Cloning And Function Study Of IL-1R And Related Family Members In Teleosts

Interleukin-1receptors belong to the TIR (IL-1R/TLR) superfamily members that play crucial roles in IL-1-and LPS-mediated NF-κB signaling pathways. In this study, IL-1RI, DIGIRR and SIGIRR were predicted and isolated from teleosts by bioinformatics and molecular cloning. Moreover, the protein characterization, molecular evolution and immune functions were further studied.In part one, IL-1RI was first cloned from Tetraodon nigroviridis, and then we performed analysis of gene structures, protein characterization, mRNA distribution and gene functions. Recombinant protein of IL-1RI was successfully purified and obtained by prokaryotic expression system, and whereafter the polyclonal antibody was preapared based on the purified IL-1RI. The data showed that IL-1R receptor had affinities with its ligands IL-1β in a dose-dependent manner by ELISA, in agreement with the results of co-evolution relationship between IL-1RI and IL-1. Moreover, when IL-1RI was blocked by the anti-IL-1RI antibodies, the relative gene expression level of IL-1β-incued inflamatory factors were significant reduced.In part two, we report a novel double-Ig-IL-1R related molecule (DIGIRR) from four model fish(Tetraodon nigroviridis, Gasterosteus aculeatus, Takifugu rubripes and Oryzias latipes), adding a previously unknown homologue to the TLR-IL-1R family. This DIGIRR molecule contains two Ig-like domains in the extracellular region, one Arg-Tyr-mutated TIR domain in the intracellular region, and a unique subcellular localization within the Golgi apparatus. These characteristics distinguish DIGIRR from other known family members. The expression of DIGIRR was significantly induced by LPS-stimulation. In vitro injection of DIGIRR into zebrafish embryos dramatically inhibited LPS-and IL-1β-induced NF-κB activation. Moreover, in vivo knockdown of DIGIRR by siRNA significantly promoted the expression of IL-1β-stimulated pro-inflammatory cytokines (IL-6and IL-1β) in DIGIRR-silenced liver and kidney tissues, and in leucocytes. These results strongly suggest that DIGIRR is an important negative regulator of LPS-and IL-1β-mediated signaling pathways and inflammatory responses. In part three, SIGIRR was first isolated from teleost zebrafish, which is considered as the homologue of mammalians SIGIRR according to the analysis of protein characterizations and the results of gene functions. Zebrafish SIGIRR was able to inhibit IL-1β-,LPS-and CpG-indued NF-kB signaling pathways.Evolutionally, we propose a hypothesis that DIGIRR might be an evolutionary "transitional molecule" between IL-1R and SIGIRR, representing a shift from a potent receptor to a negative regulator. These three molecules probably originated from a common ancient IL-1R-like molecule. Following two rounds of genome dulplications, ancient IL-1R-like molecule evolved into DIGIRR. With lineage divergence, parts of specieses retained DIGIRR, and meanwhile DIGIRR became SIGIRR in the other specieses.The identifications of IL-1RI, DIGIRR and SIGIRR demonstrat that there exsit the regulation mechanisms of IL-1R/TLR signal pathways in teleosts. All data not only contribute to better understand the evolutionary history of NF-kB signaling patyways, but also provide available data and scientific evidence for host immune and disease control in fish.