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Eukaryotic Cell, November 2007, p. 1953-1963, Vol. 6, No. 11
1535-9778/07/$08.00+0 doi:10.1128/EC.00274-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
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Infectious Disease Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021,1 Division of Infectious Diseases of the Department of Medicine and Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 104612
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INTRODUCTION |
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 Top INTRODUCTION REFERENCES |
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In immunocompromised hosts, A. fumigatus represents a major cause of morbidity and mortality. This patient population is expanding due to the increasing use of transplantation for end organ disease, the development of immunosuppressive and myeloablative therapies for autoimmune and neoplastic disease, and the human immunodeficiency virus/AIDS pandemic (38). A. fumigatus is the most common invasive mold infection in these patients, and mortality rates exceed 50% in high-risk groups, such as leukemic patients and hematopoietic stem cell transplant recipients (74). Sensitivity to A. fumigatus antigens is associated with asthma, the prevalence of which is increasing in the developed world, though proving causation has been difficult (49, 54). Regardless, this increased prevalence brings a parallel rise in the number of individuals predisposed to allergic bronchopulmonary aspergillosis, a disease associated with aberrant responses to Aspergillus antigens in the setting of chronic inflammation. The spectrum of invasive, semi-invasive, and allergic disease caused by A. fumigatus is reviewed in several outstanding articles (9, 94).
The study of A. fumigatus molecules involved in virulence has been hampered by the lack of an identifiable sexual cycle, limiting classical genetic analysis (21). A recent study, however, indicates that A. fumigatus encodes distinct mating-type loci and the pheromone machinery required for sexual mating (103). Nonetheless, within the past decade, researchers have developed and refined experimental tools to generate mutant strains by homologous recombination (21, 35, 62, 152, 154), utilized RNA interference to repress endogenous transcripts (95), and expressed heterologous genes in A. fumigatus under the control of drug-inducible regulatory elements (145). The completion of the A. fumigatus genome (98) has accelerated gene structure and function studies and made possible comparative genomic analyses with other sequenced Aspergillus species (Aspergillus oryzae and Aspergillus nidulans), as well as other genera of pathogenic (e.g., Candida albicans and Cryptococcus neoformans) and nonpathogenic (e.g., Saccharomyces cerevisiae) fungi. An important insight from the genomes has been that A. fumigatus does not share a common set of genes with other fungal pathogens (98).
The types of hosts that are susceptible to invasive aspergillosis and the lack of unique pathways conserved among pathogenic fungi underscore the importance of the host contribution to pathogenesis. Damage from A. fumigatus can result from fungal growth and tissue invasion or from inflammatory cells recruited to sites of infection (130). Included in the latter are responses that are ineffective in clearing the organism, occur in the process of immune reconstitution, or are associated with allergy. For example, in a murine model of chronic granulomatous disease, in which mice have defective phagocyte oxidase systems, administration of killed hyphae results in chronic inflammation due to persistence of fungal elements (92). From the perspective of the mammalian immune system, A. fumigatus represents an organism with continuous respiratory tract exposure that must be cleared from terminal airways with an immune response calibrated to avoid fungal tissue invasion, as well as inflammation-induced tissue damage. Here, we review our growing understanding of the interface between A. fumigatus and host defense mechanisms, with an emphasis on invasive disease in humans and small animal models.
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A. FUMIGATUS IN THE MAMMALIAN ENVIRONMENT |
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200 Aspergillus species cause human disease, and among these, A. fumigatus is the predominant isolate, a finding that remains incompletely understood. The frequency with which A. fumigatus causes disease has been ascribed to external environmental prevalence or enhanced virulence (122, 137). Supporting the former are epidemiologic studies showing that A. fumigatus predominates over other genera of filamentous fungi and/or Aspergillus species in certain environments (5, 69) and those showing a high degree of genetic diversity among A. fumigatus isolates, without clustering between clinical and environmental isolates (36). Others, however, have not found this species to predominate (100). In a hospital survey, A. fumigatus represented <1% of airborne mold spores yet accounted for nearly 50% of patient isolates (122). In contrast, A. niger constituted more than half of the airborne isolates but only 17% of patient isolates, suggesting that A. fumigatus, compared to other aspergilli, is suited to colonize and persist within the human respiratory tree. To survive within human hosts, A. fumigatus can bypass mucociliary clearance by virtue of its small airborne spores (2 to 3 µm in diameter), exhibits thermotolerant growth, and relies on biosynthetic pathways evolved to counter hostile environments within its ecological niche. A. fumigatus virulence is a polygenetic trait; single-gene virulence factors, such as C. neoformans phospholipase B (32), have not been identified. Polygenetic factors that influence the conidial size, rate of germination, and conidial resistance to host killing mechanisms likely shape the outcome of infection in permissive hosts. Once conidia have germinated, factors that influence hyphal growth rates and resistance to killing mechanisms, tissue invasion and dissemination, and production of secondary metabolites have the potential to influence pathogenesis. However, these factors are secondary to the immune status of the host.
