Immunoregulatory Function And Related Mechanisms Of Grass Carp (Ctenopharyngodon Idellus) Interleukin-21

In mammals,interleukin 21(IL-21)is a new type of cytokine with four-?-helix bundle that belongs to the common receptor?chain family cytokines.Activated CD4~+T cells and natural killer T(NKT)cells are major sources of IL-21,and it is also highly expressed in Th17 cells and follicular helper T cells(Tfh).IL-21 as a broad pleiotropic cytokine that affect different immune cells and non-immune cells,plays an important role in both innate and acquired immune system.Although the functional roles of mammalian IL-21 are well known,the function of teleost IL-21 is initially studied in rainbow trout.In order to explore the immunological function of grass carp IL-21,we isolated and cloned a coding sequence(CDS)of grass carp IL-21 gene(gcIL-21),which has a total length of 450 bp that encodes 149 amino acids.And then,recombinant grass carp IL-21(rgcIL-21)was prepared by prokaryotic expression system and renaturation method.After that,the polyclonal antibody of grass carp IL-21 was prepared by using rgcIL-21 and its specificity was verified.Using head kidney leukocytes(HKLs)of grass carp as the cell model,Western blotting(WB)analysis showed that heat-inactivated Aeromonas hydrophila(HI A.hydrophila)could significantly promote the secretion of grass carp IL-21 from HKLs supernatant,suggesting that grass carp IL-21 might participate in the regulation of immune response.Subsequently,biological activity of rgcIL-21 was proved by the finding that rgcIL-21 was effective in up-regulating the mRNA expression of IFN-?and SCOC3.Moreover,rgcIL-21 could significantly stimulate the expression of IL-10 in a time-and dose-dependent manner and also trigger the protein secretion of IL-10,indicating the immunosuppression of gcIL-21 based on its effect on IL-10 production.Further mechanisic studies found that the MEK and JNK signaling pathways partly participated in the regulation of IL-10 gene expression by rgcIL-21,and rgcIL-21 could enhance the phosphorylation levels of STAT3 in grass carp HKLs.In accordance with the regulatory effect of rgcIL-21 on IL-10,rgcIL-21 was effective in attenuating HI A.hydrophila–induced mRNA expression of the pro-inflammatory factors(TNF-?,IL-1?)in HKLs,which intensified the immunosuppressive effect of rgcIL-21.In support of this notion,in vivo studies revealed that intraperitoneal injection of rgcIL-21 was able to increase the survival rate of grass carp infected with Aeromonas hydrophila,and also reduce the pathological response caused by pathogenic bacteria infection.In conclusion,this study first elucidates the immunomodulatory function of gcIL-21,especially its immunosuppression in the inflammatory response during bacterial infection.