Carp Toll-like receptor 8 (Tlr8): An intracellular Tlr that recruits TIRAP as adaptor and activates AP-1 pathway in immune response

Toll-like receptor 8 (Tlr8) is a member of intracellular TLRs family and play a critical role in the innate immunity. In the present study, we aimed to identify tlr8 from common carp (Cyprinus carpio L.), and explored its expression profile, localization, adaptor, and signaling pathways. A novel tlr8 cDNA sequence (Cctlr8) was identified from the carp, containing a signal peptide, a LRR-N-terminal (LRR-NT), 14 leucine-rich repeats, a LRR-C-terminal (LRR-CT), a transmembrane region and a TIR domain. Phylogenetic analysis revealed that CcTlr8 exhibited closest relationship to that of Ctenopharyngodon idella and Danio. rerio. Subcellular localization analysis indicated that CcTlr8 was localized to the endoplasmic reticulum in both HeLa cells and EPC cells. Quantitative Real-Time PCR analysis demonstrated that Cctlr8 was constitutively expressed in all the examined tissues, with the highest expression observed in the spleen. After poly (I:C) injection, the expression of Cctlr8 was significantly up-regulated in all the tested tissues. In addition, the expression of Cctlr8 was up-regulated in both PBLs and HKLs following poly (I:C) stimulation. The results of immuofluorescence and coimmunoprecipitation analysis indicated that CcTlr8 might recruit TIRAP as the adaptor. Furthermore, Luciferase reporter assays revealed that CcTlr8 could activate AP-1 in 293 T cells. Taken altogether, these findings lay the foundations for future research to investigate the mechanisms underlying fish tlr8.