Macrophage colony-stimulating factor enhances phagocytosis and oxidative burst of mononuclear phagocytes against Penicillium marneffei conidia

The responses of rabbit pulmonary alveolar macrophages (PAMs) and elutriated human monocytes (EHMs) to Penicillium marneffei, an emerging dimorphic fungus that may cause fatal disease in human immunodeficiency virus-infected patients, were studied. PAMs and EHMs comparably phagocytosed conidia of two P. marneffei strains in the presence of serum. Electron microscopy showed intraphagosomal destruction of conidia after 12 h. Serum-opsonized conidia elicited significantly more superoxide anion (O(2)(-)) release from EHMs compared to non-opsonized conidia, but equivalent O(2)(-) amounts to that elicited by serum-opsonized Aspergillus fumigatus conidia. Macrophage colony-stimulating factor (M-CSF) significantly enhanced phagocytosis of P. marneffei conidia by PAMs and EHMs, as shown by light microscopy. Moreover, M-CSF enhanced O(2)(-) production by EHMs in response to both serum-opsonized (P<0.001) and non-opsonized (P=0.03) conidia of A. fumigatus as well as conidia of the P. marneffei isolates (P<0.001 and 0.03). We conclude that M-CSF enhances phagocytosis and oxidative metabolism of mononuclear phagocytes suggesting a potential role for this cytokine in host defense against pulmonary and disseminated P. marneffei infection.     

Macrophage inflammatory protein-1 alpha is a critical mediator of host defense against invasive pulmonary aspergillosis in neutropenic hosts

Invasive pulmonary aspergillosis is a devastating complication of immunosuppression that usually occurs in neutropenic patients. In this setting, augmentation of the antifungal activity of available immune cells may improve the outcome of the infection. Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is a CC chemokine with potent chemotactic activity for various subsets of mononuclear leukocytes. We therefore tested the hypothesis that the influx of mononuclear cells into the lung in invasive pulmonary aspergillosis is in part mediated by MIP-1 alpha, and the manipulation of this ligand alters the outcome of the infection. We found that in both immunocompetent and neutropenic mice, MIP-1 alpha was induced in the lungs in response to intratracheal administration of Aspergillus fumigatus conidia. In neutrophil-depleted mice challenged with intratracheal conidia, there was evidence of invasive fungal pneumonia associated with a predominantly mononuclear leukocyte infiltrate. Ab-mediated depletion of MIP-1 alpha resulted in a 6-fold increase in mortality in neutropenic mice, which was associated with a 12-fold increase in lung fungal burden. Studies of single-cell suspensions of whole lungs revealed a 36% decrease in total lung leukocyte infiltration as a result of MIP-1 alpha neutralization. Flow cytometry on whole lung suspensions showed a 41% reduction in lung monocyte/macrophages as a result of MIP-1 alpha neutralization, but no difference in other lung leukocyte subsets. These studies indicate that MIP-1 alpha is a critical mediator of host defense against A. fumigatus in the setting of neutropenia and may be an important target in devising future therapeutic strategies against invasive aspergillosis.     

CXC chemokine receptor-2 ligands are necessary components of neutrophil-mediated host defense in invasive pulmonary aspergillosis

Invasive pulmonary aspergillosis is a devastating complication of immunosuppression, which occurs in association with neutrophil dysfunction or deficiency. ELR+ CXC chemokines are a subfamily of chemokines that play a critical role in neutrophil chemotaxis and activation both in vitro and in vivo. We hypothesized that interaction of these ligands with CXC chemokine receptor-2 (CXCR2), their sole murine receptor, is a major component of neutrophil-dependent pulmonary host defense against Aspergillus fumigatus. In immunocompetent animals, neutrophils were recruited to the lung in response to intratracheally administered A. fumigatus conidia. In a model of transient in vivo depletion of neutrophils, animals developed invasive pulmonary aspergillosis, associated with delayed influx of neutrophils into the lung. In both normal and neutrophil-depleted animals, the ELR+ CXC chemokines MIP-2 and KC were induced in response to intratracheal administration of conidia. Ab-mediated neutralization of the common ELR+ CXC chemokine receptor, CXCR2, resulted in development of invasive disease indistinguishable from the disease in neutrophil-depleted animals, while control animals were highly resistant to the development of infection. CXCR2 neutralization was associated with reduced lung neutrophil influx and resulted in a marked increase in mortality compared with controls. In contrast, animals with constitutive lung-specific transgenic expression of KC were resistant to the organism, with reduced mortality and lower lung burden of fungus. We conclude that CXCR2 ligands are essential mediators of host defense against A. fumigatus, and may be important targets in devising future therapeutic strategies in this disease.     

