Immune Function Of The Ferritin And Transferrin Genes In Megalobrama Amblycephala After Infection By Aeromonas Hydrophila

Iron is an essential microelement for almost all living organisms and plays a vital roles in various physiological activity,while an excess of iron can stimulate the generation of toxic free radical and facilitate the proliferation of pathogenic microorganism.Hence,maintenance of iron homeostasis is extremely important,of which ferritin(FT)and transferrin(TF)play pivotal roles as iron storage and transporting proteins,respectively.Iron is always the centre of nutrient source strife between host and bacteria,and many iron metabolism related genes participates in host innate immune response.Currently,the immune roles of iron metabolism pathway gain increasing attentions,and a growing number of related studies have been reported.Blunt-snout bream(Megalobrama amblycephala)is one of the major freshwater aquaculture species in China,while reasons such as intensive aquiculture and environmental pollution have recently caused high frequency of various diseases,of which bacterial septicemia caused by Aeromonas hydrophila(Ah)infection is the most serious.In the present study,we cloned the Ft(M.amblycephala ferritins,MamFers)and Tf(M.amblycephala transferrin,MamTf)genes in M.amblycephala,detected their expression patterns and recombinant protein activity,conducted Digital Gene Expression Profiling of the overexpressed L8824 cells and obtained differentialy expressed genes,and finally analyzed the regulatory effect of MamFers and MamTf on iron metabolism and immune pathways.The present study aimed to explore the roles of MamFers and MamTf genes played in iron metabolism and immune system,and provide theoretical basis for the prevention and treatment of bacterial disease and disease resistance breeding in fish,the main results were as follows:1.In the present study,we cloned and characterized two Ft subunits from M.amblycephala,termed as MamFerH and MamFerM(M.amblycephala ferritin H and M subunits).The ferroxidase center structure of mammalian ferritin H subunit was identified in both MamFerH and MamFerM,and MamFerM also possessed the nucleation sites similar to that of mammalian ferritin L subunit.A predictediron-responsive element(IRE)was found in the 5' untranslated region(UTR)of MamFerH and MamFerM,where difference was observed between their secondary structures.Sequence analysis revealed that the homology among MamFerH and MamFerM was only 65%,and phylogenetic analysis revealed that ferritin H and M subunits were separately clustered,indicating that they are encoded by two different genes.MamFerH and MamFerM exhibited similar expression patterns during early development with specifically high expression at post hatching stage,while differential tissue expression patterns were observed.MamFerM was highly expressed in the spleen,liver and kidney,whereas MamFerH was predominantly expressed in the blood and brain.Both MamFerH and MamFerM were up-regulated upon A.hydrophila infection at both mRNA and protein levels,immunohistochemistry and immunofluorescence assay showed significantly increased levels of M.amblycephala ferritins in the cytoplasm and nucleus post A.hydrophila infection,of which MamFerH showed more quick and significant acute phase response upon pathogenic stress.In comparison,rMamFerH displayed more efficient bacteriostasis,while rMam FerM functioned more effectively in iron depriving.2.The MamTf gene was also cloned and characterized in the present study.The deduced MamTf protein was comprised of two homologous domains(N-terminal and C-terminal),and 4 iron binding sites were identified in each domain.MamTf gene was specifically abundant in the liver,which was significantly higher than that of other tissues.During early development,the expression of MamTf increased from 12 hpf(hours post fertilization)to 26 hpf,followed by a diminution at 32 hpf,then increased significantly to the peak level at 2 dph(days post hatching).The expression of MamTf and its receptor 1 was up-regulated in the liver,spleen and kidney at both mRNA and protein levels post A.hydrophila infection.Prussian blue staining revealed the increased intracellular iron content,Immunohistochemistry analysis showed the raised distribution of TF and TfR1 in the cytomembrane and intracellular,indicating the possible internalization of TF-receptor system with bound-iron in the liver of M.amblycephala.The iron binding activity of rMamTf was concentration dependent.Additionally,in vitro experiment showed that addition of rMamTf can inhibit the growth of A.hydrophila to some extent.3.In order to further study the regulation role of MamFers and MamTf genes,MamFerH,MamFerM and MamTf were seperately overexpressed in L8824 cells and prepared for Digital Gene Expression(DGE)Profiling Sequencing.DGE anslysis revealed that the regulatory pathway of MamFers and MamTf including immune system process and defense response,and qRT-PCR(Quantitative Real-time Polymerase Chain Reaction)analysis showed that overexpression of MamFers and MamTf would significantly up-regulate the expression of TNF?(Tumor Necrosis Factor alpha)and iNOS(Inducible Nitric Oxide Synthase),respectively.NF?B(Nuclear Factor ? B)is one of the most important transcriptional regulation factor,widely involved in host immune response,including the expression regulation of several cytokines.In the present study,MamFers and MamTf overexpression would promote the increase of transcriptional activity of NF?B and the phosphorylation level of pIKK.The addition of BAY11-7082(I?B? inhibitor)post MamFers and MamTf overexpression would suppress their induction to the expression of cytokines.Additionally,MamFers and MamTf overexpression would promote the up-regulation of the phosphorylation level of MAPK(Mitogen-activated Protein Kinase),and addition of U0126(MEK1/2 inhibitor)after MamFers and MamTf overexpression would suppress their induction on the expression of cytokines.These results indicated that MAPK and NF?B might mediate the regulation of MamFers and MamTf to the expression of proinflammatory cytokines.4.In the present study,overexpression of MamFers and MamTf would suppress the adhesion of Ah to EPC cells,while interference of MamFers and MamTf would increase the adhesion amount of Ah.qRT-PCR analysis revealed that the ECM(Extracellular Matrixc)related genes fibronectin(FN)and Integrin beta 1(Intg?1)were both significantly regulated post MamFers and Mam Tf overexpression.However,their regulation present as two different ways,the expressin of FN was up-regulated while Intg?1 was decreased when MamFers and MamTf were overexpressed,and the expression patterns were opposite when MamFers and MamTf were interfered.Further study revealed that both FN and Intg?1 participate in the regulation of MamFers and MamTf to the adhesion of Ah.5.In the present study,MamFers and MamTf overexpression would significantly up-regulated the expression of NLRC5(NOD-like Receptor Family CARD Domain Containing 5),?2M(? 2 Microglobulin)and MHC I(Major Histocompatibility Complex Class I).Co-transfection of siRNA-NLRC5 with MamFers and MamTf overexpression plasmid would suppress the induction of MamFers and MamTf to?2M/MHC I,indicating that NLRC5 might mediate the regulation of MamFers and MamTf to ?2M/MHC I expression.Furthermore,MamFers and MamTf overexpression would significantly up-regulate the expression of hepcidin gene,while co-transfection of siRNA-?2M with MamFers and MamTf overexpression plasmid would suppress the induction role.According to these results,we can speculate that MamFers and MamTf overexpression could regulate NLRC5,followed with the downstream ?2M,which can regulate the expression of hepcidin.