Identification And Functional Analysis Of Nile Tilapia CD6 And Its Ligands

In mammals,the CD6 molecule,as a lymphocyte-specific surface receptor from the scavenger receptor family,plays an important role in anti-inflammatory responses and T cell activation,refer to account both innate and adaptive immune regulation.However,the characteristics and functions of CD6 molecules in lower vertebrates represented by teleost fish are unknown.In order to explore the function of fish CD6 molecules.In this study we cloned and identified CD6 molecules and its ligands CD166,Syntenin-1,LCP2,and TARF6 in Nile tilapia.The research work was carried out in the following four aspects:（1）On the basis of obtained On CD6 extracellular domain protein through prokaryotic expression and prepared monoclonal antibodies,we explored the biological function of On CD6-ECD;（2）The function and mechanism of On CD6 molecules involved in the inflammatory response were explored through tilapia head-kidney lymphocytes and animal experiments;（3）The interaction of CD6 molecules and its ligands were identified by GST Pull-Down;（4）A preliminary investigation was conducted on the signaling pathways of CD6 molecules and their ligands.The main research results as follows:1.On CD6 molecule as a scavenger receptor has the function of pattern recognition receptorIn this study,CD6 of Tilapia was cloned and identified successfully.Subcellular localization experiments showed that On CD6 was located on the lymphocyte membrane.In vitro experiments showed that On CD6-ECD,can widely recognized various aquatic pathogens and pathogenic pattern molecules（PAMPs）under the action of Ca²⁺.It has antibacterial activities in vitro and can induce bacterial agglutination.It can promote respiratory burst effect and phagocytosis response of head-kidney lymphocytes,and regulated the expression of HKLs pro-inflammatory（IL-1βand TNF-α）and anti-inflammatory factors（IL-10 and TGF-β）.The On CD166 ligand could recognize pathogenic pattern molecules more extensively without Ca²⁺.These results indicated that the On CD6 molecule function was conserved as a scavenger receptor.2.The mechanism of On CD6 molecule involved in the acute inflammation of tilapia caused by Streptococcus agalactiaeOn CD6-ECD recombinant protein combined with Streptococcus agalactiae challenge and On CD6 overexpression in vivo challenge experiment can effectively improve the immune protection rate of tilapia against Streptococcus agalactiae infection.Further investigation found that On CD6 can effectively reduced the colonization of Streptococcus agalactiae in tilapia,and enhanced the fish total antioxidant capacity,inhibited the expression of pro-inflammatory factors such as IL-1βand TNF-α,and promoted the expression of anti-inflammatory factors such as IL-10 and TGF-β.Stimulation of HKLs by cell signaling pathway inhibitors revealed that On CD6 molecules may regulate the expression of inflammatory factors through the NF-κB and MAPK pathways.Combined with the results of in vitro studies,we have found a possible mechanism for On CD6 to participate in the inflammatory response:Through widely recognized various PAMPs and combined various pathogens to caused bacterial agglutination,increased the size of bacteria particle,enhanced antioxidant enzyme activity and enhance respiratory outbreaks,and promoted the clearance from the circulation（by,for instance,facilitating phagocyte engulfment）.At the same time,it down-regulates the expression of pro-inflammatory factors,promotes the expression of anti-inflammatory factors,and reduces the inflammatory processes that lead to injury or death by Streptococcus agalactiae infection.3.In vitro analysis the functional and interaction of On CD6 and its ligandsThe On CD6 intracellular and extracellular related ligands CD166,Syntenin-1,LCP2and TRAF6 were successfully cloned.q RT-PCR results showed that the expression characteristics of On CD6 molecules and its ligands are consistent,they mainly high expressed in immune-related tissues,and are rapidly up-regulated under the stimulation of LPS,LTA and Streptococcus agalactiae,indicating that CD6 molecules and their ligands participated in the immune response of tilapia to Streptococcus agalactiae;KLH stimulation results showed that On CD6 increased gradually after KLH stimulation,and showed a sequential expression pattern with faster expression,higher intensity and longer duration after the second immunization.We detected that On CD6 may be involved in the host defense against T cell-dependent pathogen infection in bony fish.GST Pull-Down and ELISA experiments showed that On CD6 was able to bind to the extracellular ligand On CD166,and the interaction domains were On CD6-SR₂ and On CD166-Ig₁;GST Pull-Down verified the On CD6-ICD is able to bind to On Syntenin-1 and On LCP2,and On CD6 and On TRAF6 may be bound through On Syntenin-1.4.Signal pathways involved in On CD6 and its ligandsIn this study,we found On CD6/On CD166 inhibited activation of the NF-κB pathway,and On TRAF6 significantly activated this pathway.After co-transfection of On CD6/On CD166 and On TRAF6,it was found that On CD6/On CD166 down-regulates NF-κB activation induced by TRAF6,indicating that On CD6 may participate in the negative regulation of NF-κB through binding to TARF6;In addition,On CD6/On CD166effectively activates STAT1 and ISRE interferon activates the response element and finally activates the expression of IFN3,and has no significant effect on IFN1.We speculate that the interaction between On CD6 and its ligand may play an important role in the activation of IFN3,and thus also play a role in the antiviral response.