IRAK1BP1 negatively regulated innate immune response via NF-κB signal pathway in blunt snout bream (Megalobrama amblycephala)

Interleukin receptor-associated kinase (IRAK)-1 binding protein 1 (IRAK1BP1) is a vital factor in preventing dangerous overproduction of proinflammatory cytokines by the innate immune system. The structure and function of IRAK1BP1 have been well investigated in mammals, yet the piscine IRAK1BP1 is poorly understood. In the present study, the first teleost fish IRAK1BP1 from blunt snout bream, maIRAK1BP1, was identified and characterized, which was composed of a 750 bp open reading frame encoding a protein of 249 amino acids. Phylogenetic analysis indicated that maIRAK1BP1, is a novel member of IRAK1BP1 family and showed the highest similarity to that of grass carp (96.28%). Amino acid sequence analysis revealed that maIRAK1BP1 shared a conserved SIMPL domain. Spatial expression pattern analysis revealed that maIRAK1BP1 was constitutively expressed in various normal tissues including heart, spleen, head kidneys, gill, kidney, muscle, intestine and liver. The maIRAK1BP1 expression in immune-relevant tissues (liver, head kidneys and kidney) and caudal fin cells (MAF) were significantly up-regulated after LPS and bacteria injection. The result from subcellular localization analysis indicated that maIRAK1BP1 protein was localized in both the cytoplasm and nucleus of MAF cells, but predominantly existed in cytoplasm. The activity of NF-κB promoter could be inhibited by overexpression of maIRAK1BP1 in HEK-293T cells. Overexpression of maIRAK1BP1 in MAF cells inhibited dramatically the transcriptional expression level of lipopolysaccharide-induced proinflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α). In contrast, an increased expression of these cytokines was observed when maIRAK1BP1 was knocked down via siRNA. Taken together, these results indicated that maIRAK1BP1 played an important role in mediating host innate immune response to pathogen invasion, and disclosed the involvement of maIRAK1BP1 in NF-κB-mediated production of proinflammatory cytokines, which provided foundational insights into the conserved role of IRAK1BP1 in teleost immune regulation.