The Dynamic Inflammatory Response Of Yellow Catfish (Pelteobagrus Fulvidraco) Infected With Edwardsiella Ictaluri And Function Of IL-17 Family Genes

Yellow catfish(Pelteobagrus fulvidraco)is an important commercial freshwater fish.With the enlargement of the cultivation scale,various bacterial diseases,especially Edwardsiella ictaluri,often outbroke,causing huge financial losses in China.E.ictaluri,a Gram-negative rod-shaped bacterium,was reported to cause severe ascites disease,enteric septicemia and crack-head disease in yellow catfish.In order to understand the dynamic immune response of yellow catfish against bacterial infection,the immune responses of yellow catfish at eight different time points in the spleen and liver after E.ictaluri infection were analyzed using transcriptome.Moreover,In this study,we compared and analyzed the gene sequence and protein structure of interleukin-17(IL-17)family genes using bioinformatics to show the evolution of Pf＿IL-17 family genes,we examined the m RNA and protein expressions of Pf＿IL-17 family genes after E.ictalur infection and pathogen associated molecular patterns(PAMPs)stimulation to investgate the immune response of Pf＿IL-17 family genes against invasive pathogens,we detected the effects of recombinant(r)Pf＿IL-17 protein on the expression of inflammatory cytokines,chemokines,antimicrobial peptides and downstream signaling pathway related genes of peripheral blood leucocyte(PBL)and gill leucocyte(GL),as well as the phagocytosis and chemotaxis of PBL and GL to compare and uncover similarities and differences in function of Pf＿IL-17 genes.The key results are shown below:1.The dynamic immune response of yellow catfish after E.ictaluri infection presenting the inflammation processIn this study,RNA-Seq technology was used to study the immune response in two distinct tissues of yellow catfish at eight different time points(h)after E.ictaluri infection.The number of differentially expressed genes(DEGs)in the spleen and liver was low at 3 h and 6 h after E.ictaluri infection,respectively.Afterwards,the most number of DEGs in the spleen was detected at 72 h,while the number of DEGs in the liver maintained a high level from 24 h to 120 h.The GO and KEGG enrichment analyses of DEGs at different time points uncovered that cytokines were continuously transcribed at 6 h to 120 h;whereas the liver is the main organ that secretes the components of the complement system,and complement genes were extensively transcribed from 12 h to 120 h.Moreover,an overview of the inflammation response of yellow catfish was exhibited including pattern-recognition receptors,inflammatory cytokines,chemokines,complements,and inflammation-related signal pathways.2.Sequence structure and evolutionary analysis of Pf＿IL-17 family geneThe IL-17 family genes were cloned from yellow catfish.Protein structure analysis showed that all IL-17 family genes contained one signal peptide and one IL-17 superfamily domain.Multiple sequence alignment showed that IL-17A/F1 and IL-17A/F3 genes contained four conserved cysteine residues,IL-17A/F2 and IL-17 N genes contained six conserved cysteine residues,and IL-17 B,IL-17 C and IL-17 D contained eight conserved cysteine residues.Homology and phylogenetic tree analysis showed that yellow catfish IL-17 genes are highly conserved in vertebrates.Exon and intron analysis showed that IL-17A/Fs,IL-17 B,IL-17 C and IL-17 N all contained three exons and two introns,while IL-17 D only contained two exons and one intron.Gene syntenic analysis showed that IL-17A/F1 and IL-17A/F2 were on the same chromosome,while other IL-17 genes were on different chromosomes,respectively.3.Expression analysis,prokaryotic expression and protein purification of Pf＿IL-17 family geneThe recombinant(r)proteins of Pf＿IL-17 family genes were obtained and purified by prokaryotic expression.Additionally,polyclonal antibodies of Pf＿IL-17 B and N were prepared using recombinant proteins.The tissue distribution of Pf＿IL-17 family genes showed that IL-17 family genes had the highest expression in blood of healthy yellow catfish.Three IL-17A/Fs genes had higher expression levels in mucosal tissues,but low expression levels in immune tissues.IL-17 B,IL-17 C and IL-17 D genes were highly expressed in the gonad,and the higher expression of IL-17 N gene was detected in the kidney and brain.Pf＿IL-17A/Fs,Pf＿IL-17 C,Pf＿IL-17 D and Pf＿IL-17 N genes were significantly up-regulated at certain time points in the detected tissues after E.ictaluri infection,while Pf＿IL-17 B was not sensitive against E.ictaluri infection.Furthermore,the leukocytes were isolated from peripheral blood,gill and kidney.The expression of IL-17A/Fs,IL-17 B,IL-17 C and IL-17 D m RNA was significantly up-regulated at some time points in PBLs after stimulation of lipopolysaccharide(LPS),polyinosinic-polycytidylic acid(Poly I:C),peptidoglycan(PGN)and phytohaemagglutinin(PHA),while only LPS,Poly I:C and PGN can significantly induce the expression of IL-17 N in PBLs.4.Preliminary study on the function of Pf＿IL-17 family genesThe m RNA expression of inflammatory cytokines,chemokines,antimicrobial peptides and IL-17 downstream signaling-related-genes were significantly induced in PBLs after all r Pf＿IL-17 proteins stimulation.The r Pf＿IL-17 B,C and D proteins also significantly induced the m RNA expression of inflammatory cytokines and chemokines in GLs,while r Pf＿IL-17 N protein only significantly induced the m RNA expression of chemokines in GLs.All r Pf＿IL-17 proteins notably promoted the phagocytosis of myeloid cells in PBLs,and the r Pf＿IL-17 C and D proteins also distinctly promoted the phagocytosis of lymphocytes in PBLs.Additionally,PBLs migration was significantly induced in the medium supernatants from the PBLs treated with all r Pf＿IL-17 proteins,and GLs migration was significantly induced in the medium supernatants from the PBLs treated with r Pf＿IL-17 N protein,but all r Pf＿IL-17 proteins had no chemotactic activity.The r Pf＿IL-17 N protein also significantly induced the expression of inflammatory cytokines,chemokines and antimicrobial peptides in vivo,and caused severe inflammatory infiltration in the kidney,and aggravating inflammatory infiltration in the kidney after E.ictaluri infection.Inhibitor of NFκB and MAPK signaling pathway can significantly prevent the expression of inflammatory cytokines and chemokines induced by all r Pf＿IL-17 proteins.