Aureobasidium pullulans culture supernatant significantly stimulates R-848-activated phagocytosis of PMA-induced THP-1 macrophages

Toll-like receptors (TLRs), which recognize a wide range of microbial pathogens and pathogen-related products, play important roles in innate immunology. Macrophages have a variety of TLRs, and pathogen binding to TLR resulted in the activation of macrophages. R-848, an immune response modifier, is an analog of imidazoquinoline derivative and binds to an endosome-localized TLR to exert an anti-viral response on leukocytes. In the present study, we verified that co-treatment of R-848 with other TLR agonists would enhance immune response. The culture supernatant of Aureobasidium pullulans (A. pullulans, which contains predominantly soluble β-glucan), which binds to cell membrane-localized TLR, and to C-type lectin receptor Dectin-1, was treated together with R-848 to THP-1 macrophages. Compared to R-848 treatment alone, co-treatment of R-848 with A. pullulans culture supernatant significantly augmented TNF-α and IL-12p40 cytokine expression. Next, we investigated whether or not apoptotic cell uptake would be increased by co-treatment of R-848 with A. pullulans culture supernatant. To detect engulfed apoptotic cells, we induced apoptosis in human lymphoma Jurkat cells by 5-fluorouracil and stained them with fluorescent dye 5(6)-carboxytetramethylrhodamine (TAMRA), whereas THP-1 macrophage was labeled with fluorescein isothiocyanate-anti-CD14 and determined the percentage increase in TAMRA-positive THP-1 macrophages by flow cytometric assay. Since R-848 or A. pullulan