Cloning Of Grass Carp IL-23R Gene And Its Role In Intestinal Inflammation Of Grass Carp

Objective:Interleukin-23(IL-23)is a pro-inflammatory cytokine in the IL-12 family and plays an important pathological role in inflammatory and autoimmune diseases.IL-23R(IL-23 receptor),as a specific receptor for IL-23,is required to transfer the signal produced by IL-23 stimulation in IL-23 signaling and plays an important regulatory role between innate immunity and adaptive immunity.Grass ceirp(Ctenopharyngodon idella)is one of the most important freshwater aquaculture species in China,but it is susceptible to infection by pathogens such as bacteria,viruses and parasites,causing inflammation and even death.The aim of this study was to clone the grass carp IL-23R gene,analyze its gene structure,and clarify its gene expression patterns under the physiological and pathological conditions,and reveal its expression and function in the intestinal inflammation in grass carp.Methods:(1)The full-length cDNA of grass carp IL-23R was cloned by RACE technology.Recombinant IL-23R was produced in prokaryotic E.coli cells by using pET-28a(+)expression vector.The recombinant protein labeled with 6 histidine(6 × His)tags was purified by Ni-NTA chelate affinity chromatography,and then was reconstituted by gradient dialysis to obtain the biologically active recombinant grass carp IL-23R protein(rgcIL-23R).(2)The gel-purified rgcIL-23R was used as immunogen to prepare polyclonal antibody against grass carp IL-23R(rgcIL-23R pAb).The titer and specificity of the rgcIL-23R pAb were detected by ELISA assay and verified by Western blotting analysis.The expression pattern of endogenous IL-23R protein was detected by indirect immunofluorescence assay using Ctenopharyngodon idella kidney(CIK)cells.(3)The qPCR technique was used to detect the IL-23R transcription levels in different tissues of healthy grass carp and of those challenged With Aeromonas hydrophila at 24 h following intraperitoneal injection or anal intubation.(4)Fish were challenged by anal intubation with active rgcIL-23R protein at different doses,the pathological characteristics of intestinal inflammation in grass carp were observed and the relative mRNA expression levels of IL-23R,IL-23p19,IL-23p40a,IL-23p40b,IL-23p40c,IL-17A/F1,IL-17N and IL-1β in mid-intestinal tissues during inflammation were detected using qPCR assay.Fish were challenged by anal intubation with A.hydrophila.12 h later,each of these fish was given another anal intubation with 20 ng rgcIL-23R pAb.The mRNA levels of IL-23R and other inflammatory pathway genes in intestinal tissues were detected using qPCR analysis,and histopathological examination was performed to assess the effects of rgcIL-23R pAb on pathological changes in intestinal tissues.(5)CIK cells were stimulated by active rgcIL-23R protein.The mRNA expression levels of IL-23R,IL-23p19,IL-23p40a,IL-23p40b,IL-23p40c,STAT3,RORa,RORy,IL-17A/F1,IL-17N and IL-1β in rgcIL-23R-stimulated CIK cells were detected using qPCR.LPS-stimulated CIK cells were treated with rgcIL-23R pAb,in which the expression levels of IL-23R and other genes that are involved in inflammatory pathway were detected by qPCR.Results:(1)The full-length cDNA of grass carp IL-23R is 3252 bp long with an ORF of 2589 bp.The CDS region encodes 862 amino acid residues,and the molecular weight is predicted to be 96.6 kDa.There are two type Ⅲ fibronectin(FN3)domains,one transmembrane domain.The extracellular region has multiple glycosylation sites.Phylogenetic tree analysis showed that fish IL-23R molecules formed a single cluster,separating from those from birds,reptiles and mammals,which is consistent with the closest relationship of grass carp with Carassius auratus,Sinocyclocheilus rhinocerous,Cyprinus carpio and Danio rerio.(2)An anti-IL-23R polyclonal antibody was prepared by using the purified rgcIL-23R as the immunogen.The ELISA analysis showed that the titer of the polyclonal antibody was 1:40000.The indirect immunofluorescence and Western blot analysis of CIK cells confirmed that the anti-IL-23R polyclonal antibody specifically recognized the rgcIL-23R generated by prokaryotic expression and the IL-23R protein in its native state.(3)IL-23R is widely expressed in various tissues of healthy grass carp.IL-23R mRNA levels were detected in trunk kidney,head kidney,spleen,tail fin,thymus,gill,heart,liver,brain,muscles,intestines,blood,and skin.After the fish was infected with A.hydrophila,the expression levels of IL-23R in related tissues changed.At 24 h after intraperitoneal injection with pathogenic bacteria,IL-23R was most significantly up-regulated in the tail fin and intestine,followed by that in kidney,spleen and heart,but IL-23R was significantly down-regulated in the gill.At 24 h after anal intubation with pathogenic bacteria,IL-23R was significantly up-regulated in the liver,intestine,thymus,tail fin,head kidney,gill,blood,and brain tissues,and was significantly down-regulated in the muscle.(4)After anal intubation with different doses of active rgcIL-23R protein,the transcription level of geIL-23R in the intestine was significantly up-regulated,reaching the highest stimulation at 72 h,and the highest expression at a dose of 15 μg/fish,and obvious inflammatory signs were observed in intestine and body surface.The expression levels of IL-23P19,IL-23p40a,IL-23p40b and IL-23p40c genes were significantly up-regulated 1 d after challenge,and then declined at varying degrees.Similar expression patterns were also found in IL-17A/F1,IL-17N,IL-1β and IL-23R genes within the inflammatory pathway.In fish that developed intestinal inflammation by anal intubation with A.hydrophila,another anal intubation with rgcIL-23R pAb could reduce the mRNA expression le-vels of IL-23R and other inflammation-related genes,and alleviated intestinal inflammation.(5)CIK cells could generate IL-23R.Both rgcIL-23R and rgcIL-23R pAb treatments caused changes in IL-23R mRNA expression levels.Regulation of IL-23R expression could cause changes in expression levels of inflammatory pathway genes.Conclusions:We have cloned and identified grass carp IL-23R.Recombinant IL-23R protein can induce intestinal inflammation in grass carp.The anti-IL-23R polyclonal antibody can reduce intestinal inflammation and regulate the expression of some genes in the inflammatory pathway.Therefore,IL-23R is an important inflammatory factor that plays a key role in intestinal inflammation in grass carp.