Human polymorphonuclear leukocytes inhibit Aspergillus fumigatus conidial growth by lactoferrin-mediated iron depletion

Aspergillus fumigatus, a common mold, rarely infects humans, except during prolonged neutropenia or in cases of chronic granulomatous disease (CGD), a primary immunodeficiency caused by mutations in the NADPH oxidase that normally produces fungicidal reactive oxygen species. Filamentous hyphae of Aspergillus are killed by normal, but not CGD polymorphonuclear leukocytes (PMN); however, the few studies on PMN-mediated host defenses against infectious conidia (spores) of this organism have yielded conflicting results, some showing that PMN do not inhibit conidial growth, with others showing that they do, most likely using reactive oxygen species. Given that CGD patients are exposed daily to hundreds of viable A. fumigatus conidia, yet considerable numbers of them survive years without infection, we reasoned that PMN use ROS-independent mechanisms to combat Aspergillus. We show that human PMN from both normal controls and CGD patients are equipotent at arresting the growth of Aspergillus conidia in vitro, indicating the presence of a reactive oxygen species-independent factor(s). Cell-free supernatants of degranulated normal and CGD neutrophils both suppressed fungal growth and were found to be rich in lactoferrin, an abundant PMN secondary granule protein. Purified iron-poor lactoferrin at concentrations occurring in PMN supernatants (and reported in human mucosal secretions in vivo) decreased fungal growth, whereas saturation of lactoferrin or PMN supernatants with iron, or testing in the presence of excess iron in the form of ferritin, completely abolished activity against conidia. These results demonstrate that PMN lactoferrin sequestration of iron is important for host defense against Aspergillus.     

Specificity of antigens from pathogenic Aspergillus species. I. Studies with ELISA and immunofluorescence

Studies were made by enzyme linked immunosorbent assay (ELISA) and indirect fluorescent antibody (IFA) tests on the reactivities and specificities of 13 antigens prepared from four species of Aspergillus against antisera from immunized rabbits and 64 sera from patients with aspergillosis, other systemic mycoses and nocardiosis. Although reactions in both serological tests were invariably strongest with homologous antigen: antibody systems, antisera from rabbits immunized with A. fumigatus, Blastomyces dermatitidis, Candida albicans and Paracoccidioides brasiliensis reacted in the ELISA test with all of the Aspergillus antigens. In contrast, cross-reactivity was virtually non-existent with antiserum to Histoplasma capsulatum. Of five antigens prepared from A fumigatus tested by ELISA against human sera from patients with aspergillosis and other nocardial and systemic fungal infections, sensitivities varied from 81 to 100% for sera from 32 patients with aspergillosis, and specificities from 20 to 97% for sera from 30 patients with nocardiosis and other systemic mycoses. Purified A. fumigatus C antigen reacted weakly with sera from eight of these 30 patients, but the reactions were readily distinguishable from those obtained with sera from patients with aspergillosis. At optimal serum dilutions, cross-reactivities of A. fumigatus in the IFA studies were non-existent in the sera from 28 patients with candidosis, coccidioidomycosis, cryptococcosis, histoplasmosis, paracoccidioidomycosis and nocardiosis. Sensitivities of IFA were 94% for patients with aspergilloma and 83% for patients with allergic bronchopulmonary aspergillosis.     

