IL-10 Suppresses Nile Tilapia T Cell Immune Response Via JAK/STAT-SOCS3 Axis

T cell immunity plays an important role in defense against pathogenic microorganisms,monitoring and eliminating cancer cells,as the activated T cells will build a powerful defense line for the body.However,excessive activation of T cell immunity will trigger inflammatory storms,leading to damage to organisms and autoimmune disease.Interleukin-10（IL-10）is considered one of the most important anti-inflammatory cytokines that play an inhibitory role in T cell immune response.Its activation will suppress T cell signaling and cytokines synthesis,thus limiting the immune response,and preventing cytokine storm or inflammation damage.It plays a key role in maintaining immune homeostasis.Previous studies have found functional conserved T cells in lower vertebrates and demonstrated the regulation mechanism of T cell immune activation.However,in lower vertebrates,the negative regulation mechanism of the T-cell immune response remains unknown,which is crucial to the T-cell immune homeostasis.Therefore,our study took Nile tilapia（Oreochromis niloticus）as a model.In addition to analyzing the activation characteristics of T cells in Nile tilapia,we elucidated the negative regulatory function of IL-10 on T cell immunity in the teleost and analyzed the pathway and mechanism of IL-10 in tilapia.Firstly,we analyzed the characteristics of T cell activation in Nile tilapia.The activation of tilapia T cells significantly up-regulated the phosphorylation of LCK ZAP70 and PLCγ,and further stimulated the NAFT,ERK,NF-κB,and m TOR pathways.We observed that the expression of Ca M and activation of transcription factor NF-κB P65,4EBP1,ERK1/2 were significantly upregulated,by which the expression of effector IL-2,IFN-γ,and membrane protein CD44,CD122 were remarkably promoted.Furthermore,we investigated that the phosphorylation of ZAP70 is a key event that mediates T-cell activation in Nile tilapia.Shown by immunofluorescence,ZAP70 is located in the inner side of the cell membrane of tilapia lymphocytes.The specific expression of ZAP70 in tilapia T cells was also observed in this study,confirming the feasibility of ZAP70 as a T cell marker in teleosts.As the inhibitory effect act on activated cells,these conclusions provide a proper method to demonstrate the negative regulation that IL-10 places on Nile tilapia T-cell immunity.Nile tilapia has a structure conserved IL-10,which is widely distributed in intestinal and other immune tissues.In the process of A.hydrophila infection,the transcription level and protein level of IL-10 were significantly upregulated,and the expression of IL-10 was also significantly induced in vitro activation of T cells induced by phytohemagglutinin（PHA）.The recombinant IL-10 protein was obtained through a prokaryotic expression system to test its regulatory function.The presence of IL-10 protein can effectively inhibit the transcription of pro-inflammatory cytokines such as IL-1β,IL-6,TNF-α,and IL-2.Meanwhile,IL-10 eliminates the effects of PHA stimulation.The induced expressions of IFN-γ,IL-2,CD44,and CD122,which marked activation of T cells,were attenuated upon IL-10 stimulation.During infection,IL-10 suppressed the proliferation of lymphocytes.The presence of IL-10 also inhibited the cytotoxicity of T cells of Nile tilapia,suggesting that IL-10 had a negative regulatory effect on the activation,proliferation,and immune function of Nile tilapia.Bioinformatics analysis revealed that a complete and conserved IL-10Ra/b receptor and JAK1/STAT3/SOCS3 pathway were found in Nile tilapia.The pulldown test confirmed that the outer portion of IL-10 Ra had an affinity and can specific binding to IL-10.As for the mechanism,IL-10 triggered the phosphorylation of JAK1 and STAT3,promoted the transcription of SOCS3,and,therefore,exerted negative regulation on tilapia T cell immunity by inhibiting the activity of m TOR,MAPK/ERK,and NF-κB signaling.Altogether,our study revealed the down-regulatory mechanism of IL-10 on T cells in a fish species,adding significant evidence to the mechanism of T cell immunity and the evolution of the adaptive immune system.