Thermotolerance.
Thermotolerance facilitates A. fumigatus growth over less thermotolerant aspergilli not only within decaying organic matter, its ecological niche, but also within the mammalian respiratory tree. The molecular basis of thermotolerance remains unknown. Deletion of the cgrA gene, which encodes a nucleolar protein involved in ribosome biogenesis, impairs growth in vitro at 37°C but not at 22°C (16). A 
cgrA strain is hypovirulent in a murine model of invasive aspergillosis but not in a fruit fly model of fungal disease at ambient temperature. Deletion of the thtA gene impairs growth at 48°C but does not affect virulence, an outcome that is not surprising, given that growth is maintained at 42°C (30). Both cgrA and thtA, whose function is unknown, have homologs in nonpathogenic and nonthermophilic fungi. To date, DNA microarray analyses have failed to identify a conserved set of genes that confer thermotolerance or facilitate fungal growth at different temperatures (98).
The conidial surface.
The outer conidial surface contains protrusions, termed rodlets, that impart hydrophobic properties important in conidial dispersal. rodA conidia lack rodlets and display enhanced sensitivity to alveolar-macrophage killing. However, deletion of the rodA gene does not impact virulence in a murine pulmonary-infection model (104, 138). Thus, in a complex system, lack of rodlets does not translate into a difference in pathogenicity compared to wild-type controls or reconstituted mutants. The molecular features of the conidial surface are represented in Fig. 1, together with significant interactions with mammalian factors that impact fungal growth and survival.
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The cell wall.
The cell wall consists predominately of a polysaccharide matrix comprised of 
-(1,3) glucan; ß-(1,3) glucan, some of which contains ß-(1,6) branches; linear ß-(1,3), ß-(1,4) glucan; chitin; and galactomannan (68). All three predicted -glucan synthase genes (ags1, ags2, and ags3) have been deleted. While the ags1 mutant displays the most profound -glucan synthetic deficit (10), the ags3 strain causes enhanced virulence in a murine model of invasive aspergillosis (80). ags3 conidia and hyphae contain -glucan levels similar to those of wild-type cells, suggesting that the mutant phenotype is due to other effects of ags3 disruption; these include increased conidial melanin content, rapid germination, and relative conidial resistance to reactive oxygen species (80). The ags3 mutant and two other hypervirulent mutants characterized to date exhibit one or more of these conidial phenotypes (Fig. 2).
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Afpig-a strain is hypovirulent in a neutropenic mouse model of pulmonary aspergillosis, a phenotype that may reflect slower growth. However, the absence of melanin and inability to traffic GPI-linked proteins to the plasma membrane and cell wall may contribute, as well (71).
In contrast, disruption of the GPI-linked protein Ecm33p enhances virulence in cyclophosphamide-treated mice (119). This finding may depend on delayed host clearance of ecm33 conidia (29). Though ecm33 conidia germinate more rapidly than wild-type conidia (119), which could contribute to hypervirulence, this phenotype is also present in the hypovirulent GPI anchor-deficient strain, as noted above. The ecm33 strain gives rise to large, chitin-rich conidia that form linear chains with a separation defect (29, 119). Although ecm33 conidia are more resistant to killing by alveolar macrophages and neutrophils than control cells, ecm33 hyphae are more susceptible to neutrophil-dependent killing.
