Molecular Identification And Immunefunction Of Tumor Necrosis Factor Receptor 1 And 2 In Grass Carp

Tumor necrosis factor-?(TNF-?)is a cytokine with pleiotropic effects and plays crucial roles in cell growth,proliferation,differentiation and death,as well as inflammation and auto-inflammatory disease.In mammals,it is well known that the physiological functions of TNF-? are mediated by TNFR1 and TNFR2,two cognate receptors of TNF-?.TNF-? gene has been cloned and identified in several fishes and the immunoregulatory function of TNF-? has been characterized in teleost.However,relatively little attention has been paid to fish TNFR1 and TNFR2,and their functional roles and differences need to be further studied.At present,there are no studies with respect to the exience of soluble forms of TNFR1 and TNFR2(sTNFR)and their functions in teleost.In this study,grass carp TNFR1(gcTNFR1)and gcTNFR2 were cloned and characterized,and their functional roles in mediating TNF-? signaling were clarified.In addition,the existence of soluble forms of gcTNFR1 and gcTNFR2 was determined,and the effects of extracellular regions of these two receptors aganist bacteria infection were explored in grass carp.The detailed results are showed as follows:1.Cloning and characterization of grass carp TNFR1 and TNFR2 and their tissue distribution patterns.(1)Full-length cDNA sequences of gcTNFR1 and gcTNFR2 were obtained by using homology-based cloning techniques and they were closely related to their respective homologous sequences revealed by bioinformatics analysis.Moreover,the gene organizations and protein structures of two receptors were also conserved in teleost.(2)The expression patterns of gcTNFR1 and gcTNFR2 transcripts were detected by RT-qPCR in eight tissues from healthy grass carp.Results showed that both receptors expressed in all tested tissues,but the expression levels of gcTNFR1 transcripts were significantly higher than that of gcTNFR2 in the same tissue except spleen,indicating the expression differences of two receptors.2.Expression analysis of gcTNFR1 and gcTNFR2 under inflammatory conditions.(1)The grass carp head kidney leukocytes(HKLs)were stimulated with recombinant grass carp TNF-?(rgcTNF-?)and LPS for different time points,and then RT-qPCR assay showed that rgcTNF-? rapidly up-regulated gcTNFR2 gene expression while slightly increased gcTNFR1 mRNA levels.Additionally,LPS induced two receptors gene expression in the late phase of treatment.(2)In Aeromonas Hydrophila(A.hydrophila)infected grass carp,the expression levels of gcTNFR1 and gcTNFR2 were up-regulated in gill,liver and head kidney.Besides,gcTNFR1 mRNA levels were also induced in intestine.The inductive expression of both receptors returned to basal levels in the late phase after bacterial infection.In vitro and in vivo experiments revealed the inductive expression differences of gcTNFR1 and gcTNFR2 transcripts under inflammatory conditions.(3)The customized polyclonal antibodies for gcTNFR1 and gcTNFR2 were prepared based on amino acid sequences of extracellular region of two recptors,and its specificity was validated by Western Blotting assay.Using these two antibodies,immunohistochemistry analysis showed that bacterial infection significantly increased the expression of gcTNFR1 in the trunk kidney,intestine and spleen,gcTNFR2 expression was upregulated in trunk kidney and spleen,decreased in intestine.These findings suggested the involvement of TNFR1 and TNFR2 in fish inflammatory responses.3.Functional identification of gcTNFR1 and gcTNFR2.(1)Construction of the related plasmid: the obtained grass carp IL-1b promoter sequence was subcloned into pGL3 basic vector,and the eukaryotic expression plasmids of gcTNFR1 and gcTNFR2 were constructed.(2)Overexpression of gcTNFR1 and gcTNFR2 significantly enhanced rgcTNF-?