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Genomic characterization and expression analysis of five novel IL-17 genes in the Pacific oyster, Crassostrea gigas

https://doi.org/10.1016/j.fsi.2014.07.026Get rights and content

Highlights

  • Five novel-cloned IL-17 genes are identified.

  • The expression of the CgIL-17 family genes was significantly up-regulated under PAMPs challenge.

  • This study reveals CgIL-17's diversified roles in host defense.

Abstract

Interleukin-17 (IL-17) is a proinflammatory cytokine that plays an important role in clearing extracellular bacteria and contributes to the pathology of many autoimmune and allergic conditions. In the present study, five novel IL-17 homologs were identified by searching and analyzing the Pacific oyster genome. All six CgIL-17 members (including a previously reported homolog) contained four conserved cysteines that were used in the formation of disulfide bonds. Phylogenetic analysis showed that all invertebrate IL-17s were clustered into one group, implying that invertebrate IL-17s evolved from one common ancestral gene and subsequently diversified. All CgIL-17s shared the same genomic structure, containing two exons and one intron, except for the CgIL-17-3 and CgIL-17-5 genes, which each had only one exon. The expression pattern of the CgIL-17 genes was analyzed by qRT-PCR in a variety of tissues and at different developmental stages, and these genes were highly expressed in the gill and digestive gland tissues. Moreover, the expression of the CgIL-17 family genes was significantly up-regulated in hemocytes challenged with Pathogen-Associated Molecular Patterns (PAMPs). CgIL-17-3 had a strong response to lipopolysaccharide (LPS) and heat-killed Vibrio alginolyticus (HKVA) challenge, while CgIL-17-5 and CgIL-17-6 can be activated by peptidoglycan (PGN), but not by heat-killed Listeria monocytogenes (HKLM). The distinct, up-regulated transcript levels of the CgIL-17s in response to PAMPs challenge further indicate that CgIL-17s are likely to be significant components of immune responses by playing diversified roles in host defense in the Pacific oyster. These findings suggest that CgIL-17s are involved in innate immune responses and further supports their conserved function in mollusks immunity.

Introduction

The inflammation response is a complex biological regulatory network used to protect organisms from pathogens, allergens, tissue injury, and irritants [1]. This process is coordinated by soluble effectors, primarily cytokines and chemokines, which can induce the migration of immune cells to sites of infection or injury [2]. Interleukin (IL)-17, initially identified by Rouvier et al. as cytolytic T-lymphocyte (CTL)-associated antigen 8, is a T-cell-derived cytokine with proinflammatory activity [3]. As a proinflammatory cytokine, IL-17 plays an important role in the clearance of extracellular bacteria and contributes to the pathology of many autoimmune and allergic conditions. In addition to IL-17 production by Th 17cells, it may be expressed in a variety of other cell types, including γδT cells, NKT cells, NK cells, neutrophils, and eosinophils [4], [5], [6], [7], [8]. In addition, IL-17 can potently synergize with other cytokines and activate transcription factors such as NF-κB (nuclear factor kappa-light chain enhancer of activated B cells) and activator protein 1 (AP-1), placing it in the center of the inflammatory network [9], [10], [11].

Currently, six IL-17 members (IL-17A-F) have been identified in humans [12], [13]. Among them, IL-17A, -17E (IL-25), and -17F have been well characterized and shown to be proinflammatory in nature [14]. Although IL-17 family members show low homology (16–50%) among themselves, they contain highly conserved cysteine residues that are involved in the formation of intra-chain disulfide linkages, similar to a common structural motif found in growth factors such as bone morphogenetic proteins (BMPs) and nerve growth factors (NGFs) [15], [16], [17]. Due to their unique structural features and little homology to other known cytokines or receptors, IL-17s are thought to represent a distinct ligand-receptor signaling system that appears to have been well conserved throughout their evolution [18].

IL-17 homologs from many vertebrates, including mice, chickens, Japanese pufferfish, and zebrafish, have been cloned and characterized [19], [20], [21], [22]. Although IL-17 genes have been extensively studied in vertebrates, little is known about their evolution and physiological roles in invertebrates, especially in mollusks. The first IL-17 gene released from the genome database of invertebrates was from the sea squirt, where three IL-17 homolog genes were found [23]. Soon after, Hibino et al. found 22 genes in the sea urchin genome that had similar sequences and comparable domain architecture to vertebrate IL-17 [24]. However, in mollusks, only one homolog of IL-17 was cloned from the Pacific oyster and pearl oyster [25], [26]. A thorough study of the IL-17 family genes in mollusks has not been undertaken to date; however, determining the genome sequence is a good starting point to fill this gap. With the draft genome of the Pacific oyster now available, it is possible to conduct a genomic analysis of the IL-17 genes [27]. We therefore conducted a genomic-scale search for IL-17 family members. Interestingly, we discovered five novel IL-17 homologs in the genome database, in addition to one previously reported gene [18], composing a complex IL-17 family of Crassostrea gigas.

In this study, we report the cloning and genomic characterization of these IL-17 sequences from C. gigas that have characteristics homologous to mammalian IL-17 family members. To better understand the role of the IL-17 genes in innate immunity, we investigated the expression profile of the IL-17 genes during larvae developmental stages and under Pathogen-Associated Molecular Patterns (PAMPs) challenge, in hope of providing insights into the evolution and function of the IL-17 family in oysters.

Section snippets

Sequence retrieval

Bioinformatic screening of the IL-17 genes was conducted using BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi) from the Pacific oyster genome database (http://oysterdb.cn) with amino acid sequences of reported C. gigas IL-17 (EF190193).

Cloning of full-length CgIL17s cDNA

Based on the IL-17 sequences from the genome database, their 3′ and 5′ ends were determined using the GeneRacer™ kit (Invitrogen, CA, USA) according to the manufacturer's instructions. Briefly, 5 μg of total RNA was isolated from hemocytes of the Pacific oysters

cDNA cloning and characterization of CgIL-17 genes

The Pacific oyster genome database was searched for IL-17 family genes using the reported C. gigas IL-17 gene (designated as CgIL-17-1). Five IL-17-like genes were identified and designated as CgIL-17-2 to CgIL-17-6. Based on these sequences, five full-length IL-17 cDNA sequences were obtained from the Pacific oyster by 5′ and 3′ RACE and submitted to GenBank (accession Nos. KJ531893–KJ531897). The cDNA sequences ranged from 722 to 1170 bp and each contained one ORF that varied in length, from

Discussion

The innate immune system represents the first barrier against pathogen infections in mollusks [31]. The IL-17 family contains several members, each playing a distinct role in the immune response [32]. However, only one IL-17 gene had been characterized from a mollusk species before this study [25], [26]. For the first time, we report the presence of six IL-17 homologs of oysters at the genomic scale, including CgIL-17-1 to CgIL-17-6. Structural analysis of the CgIL-17 genes showed that they

Acknowledgments

This work was supported by the National Basic Research Program of China (No. 2010CB126404), the National Natural Science Foundation of China (41176150 and 41306145), the Program of the Pearl River Young Talents of Science and Technology in Guangzhou of China (2013J2200095), and Joint Funds of NSFC-Guangdong of China (U1201215).

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    These authors contributed equally to this work.

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