Table_1_R848 Is Involved in the Antibacterial Immune Response of Golden Pompano (Trachinotus ovatus) Through TLR7/8-MyD88-NF-κB-Signaling Pathway.doc

R848 is an imidazoquinoline compound that is a specific activator of toll-like receptor (TLR) 7/8 and is often used in immunological research in mammals and teleosts. However, the immune responses initiated by R848 through the TLR7/8 pathway in response to bacterial infection remain largely unexplored in teleosts. In the current study, we investigated the antibacterial response and the participating signaling pathway initiated by R848 in golden pompano (Trachinotus ovatus). We found that R848 could stimulate the proliferation of head kidney lymphocytes (HKLs) in a dose-dependent manner, enhance the survival rate of HKLs, and inhibit the replication of bacteria in vivo. However, these effects induced by R848 were significantly reduced when chloroquine (CQ) was used to blocked endosomal acidification. Additionally, an in vivo study showed that R848 strengthened the antibacterial immunity of fish through a TLR7/8 and Myd88-dependent signaling pathway. A cellular experiment showed that Pepinh-MYD (a Myd88 inhibitor) significantly reduced the R848-mediated proliferation and survival of HKLs. Luciferase activity analysis showed that R848 enhanced the nuclear factor kappa B (NF-κB) activity, whereas this activity was reduced when CQ and Pepinh-MYD were present. Additionally, when an NF-κB inhibitor was present, the R848-mediated pro-proliferative and pro-survival effects on HKLs were significantly diminished. An in vivo study showed that knockdown of TLR7, TLR8, and Myd88 expression in golden pompano via siRNA following injection of R848 resulted in increased bacterial dissemination and colonization in fish tissues compared to that of fish injection of R848 alone, suggesting that R848-induced antibacterial immunity was significantly reduced. In conclusion, these results indicate that R848 plays an essential role in the antibacterial immunity of golden pompano via the TLR7/8-Myd88-NF-κB- signaling pathway.

R848是一种咪唑并喹啉类化合物，属于toll样受体（toll-like receptor, TLR）7/8的特异性激活剂，常被应用于哺乳动物与硬骨鱼类的免疫学研究。然而，R848通过TLR7/8通路引发的、针对细菌感染的免疫应答机制，在硬骨鱼类中仍未得到充分探究。本研究以卵形鲳鲹（Trachinotus ovatus）为实验对象，探究了R848介导的抗菌应答及其参与的信号通路。研究结果显示，R848可呈剂量依赖性刺激头肾淋巴细胞（head kidney lymphocytes, HKLs）增殖，提升HKLs的存活率，并抑制体内细菌的复制。但当使用氯喹（chloroquine, CQ）阻断内体酸化时，R848诱导的上述效应会显著减弱。此外，体内实验表明，R848可通过依赖TLR7/8与髓样分化因子88（Myd88）的信号通路增强鱼类的抗菌免疫力。细胞实验显示，Pepinh-MYD（一种Myd88抑制剂）可显著削弱R848介导的HKLs增殖与存活效应。荧光素酶活性分析结果表明，R848可增强核因子κB（nuclear factor kappa B, NF-κB）的活性，而当CQ与Pepinh-MYD共同存在时，该活性会被抑制。此外，当使用NF-κB抑制剂时，R848对HKLs的促增殖与促存活效应也会显著减弱。体内研究还显示，向卵形鲳鲹体内注射R848后，通过小干扰RNA（small interfering RNA, siRNA）敲低TLR7、TLR8及Myd88的表达，会导致鱼类组织内的细菌播散与定植量相较于仅注射R848的对照组更高，表明R848诱导的抗菌免疫力被显著削弱。综上，本研究结果表明，R848通过TLR7/8-Myd88-NF-κB信号通路在卵形鲳鲹的抗菌免疫中发挥关键作用。