CLINICAL MICROBIOLOGY REVIEWS, July 2009, p. 447���465 Vol. 22, No. 3 0893-8512/09/$08.00 0 doi:10.1128/CMR.00055-08 Copyright �� 2009, American Society for Microbiology. All Rights Reserved. Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis Taylor R. T. Dagenais1 and Nancy P. Keller1,2* Medical Microbiology and Immunology, University of Wisconsin���Madison, Madison, Wisconsin,1 and Bacteriology, University of Wisconsin���Madison, Madison, Wisconsin2 INTRODUCTION.......................................................................................................................................................447 The Human Pathogen A. fumigatus ......................................................................................................................447 Invasive Aspergillosis .............................................................................................................................................448 Infectious life cycle .............................................................................................................................................448 Risk factors and pathology................................................................................................................................448 Animal Models of Invasive Aspergillosis.............................................................................................................449 Biology of Aspergillus fumigatus.............................................................................................................................450 AIRWAY COLONIZATION ......................................................................................................................................450 Aspergillus Interactions with Soluble Lung Components ..................................................................................450 Aspergillus Interaction with Respiratory Epithelia.............................................................................................451 Aspergillus and the Alveolar Macrophage............................................................................................................452 Macrophage responses to Aspergillus ...............................................................................................................452 A. fumigatus defenses..........................................................................................................................................453 (i) Melanin.......................................................................................................................................................453 (ii) Mediators of ROS defense......................................................................................................................454 ADAPTATION TO THE MAMMALIAN LUNG ENVIRONMENT ....................................................................454 Mammalian Tissue Degradation ..........................................................................................................................454 Nutrient Biosynthesis and Acquisition ................................................................................................................455 Iron Acquisition ......................................................................................................................................................456 Additional Environmental Stresses......................................................................................................................456 ASPERGILLUS AND THE NEUTROPHIL.............................................................................................................457 Role of the Neutrophil ...........................................................................................................................................457 A. fumigatus Hyphal Defenses ...............................................................................................................................457 ROS scavengers...................................................................................................................................................457 Secondary metabolites........................................................................................................................................458 DISSEMINATION......................................................................................................................................................459 CONCLUSION............................................................................................................................................................459 REFERENCES ............................................................................................................................................................460 INTRODUCTION Aspergillus species are ubiquitous, saprophytic fungi that play a significant role in global carbon and nitrogen recycling. Although their primary ecological niche is soil or decaying vegetation, aspergilli produce small, hydrophobic conidia that disperse easily into the air and can survive a broad range of environmental conditions. The genus Aspergillus, which in- cludes almost 200 species, has a tremendous impact on public health both beneficially as the workhorse of industrial appli- cations and negatively as plant and human pathogens (71). Several Aspergillus species are utilized for their rich enzymatic profile in the industrial production of foods and pharmaceuti- cals. For example, Aspergillus niger is used for the industrial production of citric acid, amylases, pectinases, phytases, and proteases A. terreus is used for the cholesterol-lowering drug lovastatin and A. oryzae is used for the fermentation of soy- beans and rice into soy sauce and