Characterization of interleukin-1β as a proinflammatory cytokine in grass carp (Ctenopharyngodon idella)

Interleukin-1 beta (IL-beta) is a well-characterized cytokine that plays key roles in cellular responses to infection, inflammation, and immunological challenges in mammals. In this study, we identified and analyzed a grass carp (Ctenopharyngodon idella) ortholog of IL-1 beta (gclL-1 beta), examined its expression patterns in various tissues in both healthy and lipopolysaccharide (LPS)-stimulated specimens, and evaluated its proinflammatory activities. The gcIL-1 beta gene consists of seven exons and six introns. The full-length cDNA sequence contains an open reading frame of 813 nucleotides. The deduced amino acid sequence exhibits a characteristic IL-1 signature but lacks the typical IL-1 beta converting enzyme cleavage site that is conserved in mammals. In the phylogenetic tree, IL-1 beta s from grass carp and other members of the Cyprinidae family clustered into a single group. Expression pattern analysis revealed that gcIL-1 beta is constitutively expressed in all 11 tissues examined, and LPS stimulation leads to significant up-regulation in muscle, liver, intestine, skin, trunk kidney, head kidney, and gill. Recombinant grass carp IL-1 beta (rgcIL-1 beta) was generated prokaryotically as a fusion protein of Trx-rgcIL-1 beta. An anti-rgcIL-1 beta polyclonal antibody (rgcIL-1 beta pAb) was raised in mice against the purified Trx-rgcIL-1 beta. Western blot analysis confirmed that rgclL-1 beta pAb reacted specifically with gcIL-1 beta in C. idella kidney (CIK) cells. Quantitative real-time PCR data indicated that intestinal mRNA expression levels of endogenous IL-1 beta, IL-1R2, and TNF-alpha were significantly up-regulated following Trx-rgcIL-1 beta exposure. The inhibitory activities of rgcIL-1 beta pAb against the inflammatory response were confirmed in a model of Aeromonas hydrophila-induced intestinal inflammation. Our immunohistochemical study revealed that the degree and intensity of inflammatory cell infiltration are fully consistent with the observed mRNA expression patterns of these key inflammatory genes. Taken together, these data suggest that gclL-1 beta plays a critical role in the proinflammatory response in the grass carp intestine. (C) 2015 Elsevier Ltd. All rights reserved.