In this study we successfully imaged murine invasive pulmonary aspergillosis using bioluminescence recordings in a serial manner. We applied different immunosuppression regimens to elucidate their impact on the susceptibility of mice to invasive aspergillosis (IA). By combining bioluminescence imaging and histopathology we gained new insights on the impact of different immune effector cells (mainly macrophages and neutrophils) in host defense against conidial germination and tissue invasion.
Interestingly, under conditions of high inflammation, such as the cortisone acetate or RB6-8C5 treatment, bioluminescence signal intensities nicely reflected the early germination of conidia, but only showed limited correlation with the amount of alive fungal cells at later time points of infection. Quantification of the fungal DNA from late time points of cortisone acetate treated animal implied that the number of living cells stayed constant over time. This result confirmed that neutrophils, although affected in their killing capacity by the corticosteroid, limited the uncontrolled spreading of fungal mycelium through the lung tissues. However, one would have expected that the bioluminescence signal stays at a high level rather than declining. Due to the large necrotic areas (covering approximately 11% of the whole lung parenchyma), we attribute the decline of the bioluminescence signal to the development of hypoxia, as observed in tissues after stroke or myocardial infarction and for growing tumors, which become hypoxic when they outgrow the vascular supply [23
]. The limitation of bioluminescence imaging in hypoxic tissues has already been described by investigating the decrease in bioluminescence of luciferase-transfected gliosarcoma tumor cells under defined hypoxic in vitro conditions [24
]. Additionally, bioluminescent implanted tumor cells can become necrotic at a certain age with subsequent decline of bioluminescence although the tumor itself does not reduce its size [25
]. This latter scenario is likely to be comparable to our results obtained during bioluminescence imaging of invasive aspergillosis under cortisone acetate and RB6-8C5 antibody treatment. In addition, the occurrence of hypoxia has been assumed from the attenuated virulence of A. fumigatus
mutants with a defective adaptation to hypoxic conditions [26
] and seems confirmed independently by our bioluminescence measurements. Although the correlation of the bioluminescent signal at later time points with the fungal burden is limited the decline in bioluminescence under in vivo conditions in late stages of infection acts as an indicator of tissue destruction and hypoxia and indicates the imminent death of the animals.
Despite the low correlation of fungal biomass and bioluminescence at late time points after infection in the cortisone acetate and RB6-8C5 treatments, a good correlation between the increase in the fungal biomass and the bioluminescence was observed under the cyclophosphamide regimen. Under this treatment, although the growing hyphae were responsible for diffuse parenchyma lesions, the accessibility of oxygen remains possible in the absence of inflammation. At late time points, an ongoing increase of the luminescence signal reflects the increase of biomass. Therefore, cyclophosphamide immunosuppression seems best suited to follow the effect of antifungal drug treatment on clearance of fungal infections.
This study additionally allowed gaining new insight concerning the impact of different immune effector cells in the defense against invasive aspergillosis. Alveolar macrophages (AM) were assumed to play an important role in clearance of conidia from tissues and provide a "first-line of defense" against A. fumigatus
]. AM are thought to trigger the recruitment of immune effector cells to the site of infection after recognition and phagocytosis of conidia [27
] through the release of inflammatory and chemotactic mediators.
Due to the importance of AM in conidial host defense, we expected that their reduction by the clodrolip treatment would increase the susceptibility of mice to IA. This assumption was not confirmed experimentally. Intranasally clodrolip-treated mice showed a 80% reduction in the number and viability of AM [28
], but a 2.6 fold increase in the number of BAL fluid neutrophils, one day post-infection. A significant increase in the neutrophil number in BAL fluid of macrophage-depleted (clodrolip-treated) mice 24 hours after instillation of Pseudomonas aeruginosa
has already been reported. However, in this work macrophage-deficient mice showed impaired bacterial clearance [30
]. In contrast, in our model, neutrophil migration into the airways of macrophage-depleted infected mice is likely to have prevented conidial germination per se.
Supporting this idea, we found that the thoracic region or BAL fluid of AM-depleted animals only showed a slight increase in bioluminescence above control levels (Figure ). This finding correlated with survival data and histopathological findings, demonstrating an absence of conidial germination in AM-depleted mice. These results do not fully exclude the possibility that (i) there were still sufficient residual viable alveolar macrophages to trigger an immune response due to only 80% of depletion and (ii) the possibility of recruitment of peripheral monocytes or (iii) that the increase in recruited neutrophils was able to control the infection in the absence of normal alveolar macrophage counts. The first and second scenarios, however, appear rather unlikely, because hardly any macrophages or monocytes were observed in histopathologic analyses at day one after infection. The third scenario appears quite likely, because histopathological analysis revealed a strong infiltration of neutrophils encasing ungerminated conidia. In contrast, functionally attenuated neutrophils and macrophages in corticosteroid-treated mice allowed development of invasive disease despite robust cellular recruitment in the lung parenchyma.