sake, respectively. Aspergilli also have a less reputable side in the agricultural industry. Aspergillus section Flavi, particularly A. flavus and A. parasiti- cus, can contaminate several common crops with aflatoxin, a highly toxic carcinogen with immunosuppressive properties (228, 230). The consumption of contaminated crops can cause serious illness or death and is a common problem in develop- ing countries. The Human Pathogen A. fumigatus Among the human pathogenic species of Aspergillus, A. fu- migatus is the primary causative agent of human infections, followed by A. flavus, A. terreus, A. niger, and the model organ- ism, A. nidulans (54, 135). Aspergilli cause a wide range of human ailments depending on the immune status of the host (54, 107). In individuals with altered lung function such as asthma and cystic fibrosis patients, aspergilli can cause allergic bronchopulmonary aspergillosis, a hypersensitive response to fungal components. Noninvasive aspergillomas may form fol- lowing repeated exposure to conidia and target preexisting lung cavities such as the healed lesions in tuberculosis patients. Invasive aspergillosis (IA) is perhaps the most devastating of Aspergillus-related diseases, targeting severely immunocom- promised patients. Those most at risk for this life-threatening disease are individuals with hematological malignancies such * Corresponding author. Mailing address: University of Wisconsin��� Madison, 3467 Microbial Sciences Building, 1550 Linden Drive, Mad- ison, WI 53706. Phone: (608) 262-9795. Fax: (608) 263-2626. E-mail: npkeller@wisc.edu. 447

as leukemia solid-organ and hematopoietic stem cell trans- plant patients patients on prolonged corticosteroid therapy, which is commonly utilized for the prevention and/or treat- ment of graft-versus-host disease in transplant patients indi- viduals with genetic immunodeficiencies such as chronic gran- ulomatous disease (CGD) and individuals infected with human immunodeficiency virus (54, 97, 126, 133, 148, 162, 227). Mortality rates range from 40% to 90% in high-risk populations and are dependent on factors such as host immune status, the site of infection, and the treatment regimen applied (114). The severity and increased incidence of IA necessitate a better understanding of the interplay between host and fungus that contributes to A. fumigatus pathogenesis (130). Pathogen- esis and virulence are terms used here in the context of altered host immune function, as this organism is inherently an oppor- tunistic pathogen, and disease pathology and progression are the result of both fungal growth and the host response. In this review, we will thus discuss the pathogenic potential of A. fumigatus as a progression of the infectious life cycle within the context of these immunodeficiencies. Invasive Aspergillosis Infectious life cycle. Aspergilli are predominantly sapro- phytes, growing on dead or decaying matter in the environ- ment. The infectious life cycle of Aspergillus begins with the production of conidia (asexual spores) that are easily dispersed into the air, ensuring ubiquity in both indoor and outdoor environments (Fig. 1) (65, 137). The primary route of human infection is via the inhalation of these airborne conidia, fol- lowed by conidial deposition in the bronchioles or alveolar spaces. In healthy individuals, conidia that are not removed by mucociliary clearance encounter epithelial cells or alveolar macrophages, the primary resident phagocytes of the lung. Alveolar macrophages are primarily responsible for the phagocytosis and killing of Aspergillus conidia as well as the initiation of a proinflammatory response that recruits neu- trophils (one type of polymorphonuclear cell [PMN]) to the site of infection. Conidia that evade macrophage killing and germinate become the target of infiltrating neutrophils that are able to destroy hyphae. The risk of developing IA results primarily from a dysfunction in these host defenses in com- bination with fungal attributes that permit A. fumigatus sur- vival and growth in this pulmonary environment (176). Al- though other host responses have been associated with disease resistance, for this review, we will focus on fungal interactions with the primary innate components that are most important for fungal defense. Risk factors and pathology. The primary host immunodefi- ciencies that are responsible for the increased risk of IA are neutropenia and corticosteroid-induced immunosuppression, and the pathological consequences of IA under these immu- nosuppressive conditions differ, as described previously for patients and animal models (9, 17, 53, 192). Prolonged neu- tropenia is classically defined as the most dominant risk factor for IA and is often the result of highly cytotoxic therapies such as cyclophosphamide, which is used for transplant patients or those with hematological diseases. Cyclophosphamide, a DNA-alkylating agent, binds to DNA and interferes with cel- lular replication, depleting circulating white blood cells includ- ing neutrophils. In neutropenic patients and animal models of chemotherapy-induced neutropenia, IA is characterized by thrombosis and hemorrhage from rapid and extensive hyphal growth (41, 192). The lack of inflammatory infiltrates, despite the production of tumor necrosis factor alpha (TNF- ), results FIG. 1. Infectious life cycle of A. fumigatus. Aspergillus is ubiquitous in the environment, and asexual reproduction leads to the production of airborne conidia. Inhalation by specific immunosuppressed patient groups results in conidium establishment in the lung, germination, and either PMN-mediated fungal control with significant inflammation (corticosteroid therapy) or uncontrolled hyphal growth with a lack of PMN infiltrates and, in severe cases, dissemination (neutropenia). 448 DAGENAIS AND KELLER CLIN. MICROBIOL. REV.

