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Abstract: Mammalian Toll-like receptor 3 (TLR3) recognizes extracellular and intracellular viral dsRNA, and then initiates signaling cascades leading to NF-κB activation and interferon (IFN) production. To understand the roles of TLR3 in the fish immune system, TLR3 gene (JfTLR3) was identified from Japanese flounder (Paralichthys olivaceus), which consisted of 4 exons and 3 introns. Its expression in peripheral blood leukocytes increased upon stimulation with poly I:C and CpG ODN 1668. Exposure to viral hemorrhagic septicemia virus increased expression of JfTLR3 in the blood, liver, head kidney and spleen. Intracellular poly I:C stimulation in JfTLR3-overexpressing YO-K cells significantly induced IFN-inducible and NF-κB-regulated genes. NF-κB activity in JfTLR3-overexpressing YO-K cells was significantly induced by intracellular poly I:C while expression of IFN-inducible genes and NF-κB reporter activity in JfTLR3-overexpressing HINAE cells increased upon stimulation by extracellular poly I:C. These results suggest that JfTLR3 plays an important role in the induction of antiviral immune response. [Copyright &y& Elsevier]

Abstract: Type I interferon (IFN) induces the antiviral response in innate immunity. The type I IFN gene cloned from Japanese flounder (Paralichthys olivaceus) has a length of 1189bp and consisting of 5 exons and 4 introns. In a phylogenetic tree of type I IFNs, Japanese flounder grouped with other Acanthopterygii. To gain insight into the transcriptional regulation of IFN gene, the 1.36kb 5′-upstream region including numerous canonical motifs to bind transcription factors [for example, IFN regulatory factor (IRF)] was analyzed. In HINAE cells using a luciferase reporter assay, poly I:C-responsive transcriptional activity was found in the region from −634 to −179bp. This region includes several IRF motifs. In the presence of poly I:C, overexpression of IRF3 and RLR strongly enhanced transcriptional activity. These results suggest that the transcriptional regulation of Japanese flounder type I IFN is regulated by IRF3 after triggering with dsRNA sensors. [Copyright &y& Elsevier]