Gene Cloning And Immune Functional Analysis Of Tollip And MyD88 In Japanese Eel(Anguilla Japonica)

Myeloid differentiation factor(MyD88)and Toll-interacting protein(Tollip)play an important role in innate immune response as principal components involving the TLR/IL-1R immune signaling pathway.In this study,MyD88 and Tollip were cloned for the first time from Anguilla japonica,named AjMyD88 and AjTollip,respectively.Full-length cDNA sequence of AjMyD88 was 1774bp,with an 846 bp open reading frame encoding a protein of 282 aa.The predicted three-dimensional structure of AjMyD88 could be merged well with the crystal structure of the human MyD88 and shares the typical death domain and TIR(Toll-like/IL-1 receptor)domain of the MyD88 families.In the TIR domain,three conserved boxes,named box 1,box 2 and box 3,are found.According to the comparison with known MyD88 amino acid sequences,the putative protein of AjMyD88 showed the highest identities with Ictalurus punctatus(75%),and other bony fish(68%~73%).All the sequences were clustered into three main branches in which all MyD88 of fish clustered together,while the MyD88 of mammalian and amphibious formed two separate clusters.Full-length cDNA sequence of AjTollip was 1313 bp,with 828 bp open reading frame encoding a protein of 276 aa.The predicted three-dimensional structure of AjTollip could be merged well with the crystal structure of the human Tollip.AjTollip contains a typical TBD domain,C2 domain,and CUE domain and these amino acid sequences are highly conserved.The putative protein of AjTollip presented the highest identities with Atlantic salmon(92%).All the sequences were clustered into three main branches in which all Tollip of fish clustered together,while as the Tollip from mammalian and amphibious formed two separate clusters.AjMyD88 and AjTollip genes were expressed in various tissues of the Japanese eel according to the Real-time PCR results.The highest gene expression was observed with AjMyD88 in spleen and AjTollip in blood while both of the genes were found to express very weak in in muscle and heart.The temporal expression profiles of AjMyD88 and AjTollip in spleen,liver,kidney,and blood were evaluated following stimulation with LPS,poly I:C,or Aeromonas hydrophila.Expression profiling results was as following:after injection with LPS,the AjMyD88 gene was obviously up-regulated in the kidney,liver,and spleen whereas the gene expression level was down-regulated significantly in blood.When stimulated with polyI:C,the AjMyD88 gene expression was found to increase strongly from 6 h to 48 h in all the tested tissues,but down-regulation of the gene expression was detected at 12 h in the kidney.Post Aeromonas hydrophila infection,the AjMyD88 expression level was obviously up-regulated in the liver,spleen,and blood.But in the kidney,the gene expression was down-regulated significantly.After injection with LPS,the AjTollip gene was found to obviously increase in kidney and liver,and decrease in blood while no change of the gene expression was observed in spleen.When stimulated with polyI:C,AjTollip gene was up-regulated in kidney and liver but down-regulated in blood and spleen.After infection with Aeromonas hydrophila,AjTollip gene expression in kidney and liver was found to increase significantly,and the down-regulation of the gene was detected in blood.However,the gene expression in kidney was shown down-regulated at 6 h,but enhanced at 72 h.Temporal expression profile of the AjMyD88 and AjTollip transcripts in Japanese eel liver cell line after treatment with LPS,poly I:C,CpG-DNA,PGN or different concentration of Aeromonas hydrophila infection was revealed as follows:with simulation with LPS,no significant change of AjMyD88 gene expression level was observed.But the gene could be strong induced by treatment with polyI:C,CPG,and PGN.When infected by Aeromonas hydrophila,AjMyD88 gene expression was shown to increase significantly at 72 h.However,the bacteria with concentrations of 10~7 cfu/mL and 10~8 cfu/mL could down-regulate the gene expression at 3 h.AjTollip gene expression could be enhanced by LPS,polyI:C,CPG,and PGN stimulation.Up-regulation expression of the gene was also observed post Aeromonas hydrophila infection at different concentration from 3 h to 12 h.However,under the high concentration of 10~8 cfu/mL bacteria,AjTollip expression level was found to decrease from24 h to 48 h.GFP fusion gene expression vector pEGFP-N1-AjMyD88 and pEGFP-N1-AjTollip were transfected to HEK-293T cells.Then the human TNF-?,MXA and IL6 gene expression HEK-293T cells were analyzed post the stimulation with LPS and polyI:C.The overexpression of AjMyD88 was found to enhance both IL6 and MXA expression induced by LPS or TNF-?and IL6 gene expression triggered by polyI:C.All the three genes TNF-?,MXA and IL6 could be up-regulated more strongly by polyI:C after the overexpression of AjTollip in HEK-293T cells.Only MXA expression level was enhanced with the treatment of LPS.Our results revealed that both AjMyD88 and AjTollip are key signal transduction and regulatory factors of the TLR/IL-1R signaling pathway in Japanese eel and play an important role in the immune response to bacterial and viral infection.