in low levels of inflammation. Without neutrophil recovery, angioinvasion and dissemination to other organs via the blood result. A variety of nonneutropenic patients, most commonly those on corticosteroid therapy such as allogeneic transplant patients receiving corticosteroids for prophylaxis or treatment of graft- versus-host disease, are susceptible to IA, although the pathol- ogy of the disease is quite different. IA in these patients and nonneutropenic animal models is nonangioinvasive, character- ized by limited fungal development with pyogranulomatous infiltrates, tissue necrosis, and excessive inflammation. Corti- costeroids have significant consequences for phagocyte func- tion, including but not limited to the impairment of phagocy- tosis, phagocyte oxidative burst, production of cytokines and chemokines, and cellular migration (reviewed in reference 116). Several studies have shown that corticosteroids impair the functional ability of phagocytes to kill A. fumigatus conidia and hyphae (37, 92, 132, 171, 172, 214). Despite the effects of steroids on innate immune cell function, neutrophils are re- cruited to the lung and prevent hyphal invasion but create an inflammatory environment that results in tissue injury. This exacerbated inflammatory response is generally regarded as being the cause of death, in contrast to the uncontrolled fungal growth observed in neutropenic hosts. The dramatic differ- ences in both fungal development and host responses under each immunosuppressive regimen highlight the importance of studying Aspergillus pathogenesis within the context of host immune status and subsequent response to fungal infection. Animal Models of Invasive Aspergillosis Identification of the contribution of individual fungal com- ponents to overall pathogenicity requires the use of in vivo models of IA. Drosophila melanogaster (104, 115, 186) and Galleria mellonella (140, 163, 164, 166) have been applied to screen A. fumigatus mutants for virulence attributes owing to their ethical and financial advantages over the use of mamma- lian models. However, results should be interpreted with cau- tion, and interesting phenotypes should be reevaluated using a more applicable animal model. For example, the difference in temperature (flies and worms, which are unable to grow at 37��C, are grown at 25��C) is known to affect multiple fungal characteristics including growth rate and toxin production (see below), and clearly, these models cannot be used to assess pathological outcomes of infection that are relevant to human infection. Indeed, a recent study highlights the need for cau- tion in using Galleria those authors found that melanin mu- tants known to be less virulent in mammalian studies (see below) were more virulent in the G. mellonella model (86). A variety of vertebrates including rats, rabbits, birds, and guinea pigs have been used, but mouse models predominate due to the availability of genetically defined species and reagents (42). Outbred mice are commonly chosen because of their cost com- pared to that of inbred strains, but sufficient numbers should be used to establish reproducibility due to the inherent genetic variability within populations. On the other hand, although inbred mice offer the advantage of genetic reproducibility, studies between individual inbred strains can be vastly differ- ent, such that comparisons of multiple inbred strains may ben- efit studies of fungal pathogenesis. Specific genetic mouse models exist. CGD (p47phox / ) or X-CGD (gp91phox / ) mice display pathological consequences (such as peribronchiolar and alveolar necrosis) of A. fumigatus infection similar to those for humans with CGD and have been a useful model for studying aspergillosis in the context of this specific genetic disease (136, 161). The importance of pattern recognition receptors (PRRs) to fungal recognition and mod- ulation of host responses has been clarified with the use of knockout mice, such as those for dectin-1 and several of the Toll-like receptors (TLRs) (142). Cytokine-deficient mice have also been used to demonstrate the contribution of cytokines to host resistance (such as TNF- ) or susceptibility (interleu- kin-10 [IL-10]) (33, 40, 158). The most commonly used animal models of IA involve the induction of neutropenia or corticosteroid-induced immuno- suppression to mimic human infection. Neutropenia may be induced by cyclophosphamide or other chemotherapeutic agents (antibody-mediated neutrophil depletion has also been used), whereas animals treated with corticosteroids represent the nonneutropenic model used to evaluate A. fumigatus patho- genesis in the context of inflammatory responses commonly ob- served in nonneutropenic patients. The use of specific drug or depletion regimens is known to influence survival, pathology, and other outcome parameters (191). Comparison of both models can help to differentiate the fungus-host interactions responsible for pathogenesis in unique patient populations. One of the most striking examples of this is in the case of gliotoxin mutants, which demonstrate wild-type virulence in a neutropenic model but re- duced virulence in a nonneutropenic model, suggesting that glio- toxin may be important for pathogenicity only in the context of nonneutropenic hosts (Table 1). Other variables to account for when establishing an appropri- ate animal model to assess fungal pathogenesis include the amount of fungal inoculum, route of infection, and outcome anal- yses (58). Conidial inoculation may be performed intratracheally, intranasally, intravenously, or via inhalation chamber. Intranasal inoculation is commonly used because of ease of handling, al- though chamber inhalation is potentially the most useful model in terms of both reproducibility and mimicking human infection (182, 190). Outcome analyses often chosen for assessing disease development include animal survival, organ pathology, host cel- lular responses, and fungal burden, all of which can be influenced by the variables described above (191). The interaction of fungi with mammalian cells in vitro can be a useful complement to in vivo studies and can steer experi- ments toward the appropriate in vivo assays. For example, although a gliZ gliotoxin mutant displayed virulence similar to that of a wild-type strain in the neutropenic mouse model, gliotoxin production did contribute to neutrophil apoptosis in vitro, supporting the observed virulence reduction of gliotoxin mutants in a nonneutropenic model and reduced neutrophil apoptosis at sites of infection (25, 186, 197). In vitro studies with primary mammalian cells and cell lines are frequently used to assess the role of specific fungal components during fungus-host cell interactions, although A. fumigatus mutants that display altered interactions with host cells in vitro do not always correlate with virulence defects in vivo, particularly when the only in vivo assessment made is animal mortality. This is in agreement with the multifactorial nature of A. fu- migatus pathogenesis and emphasizes the significance of exam- VOL. 22, 2009 VIRULENCE OF ASPERGILLUS FUMIGATUS 449