| Interleukin(IL)-4 and IL-13 are T helper 2(Th2)cytokines with pleiotropic functions.Both IL-4 and IL-13 participate in adaptive immune responses by promoting CD4+ Th cell differentiation and a variety of Th2-mediated humoral immune responses,including B cell proliferation and activation.Grass carp(Ctenopharyngodon idella)is an endemic fish and has been widely cultivated throughout China.Whilst,recent years,the high density and intensive farming model has resulted in the weaker immunity and outbreak of the diseases,which causes extensive losses in aquaculture.Flavobacterium cloumnare and grass carp hemorrhage virus(GCRV),as the most devastating pathogenic microorganism for freshwater fish,restricted the development of grass carp aquaculture seriously.To date,IL-4/13 as a key molecule in the immune system of grass carp,but its function and role in resisting of the F.cloumnare and GCRV was still unclear.The role of IL-4/13 in polarization of grass carp macrophages and the binding of IL-4/13 with various receptor chains are not systematically studied.(1)In fish,two IL-4/13 homologs have been identified but their phylogenetic relationships with IL-4 and IL-13 are ambiguous.In this study,we identified IL-4/13 A,IL-4/13 B and six putative IL-4/13 receptor homologs in grass carp,including ?C1,?C2,IL-4R?1,IL-13R?1,IL-13R?2 and a soluble form of IL-4R?2.Two grass carp IL-4/13 genes,designated as Ci IL-4/13 A and Ci IL-4/13 B.The full length cDNA of CiIL-4/13 A was 1202 bp,including a 403 bp 5?-UTR,a 397 bp 3?-UTR,a 402 bp ORF encoding for a putative protein with 133 amino acids,with a predicted signal peptide of 16 aa.The full-length cDNA of Ci IL-4/13 B was identified to be 796 bp,consisting of a 264 bp 5?-UTR,a 124 bp 3?-UTR,and a 408 bp ORF encoding a protein of 135 amino acids with a predicted signal peptide of 16 aa.IL-4 interacts with two receptors consisting of IL-4R?/? chain receptor(?C)and IL-4R?/IL-13R?1.In contrast,IL-13 binds to IL-13R?2 but also shares the receptor complex containing IL-4R?/IL-13R?1.Two grass carp ?C genes,designated as Ci?C1 and Ci?C2,were obtained and predicted to translate into proteins of 360 aa and 335 aa respectively.The Ci?C1 contained a predicted signal peptide of 19 aa,an extracellular region of 201 aa,a predicted transmembrane domain of 23 aa and an intracellular region of 117 aa.The Ci?C2 molecule contained a predicted signal peptide of 26 aa,an extracellular region of 196 aa,a predicted transmembrane domain of 23 aa and an intracellular region of 90 aa.The extracellular region of both molecules consisted of two tandem fibronectin type?(FN?)like domains.Comparative sequence analyses revealed that these receptors possess conserved characteristic domains and the genes encoding them share conserved gene synteny with their human counterparts.All six receptors contain a cytokine binding homology domain(CHD)and two fibronectin type ?(FN?)like domains,with IL-13R?1 and IL-13R?2 harbouring an extra Ig-like domain preceding the CHD domain.Interestingly,grass carp IL-13R?1 and IL-13R?2 lack the characteristic WSXWS motif,a typical feature of mammalian type I cytokine receptors.(2)To confirm the phylogenetic relationships of two grass carp IL-4/13 with homologs from other vertebrates,an unrooted tree was constructed.The protein sequences of IL-4/13 from fish and vertebrate species were selected for analysis.Fig.2 showed that Ci IL-4/13 A and B molecules grouped closely with the IL-4/13 from fish species and formed a distinct clade with the mammals.Within the fish clade,Ci IL-4/13 A and B with their respective zebrafish and common carp homologs were well separated,indicating two separate IL-4/13 groups had diverged in teleosts.The gene synteny of IL-4/13 genes are conserved among human,zebrafish and grass carp.To confirm the phylogenetic relationships of six grass carp IL-4/13 receptors with homologs from other vertebrates,an unrooted tree was constructed.The protein sequences of ?C,IL-4R?,IL-13R?1 and IL-13R?2 from fish and vertebrate species were selected for analysis.