Preliminary Study On The Function Of IRAK-4 And IKKαs In Grouper (Epinephelus Coioides)

The innate immunity plays an important role in resisting pathogenic invasion of fish.The innate immune system mainly uses pattern recognition receptors(PRRs)expressed by innate immune cells to quickly recognize the conserved pathogen-associated molecular patterns(PAMPs),which induce innate immune defense and inflammatory response.Among the pattern recognition receptors discovered so far,toll like receptors(TLRs)are the most widely studied but there are still many issues to be revealed.In this study,two important signal transduction molecules of grouper---IKKα and IRAK-4 in the TLR pathway are studied.The results are as follows:Firstly,two IKKα homolog were cloned from the grouper,and it was determined that they were highly similar to that of other species through homology comparison and phylogenetic analysis.Ec IKKα-1 and-2 are widely distributed in different tissues of healthy grouper,and both have high expression levels in immune-related tissues.After infection with Crytocaryon irritans,the expression of Ec IKKαs was significantly up-regulated in the gill;In spleen,the expression of Ec IKKα-1 was first up-regulated and then decreased after infection,but the expression of Ec IKKα-2 did not change significantly at all tested time points.Ec IKKα-1 was localized in the cytoplasm of GS cells,while Ec IKKα-2 was localized in the entire cell.Both Ec IKKαs can significantly activate NF-κB,and the activation degree of Ec IKKα-2 was two folds as Ec IKKα-1.It is found that they can positively regulate the expression of proinflammatory cytokines such as IL-1β,IL-6,TNF-α,etc.after transfection of Ec IKKαs or their Si RNAs into GS cells respectively.Secondly,EcIRAK-4 and other four fish IRAK-4 can significantly activate NF-κB signaling in GS cells.Ec IRAK-4 can be combined with Ec My D88,and does not inhibit Ec My D88-dependent NF-κB activation.Different doses of Ec IRAK-4 can cause different degrees of nuclear translocation of the transcription factor NF-κB p65 subunit.And it can induce the transcription of multiple downstream pro-inflammatory cytokines.In addition,we also constructed some expression vectors of deletion domains or mutations at important sites of Ec IRAK-4,and found that the Ec IRAK-4 kinase domain is necessary for its signal transduction function.The conserved amino acid sites performed similar functions,while the grouper-specific amino acids like Y16 and E339 also play important roles.