Figure 1.
NET formation by human neutrophils co-incubated with resting and swollen conidia or hyphae of A. fumigatus.
CLSM fluorescence, bright field and overlay images showing NET formation of human neutrophils after co-incubation with A. fumigatus. Extracellular DNA was stained with propidium iodide (red), conidia and hyphae with calcofluor white (blue). Microscopic pictures were taken after 3 hours. Neutrophils were co-incubated with resting conidia (A), swollen conidia (B) and hyphae (C). All scale bars represent 20 µm length.
Figure 2.
Scanning electron microscopy (SEM) micrographs of conidia and hyphae trapped in NETs.
NET formation of human neutrophils after co-incubation with A. fumigatus. Microscopic pictures were taken after 3 hours. Neutrophils were co-incubated with resting conidia (A), swollen conidia (B) and hyphae (C). All scale bars represent 5 µm length. Morphological structures are indicated by labelled arrows.
Figure 3.
Time-lapse widefield microscopy of NET formation by neutrophils co-incubated with swollen conidia of A. fumigatus.
(A) Time series of NET formation by human neutrophils upon contact to swollen A. fumigatus conidia. Extracellular DNA was stained with propidium iodide (red), conidia and germ tubes with calcofluor white (blue-pink). Microscopy was carried out for 5 h after onset of co-incubation while single pictures were taken every 30 seconds (Video S1). (B) Co-incubation after 150 minutes. Black arrowheads indicate cells which had died without releasing DNA, white arrowheads point to cells undergoing preparation for NET release. The colours are the same as in (A). (C) DNase digestion of NETs after 180 minutes of co-incubation. White arrowheads indicate the preformed NET structures right before destruction by the enzyme (Video S2). The colours are the same as in (A). Where appropriate, real time is indicated in minutes. The DNase was added 7 min before onset of the visible NET digestion to the border of the microscopy chamber right before sealing. The size of scale bars is indicated directly. These movies are representative for at least 6 independent experiments that were performed.
Figure 4.
In situ 2-Photon microscopy of NET-like structures formed in a murine A. fumigatus infection model.
(A) Model system used to demonstrate NET formation and -structure in living lung-slices. 7–10 hours after infection of live mice the right lung lobe was prepared, dissected and NETs were stained with a specific DNA dye. In situ 2-photon microscopy was carried out in PBS pre-warmed to 37°C (Video S3). (B) High resolution image of a fungal mass with outgrowing hyphae (blue colour, arrowheads) within the infected lung. Red staining (DNA) shows NET-structures as well as the intact nuclei of host cells within the lung. Please also note the fine blue curvature of alveoli (white arrows). The same image is 3-D rendered in Video S8. (C) In lungs of infected mice multiple of such large accumulations of fungal masses were visible (blue colour, white arrowheads). At higher magnification
these fungal masses were surrounded by fine, red fibres demonstrating NET formation in these areas (D) In low
and especially high magnification
such structures often strongly resembled NETs observed before in vitro (red) and were mostly associated with swollen A. fumigatus conidia (blue) in lung slices freshly prepared from infected lungs (“acute lung slices”). (E) In mice treated i.t. with PBS NET formation was absent (
overview,
higher magnification). Blue: SHG signal of the lung tissue and fungal masses, red: nuclei of cells cut open during processing. The images are representative of more than 20 individual mice, which were analysed.
Figure 5.
Neutrophil motility and interaction with fungal elements in living lung slices.
Lys-EGFP mice were infected and acute lung slices were prepared as described in Figure 4. Subsequently, time-lapse 2-photon microscopy was used to generate movies of cells migrating in these lung slices. (A) A still image of a movie showing individual neutrophils (green), DNA (red) and tissue/fungal elements (blue). Tracks of migrating cells are shown in white.
Image of the tracks alone (Video S4). (B) Many neutrophils (green) can be seen migrating within the tissue and internalising conidia in slices. The red square is shown as a magnification on the right (white arrowheads denote phagocytosis events). Tissue (dark blue) fungal elements (light blue), DNA (red, Video S5). (C) A still image from the middle of a Z-stack of an infected lung in a Lys-EGFP animal. The area boxed in white is shown enlarged from the bottom and as 3-D rendering from the side to demonstrate the internalisation of a conidium (light blue) within a neutrophil (green). See also Video S9. (D) Multiple neutrophils (green) cooperate to transport a hypha (White arrowhead, hypha is light blue. The area of the red square is shown as a magnification below.), that is too big to be engulfed, to an area with more neutrophils (Video S6). Red: DNA from nuclei and NETs. Similar events can also be observed in vitro (Video S10). (E) An individual neutrophil (green, arrowhead) enters the alveolar space and migrates along the alveolar surface (Dark blue structure. The white track is the migration path of the neutrophil.). The border of the alveolus is depicted with a broken blue line in the magnification of the area identified by the red square (Video S7). The images are representative of 8 individual mice that were analysed.
