Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015;7(4):364-74.
doi: 10.1159/000369493. Epub 2015 Feb 10.

Effective Neutrophil Phagocytosis of Aspergillus fumigatus Is Mediated by Classical Pathway Complement Activation

Steven G E Braem  1 , Suzan H M Rooijakkers, Kok P M van Kessel, Hans de Cock, Han A B Wösten, Jos A G van Strijp, Pieter-Jan A Haas
Affiliations

Effective Neutrophil Phagocytosis of Aspergillus fumigatus Is Mediated by Classical Pathway Complement Activation

Steven G E Braem et al. J Innate Immun. 2015.

Abstract

Aspergillus fumigatus is an important airborne fungal pathogen and a major cause of invasive fungal infections. Susceptible individuals become infected via the inhalation of dormant conidia. If the immune system fails to clear these conidia, they will swell, germinate and grow into large hyphal structures. Neutrophils are essential effector cells for controlling A. fumigatus infection. In general, opsonization of microbial particles is crucial for efficient phagocytosis and killing by neutrophils. Although the antibodies present in human serum do bind to all fungal morphotypes, we observed no direct antibody-mediated phagocytosis of A. fumigatus. We show that opsonization, phagocytosis and killing by neutrophils of A. fumigatus is complement-dependent. Using human sera depleted of key complement components, we investigated the contribution of the different complement initiation pathways in complement activation on the fungal surface. We describe the classical complement pathway as the main initiator of complement activation on A. fumigatus swollen conidia and germ tubes. Antibodies play an important role in complement activation and efficient innate recognition, phagocytosis and killing of A. fumigatus by neutrophils.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
C3b and IgG deposition on different Aspergillus morphotypes. a Different morphotypes (dormant conidia, swollen conidia and germ tubes) were incubated with NHS, and C3b deposition was detected by flow cytometry. b The different morphotypes were incubated with 10% HI NHS and C3b deposition was measured by flow cytometry. c The morphotypes were incubated with 1% HI NHS and IgG deposition was measured by flow cytometry. a-c The assays were performed at least 3 times and the mean ± SD is shown. d The morphotypes were incubated with buffer, 10% NHS and 10% HI NHS. C3b and IgG deposition was visualized using confocal microscopy after staining with mouse-anti-C3 and protein A Alexa Fluor 488 or anti-IgG-FITC antibodies, respectively. Wheat germ agglutinin (WGA) Alexa Fluor 647 was used to visualize the fungal cell wall. The confocal microscopy assay was performed 3 times and representative images are shown. Scale bar: 5 μm.
Fig. 2
Fig. 2
Complement induces phagocytosis of A. fumigatus. a-c Different morphotypes (dormant conidia, swollen conidia and germ tubes) of a red fluorescent strain Af-mRFP1 were preopsonized with buffer, 10% NHS and 10% HI NHS. DiO-labeled neutrophils (neutro) were added in an effector-to-target ratio of 1:5. After an additional incubation of 15 min, the cells were fixed and analyzed by flow cytometry (a, b) or confocal microscopy (c). Phagocytosis is expressed as the percentage of mRFP1-positive (mRFP1-pos.) neutrophils (a) or the MFI of mRFP1 within the DiO-positive population (b). Both assays show the mean ± SD of 3 independent experiments. The ingestion of the mRFP1 morphotypes was visualized by confocal microscopy (c), showing representative images of 3 independent experiments. Scale bar: 10 μm.
Fig. 3
Fig. 3
Complement induces the neutrophil-dependent killing of A. fumigatus. a, b GFP-expressing dormant conidia were incubated with 10% NHS or 10% HI NHS with or without the addition of neutrophils (neutro) in an effector-to-target ratio of 3:1. GFP fluorescence was measured every 10 min for 33 h and plotted as relative fluorescent units (RFU). The assay was performed 3 times in duplicate and a representative plot with mean ± SEM is shown (a). The AUC was calculated over the total incubation time and the mean ± SD of 3 independent experiments is shown (b). * p < 0.05.
Fig. 4
Fig. 4
Opsonization and phagocytosis of Aspergillus morphotypes in complement-deficient (def) sera. a-c Different morphotypes, i.e. dormant conidia (a), swollen conidia (b) and germ tubes (c) were incubated with NHS, HI NHS or complement-deficient serum with or without purified complement protein. C3b deposition was detected by flow cytometry. d-f Different morphotypes of a red fluorescent strain Af-mRFP1, i.e. dormant conidia (d), swollen conidia (e) and germ tubes (f) were preopsonized with NHS, HI NHS or complement-deficient serum with or without reconstitution of complement protein. DiO-labeled neutrophils were added in an effector-to-target ratio of 1:5. After an additional incubation of 15 min, cells were fixed and analyzed by flow cytometry. Phagocytosis is expressed as the MFI of mRFP1 within the DiO-positive population. Both assays show the mean ± SD of 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig. 5
Fig. 5
Generation of chemoattractant C5a. a-c Different morphotypes, i.e. dormant conidia (a), swollen conidia (b) and germ tubes (c) were incubated with complement-deficient serum with or without reconstitution of complement protein. Supernatant was examined on the presence of C5a as explained in the Material and Methods. C5a activation is expressed as the amount of C5a generation in deficient serum compared to C5a generation in deficient serum reconstituted with purified complement protein. The assay was performed 3 times and the mean ± SD is shown.
See this image and copyright information in PMC

References

    1. Stevens DA, Melikian GL. Aspergillosis in the ‘nonimmunocompromised’ host. Immunol Invest. 2011;40:751–766. - PubMed
    1. O'Gorman CM, Fuller HT. Prevalence of culturable airborne spores of selected allergenic and pathogenic fungi in outdoor air. Atmospher Environ. 2008;42:4355–4368.
    1. Mircescu MM, Lipuma L, van Rooijen N, Pamer EG, Hohl TM. Essential role for neutrophils but not alveolar macrophages at early time points following Aspergillus fumigatus infection. J Infect Dis. 2009;200:647–656. - PMC - PubMed
    1. Winkelstein JA, Marino MC, Johnston RB, Boyle J, Curnutte J, Gallin JI, et al. Chronic granulomatous disease. Report on a national registry of 368 patients. Medicine (Baltimore) 2000;79:155–169. - PubMed
    1. Young RC, Bennett JE, Vogel CL, Carbone PP, DeVita VT. Aspergillosis. The spectrum of the disease in 98 patients. Medicine (Baltimore) 1970;49:147–173. - PubMed

Publication types