Grass carp (Ctenopharyngodon idella) interferon regulatory factor 8 down-regulates interferon1 expression via interaction with interferon regulatory factor 2 in vitro

Abstract

Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein (ICSBP), is a negative regulatory factor of interferon (IFN) and plays an important role in cell differentiation and innate immunity in mammals. In recent years, some irf8 homologous genes have been cloned and confirmed to take part in innate immune response in fish, but the mechanism still remains unclear. In this paper, a grass carp (Ctenopharyngodon idella) irf8 gene (Ciirf8) was cloned and characterized. The deduced protein (CiIRF8) possesses a highly conserved N-terminal DNA binding domain but a less well-conserved C-terminal IRF association domain (IAD). Ciirf8 was widely expressed in all tested tissues of grass carp and up-regulated following poly(I:C) stimulation. Ciirf8 expression was also up-regulated in CIK cells upon treatment with poly(I:C). To explore the molecular mechanism of how fish IRF8 regulates ifn1 expression, the similarities and differences of grass carp IRF8 and IRF2 were compared and contrasted. Subcellular localization analysis showed that CiIRF8 is located both in the cytoplasm and nucleus; however, CiIRF2 is only located in the nucleus. The nuclear-cytoplasmic translocation of CiIRF8 was observed in CIK cells under stimulation with poly(I:C). The interaction of CiIRF8 and CiIRF2 was further confirmed by a co-immunoprecipitation assay in the nucleus. Dual-luciferase reporter assays showed that the promoter activity of Ciifn1 was significantly inhibited by co-transfection with CiIRF2 and CiIRF8. The transcription inhibition of Ciifn1 was alleviated by competitive binding of CiIRF2 and CiIRF8 to CiIRF1. In conclusion, CiIRF8 down-regulates Ciifn1 expression via interaction with CiIRF2 in cells.