| Objective:Interleukin-23(IL-23)is an inflammatory cytokine that is mainly secreted by activated dendritic cells,macrophages,endothelial cells and T cells.It plays an important physiological and pathological role in the host immune response to infection.IL-23 is a heterodimer formed by the dimerization of the p19 and p40 subunits,which are connected by disulfide bonds.Previous studies have confirmed that there exist three paralogous p40 genes,namely p40a,p40b and p40c,in several fish genomes.However,it is not clear whether all p40 subunits encoded by these paralogues could equally bind to p19 subunit to form functionally active IL-23 proteins.It is well known that grass carp is one of the "four major Chinese carps" and is a farmed freshwater fish species with considerable economic significance in China.However,grass carp is usually highly susceptible to various pathogenic microorganisms,including bacteria,fungi,viruses and parasites.Microbial infections trigger excessive inflammation,cause severe inflammatory diseases,and even lead to massive death in farmed grass carp,which result in huge economic loss to farmers.This study aims to clarify the difference in expression of p40a/b/c and p19 genes in normal grass carp tissues,the response to bacterial infections,and the characteristics of heterodimer formation between p19 and the three p40 subunits.It is expected to lay the foundation for the study of the pathological mechanism of fish intestinal inflammation.Methods:(1)The complete mRNA sequences of grass carp p19 and p40a/b/c were obtained from NCBI GenBank,and corresponding qPCR primers were designed.Fish were challenged by intraperitoneal injection with Aeromonas hydrophila,and the transcription levels of the above genes in various tissues at 24 h following challenge were detected by qPCR analysis.Similarly,the mRNA levels of these genes in tissues of healthy fish were also detected.Besides,the relative expression levels of p19 and p40a/b/c genes in the intestinal tissues were detected at 1 d,3 d,and 7 d after fish were infected by intraperitoneal injection and/or anal intubation with A.hydrophila.(2)We designed specific primers based on the complete coding sequences of p19 and p40a/b/c genes,obtained ORF sequences for each gene,and constructed recombinant plasmids using prokaryotic expression vector pET-32a(+).Recombinant grass carp p19 and p40a/b/c proteins(rgcp19,rgcp40a/b/c)were generated in E.coli prokaryotic expression system.The rgcp40a/b/c proteins were isolated from E.coli cells and purified for use as immunogens.In addition,a short peptide was designed based on the complete amino acid sequence of p19 and cross-linked with the carrier KLH to rgcp19-KLH,as an immunogen to produce the p19 polyclonal antibody.Polyclonal antibodies against rgcp40a/b/c and p19(rgcp40a/b/c pAb,rgcp19 pAb)were prepared.The antibodies were assayed for their titers by performing agarose immunodiffusion and ELISA experiments,and for their specificities by Western blot analysis.(3)Using the prepared rabbit-derived rgcp19 pAb and mouse-derived rgcp40a/b/c pAb,the specific expression of the above genes in Ctenopharyngodon idella kidney(CIK)cells was verified by Western blot,and the expression changes of IL-23 subunits after bacterial lipopolysaccharide(LPS)stimulation of CIK cells were examined.(4)Using the prepared rabbit-derived rgcp19 pAb and murine-derived rgcp40a/b/c pAb,we analyzed the binding characteristics of p19 and p40a/b/c in CIK cells by Co-IP.In addition,eukaryotic fluorescent expression system for p19 and/or p40a/b/c was constructed,and the mouse embryonic fibroblast cell line(NIH-3T3)was transfected with liposomes to observe the co-localized expression of p19 and p40,which clarify the characteristics of p19 and p40a/b/c to form IL-23 heterodimer;LPS and poly(I:C)were used to stimulate NIH-3T3 cells,and the response characteristics of IL-23 proteins against infection were observedResults:(1)p19 and p40a/b/c genes are expressed in 12 tissues and blood of healthy fish,including the muscle,liver,intestine,skin,fin,heart,thymus,brain,body kidney,head kidney,gill and spleen.The highest expression levels of p19 and p40c genes were found in the gill,followed by skin and body kidney;and p40a showed its highest expression in blood,followed by body kidney and gill,while p40b has the highest transcription level in skin,followed by gill,intestine,heart,and head kidney.Twenty-four hours after the fish were infected with A.hydrophila,the transcription levels of p19 and p40 changed.Except that in heart the relative expression level remained basically unchanged,the relative expression levels of p19 were up-regulated to varying degrees in various tissues,with higher levels in the head kidney,blood,gills and body kidney.p40a was most significantly up-regulated in the gills,followed by the spleen,thymus,and head kidney,but basically unchanged in the brain and body kidney;p40b has the largest up-regulation in the head and kidney,followed by blood,skin,and thymus,but it has a significant decreasing trend in the skin and heart;the expression of p40c in head kidney,blood,gills,intestine,spleen,liver and body kidney was