(3)Quantitative RT-PCR analysis was used to quantify the gene expression of Ci IL-4/13 A,Ci IL-4/13 B and receptor subunits in various tissues.were constitutively expressed in the seven tissues analysed but the expression levels varied considerably among the tissues.The highest level of expression of Ci IL-4/13 A and B was found in gills.The lowest expression of Ci IL-4A and B was detected in the head kidney and intestine,respectively.All the receptor subunits were constitutively expressed in the eight tissues analysed but the expression levels varied considerably among the tissues.The highest level of expression of Ci?C1 and Ci?C2 was found in spleen and brain.Comparably,the Ci?C1 expression was significantly higher than that of Ci?C2 in spleen but lower in brain.Head kidney,thymus and gills also expressed high levels of Ci?C1,whilst for Ci?C2,high levels of expression were detected in liver,gills,head kidney and skin.The Ci IL-4R?1 was constitutively expressed in all the tissues examined.The expression of CiIL-4R?1 was significantly higher than that of sCi IL-4R?2 in spleen but lower in liver where the highest expression of sCi IL-4R?2 was detected.The expression of Ci IL-13R?1 was higher than CiIL-13R?2 in most tissues including instestine,gills,liver,spleen,skin and brain.The highest expression of Ci IL-13R?1 and Ci IL-13R?2 was detected in the intestine and head kidney respectively.(4)The modulation of Ci IL-4/13 A and B expression in response to GCRV-? infection or F.cloumnare was investigated.The Ciil-4/13 A was significantly upregulated(p< 0.05)in head kidney and thymus at 1 and 7 d post infection(dpi)with GCRV-?,respectively.In contrast,there was no apparent pattern of expression modulation of CiIL-4/13 B after infection GCRV-?.The Ciil-4/13 A was significantly up-regulated in head kidney at 72 hpi after infection F.cloumnare.For the CiIL-4/13 B,its expression was up-regulated at 24,48 and 72 h post infection(hpi)with F.cloumnare,whilst the expression levels of Ci IL-4/13 B decreased significantly at 12 hpi and returned to normal level at 24 hpi,then up-regulation at 72 hpi in spleen.The expression of Ci IL-4/13 A showed up-regulation at 48 hpi in spleen after F.cloumnare infection.The IL-4/13 receptor genes can be modulated by F.cloumnare,suggesting they are involved in immune response against F.cloumnare infection.(5)To confirm the bioactive of Ci IL-4/13 A and B in MO/M?s,rCi?L-4/13 A and rCiIL-4/1B were incubated with the MO/M?s deriver from head kidney described above.After incubated with rCi IL-4/13 A or rCiIL-4/13 B 7 d,the expression levels of the marker genes of M1 macrophage was dowm-regulated,and the marker genes of M2 macrophage was up-regulated.The mRNA levels of selected immune-related genes were significantly up-regulated by rCiIL-4/13 A or rCiIL-4/13 B.In conclusion,we identified two IL-4/13 homologues,Ci IL-4/13 A and CiIL-4/13 B,from the grass carp.Both CiIL-4/13 A and CiIL-4/13 B have the similar roles in promoting alternative activation of head kidney MO/M?s,although they exhibit a lower degree of sequence identity and differential expression pattern.Six IL-4/13 receptor genes were identified in grass carp.All the receptors were constitutively expressed in most of tissues of healthy fish,and their expression could be modulated by infection of F.cloumnare.The results suggest that the functions of IL-4/13 ligands could be regulated by modulation of expression of individual receptors and decoy receptors.Further studies are needed to fully elucidate the specific receptors and signaling pathways of these two Ci IL-4/13 molecules. |
PDF Full Text Request