Cloning Of Grass Carp Interleukin-1β Gene And Its Functional Characterization In Intestinal Inflammation

Objective: Interleukin-1β(IL-1β) is a proinflammatory cytokine mainly produced by activated monocytes and macrophages. IL-1β participates in a variety of physiological and pathological processes and plays an important role in the maturation, activation, proliferation and immune regulation of immune cells. Grass carp(Ctenopharyngodon idella) is a farmed fish species with highly economic importance in China, but is vulnerable to infection by many bacterial and viral pathogens. Pathogens usually get into fish body via the intestine, resulting in inflammation, or even death, thus cause substantial economic losses. In this study, we aimed to clone and identify grass carp IL-1β gene, and to assess its expression profiles in various tissues under physiological and pathological conditions, and thereby to determine its potential role in intestinal inflammation.Methods: The rapid amplification of c DNA ends(RACE) procedure was used to obtain the full length c DNA of grass carp IL-1β. Recombinant IL-1β protein fused with a Trx tag was generated in p ET32a(+) prokaryotic expression system and purified by Ni-NTA affinity chromatography. The q PCR technique was employed to quantify:(1) the transcription levels of IL-1β in various tissues of healthy grass carp;(2) the changes of relative expression levels of IL-1β in various tissues 4 h, 12 h, 24 h, and 48 h after intraperitoneal injection with endotoxin(LPS), respectively;(3) the changes relative expression levels of IL-1β, IL-1R2 and TNF-α genes in intestinal tissues during inflammatory processes after challenge by anal intubation with different doses of recombinant IL-1β, and those in healthy tissues(negative control) or Aeromonas hydrophila-infected tissues(positive control). The recombinant IL-1β protein(rgc IL-1β)purified from SDS-PAGE gel was used as immunogen to raise the polyclonal antibody(rgc IL-1β p Ab). The efficiency and specificity of the antibody were tested by double immunodiffusion assay and Western blot, respectively. Grass carp model with intestinal inflammation was generated by anal intubation with Aeromonas hydrophila. 12 h after inflammation induction, fish were treated with rgc IL-1β p Ab at 20 ng/fish via anal intubation route, and then the m RNA expression levels of IL-1β, IL-1R2 and TNF-α in intestinal tissue during inflammatory processes were quantified by q PCR assay.Immunohistochemical analysis was performed to characterize the natural IL-1β protein secretion in the intestine of grass carp 3 d after treatment with rgc IL-1β p Ab.Results: The full-length c DNA of grass carp IL-1 b(gc IL-1 b) is 1260 bp long,containing a 813-bp open reading frame that encoded a peptide of 270 amino acids with predicted molecular weight of 30.1 k Da. The amino acid sequence contained a signature that is proposed to be characteristic of the IL-1 protein family, but lacked a typical IL-1bconverting enzyme cleavage site that conserved in mammals. Genomic DNA of gc IL-1b is2863 bp in length, consisting of seven exons and six introns. Phylogenetic tree analysis showed that, IL-1βs from grass carp and other members of the Cyprinidae family clustered into a single group. The gc IL-1β gene was constitutively expressed in 11 tissues, including the muscle, liver, intestine, skin, fin, heart, brain, kidney, head kidney, gill, and spleen.Among these tissues, the higher gc IL-1β expression was observed in spleen, gill, head kidney, and kidney. Results also showed that LPS stimulation induced significant up-regulation of gc IL-1β in muscle, liver, intestine, skin, kidney, head kidney, and gill.Peak levels were found in muscle, skin, fin and spleen at 12 h post LPS stimulation, while those in other six tissues were observed at 24 h. IL-1β m RNA levels were up-regulated significantly followed anal intubation with different doses of rgc IL-1β, and reached the peak at 24 h after stimulation. The highest expression level was observed when treated with a dose of 5 μg/fish, and the level gradually decreased 72 h post stimulation.Correspondingly, the m RNA expression levels of the receptor gene IL-1R2 and the downstream TNF-α gene were also up-regulated, and changed in a similar pattern with IL-1β. The double immunodiffusion assay showed that the valence of rgc IL-1β p Ab was1:32. Western blot confirmed that the rgc IL-1β p Ab could specifically bind the gc IL-1βprotein. Our results indicated that treatment with rgc IL-1β p Ab reduced the m RNA levels of IL-1β, IL-1R2 and TNF-α genes, lowered the IL-1β secretion in intestine, and alleviated intestinal inflammation.Conclusion: We have cloned and identified gc IL-1β. Our data confirmed that IL-1βgene was constitutively expressed in various tissues of grass carp. The rgc IL-1β could induce intestinal inflammation in grass carp, and the rgc IL-1β p Ab could partially block the intestinal inflammation. Therefore, IL-1β might play an important role in intestinal inflammation in grass carp.