Figure 6.
NET formation in vivo is dependent on the presence of newly immigrating neutrophils.
(A) Mice were intratracheally injected with either swollen A. fumigatus conidia or PBS. 7 h after infection, the number of neutrophils in the bronchoalveolar lavage of these mice was measured by FACS. (B) Lys-EGFP mice were treated with the neutrophil depleting anti Gr-1 antibody RB6-8C5 24 h before infection with A. fumigatus. A lung slice of such a mouse analysed 10 h after infection shows almost no green cells (arrowhead in the magnification of the area boxed in green shows one of the very rare cells in this slice) and no NET-like structures (note only punctate red staining for DNA of nuclei) in areas of fungal masses (blue). (The area of the red square is magnified on the right) The image is representative for 3 animals that were analysed. (C) Quantification of NET formation in Gr-1- and mock-depleted mice infected with swollen conidia as well as untreated mice infected with resting conidia. Shown is a representative result of 3 independent experiments performed. For each condition 20 fungal clouds >20 µm were scored for the presence of NET structures. The 3 images are reference pictures for the type of structure scored with −/+/++.
Figure 7.
Detection of extracellular DNA by propidium iodide-staining after co-incubation of neutrophils with Aspergillus morphotypes.
The different morphotypes of A. fumigatus were co-incubated for 180 min with neutrophils and the release of extracellular DNA was determined by measuring the fluorescence intensity of propidium iodide. Hyphae (black bars), swollen conidia (grey bars) and resting conidia (white bars) of A. fumigatus were used. (A) A. fumigatus ATTC 46445 wild type strain after co-incubation with human neutrophils. DNase I or DPI were added from the beginning of the co-incubation. Asterisks indicate significant differences (*p<0.05 or **p<0.01) based on Student's t-test. (*1) Indicates comparison of each morphotype of the wild type (ATCC 46645) strain with the DNase I treated wild type strain; and (*2) with the DPI treated wild type strain. (B) Analysis of different A. fumigatus wild type and mutant strains. In some experiments, 0.07 µg RodA protein was added to the A. fumigatus mutant strain ΔrodA just 15 min prior to the co-incubation with neutrophils. Asterisks indicate significant difference (*p<0.05 or **p<0.01) in the formation of extracellular DNA by neutrophils during (*1) co-incubation of HF treated resting conidia of the DAL wild type strain in comparison to untreated resting conidia as a control, during (*2) co-incubation with the DAL wild type strain in comparison to the mutant strain ΔrodA, and (*3) during co-incubation with the mutant strain ΔrodA in comparison to ΔrodA supplemented with the spore surface protein RodA. Only the single morphotypes were compared with each other.
Figure 8.
Survival of resting and swollen conidia of A. fumigatus strains after co-incubation with neutrophils.
The number of CFU determined for conidia without co-incubation (T0) was set as 100% survival. CFUs of A. fumigatus conidia were determined as described in materials and methods. Survival of swollen (grey bars) and resting conidia (white bars) are depicted. (A) Survival of A. fumigatus strain ATCC46645 during co-incubation with neutrophils over time (T0, T60, T120 and T180). Asterisks indicate significant difference (*p<0.05 or **p<0.01) in survival in comparison to the time point T0 for each morphotype. (B) Survival of A. fumigatus strain ATCC46645 after co-incubation with neutrophils and in the presence of DNase I or DPI. Asterisks indicate significant difference (*p<0.05 or **p<0.01) in survival in comparison to the time point T180 after neutrophil co-incubation. The survival rate did not increase significantly by the addition of DNase I or the NAD(P)H-oxidase inhibitor DPI. Only the addition of cytochalasin D increased the survival of A. fumigatus conidia. (C) Analysis of the survival of the A. fumigatus strain pksP and its parental wild type strain ATCC46645 showed no significant differences in killing. Also the deletion mutant ΔrodA and its parental wild type strain DAL revealed no difference in killing. The addition of the conidial hydrophobin RodA (0.07 µg [w/well]) did not influence the survival of the Aspergillus fumigatus mutant strain ΔrodA and the wild type strain DAL during co-incubation with neutrophils.
Figure 9.
Determination of the atmospheric molecular oxygen consumption of A. fumigatus hyphae after co-incubation with neutrophils for 3 to 12 h.
Hyphae (black bars) were co-incubated with neutrophils for different periods of time. Untreated, hyphae not co-incubated with neutrophils served as controls (stipled bars). The change of the oxygen saturation in the medium (in %) over time (h) was plotted. Asterisks indicate significant difference (*p<0.05 or **p<0.01) in the change of oxygen saturation in comparison to the control. In some experiments DNase I or DPI was added to the co-incubation and the control.