The Regulatory Mechanism Of MAPK/Erk Signaling On T Cell Immunity In Nile Tilapia

Mitogen activated protein kinase/extracellular signal regulated kinase(MAPK/Erk)pathway exists widely in all eukaryotic cells.It responds to a variety of extracellular signals and acts on a series of downstream substrates to regulate cell growth,activation,and proliferation.As one of the most classical pathways induced by T cell receptor(TCR)signaling,MAPK/Erk pathway is indispensable for T cell development,activation,and effector function.However,whether and how MAPK/Erk pathway regulates T cell immunity in lower vertebrates is still a mystery.As the lowest animal with complete adaptive immune system,fish is an ideal model to study the function and evolution of T cells.In this study,we constructed a complete MAPK / ERK pathway in Nile tilapia(Oreochromis niloticus),explored the regulation of this pathway on T cell activation,proliferation and effector function,and further analyzed its related mechanism.The genome of Nile tilapia encodes all the elements of MAPK/Erk pathway,including h-Ras,c-Raf,Mek1,Mek2,Erk1,and Erk2.These elements were very conservative in amino acid sequence,domain configuration and 3-dimensional structure,and were widely distributed in lymphoid related tissues.Using specific inhibitors of the three kinases,it was found that the kinases were phosphorylated in the order of c-Raf-Mek1/2-Erk1/2,which indicated that tilapia activated MAPK/Erk pathway by a conservative "tertiary kinase cascade" reaction.The mRNA expression and protein phosphorylation levels of MAPK/Erk pathway elements in Tilapia lymphocytes were significantly up-regulated during lymphocyte activation,and during the adaptive immune stage induced by pathogen infection;meanwhile,Erk1/2 in activated lymphocytes entered the nucleus after phosphorylation.These results suggest that MAPK/Erk pathway is involved in Tilapia lymphocyte activation and antiinfectious immunity.In Tilapia T cells activated by mitogen PHA,the inhibition of MAPK/Erk pathway blocked the up-regulation of IFN-γ and CD122.During the primary immune response,the lack of MAPK/Erk down-regulated of T cell proliferation,as well as expression level of effector T cell related genes,and finally resulting in the impaired ability of pathogen clearance and increase in mortality.These results indicate that Tilapia MAPK/Erk pathway promotes the activation,proliferation,and effector function of T cells.Inhibition of MAPK/Erk pathway limited the expression of glucose transporter 1(GLUT1)and glucose uptake in PHA-activated T cells.The blockage of MAPK/Erk pathway also reduced the mRNA expression of three rate limiting enzymes during glycolysis,hexokinase 2,phosphofructokinase and pyruvate kinase,the enzyme activity of pyruvate kinase,and the expression of c-Myc.Further studies showed that the inhibition of c-Myc significantly down regulated the expression of LDHA and IFN-γ during T cell activation,and also weaken the proliferation of lymphocytes in antiinfectious immunity.These results suggest that MAPK/Erk pathway promotes the activation and proliferation of T cells through c-Myc-mediated glucose metabolism.The IL-2 gene downstream of MAPK/Erk pathway was identified from the genome sequence of Nile tilapia(Oreochromis niloticus)by syntenic analysis.Upon T cell activation or pathogen infection,the expression of IL-2 in spleen lymphocytes was dramatically induced,while the blockage of MAPK/Erk could inhibit the expression of IL-2 in activated T cells.The recombinant Nile tilapia IL-2 was prepared by prokaryotic expression system.It was found that IL-2 could activate several immune signal pathways including MAPK/Erk pathway in lymphocytes.IL-2 also regulated the expression of cytokines and transcription factors related to effector T cells.These results indicate that MAPK/Erk pathway regulate Tilapia T cell immunity by promoting the expression of IL-2.This study investigated the regulation of MAPK/Erk pathway on T cell immunity of tilapia,and elucidated in-depth regulatory mechanism.The results enrich the theory of disease prevention in fishery cultivation,the theory of fish adaptive immunity,and provide a new perspective for understanding the origin and evolution of adaptive immune system.