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.

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Available from: Constantin Felix Urban, Feb 28, 2014
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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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    ABSTRACT: X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of the immune system. It is characterized by a defect in the production of reactive oxygen species (ROS) in phagocytic cells due to mutations in the NOX2 locus, which encodes gp91phox. Because the success of retroviral gene therapy for X-CGD has been hampered by insertional activation of proto-oncogenes, targeting the insertion of a gp91phox transgene into potential safe harbor sites, such as AAVS1, may represent a valid alternative. To conceptually evaluate this strategy, we generated X-CGD patient-derived induced pluripotent stem cells (iPSCs), which recapitulate the cellular disease phenotype upon granulocytic differentiation. We examined AAVS1-specific zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for their efficacy to target the insertion of a myelo-specific gp91phox cassette to AAVS1. Probably due to their lower cytotoxicity, TALENs were more efficient than ZFNs in generating correctly targeted iPSC colonies, but all corrected iPSC clones showed no signs of mutations at the top-ten predicted off-target sites of both nucleases. Upon differentiation of the corrected X-CGD iPSCs, gp91phox mRNA levels were highly up-regulated and the derived granulocytes exhibited restored ROS production that induced neutrophil extracellular trap (NET) formation. In conclusion, we demonstrate that TALEN-mediated integration of a myelo-specific gp91phox transgene into AAVS1 of patient-derived iPSCs represents a safe and efficient way to generate autologous, functionally corrected granulocytes. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Biomaterials 08/2015; 69:191-200. DOI:10.1016/j.biomaterials.2015.07.057 · 8.56 Impact Factor
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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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    ABSTRACT: We report on a series of sequential events leading to long-term survival and cure of pediatric X-linked chronic granulomatous disease (X-CGD) patients after gamma-retroviral gene therapy (GT) and rescue HSCT. Due to therapy-refractory life-threatening infections requiring hematopoietic stem cell transplantation (HSCT) but absence of HLA-identical donors, we treated 2 boys with X-CGD by GT. Following GT both children completely resolved invasive Aspergillus nidulans infections. However, one child developed dual insertional activation of ecotropic viral integration site 1 (EVI1) and signal transducer and activator of transcription 3 (STAT3) genes, leading to myelodysplastic syndrome (MDS) with monosomy 7. Despite resistance to mismatched allo-HSCT with standard myeloablative conditioning, secondary intensified rescue allo-HSCT resulted in 100% donor chimerism and disappearance of MDS. The other child did not develop MDS despite expansion of a clone with a single insertion in the myelodysplasia syndrome 1 (MDS1) gene and was cured by early standard allo-HSCT. The slowly developing dominance of clones harboring integrations in MDS1-EVI1 may guide clinical intervention strategies, i.e. early rescue allo-HSCT, prior to malignant transformation. GT was essential for both children to survive and to clear therapy-refractory infections, and future GT with safer lentiviral self-inactivated (SIN) vectors may offer a therapeutic alternative for X-CGD patients suffering from life-threatening infections and lacking HLA-identical HSC donors.
    Current Gene Therapy 05/2015; 15(4). DOI:10.2174/1566523215666150515145255 · 2.54 Impact Factor
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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.
    European Journal of Obstetrics & Gynecology and Reproductive Biology 10/2014; 183. DOI:10.1016/j.ejogrb.2014.10.040 · 1.70 Impact Factor
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