Identification of novel interleukin 1 beta family genes in Japanese flounder Paralichthys olivaceus

Fish & Shellfish Immunology 34 (2013) 393e396

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Short sequence report

Identification of novel interleukin 1 beta family genes in Japanese flounder Paralichthys olivaceus Apichaya Taechavasonyoo a, Hidehiro Kondo a, *, Reiko Nozaki a, Yutaka Suzuki b, Ikuo Hirono a a

Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan Laboratory of Functional Genomics, Department of Medical Genome Sciences, Graduate School of Frontier Science, The University of Tokyo, 5-1-5 Kashiwanoha Kashiwa, Chiba 277-8562, Japan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 June 2012 Received in revised form 26 September 2012 Accepted 2 October 2012 Available online 13 October 2012

Gene in the interleukin 1 family plays a central role in the regulation of immune and inflammatory responses. Here, we describe two novel IL-1-like gene in Japanese flounder Paralichthys olivaceus (jfIL1-L1 and jfIL1-L2). jfIL1b-L1 was homologous to Nile Tilapia IL-1b-like gene and Arctic char IL-1, and jfIL1b-L2 showed homology to hypothetical protein LOC100699119 of Nile Tilapia and rainbow trout Oncorhynchus mykiss IL-1 receptor agonist (RA). The deduced amino acids sequences of these IL-1b-like genes showed very low identities to the Japanese flounder IL-1 (jfIL-1). Phylogenetic analysis confirmed that jfIL1b-L1 and -L2 were distinct from jfIL-1. The gene encoding the predicted ORF of jfIL1b-L1 and -L2 is divided into 6 exons and 7 exons, respectively. Transcripts of jfIL1b-L1 were detected in gills, intestine, kidney and spleen, and those of jfIL1b-L2 were detected in gills, intestine and spleen. The mRNA levels of jfIL1b-L1 and -L2 were not effect or slightly decreased by treatment with LPS and the formalin-killed cells of Edwardsiella tarda whilst mRNA levels of jfIL1b were significantly increased in the kidney and spleen at 6 h by these treatments. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Interleukin 1b Japanese flounder Paralichthys olivaceus Phylogenetic analysis

1. Introduction Interleukin 1 family genes have been well characterized in mammals. The four main members of this family are IL-1a, IL-1b, IL-1 receptor antagonist (IL-1ra) and IL-18. IL-1b is an important inflammatory cytokines with multiple functions [1] and is mainly produced by activated macrophages and blood monocytes [2]. IL-1b potentially induces the proliferation, differentiation and activation of non specific (NK cells, macrophages, etc.) as well as a specific immune response [3]. In fish, IL-1b is the most well investigated cytokine. The IL-1b genes have been well characterized in many fish species such as rainbow trout Oncorhynchus mykiss [4], Sea bass Dicentrarchus labrax [5], carp Cyprinus carpio [6,7], Atlantic cod Gadus morhua L [8], catshark Scyliorhinus canicula [9], orange spotted grouper Epinephelus coioides [10] and Japanese flounder Paralichthys olivaceus [11]. In mammals, IL-1 is produced as an inactivate precursor that must be cleaved by IL-1 converting enzyme (ICE). In contrast, the non-mammalian IL-1bs seem to lack a clear caspase-1 cleavage site [5,12].

* Corresponding author. Tel./fax: þ81 3 5463 0174. E-mail address: [email protected] (H. Kondo). 1050-4648/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2012.10.001

The same cells that produce IL-1a and often constitutively produce IL-1 receptor antagonist (IL-1ra), which antagonizes the IL-1 receptor type I-induced activity of both IL-1a and IL-1b by competitive inhibition [13,14]. The signal peptide of IL-1ra is homologous to the first exon of IL-1b (which is not translated), which suggests that IL-1ra evolved from a duplicated IL-1b gene that arose some 350 million years ago [15,16]. Here we performed EST analysis of Japanese flounder leukocytes, and identified two IL-1b-like sequences were obtained from head kidney which is a key organ for regulatory function and the central organ for immuneeendocrine interaction [17]. 2. Materials and methods 2.1. cDNA and genome sequencing of novel interleukin 1 family genes 23,045 EST sequences of Japanese flounder leucocyte cDNA library were obtained from the 454 next generation sequencer (Roche, Switzerland). By using the EST sequences database, we designed primer sets (Supplemental Table 1). For cDNA and genome sequencing of IL-1b-like gene, the total RNA from head kidney was isolated with RNAiso (Takara, Japan) following the manufacturer’s instructions. The

