The Medicinal and Antitumor Mushroom, Agaricus Blazei Murill, Activates NF-κB Via TLR2 but Not TLR4 In Monocytic Cells, and Stimulates Monocyte-Derived Dendritic Cells (MDDC) to Increased Cell Surface Marker Expression and Cytokine Production, and May Thus Have Adjuvant Effect In MDDC Cancer Vaccines

Abstract 3904

Agaricus bM (AbM) is an edible Basidiomycetes mushroom from Brazilian rain forest that is used in traditional medicine against cancer and other diseases. It is rich in immunomodulating substances like β-glucans and is shown to inhibit tumors and adjuvate hepatitis B virus DNA vaccine in mouse models. Previously, we have found that an AbM-based (82%) extract, AndoSan™, additionally containing Basidiomycetes mushrooms Hericium erinaceum and Grifola frondosa, also protected against gram positive and gram-negative sepsis and allergy in mice. Now, to examine the mechanisms behind AbM's biological effects and to see whether it is beneficial as adjuvant in DC cancer vaccines, cellular surface markers on stimulated MDDC were studied by flow cytometry and cytokine production by a 17-plex Luminex system. Transcription factor NF-kB activation, which is linked to cytokine production, cell cyclus regulation and more, was then determined by a luciferase assay system and western blotting as translocation of NF-kB from cytosol to nucleus in AbM stimulated promonocytic THP-1 cells and in HEK293 cells, transfected with CD14/MD2 and TLR2 or TLR4. In both cell lines, AbM induced NF-kB activation via TLR2, but inhibited such activation induced via TLR4. After 24h incubation of MDDC with AndoSan™, there was down-regulated CD11c, de novo CD69 and enhanced CD86 expression on the cells, as well as increased levels in the culture supernatants of cytokines TNFα, IL-1β, IL-2, IL-6, IL-8, MIP-1β, IFNγ and G-SCF. Moreover, when this mixed mushroom extract was added as adjuvant to a DC melanoma vaccine, there was increased proliferation (H³-thymidine incorporation) of cancer-peptide-specific T cells. We conclude that in monocytes and probably also MDDC, AbM activates transcription factor NF-kB via TLR2 stimulation but inhibits such activation induced via the LPS receptor, TLR4. In MDDC the AbM-based extract AndoSan™ induced differential expression of cell surface markers for cell adhesion, activation and antigen presentation, and increased production of a leukocyte growth factor and proinflammatory, chemotactic and some Th1-type but not Th2-type cytokines in vitro. This may explain some of the known antitumor properties of AbM and its effect against infection and allergy. Since preliminary data suggest that the AbM-based extract, AndoSan™, may also have positive adjuvant effect in MDDC-based cancer vaccine, it will be tested further ex vivo and in clinical trials with cancer patients subjected to such DC vaccines.