Cloning And Immune Functional Characterization Of TRIF And TRAF3 In Large Yellow Croaker Larimichthys Crocea

As an adaptor in Toll-like receptor(TLR)signaling pathway,Toll/interleukin-1 receptor(TIR)domain containing adaptor inducing interferon-β(TRIF)mediates downstream signaling cascades and plays important roles in host innate immune responses.TRAF3 is a member of the tumor necrosis factor receptor(TNFR)related factor(TRAF)family,and is an important regulator of NF-κB and type I interferon(IFN)activation.In the present study,The TRIF and TRAF3 of large yellow croaker were cloned and identified,named as Lc-TRIF and Lc-TRAF3,respectively.And the expression patterns of TRIF and TRAF3 in various tissues of healthy fish and stimulated with different PAMPs were detected by quantitative real-time PCR.The subcellular localization of TRIF and TRAF3 was revealed by using fluorescent tracer plasmids,and the role of TRIF and TRAF3 on activation of NF-λB,IFN1,IRF3 and IRF7 promoters was also determined by using dual-luciferase reporter system.In addition,the core region of TRIF promoter and the immune-related molecules associated with TRIF was also disscused.Collectively,the present study provided immune functional characterization of TRIF and TRAF3 in large yellow croaker,and disscussed the cellular signal transduction pathways mediated by TRIF,and also their roles in host immune responses.The main findings are as follows:1)The full length cDNA of Lc-TRIF was 2834 bp,of which ORF is 1806 bp,encoding a protein of 601 amino acid(aa).Sequence comparison analysis showed that Lc-TRIF has a conserved TIR domain but without TRAF6 binding motif.The genome organization of Lc-TRIF is conserved,with two exons and one intron.The gene homology search and collinear analysis found that TRIF exists in the genomes of fish,amphibians,birds,reptiles and mammals,but no homologous sequences of TRAM were found in fish,amphibians and birds.Expression analysis revealed that Lc-TRIF was broadly expressed in various tissues,with the highest expression level in gills and the weakest expression in brain,and Under the stimulation of Poly I:C,LPS,PGN and Pseudomonas plecoglossicida,the expression of TRIF in gill,intestine,spleen,head kidney and blood tissue of large yellow croaker was significantly induced.2)The cloned promoter region of Lc-TRIF was 1619 bp before the translation start site.Based on the prediction of the core promoter region and transcription factor binding sites,plasmids lacking various fragments were constructed for subsequent confirmation by using dual-luciferase reporter system.The results revealed that the region between-105 bp and-155 bp upstream of the transcriptional start site was the core regulatory region of the large yellow croaker TRIF promoter.And the transcription factors including C/EBPalp,GATA-1,AP-1,c-Jun and ISGF-3 located in the region between-60 bp to-1046 bp may be important regulatory elements of the TRIF promoter.3)The results of luciferase analysis showed that the expression of large yellow croaker TRIF could significantly induce the activation of NF-κB,IFN1,IRF3 and IRF7 promoters.and the RHIM domain of the TRIF play an important role in the activation of NF-κB,IFN1,IRF3 and IRF7.4)Base on the luciferase analysis of the association between the immune-related molecules of large yellow croaker and TRIF,it was found that TLR3 significantly inhibited TRIF-mediated NF-κB activation,but had a synergistic effect in IRF3 and IRF7-dependent signaling pathways.TLR22 significantly inhibits TRIF-ediated activation of NF-κB and IFN1.Although SARM1 and TRAF6 inhibit TRIF-mediated activation of NF-κB,they can significantly activate TRIF-mediated activation of IRF3 and IRF7.Co-transfection of TBK1 and TRIF inhibited the activation of NF-κB and IFN1,but significantly enhanced the activation of the IRF7 promoter.In addition,IRF3 co-expressed with TRIF could significantly down-regulated the activation of NF-κB,but significantly up-regulated the activation IFN1 and IRF3 promoters.And IRF7 expression significantly enhanced TRIF-mediated activation of NF-κB,IRF3 and IRF7 promoters.5)The open reading frame of Lc-TRAF3 contains 1788 bp encoding a protein of 595 amino acids.Sequence analysis indicated that Lc-TRAF3 is conserved in vertebrates,constituted with a N-terminal RING finger,two TRAF-type zinc fingers,and a C-terminal TRAF-MATH domain.The genome organization of Lc-TRAF3 is conserved in fish,with 13 exons and 12 introns.Lc-TRAF3 was identified as cytosolic protein base on fluorescence microscopy analysis.Expression analysis revealed that Lc-TRAF3 was broadly distributed in examined tissues,with the highest expression level in gill and weakest in brain,and could be Induced expression under Poly I:C,LPS,PGN,and Pseudomonas plecoglossicida stimulation in vivo.Interestingly,overexpression Lc-TRAF3 could induce the activation of NF-κB,and Lc-TRAF3 co-transfected with Lc-TRIF induced a significantly higher level of NF-κB and IRF3 promoter activity,implying that Lc-TRAF3 may function as an enhancer in Lc-TRIF-mediated signaling pathway.In conclusion,the prestent study characterized temporal and spatial expression pattern of TRIF and TRAF3 in large yellow croaker,and revealed the transcriptional regulation mechanism of TRIF.Moreover,the association between TLR3,TLR22,TRAF3,TRAF6,SARM1,TBK1 and TRIF was discussed,and the signaling pathway mediated by TRIF in large yellow croaker was elucidated.It is thus provide a foundation for further study on the regulatory role of TRIF and TRAF3 in host immune defense.