Grass Carp(Ctenopharyngodon Idella)IRAK1 And STAT3 Upregulate Synergistically The Transcription Of IL-10

In vertebrates,interleukin 10(IL-10)is an anti-inflammatory factor that serves as a key inhibitory role in a wide range of immune responses.IL-1 receptor-associated kinase 1(IRAK1),a key molecule in the inflammatory signal of IL-1R/TLR,plays an important role in regulating the body's autoimmune process.Signal transduction and transcription activator 3(STAT3)has multiple biological functions including processes involved in inflammation,tumorigenesis,metabolic disorders and immune response.In mammals,under the stimulation of LPS,IRAK1 and STAT3 interact in nucleus to regulate IL-10 gene transcription.However,the relationship between fish IRAKI and STAT3 has not been reported.To explain the anti-inflammation in fish,we amplified and identified the complete open reading frame of grass carp IRAK1(CiIRAK1)and STAT3(CiSTAT3)based on the existing sequences.After stimulation of grass carp kidney cells(CIK cells)with LPS,it was found that the nucleic acid expression levels of CiIRAK1 were significantly up-regulated from 6 h to 24 h and reached the highest level at 24 h;The expression level of CiSTAT3 was up-regulated from 6 h to 12 h and reached the highest level at 6 h.This experimental result shows that both CiIRAK1 and CiSTAT3 can respond to LPS stimulation and are involved in the signaling of LPS-TLR4 pathway.In the subcellular localization analysis of CiIRAK1 and CiSTAT3,we found that without any stimulation CiIRAK1 is only distributed in the cytoplasm,and after LPS stimulation,a part of CiIRAK1 enters the nucleus.After LPS stimulation,a part of CiIRAK1 entered the nucleus.CiSTAT3 can be detected in cytoplasm and nucleus with or without LPS stimulation.The results of co-immunoprecipitation analysis showed that CiIRAK1 and CiSTAT3 could interact with each other under LPS stimulation.In the absence of LPS stimulation,CiIRAK1 and CiSTAT3 can not interact.Subsequently,immunofluorescence co-localization experiments further demonstrated that CiIRAK1 and CiSTAT3 interacted in the nucleus under LPS stimulation.In order to explore the role of CiIRAK1 and CiSTAT3 in the transcriptional regulation of CiIL-10,first,we transfected the recombinant plasmids IRAK1-pCDNA3.1 or STAT3-pCDNA3.1 into CIK cells,respectively.The results of Q-PCR experiments showed that under the condition of overexpressing CiIRAK1 or CiSTAT3,the expression level of CiIL-10 was significantly increased;Conversely,after transfecting the relevant interfering sequences SiIRAK1 or SiSTAT3 into CIK cells respectively.Under the conditions of knocking down CiIRAK1 and CiSTAT3,the expression of CiIL-10 was significantly down-regulated.Finally,transfection of IRAK 1-pCDNA3.1 or STAT3-pCDNA3.1 respectively and co-transfection of recombinant plasmids of IRAK1-pCDNA3.1 and STAT3-pCDNA3.1 into CIK cells for dual luciferase gene report experiments.The experimental results show that both CiIRAK1 and CiSTAT3 can enhance the activity of CiIL-10 promoter.Compared with the single transfection,the promoter activity of CiIL-10 was stronger when the IRAK1 and STAT3 plasmids were co-transfected.