NF-κB Couples TCR And IL-17 Signals To Regulate Ancestral T Cell Immune Response In Nile Tilapia

T cell plays a key role in anti-infection and anti-tumor immunity and is an important part of the adaptive immune system.The activation,proliferation and effector functions of T cells are precisely regulated by many pathways,including the nuclear transcription factor kappa-B(NF-κB)signaling pathway.However,the current research on the regulation of T cell immunity by the NF-κB pathway is mainly focused on higher animals.Whether and how this pathway participates in the T cell immunity of lower vertebrates is still unknown.Fish are the earliest animals with T cells.The investigation of the immune function and regulatory mechanism of T cell can help to understand the origin and evolution of adaptive immunity,and provide theoretical basis for disease prevention in aquaculture.In this study,using Nile tilapia(Oreochromis niloticus)as a model,bioinformatics,molecular and cellular immunology,biochemistry and other methods were used to study the composition of NF-κB pathway,signal transduction pathway,and the regulatory mechanisms of T cell immunity.Nile tilapia has complete NF-κB pathway components,including PKCθ,IKKα,IKKβ,NEMO,IκBα and NF-κB.Bioinformatics analysis found that these pathway components are highly conserved in evolution.In the adaptive immune stage after Streptococcus agalactiae infection,the mRNA and phosphorylation levels of the main components of the NF-κB pathway in spleen lymphocytes were significantly upregulated,accompanied by a decrease in total protein of IκBα and the entry of NF-κB into the nucleus,suggesting that this pathway is involved in the adaptive immune response in Nile tilapia.The NF-κB pathway was significantly activated during the activation of T cells induced by P+I or PHA.However,the specific inhibitor QNZ mediated blocking of NF-κB pathway inhibited the up-regulated expression of CD122 and IFN-γ,which were markers of T cell activation,indicating that this pathway promoted the activation of T cells in Nile tilapia.At the same time,in the primary immune response against S.agalactiae infection,the lack of NF-κB pathway activity weakened the proliferation of lymphocytes,resulting in a decrease in the number of T cells,and ultimately aggravated the death of tilapia.These results suggested that Nile tilapia promotes T cell activation and proliferation by utilizing the evolutionarily conserved NF-κB pathway to resist S.agalactiae infection.Inhibition of the NF-κB pathway down-regulated the level of IL-17 A mRNA in lymphocytes,suggesting the role of this pro-inflammatory cytokine in tilapia antiinfection immunity.Focusing on IL-17,IL-17 family members including IL-17 A,IL-17 C,IL-17 D and IL-17 F were found in the Nile tilapia genome.The mRNAs of these members were widely distributed in the lymphatic tissues of Nile tilapia,and their expression in spleen lymphocytes could be significantly induced by the infection of S.agalactiae.The recombinant protein of IL-17 A was prepared by prokaryotic expression system,and the polyclonal antibody was produced.Further studies showed that activated T cells could secrete IL-17 A when stimulated by P+I or PHA in vitro.At the same time,in the adaptive immune response to bacterial infection,the protein level of IL-17 A in T cells was also significantly up-regulated.The results showed that the NF-κB pathway promotes the secretion of IL-17 A by T cells,which in turn regulates the adaptive immune response of tilapia.In terms of mechanism,tilapia IL-17 A could bind to the surface receptor IL-17 RA of lymphocytes,activated the downstream activator of NF-κB 1(ACT1),which triggered tumor necrosis factor receptor-related factor 6(TRAF6)ubiquitination and degradation.The ubiquitinated TRAF6,in turn,activated the downstream TRAF6-dependent transforming growth factor-activated kinase 1(TAK1)to initiate the NF-κB pathway and promote the activation of T cells.In addition,IL-17 A also mediated the expression of pro-inflammatory factors TNF-α,IL-6,and β-defensin through the ACT1-TRAF6-TAK1 axis,thereby regulating the anti-infection immunity of tilapia.It is suggested that IL-17 A secreted by effector T cells can regulate the NF-κB pathway and the T cell immunity of tilapia through the ACT1-TRAF6-TAK1 axis.In the present study,using Nile tilapia as a model,we investigated the mechanism of NF-κB couples TCR and IL-17 signals to regulate ancestral T cell activation and anti-infection immunity in Nile Tilapia.Our study provides new evidence for the understanding of the regulatory mechanism of T cell immunity and the evolution of the adaptive immune system in fish.It also lays a theoretical foundation for the immune prevention of cultured fish.