-induced NF-kB activity and gcIL-1b promoter activity in COS7 cells,indicating both receptors are involved in TNF-? induced NF-kB activation and gene transcription.(3)Overexpression of gcTNFR1 and gcTNFR2 further amplified rgcTNF-?-induced COS7 cell apoptosis,suggesting the overlap function of two receptors in mediating cell apoptosis.(4)The results of immunoneutralization of two receptors showed anti-gcTNFR1 antibody but not anti-gcTNFR2 antibody significantly inhibited TNF-?-induced IL-1b secretion in grass carp HKLs,indicating distinct roles of TNFR1 and TNFR2 in TNF-?-induced cytokine release.4.Determination of soluble gcTNFR1 and gcTNFR2(sgcTNFR1and sgcTNFR2)and functional characterization of recombinant the extracellular region of gcTNFR1 and gcTNFR2.(1)The sgcTNFR1 and sgcTNFR2 were discovered in the medium of HKLs by using polyclonal antibodies for gcTNFR1 and gcTNFR2,respectively.And the infection of A.hydrophila could significantly increase their secretion.(2)The high-purity recombinant proteins of extracellular region of gcTNFR1 and gcTNFR2(rgcTNFR1 and rgcTNFR2)which mimic the soluble receptors were obtained by using the prokaryotic expression system and affinity chromatography methods.(3)Binding assay in vitro showed rgcTNF-? could bind to rgcTNFR1 and rgcTNFR2 and the affinity of rgcTNFR2 was obviously stronger than rgcTNFR1.(4)Both rgcTNFR1 and rgcTNFR2 could significantly attenuate the grass carp IL-1b mRNA expression induced by rgcTNF-? in HKLs.(5)Competitive ELISA assay showed the secretion of gcIL-10 induced by rgcTNF-? was obviously inhibited by the rgcTNFR1 and rgcTNFR2.These findings demonstrated the conserved antagonism function of rgcTNFR1 and rgcTNFR2 as seen in mammals.5.Effects of rgcTNFR1 and rgcTNFR2 in the bacteria-infected grass carp.(1)The rgcTNFR1 was injected intraperitoneally to grass carp pre-infected by the pathogenic A.hydrophila.The TNF-? mRNA expression levels in head kidney and intestine,and the biochemical paramaters from serums were detected.Results showed that rgcTNFR1 could attenuate infection-induced up-regulation of TNF-? mRNA expression and improve the biochemical paramaters resulted from infection,implying its potential anti-inflammatory effects in grass carp.(2)The rgcTNFR2 was intraperitoneal injected to grass carp pre-infected by the pathogenic A.hydrophila and HE staing results showed that the rgcTNFR2 could significantly ameliorate the hemorrhage and infiltration of inflammatory cells induced by A.hydrophila in spleen,head kidney,intestine and liver.The symptom of splenomegaly was also relieved by rgcTNFR2.These results indicated that rgcTNFR1 and rgcTNFR2 could act as potencial drugs in grass carp during bacteria infection.In summary,the present study isolated and characterized gcTNFR1 and gcTNFR2,including analysis of gene organization and protein structures,their constitutive expression patterns in healthy tissues and inductive expression profiles under inflammatory conditions.The relationship and differences between gcTNFR1 and gcTNFR2 in mediating TNF-?-induced immune responses were investigated at the gene transcription,protein secretion and cell apoptosis levels.The existence of soluble forms of gcTNFR1 and gcTNFR2 was determined in HKLs.To mimic soluble receptors,rgcTNFR1 and rgcTNFR2 were prepared,and they could bind to rgcTNF-? in vitro and exerted the antagonistic role in the rgcTNF-? induced inflammation.Finally,the protective roles of rgcTNFR1 and rgcTNFR2 were determined in grass carp during bacteria infection.These results not only provide evolutional information of induced expression patterns and functional roles of gcTNFR1 and gcTNFR2,but also enriched the content of fish immune regulatory mechanisms base on TNF-? signaling and confirmed the existence of sTNFR1 and sTNFR2 in teleost.Moreover,this study provided the potencial drugs aganist bacteria infection and layed the theoretical foundion for disease prevention in fish aquaculture.