A CXCL ortholog from Hippocampus abdominalis: Molecular features and functional delineation as a pro-inflammatory chemokine

Accepted Manuscript A CXCL ortholog from Hippocampus abdominalis: Molecular features and functional delineation as a pro-inflammatory chemokine Minyoung Oh, S.D.N.K. Bathige, Yucheol Kim, Seongdo Lee, Hyerim Yang, MyoungJin Kim, Jehee Lee PII:

S1050-4648(17)30295-4

DOI:

10.1016/j.fsi.2017.05.050

Reference:

YFSIM 4603

To appear in:

Fish and Shellfish Immunology

Received Date: 23 December 2016 Revised Date:

11 May 2017

Accepted Date: 19 May 2017

Please cite this article as: Oh M, Bathige SDNK, Kim Y, Lee S, Yang H, Kim M-J, Lee J, A CXCL ortholog from Hippocampus abdominalis: Molecular features and functional delineation as a proinflammatory chemokine, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.05.050. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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A CXCL ortholog from Hippocampus abdominalis: Molecular features and functional

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delineation as a pro-inflammatory chemokine

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Minyoung Oh1,2, S.D.N.K Bathige1,2, Yucheol Kim1,2, Seongdo Lee1,2, Hyerim Yang1,2,

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Myoung-Jin Kim1,2 and Jehee Lee1,2*

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University, Jeju Self-Governing Province 63243, Republic of Korea

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Fish Vaccine Development Center, Jeju National University, Jeju Self-Governing Province

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Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National

63243, Republic of Korea

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Jehee Lee, Marine Molecular Genetics Lab, Department of Marine Life Sciences, College of Ocean Science, Jeju National University, 66 Jejudaehakno, Ara-Dong, Jeju 690-756, Republic of Korea

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Tel: +82-64-754-3472; Fax: +82-64-756-3493; E-mail: [email protected] (J. Lee)

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Corresponding author:

ACCEPTED MANUSCRIPT Abstract

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Chemokines are a family of chemotactic cytokines that regulate leukocyte migration. They

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are classified into four groups namely, CXC, CC, C and CX3C, based on the formation of a

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disulfide bridge. Among these, CXC chemokines have been identified as the largest group of

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chemokines in humans. In this study, we identified and functionally characterized a homolog

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of CXC chemokine from the big-belly seahorse, Hippocampus abdominalis, and designated

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it as ShCXCL. The cDNA of ShCXCL composed of a 342-bp open reading frame encoding

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113 amino acids (aa). The CXC family-specific small cytokine domain (SCY) was identified

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from the mature peptide region, which comprised of a conserved CXC motif. As ShCXCL

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lacks an ELR (Glutamic acid-Leucine-Arginine) motif, it belongs to ELR− subfamily. The

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recombinant ShCXCL protein strongly induced the nitric oxide (NO) production in

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macrophage cells (RAW 264.7 cell line) and showed the chemotactic effect on flounder

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peripheral blood leukocytes. Tissue profiling showed a ubiquitous expression pattern in all

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examined tissues, with a high abundance in spleen. The up-regulated mRNA expression

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pattern of ShCXCL was observed in blood and kidney tissues after immune stimulation by

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live bacteria, such as Streptococcus iniae and Edwardsiella tarda, and mitogens, such as

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lipopolysaccharides (LPS) and polyinosinic:polycytidylic acid (poly I:C), suggesting its

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important role in host immune defense against microbial infection.

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Key words: Chemokines, ShCXCL, chemotaxis, immune challenges, seahorse

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1. Introduction Chemokines are chemotactic cytokines, classified into 4 groups based on the

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arrangement of conserved cysteine residues such as CC, CXC, C and CX3C [1, 2]. Among

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these, the CXC chemokines are the largest group [2] and, are further classified into ELR+ or

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ELR− groups based on the presence of a conserved ELR (glutamic acid-leucine-arginine)

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motif [3, 4]. The ELR motif, which was identified in CXC motif ligand 1 (CXCL), CXCL2,

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CXCL3, CXCL5, CXCL6, CXCL7, CXCL8 and CXCL15, plays an important role in

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promoting angiogenesis, receptor binding and leukocyte migration in vertebrates [5-7]. The

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ELR− group exhibits anti-angiogenic [8] and chemotactic properties towards lymphocytes

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and monocytes, but shows little or no attraction towards neutrophils [9, 10]. This group

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includes CXCL4, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14 and CXCL16

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[11-13]. In contrast to the higher vertebrate CXCL8 [14, 15], the teleostean CXCL8, usually

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lacks an ELR motif [15-18], except in Melanogrammus aeglefinus [19]. The involvement of

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human CXCL8 in neutrophil migration and monocyte-macrophage growth and differentiation

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during acute inflammatory responses has been reported so far [20-22].

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Amongst teleosts, only a few number of chemokines orthologous to the mammalian

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CXC chemokines have been identified. A CXCL2 from Ictalurus punctatus [8]; CXCL12

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from Oplegnathus fasciatus [23], Epinephelus coioides [24], Cyprinus carpio [25] and I.

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punctatus [8]; a CXC chemokine which is closely related to CXCL9/CXCL10/CXCL11 from

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Miichthys miiuy [26]; a CXCL10-like chemokine from Scophthalmus maximus [27];

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CXCL13 from Paralichthys olivaceus [28]; CXCL14 from C. carpio [25], I. punctatus, [8]

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and Danio rerio [29]; a CXC chemokine, each from O. fasciatus [30] and S. maximus [31];

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and a CXCa and CXCb from C. carpio [32] have been reported. Some of the chemokines,

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which were characterized as novel CXC family members, were specifically identified from

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ACCEPTED MANUSCRIPT the teleost species and they were named as CXCL_F according to the classification described

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by J. Chen et al [33]. Further, they were categorized into five different subfamilies designated

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as CXCLF1–5 [33]. Fish-specific chemokines were found to be transcriptionally expressed in

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various tissues and the transcription could be triggered by a bacterial or viral stimulation [33-

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36]. Thus, chemokines might play a putative role in antimicrobial immune responses.

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However, little is known about the real function of chemokines and their associated elements.

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Therefore, it is important to investigate fish chemokines and their functional features in order

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to improve the knowledge of the fish immune system.

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Seahorses have been considered as one of the important candidates in the aquaculture

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industry in the recent years mainly because of their significance in the oriental medicine and

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aquarium trade. The big-belly seahorse (Hippocampus abdominalis) is one of the large

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seahorse species known [37] and is utilized in the oriental medicine in Asia, particularly in

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China, Korea and Japan. