Restoration of NET formation by gene therapy in CGD controls aspergillosis

Abstract

IntroductionActivated neutrophils kill microbes intracellularly after phagocytosis and by extracellular mechanisms, including neutrophil extracellular traps (NETs), which are composed of chromatin decorated with granular proteins. 1 NETs bind bacteria 1 and fungi 2 and expose antimicrobial molecules. Generation of NETs requires reactive oxygen species produced by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. 3 Chronic granulomatous disease (CGD) is caused by mutations in genes encoding NADPH oxidase subunits. CGD patients do not produce reactive oxygen species, kill microbes poorly, and are susceptible to recurrent life-threatening infections. 4 Aspergillus spp infections cause pneumonia and disseminated disease and are the leading cause of death in these patients. [4][5][6] It is unclear how Aspergillus infections are controlled in healthy persons. [7][8][9][10][11][12][13] In CGD patients, these infections are frequently refractory to antifungal therapy, treatment with interferon-␥, or granulocyte transfusions. 5 Here we show that the recently discovered NADPH oxidase-dependent microbicidal pathway through NETs 1-3 is efficient against Aspergillus nidulans conidia and hyphae in vitro and that restoration of NET formation by GT of X-CGD aided clearing severe invasive A nidulans infection in vivo. Methods Gene therapyWe treated an 8.5-year-old boy with X-linked gp91 phox -deficient CGD and therapy refractory A nidulans lung infection with a monocistronic long terminal repeat-driven gamma-retroviral SF71gp91 phox vector (see supplemental data, available on the Blood website; see the Supplemental Materials link at the top of the online article). The protocol for the patient's treatment was approved by the ethics review board of the University Children's Hospital Zurich and the Swiss Expert Committee for Bio-Safety, after written informed consent from his parents in accordance with the Declaration of Helsinki. For follow-up monitoring, gp91 phox expression was measured by fluorescence-activated cell sorter (FACS) on peripheral neutrophils after 30 minutes of staining at room temperature with 10 g/mL gp91 phox -fluorescein isothiocyanate (FITC) antibody (Anti-Flavocytochrome b558, clone 7D5, MBL). NADPH oxidase activity was measured by standard dihydrorhodamine and nitroblue tetrazolium tests (supplemental materials). Bone marrow colony assays and determination of proviral gp91 phox sequences in genomic DNA were performed as described. 14 NET inductionNET formation was visualized as described (supplemental materials) and quantified after stimulation of 5 ϫ 10 4 neutrophils for 3 hours with 40 nM phorbol 12-myristate 13-acetate (PMA) and staining the NET-DNA with 1 M Sytox green (Invitrogen) in a black 96-well plate (BD Biosciences). The plates were read in a fluorescence microplate reader (Victor, 3 PerkinElmer Life and Analytical Sciences) with a filter setting of 485 nm/535 nm (excitation/emission). NET antifungal activityThe A nidulans strain used was isolated from bronchoalveolar lavage fluid ...

Changing world of neutrophils

“…Furthermore, in a case of severe Aspergillosis in a CGD patient, negative correlation was found between the ability of neutrophils to form NETs and the severity of infection [10].…”

Biology, role and therapeutic potential of circulating histones in acute inflammatory disorders

“…This in turn leads to a clinical picture of recurrent and/or persistent infections, in particular with fungal pathogens. Impressively, there has been a successful report of gene therapy, where an 8‐year‐old boy was treated with a retroviral vector containing a functional gp91 (PHOX subunit) gene, resulting in neutrophils regaining the ability to NETose and leading to a termination of an intractable Aspergillus nidans infection 51. Experimentally, inhibition of NADPHO or myeloperoxidase (MPO) effectively inhibited NETosis stimulated by PMA, whereas inhibition of mitochondrial respiration or superoxide dismutase did not 52.…”

Neutrophil extracellular trap cell death requires both autophagy and superoxide generation

“…The results showed that NETs are formed during the early stages of infection. In addition to the reported induction of NETosis formation by bacteria [3,6,12,13], fungi [17][18][19] and protozoa [15], NETosis has also been shown to be induced by LPS-activated platelets [14] and by antineutrophil autoantibodies isolated from patients with SVV [16], whereas impaired degradation of NETs has been associated with systemic lupus erythematosus as well [20].…”