These examples demonstrate that fungal cell wall composition profoundly influences virulence in immunocompromised mice. While specific genetic deletions can yield hypervirulent Aspergillus strains that germinate and grow more rapidly in vivo, disruption of key biosynthetic pathways more commonly results in growth-delayed phenotypes and strains with reduced virulence in animal models of invasive aspergillosis (84, 85, 96). In addition, fungal cell wall composition has the potential to affect the host immune response, a finding that is discussed below.
Pigment biosynthesis. Many fungi synthesize melanin pigments that provide a protective barrier against UV radiation and serve to maintain the genomic integrity of long-lived cells, such as spores. In the context of infection, melanins counter host responses by diminishing fungal cell phagocytosis and intracellular trafficking to acidified compartments, as well as by increasing fungal resistance to reactive oxygen species and to cell lysis (22).
A. fumigatus conidia appear gray-green due to accumulation of 1,8-dihydroxynaphthalene-melanin (22, 156) and the loss of polyketide synthase activity involved in pigment biosynthesis (PksP) yields strains with smooth, white conidia (65, 140, 141). In vitro, pksP conidia induce neutrophils to release greater amounts of reactive oxygen species than wild-type conidia (65) and undergo phagocytosis (140) and trafficking to phagolysosomes more readily (56). In a murine model of invasive aspergillosis, the pksP strain displayed reduced virulence compared to a wild-type strain (55, 140). Although pksP expression is triggered in conidia and hyphae recovered from infected mice (66), melanization of hyphae growing in vivo has not been demonstrated (156). Interestingly, loss of pksP function alters the conidial cell surface, resulting in enhanced exposure of ß-(1,3) glucan, a polysaccharide that forms a major target of the mammalian innate immune system (discussed below) (76). Thus, pksP-deficient cells are likely to encounter more robust inflammatory responses that may account for the decrease in virulence observed with the mutant strain.
Resistance to oxidative stress.
A. fumigatus detoxifies oxidative threats via glutathione synthesis and oxidoreductase activity, pathways conserved among pathogenic and nonpathogenic fungi (137). The protein products of four catalase and four superoxide dismutase genes catalyze the breakdown of H2O2 and superoxide radicals, respectively. Catalase deletion mutants (catA, cat1, and cat2) are more sensitive to H2O2, but not to phagocyte killing in vitro (105). The catA and cat1 mutants are as virulent as wild-type strains in immunocompromised mice (26), while the cat1 cat2 strain displays diminished histopathological lesions, suggesting that H2O2 may not be the predominant reactive oxygen species responsible for fungal killing.
Enhanced resistance to reactive oxygen species was observed in a strain in which three fatty acid oxygenase genes (ppoA, ppoB, and ppoC) are transcriptionally repressed by RNA interference (142). The triple-ppo-silenced strain was hypervirulent in a murine model of invasive aspergillosis. The mechanism underlying its phenotype remains unclear but does not appear to involve changes in the synthesis of prostagalandins or gliotoxin, a mycotoxin.
Gliotoxin. Gliotoxin is readily detected in human aspergillosis (70) and exerts immunosuppressive properties on host leukocytes by blocking phagocytosis and transcription of inflammatory mediators (99) and inducing apoptosis of neutrophils and monocytes (126, 147). In vitro, gliotoxin exhibits ciliostatic properties on respiratory epithelial cells (1). Strains of A. fumigatus that do not produce gliotoxin appear less virulent than gliotoxin-producing strains (132). Thus, gliotoxin has long been suspected to contribute to the pathogenesis of invasive aspergillosis.
Three independent groups have disrupted a nonribosomal peptide synthetase encoded by gliP within the putative gliotoxin biosynthetic cluster (33, 63, 131). The gliP strains failed to produce gliotoxin, and no morphological or developmental phenotypes were detected. The first two groups reported no difference in survival after infection with the gliP, parental, or complemented strains in neutropenic mice (33, 63). Similar results were obtained with a gliZ strain that lacked a transcription factor required for gliotoxin biosynthesis (17). However, the third group recently demonstrated reduced virulence of a gliP strain in a low inoculum model of pulmonary aspergillosis in corticosteroid-treated mice (131). These data suggest that gliotoxin is dispensable for A. fumigatus virulence in some hosts, but not others, and do not preclude a role in virulence for numerous other mycotoxins produced by this organism.
