Molecular Characterization And Bioactivity Of Grass Carp Interleukin-23 Isoforms

Interleukin-23(IL-23),an important member of the IL-12 family,is a classical heterodimeric cytokine comprised of two covalently linked subunits,namely IL-23-specific p19 subunit and the IL-12p40 subunit,which is shared with IL-12.Studies in mammals have shown that IL-23 stabilizes the phenotype of Th17 cells by promoting the secretion of some cytokines such as IL-17 A and IL-22,and also mediates inflammatory response and the occurrence of autoimmune diseases.Thus,IL-23 is regarded as an important pro-inflammatory cytokine.Currently,the IL-23-related subunits p19 and p40 have been cloned from several teleosts.In contrast to mammals,many teleosts have several p40 paralogues,leading to the hypothesis that there are multiple IL-23 isoforms in fish.However,there is no direct evidence to support this hypothesis.In addition,the role of IL-23 in teleosts is still unknown,including the involvement of IL-23 in fish inflammatory response,and the regulation of IL-23 on the expression of Th17-related cytokines as seen in mammals.In this study,grass carp was used as the research model,and the results proved the existence of three grass carp IL-23 isoforms.Moreover,the data also implied the expression patterns of IL-23 subunits under bacterial infection.Besides,we obtained the recombinant proteins of grass carp IL-23 isoforms and identified their bioactivity.Furthermore,great emphasis was placed on the roles of IL-23 isoforms in mediating the expression of Th17-related cytokines as well as the related mechanisms.Meanwhile,we also explored the role of autophagy in the regulation of inflammatory response in fish as we found that grass carp IL-23 could induce the occurrence of autophagy in grass carp immune cells.The details are as follows:1.Analysis of grass carp IL-23 expression patterns under inflammatory conditions.(1)In vivo experiments showed that the infection of Aeromonas Hydrophila(A.hydrophila)could lead to the up-regulation of the transcription of p19,p40 a and p40 b subunits to some extent in different immune tissues.However,no significant change was observed for the expression of p40 c.(2)In vitro experiments showed that the classical pro-inflammatory cytokine IL-1? could induce the expression of IL-23 subunits.Taken together,these data suggest that IL-23 may be involved in the regulation of inflammatory response in grass carp.2.Evidence for the existence of three IL-23 isoforms in grass carp and the preparation of their recombinant proteins.(1)Grass carp p19 and p40 subunits were overexpressed in HEK293 cells,separatively,and then the Co-Immunoprecipitation(Co-IP)results proved that the p19 subunit could bind with either of p40 a,p40b or p40 c to form heterodimeric IL-23 molecules,indicating that there are three IL-23 isoforms in grass carp.(2)Eukaryotic expression plasmids of three IL-23 isoforms were constructed by tandem p19 and p40 subunits,and CHO(dhfr-)cell expression system was used to produce IL-23 isoform proteins.3.Bioactivity analysis of three recombinant grass carp IL-23 isoforms.(1)The results of quantitative Real-time PCR(RT-qPCR),showed that stimulation with IL-23 isoforms could up-regulate the expression of Th17-related cytokines in headkidney leukocytes(HKLs),such as IL-17A/F1,IL-22 and IL-26,etc.(2)RT-qPCR and enzyme-linked immunosorbent assay(ELISA)data implied that IL-23 could up-regulate the gene expression and protein secretion of IL-1? in HKLs.(3)The RT-qPCR data further proved each of the grass carp IL-23 isoforms could synergize with grass carp IL-1? to promote the expression of Th17-related cytokines,further indicating the role of IL-23 in inflammatory response in grass carp.4.Evaluation of grass carp IL-23 signaling pathway regulating the expression of Th17-related cytokines.(1)Western blotting(WB)data revealed that three IL-23 isoforms could induce the phosphorylation of STAT3,ERK and JNK,suggesting the activation of STAT3 and MAPK signaling pathways induced by IL-23 isoforms.(2)Inhibitors of STAT3,ERK and JNK could attentuate the inducible effect of IL-23 on the expression of Th17-related cytokines,indicating that IL-23 regulates the expression of these cytokines through STAT3 and MAPK signaling pathways.5.The induction of IL-23 isoforms on autophagy in grass carp immune cells and the preliminary study on the role of autophagy in the regulation of inflammatory response.(1)Verification of autophagy occurrence in grass carp cells.Firstly,autophagy marker molecule LC3 B was cloned from grass carp headkidney and its high identity to its counterpart in mammals implies the conservation of autophagy.Secondly,using grass carp kidney cell line(CIK)with stable expression of GFP-LC3 B as model,confocal microscopy images showed that autophagy could be induced by LPS or starvation,meanwhile,GFP-LC3 B transfectants proliferated much better than control group under LPS or starvation stimulation,suggesting the existence of autophagy in grass carp.(2)In HKLs,all of three grass carp IL-23 isoforms could enhance autophagy,indicating that IL-23 may exert its immunoregulatory effects via inducing cellular autophagy.(3)In order to test the hypothesis,we explored the role of autophagy in the regulation of inflammatory response.In vivo data showed LC3 B mRNA expression in grass carp headkidney was significantly increased under A.hydrophila infection.In vitro data proved autophagy blocker 3-MA increased the gene expression and protein secretion of IL-1? and TNF-? in grass carp CIK or HKLs,suggesting autophagy may play an important role in the regulation of inflammatory response in grass carp.Above all,this study firstly proved the existence of three IL-23 isoforms in grass carp as well as showed the inducible expression characteristics of one p19 gene and three p40 genes.Furthermore,the role and related mechanisms of three isoforms in regulating the expression of Th17-related cytokines in grass carp HKLs indicate the function conservation of IL-23.In addition,grass carp IL-23 isoforms could trigger the occurance of autophagy in grass carp immune cells,implying a new way in which IL-23 regulates inflammatory response.Furthermore,the discovery that autophagy regulated the secretion of pro-inflammatory cytokines suggests the role of IL-23 isoforms in inflammatory response in teleosts.