Study Of Interleukin-1 Associated Kinase-4 Functions On Interleukin-1β Signaling Pathways In Grass Carp

Interleukin-1 receptor-associated kinase(Interleukin-1 Receptor-Associated Kinase, IRAK) is a key factor involved in Toll-like receptor and Interleukin-1(IL-1) receptor signal transduction. In the study of mammals, four members of the IRAK family of IRAK-1, IRAK-2, IRAK-3 and IRAK-4 are identified. In IRAK family, IRAK-4 is the most important member, responsible for triggering IL-1/TLR signaling pathway. And a variety of autoimmune diseases are associated with loss IRAK-4, but in lower animals, studies of IRAK-4 and its signaling pathway are very scarce.In this study, the grass carp IRAK-4(gc IRAK-4) is successfully molecularly cloned, sequence analysis showed that gc IRAK-4 gene bears a complete open reading frame, encoding a 473 amino acid sequence. Multiple sequence alignment discovered that gc IRAK-4 amino acid sequence is highly conservative with other species. Further bioinformatics analysis showed that gc IRAK-4 has a complete death domain and kinase domain. Moreover, 3D structural models also provide the basis for its biological functions.Tissue distribution studies show that gc IRAK-4 were expressed in many tissues, where the expression level are Similar. In vitro model in grass carp Headkidney Leukocytes(HKL) by IL-1? stimulation. IRAK-4 expression levels can be induced by IL-1, and this up-regulation can be inhibited by IL-10. In vivo models by Aeromonas hydrophila infection, grass carp IRAK-4 expression was significantly increased within 24 hours, and then disappeared, and the same process also happened in IL-1.Expression vector gc IRAK-4 and its domain mutated fragments are constructed, which were successfully overexpressed in CIK cell lines. It is for first time the first time that IRAK-4 are overexpressed in the same host cell species in lower animals. Overexpression studies have found that, in contrast with mammals, overexpression of gc IRAK-4 can inhibit NF-?B activity, and impairs IL-1??pathway by down-regulating IL-1??m RNA expression with or without IL-1??challenges. And this inhibition function of IRAK-4 in grass carp was dependent on its full functional domains, because this suppression cannot be observed in IRAK-4 domain mutated fragments. From the above results, this study speculated that inhibition functions of fish IRAK-4 may be due to two mechanisms: First, in fishes, IRAK-4 might lack autophosphorylation capacity; second, there might exist negative feedback control mechanism which reacts to IRAK-4 overexpression. Our resluts can provide a basis for further research in the future.