Neutropenia enhances lung dendritic cell recruitment in response to Aspergillus via a cytokine-to-chemokine amplification loop

Current understanding of specific defense mechanisms in the context of neutropenic infections is limited. It has previously been reported that invasive aspergillosis, a prototypic opportunistic infection in neutropenic hosts, is associated with marked accumulation of inflammatory dendritic cells (DCs) in the lungs. Given recent data indicating that neutrophils can modulate immune responses independent of their direct microbial killing, we hypothesized that neutropenia impacts the host response to Aspergillus by determining the migration and phenotype of lung DCs. Inflammatory DCs, but not other DC subsets, were found to accumulate in the lungs of neutropenic hosts challenged with killed or live-attenuated Aspergillus as compared with nonneutropenic hosts, indicating that the accumulation was independent of neutrophil microbicidal activity. The mechanism of this accumulation in neutropenic hosts was found to be augmented influx of DCs, or their precursors, from the blood to the lungs. This effect was attributable to greatly elevated lung TNF expression in neutropenic as compared with nonneutropenic animals. This resulted in greater lung expression of the chemokine ligands CCL2 and CCL20, which, in turn, mediated enhanced recruitment of TNF-producing inflammatory DCs, resulting in a positive feedback cycle. Finally, in the context of neutropenic invasive aspergillosis, depletion of DCs resulted in impaired fungal clearance, indicating that this mechanism is protective for the host. These observations identify what we believe is a novel defense mechanism in invasive aspergillosis that is the result of alterations in DC traffic and phenotype and is specific to neutropenic hosts.     

A nonredundant role for plasmacytoid dendritic cells in host defense against the human fungal pathogen Aspergillus fumigatus

While plasmacytoid dendritic cells (pDCs), a natural type I interferon (IFN)-producing cell type, are regarded as critical for innate immunity to viruses, their role in defense against fungal infections remains unknown. We examined the interactions of pDCs with hyphae of the invasive human fungal pathogen Aspergillus fumigatus. Human pDCs spread over hyphae and inhibited their growth. Antifungal activity was retained in pDC lysates, did not require direct fungal contact, and was partially reversed by zinc. Incubation with hyphae resulted in pDC cytotoxicity, partly due to fungal gliotoxin secretion. Following hyphal stimulation, pDCs released proinflammatory cytokines via a TLR9-independent mechanism. Pulmonary challenge of mice with A. fumigatus resulted in a substantial influx of pDCs into lungs, and pDC-depleted mice were hypersusceptible to invasive aspergillosis. These data demonstrate the antifungal activity of pDCs against A. fumigatus and establish their nonredundant role in host defenses against invasive aspergillosis in vivo.     

Dendritic cells transport conidia and hyphae of Aspergillus fumigatus from the airways to the draining lymph nodes and initiate disparate Th responses to the fungus.

Aspergilli are respiratory pathogens and pulmonary infections are usually acquired through the inhalation of conidia, able to reach small airways and the alveolar space where the impaired host defense mechanisms allow hyphal germination and subsequent tissue invasion. The invasive pulmonary aspergillosis is the most common manifestation of Aspergillus fumigatus infection in immunocompromised patients and is characterized by hyphal invasion and destruction of pulmonary tissue. A Th1/Th2 dysregulation and a switch to a Th2 immune response may contribute to the development and unfavorable outcome of invasive pulmonary aspergillosis. Dendritic cells (DC) have a primary role in surveillance for pathogens at the mucosal surfaces and are recognized as the initiators of immune responses to them. In the present study, we assessed the functional activity of pulmonary DC in response to A. fumigatus conidia and hyphae, both in vitro and in vivo. We analyzed mechanisms and receptors for phagocytosis by DC as well as DC migration, maturation, and Th priming in vivo upon exposure to either form of the fungus. We found a remarkable functional plasticity of DC in response to the different forms of the fungus, as pulmonary DC were able to: 1) internalize conidia and hyphae of A. fumigatus through distinct phagocytic mechanisms and recognition receptors; 2) discriminate between the different forms in terms of cytokine production; 3) undergo functional maturation upon migration to the draining lymph nodes and spleens; and 4) instruct local and peripheral Th cell reactivity to the fungus.     

