Genes Involved In T-cells Differentiation Of Atlantic Cod (Gadus Morhua L.) And Ig+Cells Distirbution And Response In Japanese Flunder (Paralichthys Olivaceus)

Papers have been reported suggest that CD4+and CD8+T cell lineages arepresence in teleosts and play central roles in the function of the immune system. Theobjectives of this study were five genes involved in T-cells differentiation of Atlanticcod including NF-kappaB repressing factor, TBK1, GATA-3, Foxp3, EOMES,Moreover, expression analysis， promoter genomic structure and functionalassessments of these transcription factors have been studied with the aim to delineatethe regulation of T-cells Differentiation in Atlantic cod. For Japanese flounder, location,quantitative investigation and response of immunoglobulin-positive cells inimmunologic tissues have been studied to explore the acquired immune system.TBK1, also termed NAK or T2K, is a ubiquitous member of the IκB kinase (IKK)family that is required for innate and adaptive immune responses. The cod TBK1geneconsists of2190bp open reading frame encoding a polypeptide of729amino acids.RT-PCR showed that the largest quantity of TBK1transcripts was found in spleen.Both PMA, poly I:C and β-glucan promoted expression of TBK1transcripts in vivo.Furthermore, we determined an875bp sequence upstream of the transcriptional startsite (TSS) and found a number of sequence motifs that matched known transcriptionfactor binding sites. Activities of the presumptive regulatory regions of this gene wereassessed by transfecting different5’ deletion constructs in CHSE-214cells. After theexpression experiments, the results showed that the basal promoters and positivetranscriptional regulator activities of cod TBK1gene were dependent by sequenceslocated from-875to-425bp and from-245to+28bp upstream of TSS. This studyprovides further insights into the transcriptional regulation of cod TBK1.NF-κB is important in regulating cellular responses because it belongs to thecategory of "rapid-acting" primary transcription factors, i.e., transcription factors thatare present in cells in an inactive state and do not require new protein synthesis to beactivated (other members of this family include transcription factors such as c-Jun,STATs, and nuclear hormone receptors). This allows NF-κB to be a first responder toharmful cellular stimuli. The cod NF-κB repressing factor cDNA consisted of3397bpin length with an open reading frame of2394bp encoding a polypeptide of797aminoacids, According to a BLAST search, the cloned NRF gene has G-patch domain (656-701) and a R3H domain (706-770). NRF proteins fused with RFP-tag were identified in or close to the nuclei48h after the plasmids were transfected in CHSE-214cells,showed that the NRF performed the function in nuclei.Eomesodermin (Eomes), a T-bet paralogue expressed in activated CD8+T cellswas recently proposed to act as a master regulator of cytotoxic CD8+T cell effectorfunction and offers an exciting avenue for future exploration. Here, we have identifiedand characterized the full-length Atlantic salmon Eomes cDNA (2618bp). Promoteranalysis of the cod Eomes showed the presence of important putative transcriptionbinding sites like NF-E2, S8，GATA-1/2，HSF2, and Ets-1. The basal core regionresponsible for the promoter activity was located between base-694and-376. RT-qPCR analysis revealed that the Atlantic salmon Eomes was normally expressed in allthe tissues studied but strongly expressed in the spleen,and the head kidney.GATA-3is a master transcription factor of the Th2cells. Cod GATA-3(GmGATA-3) has a1320bp open reading frame encoding a polypeptide of440aminoacids with two zinc finger domains that are fully conserved within teleosts and highervertebrates. The GATA-3cDNA splice variant without zinc finger domains was shownto contain an828bp open reading frame encoding a polypeptide of276amino acids.Both GATA-3proteins fused with RFP-tag were identified in or close to the nuclei48hafter the plasmids were transfected in CHSE-214cells. The full length GATA-3withtwo zinc finger domains has a transcriptional function confirmed by transfection withGATA-3reporter vector along with expression constructs of GATA-3plasmids inCHSE-214cells, whereas the GATA-3splice variant without zinc finger domain didnot enhance the activity of the GATA-3reporter vector. RT-PCR analysis revealed thatthe two Atlantic cod GATA-3variants were strongly expressed in the gills.Unexpectedly, PMA increased the expression of the GATA-3splice variant in vivo andespecially in vitro, with an increase of more than100,000fold in head kidneyleukocytes at24h and48h. On the other hand, there were significant decreases at thetranscript level of full length GATA-3between PolyI:C and β-glucan treatment groupscompared to controls.Foxp3is a T cell-specific transcription factor and plays a key role in thedevelopment of Treg cells and in the immune regulatory process during inflammation.Here we report cloning and characterization of the full-length cDNA of Atlantic codFoxp3, which possesses a Forkhead domain, a zinc finger domain and a leucine-zipperdomain as its counterpart in mammals. Foxp3is highly expressed in gills. Furthermore, we determined an1615bp sequence upstream of the transcriptional start site (TSS) andfound a number of sequence motifs that matched known transcription factor bindingsites. Activities of the presumptive regulatory regions of this gene were assessed bytransfecting different5’ deletion constructs in CHSE-214cells. After the expressionexperiments, the results showed that the basal promoters and positive transcriptionalregulator activities of cod Foxp3gene were dependent by sequences located from-400to+21bp upstream of TSS.Histological, immunocytochemical immunohistochemical and flow cytometricaldetection of immunoglobulin-positive (Ig+) cells in the peripheral blood, kidney, liver-pancreas, spleen and gut associated lymphoid tissues of the flounder (Paralichthysolivaceus) were performed. Results showed that Ig+cells were found in all the sampledtissues. Ig+cells with diffusely stained cytoplasmic were observed in the peripheralblood, but did not found Ig+macrophages. There were many macrophages,granulocytes, lymphocytes and other immune-related cells in spleen and kidney, Ig+cells were mostly presented around the melanomacrophage centers (MMCs) and bloodvessels in the spleen, and the lymphoid Ig+cells in the kidney were also closelyassociated with the MMCs and existed in group or dispersedly. The pancreas wassurrounded with liver in flounder and formed liver-pancreas, Ig+cells were separatelydistributed in the hepatic tissue, not in the pancreas. Additionally, Ig+cells in the gutassociated lymphoid tissue were rarely detected in the epithelial layer although manylymphocytes were observed, but they were frequently presented in the lamina propria ingroup or separately, presumably as a part of the intestine involved in mucosal immuneresponses. The flow cytometrical results showed that the largest relative ratio is theperipheral blood, and followed by spleen, kidney, liver and gut.