Interleukin 17 And Its Signaling Components In Oyster Crassostrea Gigas

IL-17 is an important pro-inflammatory cytokine in vertebrate, functions in immune defence system and tissue repair process, and serves a critical role in balancing homeostasis and inflammatory response. Moderate inflammatory response is necessary for self-protecting against environment stress, while excessive inflammatory response would disturb immune system, leading to autoimmune disease and tissue damage. Recently, inflammatory response phenomena were also found in mollusc under pathogen infection and abiotic stress, however the role of pro-inflammatory cytokines such as IL-17 in the regulation of inflammatory response in mollusc is largely unknown. In the present study, using molecular biological, cell biological and immunological techniques, the homologs of IL-17 and its signaling components were identified and analyzed structurally and functionally in oyster, C.gigas, for clarifying their regulation roles in response against environment stress.Ten IL-17 homologs were identified in oyster genomic database, designated CgIL17-1～CgIL17-10. They shared low similarity in amino acid sequence. However, they all remained four conserved Cysteine residues in the C terminus, which can form three dimensional Cysteine-knot similar with vertebrate IL-17. Phylogenetic analysis showed markedly difference between vertebrate and invertebrate IL-17. CgIL17 genes were clustered together with other invertebrate ones. Ten CgIL17 genes owned wide tissue distribution, with higher expression in gill, mantle and hepatopancreas. And they showed different expression patterns in the developmental stages of oysters, indicating the differentiation of their roles in the regulation and protection during individual developmental process. Among them, CgIL17-5 exhibited higher expression level in adult oysters and could be markedly up-regulated under bacteria challenge. CgIL17-5 served signaling process to activate immune relative transcript factors and finally induced the synthesis of IL-6. It also showed cytotoxicity against L929 cells, PAMPs binding and bacteriostatic activities.Six molecules containing SEFIR domain were identified from oysters, namely CgSEFIR1～CgSEFIR6, which might involve in CgIL17 signaling. Structure prediction analysis showed that CgSEFIR2 and CgSEFIR4 contained the transmembrane domain, suggesting they might be membrane proteins. CgSEFIR6 possessed a special structural mode, which contained a Death domain at the N terminus and a SEFIR domain at the C terminus similar to MyD88. Multiple sequence alignment and phylogenetic analysis showed that these six CgSEFIR genes can be divided into two groups, in which CgSEFIR1, CgSEFIR2 and CgSEFIR3 were homologous to receptor IL-17RD from vertebrates, and CgSEFIR4, CgSEFIR5and CgSEFIR6 were similar to adaptor CIKS. The further syntenic analysis confirmed the first group possessed closer genetic relationship with vertebrate IL-17RD than the other types of IL-17 receptors.The tissue distribution of four CgSEFIR genes was analyzed through mRNA blot in suit. The results showed that four genes were highly expressed in the digestive tubule of hepatopancreas, and expressed in gill at a lower level. The expression levels of CgSEFIR1, CgSEFIR3 and CgSEFIR6 mRNA were all up-regulated after LPS and PGN stimulation, while CgSEFIR5 was down-regulated after LPS stimulation and up-regulated after PGN stimulation. The interaction among four CgSEFIR genes and CgTRAF6 was tested using mammalian two-hybrid system. The result showed that CgSEFIR5 could interact with CgTRAF6, while CgSEFIR6 could interact with CgSEFIR1 and CgSEFIR3 besides CgTRAF6. Dual-luciferase reporter assays showed that overexpression of CgSEFIR1 or CgSEFIR6 could both activate NF-κB signaling, CgSEFIR5 and CgSEFIR6 could suppress the activity of MyD88 on NF-κB signaling activation. When the mRNA levels of CgSEFIR1, CgSEFIR3 andCgSEFIR6 were knocked down respectively, the expression of CgAIF and CgMIF genes induced by recombinant CgIL17 was reduced. However, the expression of CgAIF and CgMIF genes showed non-significant change when the mRNA level of CgSEFIR5 was knocked down.During air exposure, the glucose content in oyster serum was markedly increased at 3 d, which was followed by a rapid up-regulation of CgIL17- and CgIL17-5. After oysters received an injection of extra glucose, the mRNA expressions of CgIL17-1 and CgIL17-5 were also significantly up-regulated. The histopathological changes of hepatopancreas were observed after the oysters were treated by the recombinant proteins of CgIL17-1 and CgIL17-5 in vivo or subjected to air exposure. And a significant decrease of GSK3β protein was also observed after the injection of CgIL17-1 and CgIL17-5 recombinant proteins in vivo. When the oysters with CgIL17-1 and CgIL17-5 genes knocked down were subjected to air exposure, the decline of GSK3β concentration was slowed down and it could still be obviously detected at 7 d compared with that in the control. Meanwhile, the expression of CgDefensin and CgDFFA was inhibited, while CgIAP was up-regulated when CgIL17-1 and CgIL17-5 genes were knocked down, and the oysters exhibited higher mortality at 3 d, whereas lower at the late stage of air exposure compared with that in the controls.In summary, IL-17 and its signaling components were also existed in oyster C.gigas. They were similar to vertebrate ones in both structure and function, could also involve in immune defence and response to environmental stress and served a critical role in sustaining homeostasis. CgIL17 genes could be used as an important monitoring indicator of oyster health. This study provided an important theoretical basis for oyster disease prevention and cure.