Aspergillus fumigatus is a saprophytic filamentous fungus whose asexual spores called conidia are widespread in the environment, acquired through inhalation and small enough to reach the distal airways. Approximately 10% of patients with hematopoietic stem cell transplants or solid organ transplants will develop invasive aspergillosis, a life-threatening infection. Despite the development of effective fungicidal agents the prognosis for disseminated infection is quite poor, indicating that knowledge of the rules that govern the host defense against A. fumigatus is critical for development of new prevention and therapeutic strategies. Evidence for the importance of innate immune mechanisms in fungal defense is mounting. The fungal ?-1,3 glucan receptor Dectin1 is essential in pulmonary defense against A. fumigatus and collaborates with TLR2 and TLR4 in providing critical immune signals that coordinate cytokine secretion and development of the adaptive immunity. When challenged with A.fumigatus, TLR9-deficient mice are incapable of mounting an antigen-specific T cell response, directly implicating the involvement of TLR9 in the host defense against this fungal pathogen. However, the cell biological processes underlying TLR9-mediated A.fumigatus immune responses are still largely unresolved. In order to understand better the role of TLR9 in host defense against A. fumigatus, we have made the following key observations that are the rationale for our proposed work: 1) the presence of A. fumigatus phagosomes in macrophages results in a dramatic change of the subcellular distribution of TLR9 from the ER to a bright, ring-shaped compartment around the fungus 2) macrophages lacking Dectin-1 fail to recruit TLR9, indicating that the presence of Dectin-1 is required for proper TLR9 trafficking to the fungal phagosome 3) polystyrene beads with purified, fungal-derived ?-1,3-glucan attached covalently to the surface can mimic fungal particles during phagocytosis. We hypothesize that a component of the A. fumigatus cell wall mediates TLR9 recruitment and that Dectin-1 controls the trafficking of TLR9 to the fungal phagosome. We propose to: 1) determine the role of Dectin-1 in TLR9 recruitment to A. fumigatus phagosomes in APCs. 2) determine the molecular requirement for TLR9 recruitment to phagosomes containing fungal-like particles. 3) dissect the differences in immunological responses to RC, SC, H, ?-1,3 glucan and galactomannan beads by macrophages using optical trap. We will apply advanced imaging modalities including live cell imaging using spinning disk confocal microscopy and optical trapping to control spatial interactions between host cell and pathogen. Knowledge gained regarding the mechanism of regulation of TLR9 trafficking to fungal phagosomes will be important in furthering our understanding of the innate immune response to A. fumigatus, and could lead to novel insights on how to modulate the host defense against this deadly pathogen.
- Invasive aspergillosis is one of the most dreaded infections for immunocompromised patients. The initial contact between fungus and host typically occurs in the lung and innate phagocytic immune cells are usually the first cells to respond. This application seeks to understand the rules that govern the changes that occur to these cells upon uptake of these fungal pathogens.
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