Cryptococcus neoformansEnters the Endolysosomal Pathway of Dendritic Cells and Is Killed by Lysosomal Components

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen that primarily causes disease in immunocompromised individuals. Dendritic cells (DCs) can phagocytose C. neoformans, present cryptococcal antigen, and kill C. neoformans. However, early events following C. neoformans phagocytosis by DCs are not well defined. We hypothesized that C. neoformans traffics to the endosome and the lysosome following phagocytosis by DCs and is eventually killed in the lysosome. Murine bone marrow-derived DCs (BMDCs) or human monocyte-derived DCs (HDCs) were incubated with live, encapsulated C. neoformans yeast cells and opsonizing antibody. Following incubation, DCs were intracellularly stained with antibodies against EEA1 (endosome) and LAMP-1 (late endosome/lysosome). As assessed by confocal microscopy, C. neoformans trafficked to endosomal compartments of DCs within 10 min and to lysosomal compartments within 30 min postincubation. For HDCs, the studies were repeated using complement-sufficient autologous plasma for the opsonization of C. neoformans. These data showed results similar to those for antibody opsonization, with C. neoformans localized to endosomes within 20 min and to lysosomes within 60 min postincubation. Additionally, the results of live real-time imaging studies demonstrated that C. neoformans entered lysosomal compartments within 20 min following the initiation of phagocytosis. The results of scanning and transmission electron microscopy demonstrated conventional zipper phagocytosis of C. neoformans by DCs. Finally, lysosomal extracts were purified from BMDCs and incubated with C. neoformans to determine their potential to kill C. neoformans. The extracts killed C. neoformans in a dose-dependent manner. This study shows that C. neoformans enters into endosomal and lysosomal pathways following DC phagocytosis and can be killed by lysosomal components.

Interleukin-17 Is Not Required for Classical Macrophage Activation in a Pulmonary Mouse Model ofCryptococcus neoformansInfection

“…Macrophages and neutrophils work in concert to generate densely packed granulomata, which function in the containment of inflammatory . Neutrophil infiltration is crucial to the protective responses to C. neoformans infections (36,37,41). Though we do not see a significant difference in neutrophil influx following depletion of IL-17A during infection with C. neoformans strain H99␥ (unpublished observations), we do see a prolonged iNOS response (Fig.…”

Role of Dendritic Cells and Alveolar Macrophages in Regulating Early Host Defense against Pulmonary Infection withCryptococcus neoformans

“…This (29,34,63,71,77,78). Phagocytosis is associated with the production of numerous inflammatory cytokines, including tumor necrosis factor alpha, and expression of inducible nitric oxide synthase, a potent mediator of fungal cytotoxicity (2,17,20,41,46).…”

“…Both DC and AM express lectin receptors, including macrophage mannose receptor and DC-specific non-ICAM3 grabbing nonintergrin (DC-SIGN) (14, 15), which bind C. neoformans glycoantigens, including mannoproteins (42, 55). DC and AM phagocytose the organism in vitro and in vivo (29,34,63,77,78), and phagocytosis (and/or exposure to soluble glycoantigens or cryptococcal DNA) is associated with cytokine and chemokine production (5,29,40,48,49,55,61) and yeast lysis (77). It is unclear whether phagocytosis by resident DC and AM contributes to early clearance and/or the later development of adaptive immunity.…”

Distinct Patterns of Dendritic Cell Cytokine Release Stimulated by Fungal β-Glucans and Toll-Like Receptor Agonists

“…After 2 h of incubation at 4°C for the binding assay or 1 h of incubation at 37°C for the uptake assay, the wells were washed three times with Dulbecco's PBS (DPBS) (ice-cold DPBS for binding assay and room temperature DPBS for uptake assay) (Lonza, Allendale, NJ) to remove free YGPs and then examined under bright-field and epifluorescence microscopy. Confocal microscopy was performed on a Leica SP2 acousto-optical beam splitter confocal microscope (Leica Microsystems, Bannockburn, IL) using 35-mm glass bottom dishes (MatTek Corp., Ashland, MA), as in previous studies (52).…”

“…Human myeloid DCs were obtained as previously described (39,52). Briefly, peripheral blood samples were obtained from healthy volunteers following informed consent using a protocol approved by the University of Massachusetts Medical School Institutional Review Board.…”