Identification, expression and pro-inflammatory effect of interleukin-17 N in common carp (Cyprinus carpio L.)

Highlights

• Two IL-17 N genes were retrieved and cloned from Cyprinus carpio.

• Evolution analyses of fish IL-17Ns were performed.

• Gene expression patterns of CcIL-17Ns were determined.

• Recombinant CcIL-17Ns protein was proved to be involved in the proinflammatory process.

Abstract

Two interleukin (IL)-17 N genes (CcIL-17Na and b) present on different linkage groups were identified in the common carp (Cyprinus carpio) genome and confirmed by polymerase chain reaction (PCR) and real time (RT)-PCR in this experiment. Synteny analysis revealed that IL-17 N is transcribed by the complement sequence of TOP3B's intron 2. It is flanked by SDF2L and PPM1F in all fish studied to date, except fugu (Takifugu rubripes). The open reading frames of the two CcIL-17Ns are 411 base pairs long and encode 136 amino acids. The amino acid identity/similarity between CcIL-17Na and b is 91.2%/97.1%. The CcIL-17Ns share identity (46.8–90.4%) with their orthologs from other teleosts. Identities/similarities to other members of the IL-17 family in common carp were low at 21.4–30.2%/31.4–51.4%. In the phylogenetic tree, IL-17Ns from spotted gar (Lepisosteus oculatus, the ancestor of teleosts) and coelacanth (Latimeria chalumnae, the ancestor of tetrapods) were grouped within the same branch with a high bootstrap value of 97%, which indicates that IL-17 N is an ancient and conserved gene. Quantitative RT-PCR results showed that CcIL-17Ns were most highly expressed in the brain of healthy individuals. The expression in brain was significantly induced at 6 h post Aeromonas hydrophila infection; at 1 day post infection, expression in liver, muscle, skin, spleen, and head kidney was up-regulated. In addition, the upregulated expression of proinflammatory cytokines IL-1β, IFN-γ, IL-6, chemokine CCL20, NF - κ B and TRAF6 in kidney tissue by ccIL-17 N recombinant protein also indicate that IL-17 N can promote inflammation through NF-κB pathway and induce the expression of chemokines and inflammatory factors.

Introduction

Interleukin (IL) 17 family cytokines are important mediators of mucosal immune responses, which are tightly regulated by signals from the complex milieu of pathogenic and commensal microbes, epithelial cells, and innate and adaptive leukocytes found at tissue barriers [1]. In mammals, six members of the IL-17 cytokine subfamily (IL-17A to IL-17F) have been identified. IL-17 cytokines share a conserved cysteine-knot structure but diverge at the N-terminus [2]. With exception of Th17 cells, multiple cell types, including natural killer cells, neutrophils, mast cells, and epithelial cells, express IL-17 cytokines [3]. In humans and mice, IL-17A, IL-17F, and IL-17E are the best studied members of this cytokine family [3,4].

The IL-17 receptor family consists of five members (IL-17RA to IL-17RE). These receptors contain a conserved region known as the SEFIR (similar expression of fibroblast growth factor genes and IL-17Rs) domain at the C-terminus [5]. IL-17RA is a common receptor that forms heterodimeric complexes with IL-17RB, IL-17RC, and IL-17RE. The homo- or heterodimer of IL-17A and IL-17F signals through the IL-17RA-RC complex and triggers TRAF6-dependent target gene transcription and TRAF6-independent IKKi-dependent mRNA stabilization, and both of which are important for host defense and contribute to the pathogenesis of autoimmune diseases and cancer. Both IL-17A and IL-17F have been shown to contribute to the host response to extracellular bacteria and fungi [3,6]. The homodimer of IL-17E binds to the IL-17RA-RB receptor complex and induces Th2 responses by activating MAPK and NF-ƙB pathways to play a role in parasitic infections [3,6]. IL-17C signals through the IL-17RA-RE complex to mediate host defense and, like IL-17A, contributes to the pathogenesis of autoimmune diseases. IL-17B has been shown to interact with IL-17RB, but its biological function remains unclear [5]. IL-17RDcan disrupt the interaction of Act1 and TRAF6 to inhibit NF-ƙB activation and enhance IL-17A-dependent MAPK signaling and neutrophil recruitment through MIP-2 [7,8]. To date, the receptor of IL-17D is unknown.

The IL-17 gene family is regarded to be ancient. It presents in invertebrates such as the Pacific oyster (Crassostrea gigas) [9,10] and the ascidian Ciona intestinalis [11], also has been found in agnathans (jawless vertebrates) [12]. Gunimaladevi et al. (2006) were the first to report five IL-17 molecules in the zebrafish (Danio rerio) genome [13]; three showed homology to IL-17A and IL-17F (designated IL-17A/F1 to 3), and one each showed homology to IL-17C and IL-17D. Subsequent analysis of the fugu (Takifugu rubripes) genome revealed the presence of three IL-17A/Fs, two IL-17Cs, one IL-17D, and one IL-17 N [14]. In catfish (Ictalurus punctatus), seven IL-17 family members (IL-17A/F1 to 3, IL-17B, IL-17C, IL-17D, and IL-17 N) were discovered through database mining and cloning [1]. In salmonid fish, IL-17A/F1a/b, IL-17A/F2a/b, IL-17A/F3, two IL-17Cs, one IL-17D, and IL-17 N genes have been reported to date [[15], [16], [17], [18]]. In addition, IL-17D in grass carp (Ctenopharyngodon idella) [19] and IL-17A/F2a/b in common carp (Cyprinus carpio) [20] have been studied.

Kono et al. (2011) reviewed data of fish IL-17 ligand and receptor genes and reported IL-17 family members (IL-17A/F1 to 3, IL-17C, IL-17D and IL-17 N) in the medaka (Oryzias latipes) genome database and identified IL-17 N in the stickle back (Gasterosteus aculeatus) and zebrafish. Synteny and phylogenetic analyses suggested that IL-17 N may be a teleost specific ligand [21]. Although IL-17 N has been cloned and studied in some fish species, its phylogeny remains controversial and knowledge of its function in immunity is limited [1,15,20,21].

In this experiment, we identified IL-17 N genes in common carp, grass carp, and Sinocyclocheilus graham, a tetraploid fish that shares an ancestor with common carp and goldfish, and confirmed that IL-17 N is also present in the spotted gar (Lepisosteus oculatus) and coelacanth (Latimeria chalumnae). Using these data together with known IL-17 sequences of other fish species, we elucidated the phylogeny of fish IL-17 family members, detected the expression of CcIL-17Ns in individuals challenged with bacterium Aeromonas hydrophila, and showed the involvement of 1l-17 N genes in immunity.