| As a pleiotropic cytokine,Interleukin（IL）-2 plays a key role in innate and acquired immunity.Due to poor sequence similarity with mammalian IL-2 genes and proteins,the first fish IL2 gene was cloned until 2005.Recently,with the increasing number of fish genome databases,some fish IL2 genes were discovered and investigated,However,the physiology structure and immune organs of teleost are different from those of mammals,so the understanding of IL-2 in teleost immunity is still insufficient.In order to systematically elucidate the immunological function and properties of IL-2 in the teleost,using grass carp as the model,we firstly obtained biologically active recombinant grass carp IL-2 protein（rgc IL-2）,then identified the signal transduction ability of IL-2 receptor（IL-2R）complex,and finally explored the regulation and mechanism of IL-2 on grass carp acquired and innate immune cells.The detailed contents are shown as the follows:1.Cloning,bioinformatics analysis,recombinant protein preparation,and biological activity identification of grass carp IL-2:（1）A gene suspected to be IL2 was found in grass carp by collinearity analysis of the conserved genes along the IL2 locus in various vertebrates.The full-length coding sequence of grass carp IL2 was obtained via bioinformatics analysis and RT-PCR.The amino acid sequence analysis showed that grass carp IL-2 had four α helices,which was the structural characteristics of the IL-2 family members.（2）Tissue distribution results showed that grass carp IL2 m RNA was expressed at a high level in some immune tissues including the thymus and head kidney,suggesting that IL-2 may be involved in the regulation of immune cells in grass carp.（3）After rgc IL-2 was prepared,the ability of this protein to up-regulate the STAT5 phosphorylation in grass carp peripheral blood leukocytes（PBLs）was demonstrated,implying that the recombinant protein is able to activate signal transducer and activator of transcription 5（STAT5）pathway similar to that in mammals and can be used in subsequent investigations.2.Molecular cloning of grass carp IL-2 receptor（IL-2R）subunits and identification of its function to mediate IL-2 signaling:（1）In the present study,a new IL2 RBL gene was identified in the grass carp genome by collinearity analysis of the conserved genes along the IL2 RB locus in various vertebrates.The secondary structure of grass carp IL-2RBL protein was highly similar to that of mammalian IL-2RB,but it lacked a YLXL motif.Phylogenetic tree analysis further confirmed that the gene was a paralog of IL2 RB in grass carp.（2）Tissue distribution experiments revealed that the m RNA expression of grass carp IL2 RB,IL2RBL,IL2RG1,and IL2RG2 were tissue-specific,indicating that they may have distinct functions in different tissues and organs.（3）Moreover,the induced expression patterns of four IL-2R subunits m RNA in PBLs were different in response to lipopolysaccharide（LPS）and heat-inactivated Aeromonas hydrophila（A.hydrophila）,suggesting that the IL-2R subunits mediate different signal transductions in the immune responses.（4）In mammals,two subunits of the IL-2R complex are encoded by the IL2 RB and IL2 RG genes,respectively.In order to compare the function of different IL-2R subunit combinations to mediate the signal transduction in grass carp,two types of receptor subunits（IL-2RB and IL-2RG）were overexpressed in pairs in COS-7 cells,and the phosphorylation of STAT5 was detected under the rgc IL-2 treatment.The results showed that only IL-2RB and IL-2RG1 dimeric receptor complex could mediate the phosphorylation of STAT5 stimulated by IL-2,thus indicating that consistent with mammals,dimeric IL-2R complex is required for IL-2 signal transduction in fish species.3.Effects of IL-2 on the proliferation of head kidney leukocytes（HKLs）and gene expression of immune cell markers in grass carp:（1）The leucocytes from the head kidney,an important immune organ of grass carp,were used as the model to explore the regulation of IL-2 on immune cells.The results showed that long-term（72 h）treatment of rgc IL-2significantly promoted the proliferation of HKLs,while the expression of CD4 L gene,a helper T cell marker,was significantly up-regulated.These results indicated that fish IL-2 can promote the proliferation of immune cells,especially helper T cells,which is similar to mammals.