Supplementary Material for: Toll-like receptor 8-mediated Interleukin-12 production in human dendritic cells triggered by RNA of Lacticaseibacillus paracasei KW3110

Introduction: The immune system is divided into innate and adaptive immunity, with antigen-presenting cells such as macrophages and dendritic cells (DCs) playing crucial roles in immune regulation. Among lactic acid bacteria, Lacticaseibacillus paracasei KW3110 (KW3110) has been reported to strongly induce interleukin-12 (IL-12) production, particularly in monocytes and macrophages, thereby improving various allergic symptoms. However, the effects of KW3110 on other immune cells and its underlying mechanisms are not well understood. This study investigated the effects and molecular mechanisms of KW3110 on monocytes, DCs, T cells, natural killer cells, and B cells isolated from human peripheral blood mononuclear cells (hPBMCs).
Methods: hPBMCs from healthy donors were fractionated into the major immune cell types described above and stimulated with heat-killed KW3110. IL-12p70/p40 levels were measured by ELISA, and phagocytosis was assessed by live-cell imaging, confocal laser scanning microscopy, flow cytometry, and by treatment with the phagocytosis inhibitor, cytochalasin D (Cyt-D). KW3110-derived nucleic acids, with or without RNase or DNase treatment, were transfected using a transfection reagent, and intact KW3110 was treated with RNase. Endosomal signaling was evaluated using specific inhibitors.
Results: KW3110 induced IL-12 production in monocytes and DCs, especially myeloid dendritic cells (mDCs), whereas T, NK, and B cells did not respond. Uptake of KW3110 into mDCs was confirmed, and both phagocytosis and IL-12 production were abolished by Cyt-D treatment. KW3110-derived nucleic acids induced IL-12 production in a dose-dependent manner in DCs, which was abolished by RNase A treatment, indicating the importance of RNA components. Endosomal recognition was supported by inhibition of endosomal acidification, and a selective Toll-like receptor 8 (TLR8) inhibitor abolished KW3110-induced IL-12 production in mDCs, implicating TLR8 as the primary receptor recognizing KW3110-derived single-stranded RNA (ssRNA).
Conclusion: These findings suggest that KW3110 induces IL-12 in human DCs, especially mDCs, via phagocytic uptake and ssRNA recognition through TLR8. These findings provide novel insights into the actions of KW3110 in human immune cells.