Characterization of TLR14 ligands, adaptor proteins and immune mechanisms in Japanese flounder (Paralichthys...

Toll-like receptors (TLRs) play a pivotal role in innate immune recognition. Although the sequence and protein structural features of TLR14 of Japanese flounder (Paralichthys olivaceus, PoTLR14) have been extensively documented, its immunological functions remain largely unexplored. In this study, the expression profiles of Potlr14 in various tissues of healthy and Vibrio anguillarum challenged Japanese flounder were detected. Subcellular localization analysis revealed that PoTLR14 is predominantly distributed on the cell membrane and cytoplasm. Enzyme-linked immunosorbent assay (ELISA) assay identified lipopolysaccharide (LPS) as a ligand for PoTLR14, while pull-down experiment demonstrated its interaction with both myeloid differentiation factor 88 (MyD88) and TIR-domain-containing adaptor inducing IFN-β (TRIF). The function of PoTLR14 in the innate immune response was further elucidated through LPS stimulation, RNA interference (RNAi) and overexpression. The results suggest that Potlr14 responds to LPS stimulation by regulating the expression of genes related to pro-inflammatory cytokines and costimulatory molecules. Furthermore, Potlr14 participates in the regulation of genes associated with the apoptosis signaling pathway. Our research provides a theoretical basis for elucidating the function and mechanism of PoTLR14 in innate immunity.