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.

引言：免疫系统可分为固有免疫与适应性免疫，巨噬细胞、树突状细胞（dendritic cells，DCs）等抗原呈递细胞在免疫调控中发挥关键作用。在乳酸杆菌中，副干酪乳杆菌Lacticaseibacillus paracasei KW3110（简称KW3110）被报道可强力诱导白细胞介素-12（interleukin-12，IL-12）的产生，尤其在单核细胞与巨噬细胞中，进而改善多种过敏症状。但目前KW3110对其他免疫细胞的作用及其潜在分子机制仍不甚明确。本研究探讨了KW3110对从人外周血单个核细胞（human peripheral blood mononuclear cells，hPBMCs）中分离得到的单核细胞、DCs、T细胞、自然杀伤细胞及B细胞的作用及其分子机制。
材料与方法：从健康供者体内分离的hPBMCs被分选为上述主要免疫细胞类型，并用热灭活的KW3110进行刺激。采用酶联免疫吸附试验（ELISA）检测IL-12p70/p40水平；通过活细胞成像、共聚焦激光扫描显微镜、流式细胞术，以及使用吞噬抑制剂细胞松弛素D（cytochalasin D，Cyt-D）处理的方式评估吞噬作用。将KW3110来源的核酸经或不经核糖核酸酶（RNase）或脱氧核糖核酸酶（DNase）处理后，使用转染试剂进行转染；同时对完整的KW3110施以RNase处理。采用特异性抑制剂评估内体信号通路。
结果：KW3110可诱导单核细胞与DCs，尤其是髓系树突状细胞（myeloid dendritic cells，mDCs）产生IL-12，而T细胞、自然杀伤细胞及B细胞无此响应。研究证实KW3110可被mDCs摄取，且Cyt-D处理可同时阻断吞噬作用与IL-12的产生。KW3110来源的核酸可剂量依赖性地诱导DCs产生IL-12，该效应可被RNase A处理阻断，表明RNA成分发挥了关键作用。内体酸化抑制剂实验结果支持内体识别的作用，而选择性Toll样受体8（Toll-like receptor 8，TLR8）抑制剂可阻断KW3110诱导的mDCs产生IL-12，提示TLR8是识别KW3110来源的单链RNA（single-stranded RNA，ssRNA）的主要受体。
结论：上述研究结果表明，KW3110可通过吞噬摄取以及TLR8识别ssRNA的途径，在人DCs尤其是mDCs中诱导IL-12产生。本研究为KW3110在人免疫细胞中的作用机制提供了全新的见解。