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corresponding full-length cDNA sequences were obtained by SMARTÔ RACE cDNA Amplification Kit (Clontech, Mountain view, CA) according to the manufacturer’s instructions. The primers used were listed in the Supplemental Table 1. The genomic DNA encoding the IL-1b family genes were amplified by those primers. The cDNA sequences were aligned against the corresponding genome sequences and exon-intron structures were determined. 2.2. Expression analysis of novel IL-1b-like genes Japanese flounder were injected with Edwardsiella tarda formalin-killed cells at 1  106 cells per fish. Various tissues (brain, gill, intestine, kidney, liver and spleen) were taken from the treated fish and from non-injected fish. We also examine the expression of novel IL-1b gene in peripheral blood lymphocytes (PBLs). PBLs from three apparently healthy Japanese flounder (average weight 1 kg) were isolated using Percoll (GE Healthcare, Sweden) following Hirono et al. [18]. The isolated PBLs were re-suspended in primary culture medium (RPMI1640 supplemented with 10% fetal bovine serum, 100 UI/ml of penicillin and 100 mg/ml of streptomycin). Subsequently, primary cultured PBLs (6.3  107 cells) were treated with 500 mg/ml LPS (from Escherichia coli 0127:B8, SigmaeAldrich, USA), 50 mg/ml polyI:C (SigmaeAldrich, USA) and PBS for a control at 20  C. The PBLs were harvested at 1 and 3 days post-treatment.

A

Total RNA was extracted from PBLs using RNAiso (Takara, Japan) according to the instructions of the manufacturer. Two mg purified total RNA was used for cDNA synthesis using MMLV reverse transcriptase (Invitrogen, USA) following the manufacturer’s protocol. The cDNA samples were diluted 5-times with distilled water. The changes of mRNA level of the IL-1b were determined by real-time PCR which was performed with power SYBR green PCR master mix (Applied Biosystems, USA) using an ABI7300 Real-time PCR system (Applied Biosystems, USA) following the manufacturer’s instructions. Relative quantitative value was calculated according to the generated standard curve from a serial dilution of references run in the same plates. The expression levels of target genes were normalized to the expression level of elongation factor-1 (EF-1) as an internal control gene [19] and were expressed relative to the average level in the groups at 1 day post-treatment. The significance of differences between groups was determined with Student’s t test. 3. Results and discussion 3.1. Structural characterization of novel IL-1b-like genes The predicted ORFs were determined by 50 -RACE PCR using the primers listed in Supplemental Table 1. One of the ORFs was most homologous to an IL-1b-like gene in Nile Tilapia Oreochromis

Xenopus (NP_001079074: 154-278) 753

Rat (NP_113700: 148-263) Chicken (NP_989855: 142-256)

424

JF nIL1β-L2 (AB720987: 238-327)

584

Tilapia LOC100699119 (XP_003445852: 229-338)

1000

Rainbow trout RA (NP_001117868: 247-346) JF IL-1β (AB720983: 121-231)

770

753

Sea Bream (CAD11603: 126-240)

976

Rainbow trout 1 (NP_001117819: 154-238) 1000

Rainbow trout 2 (CAB53541: 145-239)

584 1000

Rainbow trout 3 (AJ557021: 157-280) Atlantic salmon 3 (HE817773: 151-272)

Catfish A (NP_001187148: 148-261)

776

1000

Catfish B (NP_001187149: 149-261) 454

1000 1000

B

Carp 2-2 (CAC19888; 152-269) Carp 1 (CAB52366: 153-261)

356 0.06

Carp 2-1 (CAC19887: 152-269)

739

JF nIL1β-L1 (AB720985: 147-249) Tilapia IL-1β like (XP_003453043: 120-239)

nIL1β-L1

nIL1β-L2

IL-1β

1,000 Bp

Fig. 1. Phylogeny and structure of Japanese flounder nIL-1 genes. A) Phylogenetic tree showing the relationship between the full-length Japanese flounder nIL-1F amino acid sequence, with other known vertebrate of IL-1b. The tree was constructed by neighbor-joining method using the CLUSTALW and was bootstrapped 1000 times. B) Comparison of genomic structure compare between Japanese flounder nIL-1b-L1, nIL-1b-L2 and IL-1b (B). The coding regions of the gene are shown to scale. Exons are indicated by gray boxes. The black and white arrowheads show the start and stop codons. The accession numbers are as follows: nIL-1b-L1, AB720984; nIL-1b-L2; AB720986 and IL-1b, AB720982.

