The Mechanism Of Ca²⁺ Mediated NFAT Nuclear Transportation On T Cell Immunity Regulation In Nile Tilapia

T cell plays an essential role in adaptive immunity.The development,activation,proliferation and differentiation of T cell are precisely modulated by many signaling pathways.Nuclear factor of activated T cells?NFAT?plays an important role in T cell immunity by regulating the transcription of many cytokines.NFAT is mainly regulated by calcium/calcineurin(Ca²⁺/CaN)axis,which is an important part of calcium signal transduction.The regulatory mechanism of Ca²⁺-NFAT pathway on T cell immunity is limited to mammalian model,and it is little known whether and how Ca²⁺-NFAT regulates T cell immunity in primitive vertebrates at present.Nile tilapia?Oreochromis niloticus?is an ideal material to investigate the regulation of ancestral T cell immunity by Ca²⁺-NFAT signaling in jawed fish.In this study,the composition,activation and regulation of Ca²⁺-NFAT pathway and its regulatory mechanism on fish T cell immunity were explored by immunology,cell biology and biochemistry methods.Nile tilapia encodes complete and conserved Ca²⁺-NFAT pathway elements,including IP3 recepter?IP3R?,Calmodulin?CaM?,CaN and NFAT.The analyzation of amino acid sequence,domains,tertiary structure and phylogeny of the pathway elements indicated that the Ca²⁺-NFAT pathway of Nile tilapia has the similar regulatory function in T cell.QRT-PCR and western-blot analysis of Ca²⁺-NFAT signaling components expression illustrated that,during Streptococcus agalactiae infection,Ca²⁺-NFAT signaling pathway is involved in the primary adaptive immune response.When lymphocytes of Nile tilapia were stimulated with PHA or PMA plus ionomycin in vitro,it was found that the phosphorylation level of IP3R was up-regulated,forming a continuous calcium influx,and then the protein level of CaM was increased,which activated CaN,and finally led to the entry of NFAT into the nucleus.Co-Immunoprecipitation showed that NFAT1 of Nile tilapia form homodimer in HEK293T cells,and combine with c-Fos,NIP45,T-bet,IRF4,Foxp3 to form transcription regulatory complexes,which suggested that NFAT of Nile tilapia can regulate gene expression by transcription regulatory complexes after entering the nucleus.The nuclear transportation of NFAT is regulated precisely in Nile tilapia.Nile tilapia Codes evolutionarily conserved Cabin1 and DYRK1A,and their sequences and structures are highly conserved with homologous molecules in mammals.Concordantly,the phosphatase activity of calcineurin was enhanced obviously,and we also found strengthened NFAT nuclear import when Cabin1was interfered with siRNA.It indicates Cabin1 negatively regulates NFAT nuclear import and T cell activation by inhibiting the activity of CaN.The inhibition of DYRK1A by harmine resulted in the decrease of NFAT1 nuclear export and high activation state of T cells,indicating that DYRK1A regulates the activation of T cells by promoting NFAT nuclear export.At the same time,the phosphorylation level of GSK3?was enhanced by harmine,which speculated that DYRK1A regulate NFAT nuclear export by GSK3?probably.More critically,suppression of Ca²⁺-NFAT signaling by the CaN inhibitor cyclosporine A impairs weight of immune organs,T cell activation,T cell clonal expansion,and pathogen clearance during the S.agalactiae infection.Meanwhile,the expression of pro-inflammatory cytokines,pro-apoptotic genes and cytotoxic genes in lymphocytes were also reduced,which intensified infection and death.It is suggested that Ca²⁺-NFAT pathway plays an important role in T cell immunity in Nile tilapia.This study is the first to describe the regulatory mechanism of Ca²⁺-NFAT signaling on T cell immunity in teleost fish.We suggest that modulation of T cell immunity by Ca²⁺-NFAT signaling is a primitive strategy that already existed in teleost fish.The findings of this study provide valuable perspectives for understanding the evolution of adaptive immune system.It also provides theoretical basis for disease control in aquaculture of Nile tilapia.