T cell vaccination in mice with invasive pulmonary aspergillosis

Aspergillus fumigatus, an opportunistic fungal pathogen, is responsible for multiple airway diseases of an allergic and a nonallergic nature. In a murine model of invasive pulmonary aspergillosis, resistance is associated with a decreased lung inflammatory pathology and the occurrence of an IL-12-dependent Th1-type reactivity that are both impaired by IL-4. In the present study we assess the ability of Aspergillus crude culture filtrate Ags and the recombinant allergen Asp f 2 to induce protective antifungal responses in mice with invasive pulmonary aspergillosis. Similar to what occurred upon nasal exposure to viable A. fumigatus conidia, treatment of immunocompetent mice with Aspergillus crude culture filtrate Ags resulted in the development of local and peripheral protective Th1 memory responses, mediated by Ag-specific CD4+ T cells producing IFN-gamma and IL-2 capable of conferring protection upon adoptive transfer to naive recipients. Protective Th1 responses could not be observed in mice deficient of IFN-gamma or IL-12 and did not occur in response to Asp f 2, which, on the contrary, elicited high level production of inhibitory IL-4. The results show that Ags of Aspergillus exist with the ability to induce both Th1- and Th2-type reactivity during infection, a finding that suggests a possible mechanism through which potentially protective immune responses are inhibited in mice with the infection. However, the occurrence of Th1-mediated resistance upon vaccination with Aspergillus crude culture filtrate Ags, suggests the existence of fungal Ags useful as a candidate vaccine against invasive pulmonary aspergillosis.     

Aspergillus fumigatus induces innate immune responses in alveolar macrophages through the MAPK pathway independently of TLR2 and TLR4

Aspergillus fumigatus causes invasive aspergillosis in immunosuppressed patients. In the immunocompetent host, inhaled conidia are cleared by alveolar macrophages. The signaling pathways of the alveolar macrophage involved in the clearance of A. fumigatus are poorly understood. Therefore, we investigated the role of TLRs in the immune response against A. fumigatus and their contribution to the signaling events triggered in murine alveolar macrophages upon infection with A. fumigatus conidia. Specifically, we examined the MAPKs and NF-kappaB activation and cytokine signaling. Our investigations revealed that immunocompetent TLR2, TLR4, and MyD88 knockout mice were not more susceptible to invasive aspergillosis as compared with wild-type mice and that the in vitro phosphorylation of the MAPKs ERK and p38 was not affected in TLR2, TLR4, or MyD88 knockout mice following stimulation with conidia. In vivo experiments suggest that ERK was an essential MAPK in the defense against A. fumigatus, whereas the activation of NF-kappaB appeared to play only a secondary role. In conclusion, our findings demonstrate that TLR2/4 recognition and MyD88 signaling are dispensable for the clearance of A. fumigatus under immunocompetent situations. Furthermore, our data stress the important role of ERK activation in innate immunity to A. fumigatus.     

In vivo hypoxia and a fungal alcohol dehydrogenase influence the pathogenesis of invasive pulmonary aspergillosis

Currently, our knowledge of how pathogenic fungi grow in mammalian host environments is limited. Using a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA) and (1)H-NMR metabolomics, we detected ethanol in the lungs of mice infected with Aspergillus fumigatus. This result suggests that A. fumigatus is exposed to oxygen depleted microenvironments during infection. To test this hypothesis, we utilized a chemical hypoxia detection agent, pimonidazole hydrochloride, in three immunologically distinct murine models of IPA (chemotherapeutic, X-CGD, and corticosteroid). In all three IPA murine models, hypoxia was observed during the course of infection. We next tested the hypothesis that production of ethanol in vivo by the fungus is involved in hypoxia adaptation and fungal pathogenesis. Ethanol deficient A. fumigatus strains showed no growth defects in hypoxia and were able to cause wild type levels of mortality in all 3 murine models. However, lung immunohistopathology and flow cytometry analyses revealed an increase in the inflammatory response in mice infected with an alcohol dehydrogenase null mutant strain that corresponded with a reduction in fungal burden. Consequently, in this study we present the first in vivo observations that hypoxic microenvironments occur during a pulmonary invasive fungal infection and observe that a fungal alcohol dehydrogenase influences fungal pathogenesis in the lung. Thus, environmental conditions encountered by invading pathogenic fungi may result in substantial fungal metabolism changes that influence subsequent host immune responses.     

