The objectives of this study were to examine the essential role and temporal requirements for alveolar macrophages and neutrophils in host defense against A. fumigatus. An important finding is the redundancy of alveolar macrophages for murine survival, neutrophil recruitment, and inflammatory responses in a pulmonary infection model. This conclusion is based on a compartmentalized macrophage depletion strategy rather than systemic impairment with a broadly immunosuppressive agent. A second finding relates to the brief period of host susceptibility to neutrophil depletion after infection. Neutrophil influx to the lungs in the first 6 h after infection is sufficient to protect mice from invasive disease.
The finding that alveolar macrophage-depleted mice sustain neutrophil recruitment and the synthesis of inflammatory mediators may appear surprising. However, this finding has been reported in other pulmonary infection models, such as those for infection due to Klebsiella pneumoniae
and Pseudomonas aeruginosa
]. Preserved neutrophil recruitment may reflect a role for epithelial cells and endothelial cells in phagocytic and inflammatory responses against A. fumigatus
]. In addition, natural killer cells and CCR6+
myeloid dendritic cells participate in inflammatory responses in neutrophil-depleted mice [20
]. The saturable uptake of conidia by alveolar macrophages in vitro [11
] and the dispersal of conidia observed in lung sections suggest that it is highly unlikely that the small number of alveolar macrophages that escape in vivo depletion can compensate for the profound loss of these cells in the generation of inflammatory responses. More likely, alveolar macrophage depletion may result in a transient reduction in the synthesis of inflammatory mediators in the period immediately after infection (<24 h). However, any alveolar macrophage-dependent reduction is readily compensated within a time frame that permits intact neutrophil airway recruitment and control of conidial germination.
Although these studies argue against an essential, nonredundant role for alveolar macrophages against A. fumigatus,
this conclusion does not preclude a role for these cells in optimal responses against A. fumigatus.
Because these studies did not employ broadly immunosuppressive or myelotoxic strategies to render mice susceptible to low conidial inocula (at the cost of damaging multiple leukocyte subsets), we do not exclude the possibility that alveolar macrophages have the capacity to clear small inocula without participation of other cell subsets. Consistent with this possibility, there is evidence that mice administered cytotoxic chemotherapy may become susceptible to targeted macrophage depletion [42
]. Thus, in patients with significant myeloid injury, the slow turnover and relative resistance of alveolar macrophages to irradiation [43
] may enable these cells to maintain a cellular defense against inhaled conidia after the production, trafficking, or function of short-lived leukocytes (eg, neutrophils) has been impaired.
The requirement for neutrophils in the period immediately after infection period is consistent with an essential role for these cells in preventing conidial germination at the earliest stage after infection [17
]. Our results suggest that there is a critical time window for neutrophil recruitment for protection to occur. Induction of neutropenia beyond 6 h after infection did not result in murine mortality, even when inocula were standardized for the time point of depletion. Neutrophils inactivate conidia and prevent their germination by phagocytic and nonphagocy tic mechanisms [17
]. Although extravasated neutrophils undergo apoptosis rapidly, the rapid establishment of neutrophil-dependent extracellular defense mechanisms (eg, lactoferrin release, the formation of BAL aggregates, and DNA extracellular traps) may serve to control conidial germination and hyphal tissue invasion, even with subsequent interruption in neutrophil influx.
The results presented in this study lend a cautionary note to cell depletion strategies that target macrophages and neutrophils. We found that clodronate liposomes administered systemically eliminate phagocytic cells that promote the clearance of antibody-coated neutrophils. Neutrophil and macrophage depletion strategies are often combined in animal models of inflammatory and infectious disease [46
]. Therefore, it is imperative to verify that the depletion strategy of a particular cell type does not unintentionally interfere with depletion of a second cell subset.
Our findings relate to infection control practices in high-risk patient populations. A major clinical goal is to prevent opportunistic infections in these populations, in part through reduction in exposure and administration of antimicrobial prophylaxis. Acute infection in high-risk hosts is a likely route to the development of invasive aspergillosis. However, a second possibility is that exposure prior to the onset of immune suppression or myeloblation may enable conidia to persist long enough in a viable form to establish invasive disease in the context of incipient or progressive myeloid injury [48
]. The results presented in this study are most consistent with the first possibility, because of the rapid resistance of mice to transient neutrophil depletion after infection, despite a significant viable burden of spores in the lung. Although a prior history of invasive aspergillosis has a deleterious effect on the outcome of allogeneic stem cell transplantation [49
], the window of susceptibility following conidial exposure to administration of corticosteroids or cytotoxic agents remains unknown. Understanding both the specific requirements for and the critical timing of innate immune cell action in relation to exposure is highly relevant to the formulation of strategies to protect vulnerable hosts during periods of highest risk.