Systems Analysis of Innate Immune Responses to Fungal-Derived Carbohydrates

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 DESCRIPTION (provided by applicant): 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 ß-1,3 glucan receptor Dectin-1 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. The fungal cell wall serves as the initial point of contact in the pathogen-host relationship. Despite elegant ultra-structural studies on the topology of the fungal cell wall, our understanding of the polysaccharide constituents that interact with mammalian cellular receptors is poorly understood. ß-1,3 glucan, a proinflammatory polysaccharide found on numerous pathogenic fungi, is the best studied fungal carbohydrate. The precise response triggered by other fungal carbohydrates including chitin, mannan, galactoaminogalactan, galactomannan and α-1,3 glucan remains incompletely understood. In order to understand better the contribution of these fungal polysaccharides to the innate immune response, we have made the following key observations that are the rationale for our proposed work: 1) when present in phagosomes, fungi trigger specific recruitment of multiple proteins of immunologic interest including CD63, CD82, class II MHC, LC3, LAMP-1 and TLR9. In sharp contrast, polystyrene beads failed to do so. 2) we generated size-matched polystyrene beads that possess a uniform coat of a single fungal-derived carbohydrate covalently attached to probe the immune response. These fungal like particles are biologically active and trigger cytokine responses 3) using fungal like particle, we generated phagosomes in macrophages and determined that the phagosomal proteome by mass spectrometry. We hypothesize that the content of the phagosome triggers the assembly of specific mammalian proteins to this compartment and that this assembly of proteins orchestrates the net immunologic output from this cell. We propose to: 1] Determine the mammalian proteins recruited to the phagosome containing different fungal like particles and 2] Profile the cytokine signature in human monocytes to fungal-like particles. We will apply advanced proteomics and multiplex cytokine assay to achieve these aims. Knowledge gained regarding the mechanism of carbohydrate recognition, its influence on the phagosome formation and cytokine signature will be important in furthering our understanding of the innate immune response to A. fumigatus, and could lead to novel insights to vaccine development against this deadly pathogen.