Pathways that regulate fungal growth and morphogenesis.
Hyphal growth underlies tissue invasion in susceptible hosts and is subject to regulatory control by pathways that sense and respond to stress conditions, nutrient availability, and other environmental conditions. For example, the Ca2+-calmodulin-activated protein phosphatase calcineurin, a regulator of eukaryotic stress responses, is required for virulence in C. albicans and C. neoformans. In A. fumigatus, deletion of the calcineurin catalytic subunit (cnaA) is associated with a conidiation defect and slowed hyphal growth at 37°C (129). In vivo, the cnaA strain was markedly hypovirulent in two models of invasive aspergillosis.
Cyclic-AMP-dependent signals regulate the growth, development, and morphogenesis of several fungal pathogens and underlie, in part, the synthesis of virulence factors, such as the pigment and capsule in C. neoformans. In A. fumigatus, this pathway includes a regulatory heterotrimeric G protein (gpaB) (72), adenylate cyclase (acyA) (72), and the regulatory (pkaR) (159) and catalytic (pkaC1) subunits of protein kinase A (PKA) (73). All mutants in this signaling pathway (gpaB, pkaC1, and pkaR) examined in murine models of invasive aspergillosis were less virulent than wild-type strains. Germination was delayed in the gpaB, acyA, pkaC1, and pkaR strains, while hyphal growth was impaired in the acyA, pkAC1, and pkAR strains. The gpaB, acyA, and pkaR strains were more sensitive to oxidants in vitro than the parental strains, though all mutant strains contained melanin. In sum, these results implicate the cyclic AMP-dependent PKA signal cascade as a critical regulator of conidiation, development, growth, and stress responses.
Filamentous growth depends on fungal homologs of the Ras family of proteins with GTPase activity. In A. fumigatus, the nonessential rasB gene regulates germination, radial growth, and hyphal branching (41). rasB conidia germinate slowly, and rasB hyphae display increased and aberrant branching. As expected, the rasB strain was less virulent in vivo than control and complemented strains (41).
Nutrient uptake.
Acquisition of essential nutrients within the host environment is required for invasive growth. A. fumigatus acquires iron in mammalian hosts through the action of siderophores, negatively charged molecules that bind ferric iron and facilitate iron uptake and storage. The sidA mutant lacks the enzyme that catalyzes the first committed step in siderophore biosynthesis and displays reduced virulence in immunosuppressed mice (124). Deletion of the zinc-responsive transcriptional activator gene zafA impaired zinc homeostasis and uptake during in vivo fungal growth, resulting in loss of virulence in the mutant strain (91). Nitrogen uptake is another essential process for fungal growth within mammalian tissues. The A. fumigatus rhbA gene enhances growth on nitrogen-poor sources (101, 113) and its deletion diminished virulence in vivo (102).
A. fumigatus secretes catabolic enzymes, including peptidases, to degrade macromolecular polymers for nutrient uptake. Since the pulmonary parenchyma contains large quantities of elastin and collagen, the proteolytic breakdown of these polymers may represent a fungal virulence mechanism. Two secreted collagenolytic peptidases have been isolated, an alkaline serine protease of the subtilisin subfamily (AfAlp) that is expressed in the lungs of neutropenic mice (133) and a metalloprotease (MEP) (87). Single and double gene deletion mutants all display virulence similar to that of control strains in immunosuppressed mice (57, 88, 134). Similar results have been obtained with strains defective for a secreted aspartic peptidase (112) and an intracellular metallopeptidase (MepB) (52). Although a number of other proteases have been characterized (4, 11, 12, 98, 110, 111), there is no evidence to date that loss of specific protease genes impacts A. fumigatus virulence, a feature that may reflect redundancy.
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THE HOST RESPONSE TO A. FUMIGATUS |
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Experimental studies performed in the early 1980s in immunocompromised mice indicate a critical role for macrophages in conidial defense and postulate that neutrophils act primarily against products of germination (121). In these studies, corticosteroid treatment interfered with macrophage killing of resting conidia, suggesting that macrophage function is a critical conidial clearance mechanism. In contrast, nitrogen mustard-induced neutropenia rendered mice highly susceptible to intravenous administration of swollen conidia (121). The animals were relatively less susceptible to resting conidia, implying that neutrophils play a predominant role in killing hyphae. Neutrophils attach to and degranulate on hyphal surfaces in vitro (39).