（2）Furthermore,short-term（<48 h）treatment of rgc IL-2 could promote the expression of markers of helper T cells（IFNg2,IL12p35 a,IL12p35b,IL2,TBET,TNFA,IL4/13 A,IL4/13 B and GATA3）,regulatory T cells（FOXP3 and CD25L）and cytotoxic cells（PRFL,GZMBL,and FASL）in grass carp HKLs,suggesting that IL-2 may be involved in the development and expansion of acquired immune cells in fish.4.IL-2 augments the ability of grass carp neutrophils to kill A.hydrophila via stimulating granzyme B-like（GZMBL）and perforin-like（PRFL）protein expression:（1）Firstly,neutrophils from grass carp peripheral blood were isolated by density gradient centrifugation method,the cells were identified by Giemsa and myeloperoxidase（MPO）staining.（2）Western Blotting（WB）and immunofluorescence staining results showed that GZMBL and PRFL protein were expressed in grass carp neutrophils with spot-like distribution,suggesting that these cytotoxic granular proteins may play a role in the function of grass carp neutrophils.（3）Subsequent experiments confirmed that rgc IL-2significantly upregulated the m RNA and protein expressions of GZMBL and PRFL in neutrophils through ERK,NF-κB,STAT5 signaling and glycolysis pathways.It thus provides robust evidence for the involvement of IL-2 in regulating neutrophils function.（4）Furthermore,antibody neutralization experiments demonstrated that rgc IL-2 upregulated GZMBL and PRFL protein expression could effectively improve the ability of neutrophils to kill A.hydrophila.The results imply granzyme B and perforin is not only the effector molecules of NK and CTL cells,but also a weapon for neutrophils to kill bacteria.At the same time,the results indicate that IL-2 is an effective regulator of these effector molecules,facilitating the anti-bacteria function of innate immune cells in immune defense.5.The mechanisms of the regulation of IL-2 on the death of grass carp neutrophils:（1）Annexin V/PI double staining and flow cytometry results showed that rgc IL-2significantly inhibited the early apoptosis of grass carp neutrophils.Further study revealed that rgc IL-2 stimulated extracellular trap death（NETosis）of the same cell model.These results suggested that IL-2 can inhibit apoptosis and induce NETosis of grass carp neutrophils.（2）Next,both NADPH oxidase and mitochondria-derived ROS were proved to be involved in rgc IL-2-induced NETosis.（3）In addition,inhibition experiments confirmed that the autophagy pathway was also involved in rgc IL-2-induced NETosis.（4）WB and Immunofluorescence results showed that rgc IL-2 promoted the nucleocytoplasmic transport of high mobility group box B（HMGB）1.The inhibitors of HMGB1 significantly inhibited the up-regulation of IL-2-induced autophagy marker microtubule-associated protein light chain 3（LC3）II and the occurrence of NETosis.These results reveal that IL-2 could regulate NETosis through the HMGB1-autophagy pathway.Further,the change in subcellular localization of HMGB1 may trigger the signal of autophagy initiation.（5）As mentioned above,IL-2-induced NETosis is dependent on ROS and autophagy pathways,but further experiments verified that there was no crosstalk between these two pathways in NETosis.These findings elucidated the roles of ROS and autophagy pathways in IL-2-induced NETosis,and provided evidence that IL-2 could precisely regulate neutrophil death in grass carp.6.Effect of rgc IL-2 pretreatment on the defense of grass carp against A.hydrophila infection: rgc IL-2 pretreated reduced the bacterial load in peripheral blood and increased the survival rate of grass carp infected with A.hydrophila.The above experiments verified the protective effects of IL-2 on grass carp from A.hydrophila infection,and the regulation of IL-2 on various immune cells mentioned above may play a positive role in this protection.In conclusion,we cloned the grass carp IL2 gene and obtained the biologically active rgc IL-2.On this basis,we studied the signal transduction of IL-2 and its regulatory roles in grass carp HKLs and neutrophils.These findings revealed that IL-2 dimeric receptor could transduce the signaling of IL-2,and the regulation of IL-2 on the markers of T lymphocyte subpopulations in grass carp HKLs.More importantly,we also demonstrated that grass carp IL-2 could enhance the bactericidal activity of neutrophils by inducing the expression of cytotoxic proteins and the occurrence of NETosis.These results are helpful to support the crucial roles of fish IL-2 in innate and acquired immunity,and provided a novel and potential method to use IL-2 as an immunomodulator to protect the fish against bacterial diseases. |
