In vivo bioluminescence imaging and histopathopathologic analysis reveal distinct roles for resident and recruited immune effector cells in defense against invasive aspergillosis

Abstract

Background: Invasive aspergillosis (IA) is a major cause of infectious morbidity and mortality in immune compromised patients. Studies on the pathogenesis of IA have been limited by the difficulty to monitor disease progression in real-time. For real-time monitoring of the infection, we recently engineered a bioluminescent A. fumigatus strain.

Results: In this study, we demonstrate that bioluminescence imaging can track the progression of IA at different anatomic locations in a murine model of disease that recapitulates the natural route of infection. To define the temporal and functional requirements of distinct innate immune cellular subsets in host defense against respiratory A. fumigatus infection, we examined the development and progression of IA using bioluminescence imaging and histopathologic analysis in mice with four different types of pharmacologic or numeric defects in innate immune function that target resident and recruited phagocyte subsets. While bioluminescence imaging can track the progression and location of invasive disease in vivo, signals can be attenuated by severe inflammation and associated tissue hypoxia. However, especially under non-inflammatory conditions, such as cyclophosphamide treatment, an increasing bioluminescence signal reflects the increasing biomass of alive fungal cells.

Conclusions: Imaging studies allowed an in vivo correlation between the onset, peak, and kinetics of hyphal tissue invasion from the lung under conditions of functional or numeric inactivation of phagocytes and sheds light on the germination speed of conidia under the different immunosuppression regimens. Conditions of high inflammation -either mediated by neutrophil influx under corticosteroid treatment or by monocytes recruited during antibody-mediated depletion of neutrophils- were associated with rapid conidial germination and caused an early rise in bioluminescence post-infection. In contrast, 80% alveolar macrophage depletion failed to trigger a bioluminescent signal, consistent with the notion that neutrophil recruitment is essential for early host defense, while alveolar macrophage depletion can be functionally compensated.

Figure 1

Clodrolip treated mice are not susceptible to A. fumigatus intranasal infection. In each experiment, groups of 5 mice were treated either with cortisone acetate, cyclophosphamide, RB6-8C5 antibody, or clodrolip prior to intranasal infection with 2 × 10⁶ conidia of the luminescent A. fumigatus strain C3. Untreated infected mice are designated as immnocompetent (IC). Weight loss and survival were monitored for 8 days (A and B). (C): Time response study of luminescence emission from chest region 10 min after intraperitoneal injection of D-luciferin. Light emission from live animals was recorded for 5 min. Each point represents the average from 3 independent experiments of the total photon flux measured from a defined thoracic region from each individual animal of the respective cohort (5 mice). (D): Light emission from the lung of a dead animal immunosuppressed with cortisone acetate following direct injection of D-luciferin. A total photon flux/second of 3.744 × 10⁶ has been measured using the living image software 3.1 after 1 min exposure.

Figure 2

Quantitative real-time PCR of fungal DNA enables the correlation between fungal burden and bioluminescence signals. Mice were immunosuppressed either with cortisone acetate or cyclophosphamide. Two mice from each group were sacrificed at day one and the other two animals from each group at day three after infection. An uninfected mouse was used as a negative control and revealed no signal in the qRT-PCR and is, therefore, omitted from the graph. The bars represent the amount of fungal DNA per microgram of total DNA isolated from the infected tissues with standard deviations from six data points for each individual animal. The two animals investigated for each time point and immunosuppression regimen show the general tendency that at day one after infection the cortisone acetate treated animals show a higher burden than the cyclophosphamide treated animals. Three days after infection, the burden with alive fungal cells seems to stay rather constant under the coticosteroid treatment, but strongly increases under the regimen with cyclophosphamide. The inlet shows the time response of bioluminescence from alive animals with high values for the cortisone acetate treated mice early after infection followed by a decline of the signal intensity at later time points. Under cyclophosphamide regimen the bioluminescence steadily increases. The small photographs above the bars from mice sacrificed at day three show the explanted lungs with an overlay of the emitted light intensities. Numbers above the photographs give the photons/s × cm². The high bioluminescence from lungs explanted from cyclophosphamide treated animals reflects the higher burden with living fungal cells as determined by the fungal DNA quantification.

Figure 3

Neutrophil recruitment inhibits the conidial germination in alveolar macrophages-depleted mice one day after infection. (A): Alveolar macrophage and neutrophil populations were counted in BAL fluids one day after infection of mice treated with the liposome control and clodrolip. N = 5 mice per group. One of three independent experiments is shown. * denotes a p-value < 0.05. (B): Light emission in BAL-fluids one day after infection of mice treated with liposome control (upper cell well), clodrolip (middle cell well) and cortisone acetate (lower cell well). BAL cells were collected by cytospin centrifugation using labtek chamber slides. D-luciferin was incorporated to the medium and luminescence acquired after 10 min with the IVIS 100 system. The graph shows the total luminescence evaluated by using the living image software 3.1.

Figure 4

At the early stage of pulmonary colonisation, neutrophil influx limits fungal germination after clodrolip treatment. (A): Multifocal inflammatory lesion centred on bronchi/bronchioles and blood vessels (arrowheads). (B): Inflammatory cells observed in alveolar spaces (arrowheads). (C, E): Inflammatory infiltrates containing fragmented neutrophils (suppuration, white stars). (D, F): In the inflammatory infiltrates (black star) only non-germinated conidia (arrowheads) were observed. A, B, C, E: HE staining; D, F: GMS staining.

Figure 5

Eight days post-inoculation, hyphal growth was not observed in clodrolip treated mice. (A): At low magnification, very few lesions (hemorrhages and small inflammatory infiltrates) were observed (arrowheads). (B, C): Inflammatory infiltrates were characterised by perivascular and peribronchiolar accumulation of lymphocytes and plasma cells. (D, E): A small number of non-germinated conidia, located in the cytoplasm of alveolar macrophages were observed. A, B, C: HE staining; D, E: GMS staining.

