Phagocytosis and Intracellular Fate ofAspergillus fumigatusConidia in Alveolar Macrophages

Abstract

Aspergillus fumigatus is the most prevalent airborne fungal pathogen responsible for fatal invasive aspergillosis in immunocompromised patients. Upon arrival in the lung alveolus, conidia of A. fumigatus are phagocytosed by alveolar macrophages, the major phagocytic cells of the lung. Engulfment and intracellular trafficking of A. fumigatus conidia in alveolar macrophages of two different origins, the murine cell line MH-S and human pulmonary alveolar macrophages, were analyzed by electron microscopy and immunofluorescence. Phagocytosis of A. fumigatus conidia required actin polymerization and phosphatidylinositol 3-kinase activity. Fusion of A. fumigatus phagosomes with early and late endosomes was shown by immunolabeling with specific markers for the transferrin receptor, early endosome antigen, and Rab7. Maturation of A. fumigatus phagolysosomes was monitored by using a fixable acidotropic probe, LysoTracker Red DND-99, and an anti-cathepsin D antibody. Bafilomycin A-induced inhibition of lysosomal acidification abolished the conidial killing by the macrophages. These data suggest that the maturation of A. fumigatus phagosomes results from fusion with the compartments of the endocytic pathway and that the killing of conidia depends on phagolysosome acidification. A model for the phagocytosis of A. fumigatus conidia by alveolar macrophages is proposed on the basis of these results.

Dectin-1 and TLRs Permit Macrophages to Distinguish between Different Aspergillus fumigatus Cellular States

“…Aspergillus spores (resting conidia) are ubiquitous in the environment, are inhaled frequently, and are normally cleared without pathology in immunocompetent people. Alveolar macrophages constitute a primary line of innate cellular defense, because they bind, ingest, and kill conidia (2,3). Organisms that escape macrophage killing are able to mature into germ tubes and hyphae, which can invade vessels and disseminate hematogenously; invasive pneumonia and disseminated infection is particularly common in people who lack effective hyphal defense by polymorphonuclear phagocytes (2).…”

Aspergillus fumigatus Induces Innate Immune Responses in Alveolar Macrophages through the MAPK Pathway Independently of TLR2 and TLR4

“…Unbound conidia were removed by washing with cold RPMI complete medium, and phagocytosis was initiated at 37°C in an atmosphere of 5% CO 2 . Phagocytosis was stopped after 2 h by washing the AMs with ice-cold PBS and subjecting them to fixation with 3% formaldehyde in PBS (10 min at room temperature) followed by three washes with 50 mM NH 4 Cl in PBS. The number of A. fumigatus conidia engulfed by macrophages was determined using a polyclonal anti-conidia Ab and a secondary Ab conjugated to Texas Red.…”

“…Phagocytosis assays were performed as previously described (4). AMs were challenged with two conidia per macrophage and were subsequently incubated at 4°C for 30 min.…”

“…The contribution of phagolysosomal acidification as well as NADPH oxidase to the conidicidal activity has been demonstrated. This activity is inhibited by corticosteroids via impairment of the production of reactive oxidant intermediates (3,4). If end-killing mechanisms are documented, the signaling pathways controlling these mechanisms are not known.…”

Toll-like receptor 2 and dectin-1 function as promising biomarker for Aspergillus fumigatus infection

“…Previous in vivo and in vitro studies have demonstrated that endothelial cells can adhere or endocytose the Aspergillus fumigatus spores, resulting in increased expression of the inflammatory factors interleukin (IL)-1, tumor necrosis factor (TNF)-α, IL-6 and IL-8 (3)(4)(5). In vascular endothelial cells, the receptor that recognizes the Aspergillus fumigatus fungus is not clear to date.…”