PURPOSE: The cell wall of aspergillus fumigatus consists predominantly of β1,3 and branched β1,3-1,6 glucan. Dectin-1 is a β1,3 receptor found on host defense cells including macrophages, neutrophils, and dendritic cells. We’ve isolated whole cell wall glucan particulate from aspergillus fumigatus and tested its inflammatory and immunogenic activity in human dendritic cells through dectin-1 and NF-κB upregulation.
METHODS: Whole cell wall glucan particulate was isolated by Manners protocol involving alternating hot alkali and acid extraction and stored at 2mg/ml stock in normal saline. Bone marrow derived human dendritic cells were harvested from blood donor cones by Ficoll plaque spin down and CD14 magnetic bead isolation with GM-CSF and IL-4 incubation over 5-7 days. Cells were then counted and plated at approximately 5-10million cells per well and challenged accordingly with aspergillus glucan particulate. For dectin-1 phosphorylation, cells were challenged for 30 minutes, RIPA-extracted for protein, then immunoprecipitated for dectin-1 with mouse monoclonal anti-dectin IgG and protein A Sepharose beads overnight. Western blot was done with anti-phosphotyrosine antibody to confirm dectin-1 phosphorylation from immunoprecipitated samples. For anti-dectin blocking, cold room incubation with mouse polyclonal anti-dectin antibody was done prior to overnight glucan challenges. Supernatant was collected and tested by ELISA for cytokine production of TNF-α and IL-23. NF-κB analysis was done in dendritic cells challenged for 4 and 24hrs followed by nuclear protein extraction, then assessed by a DNA binding assay (ActivMotif).
RESULTS: Phosphorylation of dectin-1 occurs after glucan challenge in human dendritic cells as noted by anti-phosphotyrosine Western blot. Anti-dectin blocking mitigates cytokine activity in glucan challenged cells as measured by TNF-α and IL-23 ELISA. Upregulation of NF-κB occurs after glucan challenge, both at 4 and 24hrs of incubation.
CONCLUSIONS: Dectin-1 is involved in cytokine production of TNF-α and IL-23 in human dendritic cells exposed to aspergillus glucan particulate. NF-κB upregulation in human dendritic cells is also increased after aspergillus glucan exposure.
CLINICAL IMPLICATIONS: Understanding the immunogenic and inflammatory pathways initiated by aspergillus cell wall glucan may be helpful in augmenting the host response of immunocompromised patients.
DISCLOSURE: The following authors have nothing to disclose: Teng Moua, Eva Carmona, Ted Kottom, Deanne Hebrink, Jeff Lamont, Andrew Limper
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