Anidulafungin: experimental therapy of fungal infections in animal models

We have reviewed the existing data on the efficacy of anidulafungin, which is the most recent echinocandin in the experimental treatment of fungal infections. The scarce published data practically only refers to disseminated and pulmonary aspergillosis and to disseminated candidiasis. Anidulafungin shows fungistatic activity against Aspergillus fumigatus, and fungicidal activity against Candida albicans and Candida glabrata.     

Systematic identification of substrates for profiling of secreted proteases from Aspergillus species

Reliable and early diagnosis of life-threatening invasive mycoses in neutropenic patients caused by fungi of the Aspergillus species remains challenging because current clinical diagnostic tools lack in sensitivity and/or specificity. During invasive growth a variety of fungal proteases are secreted into the bloodstream and protease profiling with reporter peptides might improve diagnosis of invasive aspergillosis in serum specimens. To characterise the specific protease activity of Aspergillus fumigatus and Aspergillus niger we analyzed Aspergillus culture supernatants, human serum and the mixture of both. A systematic screening for optimised protease substrates was performed using a random peptide library consisting of 360 synthetic peptides featuring fluorescence resonance energy transfer (FRET). We could identify numerous peptides that are selectively cleaved by fungus-specific proteases. These reporter peptides might be feasible for future protease profiling of serum specimens to improve diagnosis and monitoring of invasive aspergillosis.     

Signature-tagged and directed mutagenesis identify PABA synthetase as essential for Aspergillus fumigatus pathogenicity

Signature-tagged mutagenesis (STM) is a method that has been used to screen for genes required for in vivo survival of pathogenic bacteria, but has not been used to investigate a eukaryotic pathogen in an animal model of disease. We have adapted STM to identify genes required for in vivo growth of the opportunistic fungal pathogen Aspergillus fumigatus. Using a mouse model of invasive pulmonary aspergillosis, we have isolated several mutant strains with defects in their ability to replicate in vivo. One strain unable to cause lethal infection was further characterized and found to have an insertion into the promoter of a gene (pabaA) encoding para-aminobenzoic acid synthetase, an enzyme catalyzing a late step in the biosynthesis of folate. The complete inability of this strain, and other pabaA- strains constructed in this study by targeted gene deletion, to cause lethal infection in mice confirms the importance of the folate synthesis pathway for in vivo survival of this pathogen. The successful application of STM to A. fumigatus demonstrates that in vivo genetic analysis of eukaryotic pathogens is feasible and could result in the identification of potential targets, such as para-aminobenzoic acid synthetase, for novel antifungal therapies.     

Influence of polymorphisms in innate immunity genes on susceptibility to invasive aspergillosis after stem cell transplantation

The innate immune system plays a pivotal role in the primary defence against invasive fungal infection. Genetic variation in genes that regulate this response, initiated by pulmonary macrophages, may influence susceptibility to invasive aspergillosis in patients at risk. We investigated in a clinical setting whether common polymorphisms in Toll-like receptor (TLR) and cytokine genes involved in macrophage regulation are associated with susceptibility to invasive aspergillosis. Forty-four allogeneic stem cell transplantation recipients diagnosed with probable or proven IA according to the criteria of the European Organization for Research and Treatment of Cancer/Mycoses Study Group, were enrolled. The control group consisted of 64 allogeneic stem cell transplantation recipients without invasive aspergillosis. The TLR4 1063A>G single nucleotide polymorphism was associated with invasive aspergillosis when present in donors of allogeneic stem cell transplantation recipients (unadjusted OR 3.77 95%CI 1.08–13.2, p = 0.03). In a multivariate analysis, adjusted for occurrence of graft-versus-host-disease, Cytomegalovirus serostatus and duration of neutropenia, paired presence of the TLR4 1063A>G and IFNG 874T>A single nucleotide polymorphisms showed a trend towards increased susceptibility to invasive aspergillosis (p = 0.04). These findings point to the relevant immunological pathway involved in resistance to invasive aspergillosis and warrant further study of the effects of TLR and cytokine polymorphisms and their interaction, which may occur on different levels of the complex biological interplay between the immunocompromised host and Aspergillus sp.