NETs showed a significantly higher amount of NETs in endometriosis compared to the controls (0.097 vs. 0.02, p = 0.04).Conclusion This is the first study, which evaluated and described the presence of NETs in the PF of endometriosis patients. Our study shows, that NETs may be involved in the complex pathophysiology of endometriosis.
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    • "Remarkably, gene therapy in an X-CGD patient was monitored with respect to NET formation and reconstitution of Phox function was found to correlate with restored NET formation (Bianchi et al., 2009). Neutrophils from this patient were also used to implicate calprotectin in Phox-dependent human NET formation (Bianchi et al., 2011). The recent work from Urban and colleagues further demonstrated that Phox is required for NET induction in a pulmonary aspergillosis model (Rohm et al., 2014). "
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    Cellular Microbiology 06/2014; 16(8). DOI:10.1111/cmi.12320 · 4.82 Impact Factor
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    • "Calprotectin (CP) is a heterodimer of the two S100 proteins, S100A8 and S100A9, and exhibits high affinity binding for zinc and manganese. Importantly, CP displays antimicrobial activity against several pathogens, including A. baumannii, via its metal-sequestering properties (Corbin et al., 2008; Urban et al., 2009; McCormick et al., 2010; Bianchi et al., 2011; Damo et al., 2013). CP is frequently identified at sites of inflammation, including in the lungs during A. baumannii pneumonia where CP expression tracks with the progression and resolution of infection (Hood et al., 2012; Moore et al., 2013). "
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