Large yellow croaker(Larimichthys crocea)is an economically important marinecultured fish with the highest annual yield in China.With the continous expansion of farming scale and the deterioration of breeding environment,infectious diseases become more and more severe,which has become a major factor restricting the sustainable and healthy development of large yellow croaker culture industry.In recent years,immunoprophylaxis receives extensive attentions because of the serious problems of drug residues,environmental pollution and drug resistance caused by drug treatment.Therefore,it is necessary to study the immune response and its regulatory mechanism in resisting pathogens infection of large yellow croaker.Interleukin-2(IL2)is a pleiotropic cytokine that plays pivotal roles in regulation and differentiation of CD4+T helper subsets,as well as in promoting B cell proliferation and antibody production.Thus,IL-2 plays a critical role in regulating immune response and fighting against pathogen invasion.An intensive study on the functions,regulatory mechanisms and signal pathways of LcIL-2 will help to provide comprehensive insights into the role of teleost IL-2 in immune response,and provide a theoretical basis for the selection of potent immunopotentiator for large yellow croaker.An IL-2 homologue(LcIL-2)was obtained by analysis of genome sequence of large yellow croaker and RACE-PCR techniques.The open reading frame(ORF)of LcIL-2 gene is 426 bp long encoding a precursor peptide of 141 amino acids(aa),with a 20-aa signal peptide and a 121-aa mature peptide containing the IL-2/IL-15 family signature"L-X-C-X(3)-E-[LVI]-X(2/3)-[LVM]".The LcIL-2 locates closely adjacent to IL-21 and BBS12 on chromosome of large yellow croaker,and its genomic DNA sequence consists of 4 exons and 3 introns,which is similar to mammalian IL-2.The LcIL-2 gene was constitutively expressed in all examined tissues from healthy large yellow croaker,preferentially expressed in lymphocytes-rich tissues,such as spleen and blood.The LcIL-2 expression increased in head kidney and spleen upon inactivated trivalent bacterial vaccine or Poly(I:C)stimulation.Also its expression could be detected in primary head kidney leukocytes(PKL),primary head kidney macrophages(PKM)and primary head kidney granulocytes(PKG),with the highest level in PKL.In addition,the expression of LcIL-2 was increased 1.5-fold and 2.1-fold in PKL stimulated with LPS or Poly(I:C),respectively.While it could increased up to 89.4-fold or 8.3-fold in PKL stimulated with PHA or Con-A,respectively.The promoter of LcIL-2 was cloned and characterized.The activity of LcIL-2 promoter in Jurkat-T cell could be enhanced by T cell mitogen or activator,such as PHA,Con-A,PMA,CI,PMA+CI,but refractory to LPS or Poly(I:C)stimulation.In HEK-293T cell,over expression of LcNFAT or subunits of LcNF-κB could induce the activity of LcIL-2 promoter,but Lcc-Jun,the subunit of AP1 could not(P>0.05).The site-directed mutatgenesis of the transcription factor binding sequence on LcIL-2 promoter significantly hampered the induction of LcIL-2promoter activityby LcNFAT,Lcc-Rel and LcRelA.These result suggested that the expression of LcIL-2 could be induced by T cell mitogen or activator,as well as regulated by the transcription factor such as LcNFAT and NF-κB.In vivo administration of recombinant LcIL-2(rLcIL-2)produced in Pichia pastoris could significantly promote the proliferation of ZAP70+T cells,accompanied with increasing the expression of genes involved in Thl and Th2 development and differentiation,and of transcription factor STAT5B involved in IL-2 signal pathway,but inhibit the expression of genes related to Th17(IL-17A/F2 and IL-17A/F3)development and differentiation,suggesting that LcIL-2 may play an important role in regulating both Th1,Th2 and Th17 cell responses.Furthermore,rLcIL-2 remarkably enhance the B cell proliferation,increases the production of anti-DLD specific IgM,and raised the survival percent of large yellow croaker against bacterial infection.These result suggest that LcIL-2 also play a role in regulating humoral immune response.The receptors of LcIL-2,including LcIL-15Rα、LcIL-2Rβ and LcγC,have been identified in large yellow croaker.Co-IP assays demonstrated that LcIL-2 can directly bind to LcIL-15Rα and LcIL-2Rβ but not to LcγC.LcIL-2Rβ can bind to LcγC whereas LcIL-2Rβ and LcγC can’t directly bind to LcIL-15Rα.The LcIL-2 with LcIL-15Rα and LcIL-2Rβ,or with LcIL-2Rβ and LcγC could form heterotrimer,respectively.These results suggest that LcIL-2 binds to LcIL-15Rα and LcIL-2Rβ to form an LcIL-2/LcIL15Rα/LcIL-2Rβ heterotrimer,the LcγC was recruited by LcIL-2Rβ,then the LcIL2/LcIL-15Rα/LcIL-2Rβ/LcγC signal transduction complex was formed.Mammalian IL-2 exerts its function by activating the STAT5-dependent JAK-STAT,MAPK and PI3K-mTOR signaling pathways in target cells.The phosphorylation of STAT5,ERK,AKT and 4EBP1 in LcIL-2-stimulated PKL were increased,blockade of these signaling pathways with corresponding inhibitor or with anti-LcIL-15Rα or/and anti-LcIL-2Rβpolyclonal antibody impairs the LcIL-2-elicited phosphorylation of STAT5,ERK and 4EBP1,and LcIL-2 induced expression of genes involved in T cells differentiation,emphasizing that LcIL-2 stimulates a cascade the STAT5-dependent JAK-STAT,MAPK and PI3K-mTOR signaling pathways by LcIL-15Rα/LcIL-2Rβ/LcγC heterotrimer.These results first highlighting the functional and signal transduction conservation of teleost IL-2 with mammalian IL-2.To our knowledge,this is the first comprehensive studies of biological functions,regulations and signaling pathways of teleost IL-2. |