Reduced Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Independent Resistance to <i>Aspergillus fumigatus</i> in Alveolar Macrophages

Cornish, E. Jean; Hurtgen, Brady J.; McInnerney, Kate; Burritt, Nancy L.; Taylor, R. M.; Jarvis, James N.; Wang, Shirley Y.; Burritt, James B.

doi:10.4049/jimmunol.180.10.6854

Cited by 45 publications

(42 citation statements)

References 82 publications

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“…7,9,12,13,[16][17][18][19][20][21] CGD patients are unable to make NETs. 3 Indeed, neutrophils of our X-CGD patient could only make NETs and inhibit growth of A nidulans after genetic complementation by GT.…”

Section: Resultsmentioning

confidence: 99%

“…In neutrophils, however, conidia resist intracellular killing because of their relative tolerance to reactive oxygen species. 7,9,12,[19][20][21][22] Our results suggest that conidia are killed mainly extracellularly rather than after phagocytosis. Both conidia and hyphae get ensnared by neutrophils and probably killed within NETs by concentrated antimicrobials.…”

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confidence: 99%

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Restoration of NET formation by gene therapy in CGD controls aspergillosis

Bianchi

Hakkim²,

Brinkmann

et al. 2009

Blood

510

451

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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 ...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Restoration of NET formation by gene therapy in CGD controls aspergillosis

Bianchi

Hakkim²,

Brinkmann

et al. 2009

Blood

510

451

View full text Add to dashboard Cite

show abstract

“…Furthermore, they could not find evidence of the importance of NADPH oxidase in AM by using transcriptional microarray analysis, luminometry, or nitroblue tetrazolium (NBT) staining (6). Instead, they found that the most strongly upregulated transcripts in vivo encoded chemokines and additional factors that play critical roles in neutrophil and monocyte recruitment.…”

Section: Discussionmentioning

confidence: 96%

“…These results suggest that NADPH oxidaseindependent mechanisms in murine alveolar macrophages contribute to the inhibition of conidial germination (6). In order to unravel the unique interaction of the CGD host and A. nidulans and to evaluate the role of the NADPH oxidase complex and ROS in resistance to Aspergillus spp., we investigated the ability of various phagocytic cells to generate ROS in response to fungal invasion.…”

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confidence: 99%

Human Leukocytes Kill Aspergillus nidulans by Reactive Oxygen Species-Independent Mechanisms

et al. 2011

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Invasive aspergillosis is a major threat for patients suffering from chronic granulomatous disease (CGD).Although Aspergillus fumigatus is the most commonly encountered Aspergillus species, the presence of A. nidulans appears to be disproportionately high in CGD patients. The purpose of this study was to investigate the involvement of the NADPH oxidase and the resulting reactive oxygen species (ROS) in host defense against fungi and to clarify their relationship toward A. nidulans. Murine CGD alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells (PBMC) from healthy controls and CGD patients were challenged with either A. fumigatus or A. nidulans. Analysis of the antifungal effects of ROS revealed that A. nidulans, in contrast to A. fumigatus, is not susceptible to ROS. In addition, infection with live A. nidulans did not result in any measurable ROS release. Remarkably, human CGD PMN and PBMC and murine CGD AM were at least equipotent at arresting conidial germination compared to healthy controls. Blocking of the NADPH oxidase resulted in significantly reduced damage of A. fumigatus but did not affect A. nidulans hyphae. Furthermore, the microbicidal activity of CGD PMN was maintained toward A. nidulans but not A. fumigatus. In summary, antifungal resistance to A. nidulans is not directly ROS related. The etiology of A. nidulans infections in CGD cannot be explained by the simple absence of the direct microbicidal effect of ROS. In vivo, the NADPH oxidase is a critical regulator of innate immunity whose unraveling will improve our understanding of fungal pathogenesis in CGD.Chronic granulomatous disease (CGD) is a rare (prevalence, 1/250,000 individuals) inherited immunodeficiency disorder of the NADPH oxidase in which phagocytes fail to generate the microbicidal reactive oxidant superoxide anion and its metabolites due to a mutation in any of the five structural components of the NADPH oxidase complex. As a result, CGD patients are characterized by recurrent life-threatening bacterial and fungal infections as well as chronic inflammatory diseases due to dysregulated inflammatory pathways. The lifetime incidence of invasive aspergillosis (IA) in children with CGD varies between 25% and 40%, and IA has been the main cause of death (35%) (7,24).The etiology of IA differs remarkably between patients with CGD and those with other underlying immunodeficiencies.

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“…Macrophage colony-stimulating factor-induced Aspergillus hyphal damage was observed in conjunction with enhanced superoxide anion production in both human monocyte-derived macrophages and rabbit alveolar macrophages (140). In addition, alveolar macrophages from mice deficient in gp91 phox (analogous to human X-linked chronic granulomatous disease) inhibited conidium germination as efficiently as wild-type alveolar macrophages (19,45,113). In contrast, alveolar macrophages from mice lacking p47 phox , another component of the NADPH oxidase complex and mimicking an autosomal recessive form of human chronic granulomatous disease, have been reported to phagocytose conidia normally but are unable to kill them (126).…”

Section: Innate Effector Mechanisms Alveolar Macrophagesmentioning

confidence: 95%

Innate Immunity toAspergillusSpecies

Park¹,

2009

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SUMMARY All humans are continuously exposed to inhaled Aspergillus conidia, yet healthy hosts clear the organism without developing disease and without the development of antibody- or cell-mediated acquired immunity to this organism. This suggests that for most healthy humans, innate immunity is sufficient to clear the organism. A failure of these defenses results in a uniquely diverse set of illnesses caused by Aspergillus species, which includes diseases caused by the colonization of the respiratory tract, invasive infection, and hypersensitivity. A key concept in immune responses to Aspergillus species is that the susceptibilities of the host determine the morphological form, antigenic structure, and physical location of the fungus. In this review, we summarize the current literature on the multiple layers of innate defenses against Aspergillus species that dictate the outcome of this host-microbe interaction.

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Reduced Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Independent Resistance to Aspergillus fumigatus in Alveolar Macrophages

Cited by 45 publications

References 82 publications

Restoration of NET formation by gene therapy in CGD controls aspergillosis

Restoration of NET formation by gene therapy in CGD controls aspergillosis

Human Leukocytes Kill Aspergillus nidulans by Reactive Oxygen Species-Independent Mechanisms

Innate Immunity toAspergillusSpecies

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