Functional Study Of Interleukins 18,21,and 34 In The Defense Against Bacterial Infection In The Snakehead(Channa Argus)

Interleukin(IL)is a messenger molecule secreted by monocytes-macrophages,lymphocytes,etc.It is involved in a series of processes such as the activation and regulation of immune cells,the activation,proliferation and differentiation of T and B cells,and the inflammatory responses,and is involved in a wide range of biological responses.As a member of the IL-1 family of cytokines,interleukin 18(IL-18)is expressed in a variety of immune and non-immune cells and plays an important role in host defence against bacteria,fungi,and viruses.Interleukin 21(IL-21)belongs to theγ-chain(γc)family of type I cytokines and is a key cytokine in the normal differentiation and functioning of lymphatic and myeloid cells.Interleukin 34(IL-34)is a novel cytokine that maintains the growth of monocytes and has biological functions such as regulating the value-added,differentiation and migration of monocyte-macrophages and inducing the expression of inflammatory cytokines.In order to investigate the role of IL-18,IL-21,and IL-34 in the defense of fish against pathogens,the IL-18,IL-21,IL-1RAc P,and IL-34 genes(named sh IL-18,sh IL-21,sh IL-1RAc P,and sh IL-34)of snakeheads(Channa argus)were cloned in this study,and its immune response to the infection with two pathogenic bacteria,Aeromonas schubertii and Nocardia seriolae,and the stimulation with three pathogen analogues,which include lipopolysaccharides(LPS),lipoteichoic acid(LTA)and polyinosinicpolycytidylic acid [Poly(I:C)],was preliminarily analyzed.At the same time,the prokaryotic expression plasmids were constructed to express and purify recombinant sh IL-18,sh IL-21,and sh IL-34 proteins(named as rsh IL-18,rsh IL-21,and rsh IL-34),respectively.The dynamic expressions of endogenous sh IL-18,sh IL-21,and sh IL-34 genes as well as various immune factors such as IFN-γ,TNF-α,and IL-1β in downstream signaling were examined in primary head kidney leukocytes after stimulation with rsh IL-18,rsh IL-21,and rsh IL-34.The proliferative effects of rsh IL-18,rsh IL-21,and rsh IL-34 on head kidney leukocytes were measured with flow cytometry to detect whether they promote the proliferation of leukocytes.Bioinformatics,amino acid sequence comparison and phylogenetic tree analysis showed that the complete open reading frame(ORF)of the sh IL-18 gene was 609 bp in length,predicted to encode 202 amino acids,contained typical IL-1 superfamily structural domain features,and was most closely related to the IL-18 proteins of Micropterus salmoides and Takifugu rubripes.The sh IL-1RAc P gene,with an ORF length of 1 848 bp and predicted to encode 615 amino acids,possessed a typical IL-1structural characterization composed of three extracellular Ig-like domains,one transmembrane domain,and one intracellular Toll/IL-1 receptor(TIR)domain.The sh IL-21 gene,with an ORF length of 438 bp and predicted to encode 145 amino acids,had typical IL-21 structural features,including four conserved cysteine residues and four α-helix structures,and was most closely related to the IL-21 proteins of Lates calcarifer and Trachinotus ovatus.The sh IL-34 gene has an ORF length of 657 bp and was predicted to encode 218 amino acids,contained four conserved cysteine residues predicted to form two pairs of disulfide bonds,and was most closely related to that of the swordfish(Xiphias gladius).q PCR showed that sh IL-18,sh IL-1RAc P,sh IL-21,and sh IL-34 were widely expressed in all tissues examined in healthy snakeheads,with higher expression in systemic and mucosal immune tissues.The immune response analysis of two pathogenic bacteria showed that both A.schubertii and N.seriola were able to induce sh IL-18,sh IL-1RAc P,sh IL-21,and sh IL-34 expression in the head kidney and spleen of snakeheads in vivo.LPS,LTA,and Poly(I:C)were able to induce the expression of sh IL-18,sh IL-1RAc P,sh IL-21,and sh IL-34 in the head kidney leukocytes of snakeheads in vitro.The rsh IL-18,rsh IL-21,and rsh IL-34 proteins not only promoted the proliferation of head kidney leukocytes,but also induced endogenous sh IL-18,sh IL-21,and sh IL-34 genes and immune related genes such as IFN-γ,TNF-α,IL-1β.The results of this study confirm that sh IL-18,sh IL-21 and sh IL-34 play an important role in the immune response and leukocyte proliferation in the defense against pathogenic bacterial infections,and provide further insight into the role of sh IL-18,sh IL-21,and sh IL-34 in antibacterial immunity.