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niloticus and less homologous to IL-1b from Arctic char Salvelinus alpinus. The other ORF showed homology to hypothetical protein LOC100699119 of Nile Tilapia and rainbow trout IL-1 receptor agonist (RA) and whose closet matches are other fish IL-1b. The deduced amino acids sequences of these IL-1b-like genes showed very low identities to the Japanese flounder IL-1 (JF IL-1). Therefore, the two sequences were designated as Japanese flounder novel IL-71b-like1 (nIL1b-L1, AB720983) and -like2 (nIL1b-L2, AB720985). Some fish such as carp, channel catfish Ictalurus punctatus and rainbow trout possess some IL-1b isoforms, which show high homology and are likely to have recently

A B

diverged. In contrast, phylogenetic analysis using the IL-1b domains clearly showed that nIL1b-L1 and -L2 were distinct from JF IL-1b (Fig. 1A). A recent report suggests that there are 2 types of IL-1b [20]. Moreover, JF nIL1b-L1 was clustered with type 1 IL-1b and JF IL-1b was closer to type 2. The nIL1b-L2 meanwhile, clustered with rainbow trout RA (NP_001117868). The predicted ORFs of nIL1b-L1 and nIL1b-L2 contain 6 and 7 exons (Fig. 1B). The IL-1b gene contains 4 exons, and IL-1bs from other fish have different exon-intron structures [20e22]. Thus, nIL1b-L1 and -L2 might have diverged from IL-1b at very ancient times [1].

Control G

I

K

395

E. tarda L

B

S

G

I

K

L

S

nIL1β-L1 nIL1β-L2 IL-1β EF-1α

B

10

nIL1β-L1

Relative mRNA levels

1

10

*

nIL1β-L2

1

100

*

*

IL-1β

*

10 1

1

3

PBS

1

3

PolyIC

1

3

(hr)

LPS

Fig. 2. Expressions of nIL-1b-L1, nIL-1b-L2 and IL-1b in Japanese flounder. A) In vivo expressions of Japanese flounder injected and non-injected with Edwardsiella tarda formalinkilled cells as determined by RT-PCR. B, brain; G, gill; I, intestine; K, kidney; L, liver and S, spleen. B) Expression in peripheral blood leukocytes stimulated with PolyI:C or lipopolysaccharide at 1- and 3-h intervals. PBS was used as control. Expression levels are expressed in arbitrary units normalized to the expression level of EF-1a. The asterisks represent statistical significance (p < 0.05) compared with the value for PBS at 1 h.

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3.2. Expression of IL-1b-like genes Transcripts of nIL1b-L1 were detected in gills, intestine, kidney and spleen, and those of nIL1b-L2 were detected in gills, intestine and spleen (Fig. 2A). The IL-1b transcripts were significantly increased in the kidney and spleen at 6 h after treatment with formalin-killed cells of E. tarda, a pathogen of Japanese flounder. In contrast, the treatments had no influence on or slightly decreased the mRNA levels of nIL1b-L1 and -L2. IL1 mRNA levels in peripheral leukocytes were strongly induced by lipopolysaccharide (LPS, 10 m/ml) but also slightly induced by polyinosinic-polycytidylic acid (polyI:C, 10 mg/ml) (Fig.2B, bottom panel). LPS stimulation slightly induced nIL1b-L2 mRNA levels (middle panel), but neither treatment affected nIL1b-L1 mRNA levels (top panel). Because LPS and polyI:C are known to induce the expression of pathogenassociated molecular patterns (PAMPs), these results suggest that PAMPs do not have a strong role in the gene expression. In conclusion, we identified 2 IL1b-like genes, nIL1b-L1 and -L2 in Japanese flounder like those found in other fish [23]. Phylogenic analysis and genomic sequencing showed that the genes diverged at very ancient times. Although the transcripts were constitutively detected in tissues involved in immunity, such as gills, intestine and spleen, their mRNA levels were not changed significantly by a pathogenic bacterium and PAMPs. We therefore speculate that the function of IL-1b-like proteins have distinct from the function of IL-1b, even though they have a conserved IL-1b domain. Acknowledgment This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.fsi.2012.10.001. References [1] Dinarello CA. Immunological and inflammatory functions of the interleukin-1 family. Annu Rev Immunol 2009;27:519e50. [2] Bird S, Zou J, Wang T, Munday B, Cunningham C, Secombes CJ. Evolution of interleukin-1b. Cytokine Growth Factor Rev 2002;13:483e502. [3] Oppenheim J, Kovacs E, Mastsushima K, Durum S. There is more than one interleukin-1. Immunol Today 1986;7:45e56.

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