The treatment of mice with cortisone acetate or the combination of clodrolip and cortisone acetate led to 100% mortality and invasive fungal growth within the lung tissue. Although systemic administration of corticosteroids increases the number of circulating neutrophils by three- to fivefold [31
], their ability to damage A. fumigatus
hyphae is strongly reduced [32
]. One day post-infection, the lung tissue showed an extensive neutrophilic infiltration that surrounded germinating conidia. These neutrophils were able to delay uncontrolled tissue invasion by killing some proportion of fungal hyphae. As a consequence of the neutrophil infiltration severe tissue damage accompanied by parenchymal destruction (necrosis) was observed, leading to a decreased bioluminescence as described above. It is also noteworthy that under cortisone acetate treatment the efficiency of alveolar macrophages in inhibiting conidial germination after phagocytosis was strongly defective. None of the other treatment groups yielded hyphal germlings as early as one day post-infection. It could be assumed that this rapid germination is due to growth stimulation via A. fumigatus
corticosteroid receptors [33
]. However, experiments, in which different concentrations of cortisone acetate were added to A. fumigatus
cultures, neither stimulated conidia germination, nor increased the light emission (data not shown). Since cortisone acetate itself constitutes an "inactive" corticosteroid derivative, which is converted into "active" cortisol during metabolism in the liver [34
], it might be possible that a stimulation of germination is only mediated by this metabolite rather than by cortisone acetate.
Another possibility for the rapid germination of conidia is given by a neutrophil mediated tissue destruction releasing large amounts of nutrients from tissue cells, which enhanced the germination speed under this immunosuppresive regimen. The mild inflammation under RB6-8C5 treatment one day post infection and the absence of inflammation under cyclophosphamide treatment may not provide the same nutritional conditions leading to a delayed germination when compared to the cortisone acetate treatment.
Another piece of evidence that supports the dependence on the number and functional integrity of neutrophils in the clearance of A. fumigatus
is the observation that RB6-8C5 treatment renders mice highly susceptible to IA. Our results confirm earlier studies [17
] and clearly demonstrate that AM and monocytes cannot functionally compensate for loss of neutrophils. RB6-8C5 treated mice succumbed to IA with a similar time course as cortisone acetate-treated mice. However, a notable difference between both models was the absence of neutrophils and the severe tissue infiltration by mononuclear cells (mainly macrophages) seen in RB6-8C5-treated mice at days three to four after infection. This tissue infiltration covered approximately 19% of the total lung surface and was more severe than observed in the cortisone acetate treatment group (approximately 11%).
Treatment with cyclophosphamide was assumed to have the strongest impact on the development of IA. It results in: (i) a reduction in the number of monocytes and neutrophils in the peripheral blood by 64 and 88%, respectively [37
], (ii) a reduction in the number of AM and neutrophils in an experimental lung infection with Streptococcus pneumoniae
], (iii) an impairment of phagocytosis [41
], (iv) an immune dysfunction through reactive oxygen intermediate-induced damage to the immune system cells [42
] without alteration of the degranulation process [38
] and finally (v) a failure in neutrophil chemotactic function [45
]. As expected, under this treatment, we did not observe inflammation within the infected tissues. Therefore, mice treated with cyclophosphamide succumb to uncontrolled infection resulting in tissue destruction and blood vessel infiltration by the fungal mycelium and the fungal biomass produced under this regimen was by far most pronounced at late time points (Figure and ). In contrast, cortisone acetate and RB6-8C5 treatment likely results in additional tissue injury due to the strong, but ineffective host inflammatory response.
Interestingly, the luminescence additionally enabled us to detect and monitor extrathoracic growth of A. fumigatus in particular in the sinus area even in cortisone acetate treated mice. The resulting suppurative sinusitis may indicate a defect in the innate immune response in the upper respiratory airway rather than dissemination.
Reflecting on the outcome of aspergillosis from the different infection models, we conclude that AM are likely to be important in orchestrating the early immune response to recruit other immune effector cells. However, although able to slow fungal outgrowth, AM are insufficient to clear the infection in the absence of neutrophils. Neutrophil depletion by the RB6-8C5 antibody leads to a predominately monocyte infiltration to the site of infection. Influx of mononuclear cells is insufficient to replace neutrophil function. Corticosteroid treatment leads to the most rapid germination of conidia, which may reflect functional inactivation of alveolar macrophages followed by the ongoing influx of neutrophils, which are attenuated in their conidial and hyphal killing mechanisms. Although after corticosteroid-treatment, neutrophils were not able to inactivate growing hyphae completely, they retarded hyphal tissue invasion at the great cost of massive tissue inflammation and necrosis. Therefore, preservation of neutrophil number and function is indispensable for the control and clearance of A. fumigatus infections. Macrophages may play an important role in orchestrating the immune response, but their action alone is not sufficient to combat A. fumigatus.
Our data suggest that the early neutrophil recruitment is crucial to form an efficient immune response against A. fumigatus
. This assumption is supported by two previous studies, which have reported that mice deficient in the chemokine receptor CXCR2 (CXCR2-/- mice) display a defect in neutrophil recruitment and were more susceptible to IA [36
]. Therefore, we conducted a preliminary investigation, in which we used a bioluminescent A. fumigatus
strain to monitor the pathogenesis of CXCR2-/- mice. This experiment revealed an overall average of 3-fold increase of bioluminescence signal within the thoracic region of knockout compared to wild type mice. At day 6 post infection, a 12 fold-increase in luminescence was observed in knockout animals with a mortality rate of more than 60%, whereas all immune competent wild-type mice survived (data not shown). Although this experiment has to be confirmed by characterizing the histological lesions, it fits well with the assumption that the early recruitment of immunocompetent neutrophils is one of the most important factors to combat the initial onset of invasive aspergillosis.