| Objective:The interleukin-17(IL-17)family members are proinflammatory cytokines mainly produced by T_H17 cells,and play critical roles in host defense and various autoimmune diseases.Grass carp(Ctenopharyngodon idella)is a kind of herbivorous teleost fish,which is an important species of freshwater aquaculture in China,but it is susceptible to pathogenic bacteria,causing inflammation and even death.The intestine is an important mucosal immune tissue.Due to extensive contact with endovascular pathogenic factors in the lumen,the intestinal mucosa is susceptible to infection.In this study,we aimed to clone and identify grass carp IL-17 family genes,and to assess its expression in various tissues under physiological and pathological conditions,and thereby to determine their potential roles in intestinal inflammation.Methods:The rapid amplification of cDNA ends(RACE)and conventional PCR procedure were used to identify the coding sequences(CDS)of grass carp IL-17 s.Bioinformatics methods were used to analyze the structure of genes,coding products and evolutionary relatives.The expression levels of IL-17 s in 13 grass carp tissues were detected by quantitative real time PCR(qPCR).The intestinal inflammation model was established through the method of anal injection and intraperitoneal injection of Aeromonas hydrophila,and the relative expression of IL-17 s in 1 d,3 d and 7 d was detected by qPCR.The histopathological changes in the intestine were examined by light microscopy.Results:The full-length cDNA sequences of grass carp IL-17A/F2,IL-17A/F3,IL-17 C and IL-17 N genes were successfully cloned using RACE technology.All these IL-17 family genes consist of 3 exons and 2 introns.The coding sequences for IL-17A/F2,IL-17A/F3,IL-17 C,and IL-17 N are 420,474,489,and 411 bp,respectively,encoding 139,157,162,and 136 amino acids,respectively.The phylogenetic tree constructed based on the amino acid sequence showed that the grass carp IL-17 s correspond to other vertebrates,and share high identity to those of zebrafish and carp.The qPCR results showed that IL-17 s were constitutively expressed at varying levels in different tissues of healthy grass carp.In addition,results also indicated that bacterial infection via different routes differentially regulated the intestinal IL-17 s expression levels.The highest expression levels of IL-17A/F1,IL-17A/F2,IL-17A/F3,and IL-17 D were detected in the blood,while the highest expression levels of IL-17 C and IL-17 N were detected in heart and in muscle,skin,and brain,respectively.All these genes were expressed at relatively low levels in spleen,head kidney,and trunk kidney.At 24 h following intraperitoneal injection with A.hydrophila,the expression levels of IL-17 s in different tissues changed,and all those in intestine were significantly up-regulated.In addition,both the anal intubation and intraperitoneal injection of A.hydrophila can cause grass carp inflammation,but the two routes of infection differentially regulated the expression levels of IL-17 s in the intestine.The bacterial infection via intraperitoneal injection induced systemic inflammation and led to the formation of ascites,whereas that via anal intubation caused inflammation mainly in the intestine.Pathological sections of the intestinal tissues showed that the most pronounced intestinal inflammation occurred at 3 d following intraperitoneal infection,and the defect of intestinal villi were observed.7 d after the infection,there was only a very small amount of inflammatory cells in the intestinal mucosa.Compared with intraperitoneal infections,anal intubation infection caused the mild inflammation,and obvious inflammation was occurred at 3 d following anal intubation.The qPCR analysis indicated that after intraperitoneal infection with A.hydrophila,the IL-17A/F2,IL-17 C were continuously up-regulated,IL-17 D expression level is high in 7 days,while IL-17A/F1,IL-17A/F3 and IL-17 N were significantly up-regulated at 1 d,and 7 d,and no significant changes in expression levels even down-regulated at 3 d.In addition,with regard to bacterial infection by anal route,except IL-17 D,these genes were significantly up-regulated at 1 d,while they were different at 3 d,significant up-regulated was detected for IL-17A/F1,IL-17A/F3,IL-17 C,and IL-17A/F2?IL-17D?IL-17 N were back to normal levels.By 7 d,all genes were up-regulated.Conclusions:We cloned and characterized four grass carp genes of IL-17 family,including IL-17A/F2,IL-17A/F3,IL-17 C,and IL-17 N.Six IL-17 family genes,including IL-17A/F1,IL-17A/F2,IL-17A/F3,IL-17 C,IL-17 D,and IL-17 N,were involved in immune responses and played important regulatory roles in different manners during the intestinal inflammation that was induced through either intraperitoneal or anal route.Among them,IL-17 N had the highest significant up-regulation after 24 h of infection with A.hydrophila.Thence,we suppose it playes a significant proinflammatory role in the early stage of intestinal inflammation of grass carp.IL-17 N gene can be used for early diagnosis of inflammation. |
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