significantly up-regulated,and it was significantly down-regulated in various tissues such as the skin,heart,caudal fin,muscle,and brain.After intraperitoneal injection of A.hydrophila to healthy fish,the transcription levels of p19 and p40a/b/e in intestinal tissues were significantly increased one day post injection(dpi),the degree of up-regulation slightly decreased at 3 dpi,and the mRNA levels were significantly decreased,and were lower than the normal level at 7 dpi.Under anal intubation,the p19 expression showed a significant up-regulation at three time points.The transcription levels of p40a and p40b were both significantly up-regulated one day after anal intubation,down-regulated on the third day,and significantly lower than the normal expression level in the intestine,and showed an upward trend on the 7th day.The p40c expression level increased significantly on the 1st day,and returned to the basic level on the 3rd and 7th days.(2)The ORF lengths of p19 and p40a/b/c were 567,990,939,and 897 bp,respectively.Correspondingly,the calculated molecular weights of the prokaryotically produced fusion proteins were 37.67,54.28,52.71,and 50.71 kDa.Agarose immunodiffusion experiments showed that the titer of the p40a/c polyclonal antibody was 1:32 and that of the p40b polyclonal antibody was 1:64.The titer of p19 polyclonal antibody detected by ELISA was 1:80,000.Western blot analysis showed that the prepared polyclonal antibodies could specifically recognize recombinant grass carp p19 and p40a/b/c proteins generated in E.coli cells.(3)After LPS stimulation of CIK cells for 24 h,the production of p40b and p40c proteins increased significantly,while that of p19 and p40a proteins did not change significantly,suggesting that p19 and p40a act together on the upstream and downstream inflammatory signaling pathways in early antibacterial response,while p40b and p40c play an immune function at the later stage.(4)Co-immunoprecipitation studies using CIK cells showed that both p19 and p40a/b/c could interact to form heterodimers IL-23a,IL-23b,IL-23c;NIH-3T3 cell localization showed that both p19 and p40a/b/c were expressed separately in the cytoplasm.Co-transfection experiment showed that p19 and p40a/b/c were co-localized in the cytoplasm respectively,forming IL-23a and IL-23b,IL-23c;however,their expression patterns are different.IL-23a and IL-23c are granular,with a regional distribution in the cytoplasm.According to the granular size and abundance,there was more IL-23a generated than IL-23c,and IL-23b was evenly distributed throughout the cytoplasm.The antibacterial and antiviral tests showed that the number of NIH-3T3 cells in IL-23a-transfected group decreased significantly after LPS stimulation.It is speculated that IL-23a or its downstream genes exert cytotoxicity in antibacterial immunity;and the nuclear entry behavior revealed that IL-23a might play a role in the antiviral response pathway.The response of IL-23b to LPS and poly(I:C)stimulation showed a significant difference.After LPS stimulation,the IL-23b+cells and NIH-3T3 cells decreased slightly(not significant),indicating that it was toxic to host cells;however,after poly(I:C)stimulation,the IL-23b+ cells decreased significantly,while the number of NIH-3T3 cells increased significantly,indicating that IL-23b played a protective role in host response to antiviral infection.When the p19/p40c-transfected NIH-3T3 cells were stimulated by LPS,the IL-23c+cells and the number of NIH-3T3 cells increased,indicating that IL-23c played roles in host defense by inhibiting inflammation during antibacterial process.After poly(I:C)stimulation,the IL-23c+cells increased significantly,but the number of NIH-3T3 cells decreased significantly,indicating that during the antiviral process,IL-23c or its downstream genes affect cytotoxicity.The above results indicate that both p19 and p40a/b/c can form heterodimers,but they are expressed differentially in cells and play different functions in preventing bacterial and viral infections.Conclusions:In this study,we analyzed the expression patterns of p19 and p40a/b/c genes,and successfully constructed recombinant plasmids containing p19 and/or p40a/b/c for prokaryotic expression and eukaryotic fluorescent expression plasmids.The mouse anti-p40a/b/c antibody and rabbit anti-p19 antibody were prepared,and it was verified that p19 could interact in vivo with p40a/b/c proteins.IL-23a showed obvious cytotoxicity in response to bacterial and viral infections.IL-23b also had a certain cytotoxic effect during the anti-bacterial process;but it showed a certain protective effect on the host during viral infection.IL-23c expression was up-regulated during bacterial and viral infections,and it played a role in protecting the host in response to antibacterial infections,and played a cytotoxic role in response to antiviral infections.The findings of this study provide ideas for increased understanding of the immune response function of the three IL-23 isoforms(IL-23a,IL-23b,IL-23c),and lay the foundation for elucidating the pathological mechanism of fish intestinal inflammation. |
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