Restoration of NET formation by gene therapy in CGD controls aspergillosis

Abstract

Chronic granulomatous disease (CGD) patients have impaired nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function, resulting in poor antimicrobial activity of neutrophils, including the inability to generate neutrophil extracellular traps (NETs). Invasive aspergillosis is the leading cause of death in patients with CGD; it is unclear how neutrophils control Aspergillus species in healthy persons. The aim of this study was to determine whether gene therapy restores NET formation in CGD by complementation of NADPH oxidase function, and whether NETs have antimicrobial activity against Aspergillus nidulans. Here we show that reconstitution of NET formation by gene therapy in a patient with CGD restores neutrophil elimination of A nidulans conidia and hyphae and is associated with rapid cure of preexisting therapy refractory invasive pulmonary aspergillosis, underlining the role of functional NADPH oxidase in NET formation and antifungal activity.

Figures

Figure 1

Restoration of NADPH oxidase function. (A) Hematopoietic reconstitution and gene marking after GT. Absolute neutrophil counts (left y-axis), quantification of gene-modified cells in peripheral neutrophils by quantitative polymerase chain reaction and quantification of neutrophils with NADPH oxidase activity by DHR test (right y-axis) are shown. When the percentage of transduced neutrophils decreased, granulocyte colony-stimulating factor (5 μg/kg per day subcutaneously) was administered on days 49 to 57 and on day 64. (B) Reconstitution of NADPH oxidase activity. Before and 6 weeks after GT, gp91phox protein expression was measured by FACS analysis after 30 minutes of staining with 10 μg/mL gp91phox-FITC antibody. Superoxide production was assessed by oxidation of DHR on stimulation with PMA and by reduction of nitroblue tetrazolium to formazan (dark precipitate) after stimulation with opsonized zymosan. The thresholds were determined using unstained (FACS) or unstimulated (DHR) cells for each experiment. (C) PET-CT scan. Before GT, PET-CT scan showed several active infectious foci with fluorine-18-fluoro-2-deoxy-D-glucose uptake in both lungs of the patient (red arrows); the infection cleared 6 weeks after administration of gene-corrected cells. In green, physiologic FDG uptake in heart (arrow), kidneys (arrowheads), bladder (*), and brain (diamond) are indicated for reference.

Figure 2

NET formation and inhibition of A nidulans growth. Control (A,D), but not CGD (B,E), neutrophils made NETs on 3-hour PMA stimulation. For immunofluorescence, NETs were stained with an antibody that recognizes neutrophil elastase (green; A-C). NETs were clearly visible also by scanning electron microscopy (SEM; D-F). Neutrophils isolated from the CGD patient before GT could be activated because they flattened out (E) but did not make NETs. The ability to form NETs was partially restored by GT 6 weeks after GT (C,F white arrows). (G) Quantification of NET-DNA released after 3 hours of PMA stimulation of control neutrophils, CGD neutrophils before and 6 weeks after GT or (H) after stimulation of CGD gp91phox+ and CGD gp91phox− FACS-sorted neutrophils. CGD gp91phox+ neutrophils showed normal NET formation, whereas CGD gp91phox− neutrophils showed only residual NET formation. FACS-sorting efficiency was 90% to 92% for CGD gp91phox− and 95% to 96% for CGD gp91phox+ cells. (I-J) NET inhibition of A nidulans conidia and hyphae. (I) Conidia were plated on FACS-sorted neutrophils prestimulated with PMA plus or minus MNAse (ie, when NET formation was complete, cells were dead and therefore incapable of phagocytosis). Hyphal outgrowth was measured after 16 hours. (J) Hyphae were coincubated with FACS-sorted neutrophils, and PMA plus or minus MNAse and hyphal viability was assessed after 5 hours. (G-J) Data are mean ± SD of a representative triplicate experiment. Inhibition of fungal growth is expressed as percentage of control values (A nidulans conidia or hyphae incubated in media). The differences between −MNase and +MNase were significant (for control and CGD gp91phox+ cells) by Student t test: **P < .01; ***P < .001.