Neutropenia has long been an associated risk factor for the development of invasive aspergillosis in humans, and several animal models have been utilized to study invasive aspergillosis under these conditions. Recent work has expanded the view of neutrophils in host defense against A. fumigatus and has indicated a key role for these cells in anticonidial defense (Fig. 3). In mice treated with a blocking antibody to CXCR2, the receptor for the neutrophil chemoattractants CXCL2/MIP-2 and CXCL3/KC, or that are genetically deficient in it, neutrophil recruitment into the airways is delayed, enabling conidia to escape alveolar macrophage control, germinate, and establish invasive disease (18, 81). Neutrophils form aggregates around conidia in the airways of immune competent mice in vivo; these structures prevent conidial germination without requiring conidial uptake (18).
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Molecular recognition of fungal cells.
Microbial recognition through secreted and membrane-bound germ line-encoded receptors is central to the function of innate immune cells and triggers effector responses, such as the release of microbicidal molecules, cytokines, and chemokines. Conidia, but not hyphae, bind the soluble receptor pentraxin-3 within terminal airways via galactomannan, an interaction that enhances uptake by alveolar macrophages and dendritic cells (43). Pentraxin-3–/– mice are highly susceptible to invasive aspergillosis, even without exogenous immune suppression (43). In vitro, recombinant pentraxin-3 enhances conidial killing by alveolar macrophages. In vivo, pentraxin-3 drives the pulmonary production of protective Th1-biased cytokines, such as interleukin 12 (IL-12) and gamma interferon (IFN-
), and enhances murine survival in a bone marrow transplantation model of invasive aspergillosis (44). Lung surfactant protein D interacts with conidia to augment neutrophil oxidative responses and confers protective effects in a corticosteroid-induced model of invasive aspergillosis (77) (Fig. 3).
Binding and uptake of conidia occurs through a variety of receptors (19, 58, 59, 107) with distinct leukocyte expression patterns. The signaling receptor DC-SIGN on human alveolar macrophages and lung dendritic cells binds conidial galactomannan and participates in conidial uptake, though the significance of this interaction for subsequent immune responses remains unclear (125). Host immune responses to fungal cells depend on several classes of transmembrane receptors that trigger signaling cascades. The mammalian Toll-like receptor (TLR) family consists of 11 members, and at least 2, TLR2 and TLR4, are thought to mediate inflammatory responses to A. fumigatus conidia. TLR2 and TLR4 both signal through the common soluble adaptor protein MyD88 to induce the production of cytokines and chemokines (e.g., tumor necrosis factor [TNF] and macrophage inflammatory protein 2/CCL2) and reactive oxygen intermediates (ROIs) (Fig. 3).
TLR2-deficient alveolar macrophages secrete 30 to 40% less TNF, a key cytokine mediator of anti-Aspergillus responses in mice and humans (82, 148), than control cells stimulated with resting or swollen conidia (7, 128). This has also been observed for macrophages isolated from other sources (50, 78, 97). Killed hyphae and soluble hyphal antigens stimulate TLR2-dependent inflammatory responses, as well (20). In vivo, TLR2–/– mice treated with vinblastine are more susceptible to invasive aspergillosis than wild-type counterparts (6). TLR2–/– mice treated with cyclophosphamide demonstrate an elevated lung fungal burden compared to control mice (14).
Human monocytes recognize hyphae in a TLR4-dependent manner, as judged by antibody blockade (146). Both TLR4-independent and -dependent inflammatory responses to hyphae have been reported for murine macrophages (78, 83, 97). However, loss of TLR4 function increases mortality in cyclophosphamide-treated mice compared to control animals (14). TLR2- and TLR4-dependent signaling pathways activate neutrophil antifungal responses (15), though the fungal ligands recognized by these receptors remain unknown.
Although conidia and hyphae activate TLR-dependent signals, loss of the TLR adaptor protein MyD88 is not associated with enhanced susceptibility to invasive aspergillosis in otherwise immune-competent mice (14). Cyclophosphamide-treated MyD88–/– mice die more rapidly of invasive aspergillosis than control animals. These data suggest that TLR/MyD88-independent signaling is critical for murine antifungal defense.
