Insights into the role of TLR21 in innate immunity of Japanese flounder (Paralichthys olivaceus)

As an essential immune receptor in Japanese flounder (Paralichthys olivaceus), Toll-like receptor 21(TLR21) plays a pivotal role in recognizing pathogen-associated molecular patterns (PAMPs) and activating innate immune signaling pathways. In this study, the functions of Japanese flounder TLR21 (PoTLR21) were studied. Expression pattern analysis revealed that Potlr21 was ubiquitously expressed in various tissues of Japanese flounder and induced following lymphocystis disease virus (LCDV) infection. PoTLR21 was subcellularly localized in cytoplasm with weak fluorescence signals detected on cell membrane. Ligand identification results demonstrated that PoTLR21 possessed the strongest binding affinity toward CpG oligodeoxynucleotide (CpG ODN). Pull-down assay revealed that PoTLR21 interacted with TIR-domain-containing adaptor inducing IFN-β (TRIF), and TRIF knockdown suppressed the expression of TLR signaling pathway downstream effectors induced by Potlr21 overexpression. Moreover, overexpression of Potlr21 reduced the copy number of LCDV in flounder gill (FG) cells and enhanced cellular resistance to LCDV infection. Based on ligand and adaptor protein identification along with overexpression, RNA interference (RNAi) and transcriptome data, we gained preliminary insights into the immune mechanism of PoTLR21. We propose that PoTLR21 may sense viral unmethylated CpG DNA and transduce signals via TRIF, with its downstream signaling exhibiting tight crosstalk with nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Within endosomes, PoTLR21 signaling may connect to the TNF receptor-associated factor 3 (TRAF3)-dependent pathway via regulating cathepsin K (CTSK). This research offers new perspectives and foundation for further elucidation of the functional roles and mechanisms of PoTLR21 in innate immune responses.