Figure 6

Bioluminescence enables detection of thoracic and extra thoracic signals in cortisone acetate treated mice. (A): Time response study of luminescence emission from mice immunosuppressed either with cortisone acetate (A, B) or with a combination of cortisone acetate and clodrolip (C-E). Mice were intranasally infected with 2 × 10⁶ conidia. A cohort of 10 mice received liposomes as a control prior to infection (F). Images of day one (D1) and two (D2) post-infection are shown. Luminescence was monitored 10 min after intraperitoneal injection of D-luciferin. Images from ventral (V) and dorsal (D) views of the sinus region, six days after infection (D6) of mice treated with both, cortisone acetate and clodrolip, are shown (E). The graph in (G) represents the average of the total photon flux measured from a defined thoracic region from each individual animal of the respective cohort. (H): Time course of total luminescence from chest, abdomen and head regions from animals receiving the combination of cortisone acetate and clodrolip.

Figure 7

The cortisone acetate mediated neutrophil infiltration did not prevent conidia germination even one day after infection. (A): Multifocal inflammatory lesion extending from bronchi/bronchioles to alveoli (arrowheads). (B): Numerous fungal cells can be detected in the inflammatory infiltrates (arrowheads). (C, E): In the bronchioles (C) as well as in the alveoli (E), inflammatory infiltrates contained numerous neutrophils, which were very often fragmented (suppuration). (D, F): Bronchiolar spaces contained mature hyphae (D) in contrast to alveolar spaces that contained poorly mature hyphae and early germinating conidia (F). A, C, E: HE staining; B, D, F: GMS staining.

Figure 8

Despite strong infiltration of neutrophils under cortisone acetate treatment, growth of the fungus in bronchiolar and alveolar spaces is not prevented in the late stage of infection. (A): Multifocal to coalescing inflammatory lesion centred on bronchioles (black stars) and extending to alveoli and blood vessels. (B): Mycelium growing mainly in the bronchiolar spaces (black stars), but also extending to alveoli (arrowheads). (C): Lesions displayed a concentric organisation: in the centre, neutrophils accumulated and infiltrated bronchioles (arrowhead) and blood vessel walls (arrow). (D): Neutrophils (black star) were circled by a peripheral rim of activated macrophages (Δ). (E, F): Fungi displayed a high infiltrative potential, extending from bronchiolar spaces to alveoli. A, C, D, E: HE staining; B, F: GMS staining.

Figure 9

After intranasal inoculation, mice treated by cortisone acetate could develop a suppurative sinusitis. (A): The nasal sinus cavities were filled by a suppurative exudate containing fragmented neutrophils (black stars). (B): A high number of intralesional mature hyphae were detected in the exudate as well as along the nasal sinus epithelium (inlay, Δ).

Figure 10

In the early stage after RB6-8C5 treatment, although immunocompetent, macrophages were not sufficient to avoid conidial germination. (A): Multifocal small inflammatory infiltrates randomly scattered in the pulmonary parenchyma. (B): Small clusters of fungi were observed in the inflammatory infiltrates. (C): Inflammatory infiltrates were located in alveolar spaces or interalveolar interstitial tissue. They contained mononucleated cells (mainly macrophages but also rare lymphocytes and plasma cells). (D, E, F): Early germinating conidia were observed in the inflammatory infiltrates either free or in the cytoplasm of alveolar macrophages (arrowheads). Note that the conidia and hyphae were less mature than under cortisone acetate treatment (Figure. 6). A, C: HE staining; B, D, E, F: GMS staining.

Figure 11

In the late stage after RB6-8C5 treatment, macrophages and recruited monocytes were unable to prevent fungal lung colonisation. (A): Multifocal large inflammatory infiltrates centred on bronchioles but extending to alveoli and blood vessels (arrowheads). (B): Fungi displayed a high infiltrative potential with a marked extension to alveoli (arrowheads). (C): Inflammatory infiltrates were composed of mononucleated cells; mainly macrophages (inlay). (D): Hyphae were mature and displayed a high invasive potential. A, C: HE staining; B, D: GMS staining.

Figure 12

In the early stage, A. fumigatus germination was delayed after cyclophosphamide treatment. (A): At a low magnification, no significant histological lesion was observed. B: Only small clusters of conidia were multifocally detected (arrowheads). C. At a high magnification, only small infiltrates of neutrophils were noted in bronchiolar and alveolar spaces. (D): Non-germinated and early germinating conidia were observed in these inflammatory infiltrates. (E): Intra-alveolar conidia at a very early stage of germination (swollen conidia). Some conidia were observed in the cytoplasm of alveolar macrophages (arrowhead). (F): Intra-bronchiolar conidia were either swollen or started to form hyphae. Note that this stage of maturation is much less pronounced than observed in the early stage of cortisone acetate (Figure 6D) and RB6-8C5 treatment (Figure 9D). A, C: HE staining; B, D, E, F: GMS staining.

Figure 13

In the late stage after cyclophosphamide treatment no inflammatory response was observed and A. fumigatus rapidly colonised the pulmonary parenchyma. (A): Diffuse lesion characterised by a total absence of inflammatory response with a severe destruction of the bronchoalveolar structures (black star: bronchiole; white star: pulmonary artery). (B): Severe parenchyma colonisation by the fungus with infiltration of bronchioles (black star) as well as pulmonary arteries (white star). (C): Destruction of the bronchiolar (black star), alveolar, and vascular (white star) walls by hyphae. (D): Branched mature hyphae were observed, displaying a high infiltrative potential. A, C: HE staining; B, D: GMS staining.