Antibody-blocking experiments and in vitro studies with genetically deficient macrophages demonstrate a TLR-independent A. fumigatus conidial and hyphal recognition mechanism via dectin-1 (45, 50, 128, 136), an NK-like C-type lectin-like receptor with an intracellular immunoreceptor tyrosine-based activation motif-like motif (3, 23, 24). Dectin-1 binds ß-(1,3) glucans from a variety of other human-pathogenic fungi (42, 127, 144), is phosphorylated, and signals through p72Syk kinase and CARD9, a caspase recruitment domain protein, to trigger inflammatory responses that include the release of TNF and IL-12, neutrophil chemoattractants, and ROIs (117, 143). CARD9–/– mice are highly susceptible to systemic infection with C. albicans (47), while dectin-1–/– mice are susceptible to Pneumocystis carinii (120) and, in one of two reported studies, to C. albicans (136).
Inhaled A. fumigatus conidia contain little surface ß-glucan and trigger marginal inflammatory responses (Fig. 3). The process of conidial germination alters the cell wall composition, and ß-(1,3) glucan is exposed during conidial swelling and hyphal growth (45, 50, 128). Thus, dectin-1 signaling is triggered in a stage-specific manner and provides a mechanism to focus fungicidal responses on spores that pose an invasive threat and to ignore resting conidia. This division may represent a mechanism to restrict inflammatory responses spatially, for example, to phagocytosed swollen conidia, and temporally in the face of ongoing exposure to airborne A. fumigatus spores.
Killing of fungal cells. Following conidial phagocytosis, swelling proceeds within alveolar macrophages and is required to activate NADPH oxidase-dependent killing mechanisms (109). This process may depend on fungal ß-(1,3) glucan exposure in the phagosome. Consistent with this notion, conidial killing by alveolar macrophages occurs independently of MyD88 function (79). Phagosomes that contain conidia fuse with endosomes and mature into phagolysosomal compartments. Phagolysosomal acidification leads to killing of internalized conidia, a process disrupted by the inhibitor bafilomycin A (53).
Deletion of the gp91 subunit of the NADPH oxidase complex results in a murine model of X-linked chronic granulomatous disease, and gp91phox–/– mice are uniformly susceptible to intratracheal challenge with a low-dose (<104) conidial inoculum (92), highlighting the critical importance of the superoxide anion (O2·–) and its derivatives, for example, the hydroxyl radical (OH·), in host defense against A. fumigatus. Neutrophils deficient in Rac2, a hematopoietic cell-specific Rho family GTPase, have multiple deficits that include cytoskeletal abnormalities and deficiency in superoxide production. Accordingly, Rac2–/– mice are more vulnerable to intravenous A. fumigatus challenge than wild-type mice (116), although this experimental model does not recapitulate the natural route of infection.
Myeloperoxidase (MPO)-deficient mice survived intranasal administration of 2 x 105 conidia, while gp91phox–/– mice did not (2). However, MPO–/– mice showed a higher lung fungal burden than wild-type mice, suggesting that MPO-dependent HOCl production may contribute to host defense. The in vivo contributions of reactive nitrogen intermediates to anti-Aspergillus defense remain undefined. Although conidia stimulate macrophage nitric oxide production in vitro (46), neutrophils, monocytes, and alveolar macrophages may kill conidia in a nitric oxide-independent manner (86).
Human neutrophils display an NADPH oxidase-independent conidial defense mechanism through the release of lactoferrin, a chelator that sequesters bioavailable iron and thus limits conidial germination (158) (Fig. 3). In light of ongoing conidial exposure, this pathway may be particularly important in patients with chronic granulomatous disease or high-risk patient populations with increased systemic iron stores (61).
Adaptive responses: T lymphocytes. T-cell responses to Aspergillus antigens are present in healthy individuals, likely due to universal exposure to the organism. Lymphoproliferative responses to conidia and hyphae reside mainly in the CD4+ T cell fraction (48, 106). While the full antigenic specificity of these cells has not been defined, responses to recombinant catalase and DPP V proteins, both of which are potent stimuli of antibody production, have been detected (11, 48, 75). However, neither nude nor SCID mice, which lack functional T cells or both T and B cells, respectively, are more susceptible to invasive aspergillosis than wild-type mice (153; M. Feldmesser, unpublished data), demonstrating the primacy of innate immune responses in preventing this disease. Nonetheless, more recently, roles for adaptive immunity have been examined.
