Aureobasidium pullulans culture supernatant significantly stimulates R-848-activated phagocytosis of PMA-induced
Hidekazu Tamegai1, Yuka Takada, Mitsuyasu Okabe
1Department of Biotechnology, College of Science and Engineering, Tokyo Denki University, Hatoyama, Hiki-gun, Saitama, Japan.
Abstract:
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. pullulans treatment alone stimulated THP-1 macrophages to induce phagocytosis, co-treatment of R-848 with A. pullulans culture supernatant significantly augmented phagocytosis of apoptotic Jurkat cells. These results suggest that the activation of several different innate immune receptor pathways may enhance the immune response of R-848 significantly.
Insights
Combining R-848 with Aureobasidium pullulans enhances immune responses. This co-treatment significantly boosts cytokine production and the uptake of apoptotic cells by macrophages, indicating a potentiation of innate immunity.
Area of Science:
- Immunology
- Innate Immunity
- Cellular Immunology
Background:
- Toll-like receptors (TLRs) are crucial for innate immunity, recognizing microbial products and activating macrophages.
- R-848, an immune response modifier, targets endosomal TLRs for antiviral effects.
- Aureobasidium pullulans (A. pullulans) contains beta-glucans that activate cell membrane TLRs and Dectin-1.
Purpose of the Study:
- To investigate if co-treatment with R-848 and A. pullulans culture supernatant enhances immune responses in macrophages.
- To determine the effect of this co-treatment on cytokine production (TNF-α, IL-12p40).
- To assess whether co-treatment augments the phagocytosis of apoptotic cells.
Main Methods:
- THP-1 macrophages were treated with R-848 alone or co-treated with R-848 and A. pullulans culture supernatant.
- Cytokine expression (TNF-α, IL-12p40) was measured.
- Apoptosis was induced in Jurkat cells, stained with TAMRA, and co-cultured with labeled THP-1 macrophages.
- Phagocytosis was quantified using flow cytometry to detect TAMRA-positive THP-1 macrophages.
Main Results:
- Co-treatment significantly augmented TNF-α and IL-12p40 cytokine expression compared to R-848 alone.
- Co-treatment significantly enhanced the phagocytosis of apoptotic Jurkat cells by THP-1 macrophages.
- Both R-848 and A. pullulans alone stimulated some level of phagocytosis, but co-treatment showed a synergistic effect.
Conclusions:
- Simultaneous activation of multiple innate immune receptors (TLR and Dectin-1) potentiates immune responses.
- Co-treatment with R-848 and A. pullulans culture supernatant offers a significant enhancement of macrophage activation and phagocytosis.
- This suggests a promising strategy for boosting innate immune responses through combined receptor pathway stimulation.