Several lines of evidence support T cell participation in host defense. Allogeneic hematopoietic stem cell transplantation results in protracted susceptibility to invasive aspergillosis. After transplantation, the time to recovery of specific T cell responses is prolonged in comparison to that for other pathogens, such as cytomegalovirus (48). The presence of Th1-associated responses correlates with resistance to disease in animal models of pulmonary and systemic infection (27, 28, 48). Correlation of cytokine stimulation patterns from peripheral blood mononuclear cells with clinical outcome in human disease suggests that a high ratio of IFN- to IL-10 may be beneficial (48). In the first demonstration of clinical benefit of adoptive immunotherapy for invasive aspergillosis in humans, transfer of CD4+ T cell clones was associated with not only earlier recovery of Aspergillus-responsive CD4+ and CD8+ T cells, but a higher ratio of IFN- to IL-10 than was present in spontaneously recovering CD4+ T cell populations in untreated patients (106).
The mechanism by which these T cell responses benefit the host is incompletely understood. However, hyphal damage is significantly higher when the organism is incubated with T cells, antigen-presenting cells (APCs), and neutrophils than with neutrophils alone or with either T cells or APCs (13, 48). These findings support the ability of Aspergillus-responsive T cells to enhance neutrophil function.
The development of T cell responses following pulmonary infection has been examined in murine models and has yielded conflicting results. Following intratracheal administration, CD4+ T cells were detected in lung and mediastinal lymph nodes 3 days after infection, with peak responses at 7 to 10 days postinfection. While heat-killed conidia primed CD4+ T cells for Th2 development, with induction of IL-13 and IL-4 following subsequent stimulation, Th1-associated skewing of the response was seen following inoculation with live organisms, marked by a predominance of IFN- and TNF production (115). Dendritic cells exposed to hyphae in the lung primed CD4+ T cells in the thoracic lymph node and spleen to produce IL-4, a Th2-biased cytokine (19). These results suggest that the development of a beneficial Th1- or deleterious Th2-biased response depends, in part, on the fungal growth stage encountered by APCs.
However, in a second model examining Th polarization, also in immunocompetent mice, infection with live conidia via aerosol resulted in IL-25 expression in the lung. This Th2 cell- and mast cell-derived cytokine, which may be an important mediator of allergic responses, resulted in upregulation of other Th2-associated cytokines and development of eosinophilia, but through stimulation of a CD4– cell population (51). The variance in finding the development of Th1- or Th2-like environments following infection with live conidia may reflect differences in mouse strains, inocula, routes of administration, or A. fumigatus strains. Further, in the second study, CD4+ T cell phenotypes following infection were not directly studied.
In elegant studies using an A. fumigatus-specific T cell transgenic mouse, Rivera and coworkers demonstrated that development of CD4+ T cell responses is incremental following pulmonary infection, with rapid recruitment to regional lymph nodes and, following extensive proliferation, trafficking to the airways via MyD88-independent mechanisms (114). Though MyD88-mediated signaling enhances Th1 differentiation of CD4+ T cells in the regional lymph nodes, as determined by identification of cells that secrete IFN- upon further stimulation, IFN- secretion by Th1 cells in the airway is MyD88 independent (114). The contribution of dectin-1 signaling to the priming and differentiation of A. fumigatus-specific CD4+ T cell responses remains unknown.
A role for CD8+ T cells in the naturally occurring host response has not been demonstrated. In ovalbumin-specific T cell-transgenic mice, a commercial A. fumigatus extract can function as a selective CD8+ cell adjuvant, with induction of proliferation, IFN- production, and cytolytic activity (135). However, the demonstration that gliotoxin suppresses perforin-mediated cytolytic activity by a CD8+ cell clone in vitro suggests a potential means by which the organism may limit the efficacy of this cell population (155).
At present, our knowledge regarding the roles of regulatory T cells in host response is very limited, though given the prominent role played by inflammation in pathogenesis, these cells likely are very important. Lung CD25+ regulatory T cells reduce neutrophil TNF and ROI production in response to conidia (118). In murine models of invasive disease or allergy, two distinct CD4+ CD25+ T cell populations in the lung and thoracic lymph nodes play different roles in dampening inflammation (90). An early population in the lung, whose development is dependent upon the presence of the costimulatory molecule B7-2 (CD86), dampens the inflammatory response via IL-10 production and reduces neutrophil antihyphal activity at the expense of allowing fungal growth in response to invasive disease. In contrast, a late transforming growth factor ß-producing population found in the lymph nodes, whose development is dependent upon the presence of B7-1 (CD80), decreases allergic Th2-associated responses. Dendritic cell production of indolamine-2,3-dioxygenase, an enzyme involved in tryptophan catabolism in response to regulatory T cell conditioning, contributes to the suppressive activity of regulatory T cells and to development of the late regulatory T cell population (90). Thus, T cell populations may be important both for mounting optimal defenses against invasion that is associated with Th1-like polarization and for regulation, so that inflammation is limited when the organism is present in a form that can be handled by resident defenses. Much remains to be uncovered about the mechanisms for both functions.
B cells and antibody responses. Antibody responses to A. fumigatus result from environmental exposure in the absence of disease, occur in the course of either aspergilloma or invasive disease, or participate in hypersensitivity responses. Serum antibodies that bind Aspergillus components, particularly those of the immunoglobulin G (IgG) and IgM subclasses, are broadly found in patients without aspergillosis (8). In addition to systemic antibodies, isolation of Aspergillus-specific IgA with secretory component from bronchial lavage fluid suggests that significant mucosal responses also are made in those without aspergillosis (123). Many A. fumigatus hyphal molecules are potent B cell antigens, which are present among secreted, cell wall, and cytosolic fractions (37, 64, 157). Serum antibodies that bind these molecules are more commonly seen among patients with aspergilloma than among those with invasive disease, a finding that likely reflects differences in the predisposing conditions between these groups. In addition, over 80 proteins can bind IgE from sensitized patients (34), demonstrating the degree to which A. fumigatus can induce allergy, further discussion of which is beyond the scope of this review. Killed conidia do not elicit IgG responses to hyphal antigens following intratracheal inoculation of mice (115), a finding that could reflect the host's ability to limit the immune response when the organism cannot pose the threat of invasive disease but could also indicate differences in specificity. Conidial surface-specific antibody responses have not been studied.
However, antibodies are not believed to play a role in protection against invasive disease, and study of B cell-deficient µMT mice following induction of neutropenia suggests that antibody may be harmful (60, 89). Nonetheless, immune serum administered to B cell-deficient mice prolongs survival, suggesting a potential role for antibody in the initial host defense. More recently, monoclonal antibodies to laminarin [a ß-(1,3) glucan] and to an unidentified 97-kDa glycoprotein have demonstrated that antibody can prolong survival in experimental murine infection (31, 139).
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CONCLUSIONS |
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The fungal cell wall composition represents a critical interface for the host innate and adaptive immune responses. Both the fungal surface and the products secreted by the organism change substantially during the course of germination, resulting in dynamic interactions that modulate the host response. One very important example, and perhaps, the best understood, is that of stage-specific recognition of fungal ß-(1,3) glucan. This polysaccharide functions as an immunologic indicator of microbial growth and enables the immune system to focus potentially tissue-damaging inflammatory responses on fungal cells with invasive potential. Host defense mechanisms show striking redundancy at the levels of molecular recognition, antifungal effector functions, and cellular components. This redundancy likely contributes greatly to the requirement for host damage for development of invasive disease due to A. fumigatus.
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ACKNOWLEDGMENTS |
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T.M.H. received fellowship support from the Charles H. Revson Foundation. M.F. is supported by NIH grant R01AI059663.
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FOOTNOTES |
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Published ahead of print on 22 September 2007.
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REFERENCES |
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(1-3) glucan synthases with different functions in Aspergillus fumigatus. Appl. Environ Microbiol. 71:1531-1538.(1-3)glucan synthase gene family member of Aspergillus fumigatus, modulates mycelium growth in the lung of experimentally infected mice. Fungal Genet. Biol. 43:366-375.[CrossRef][Medline]
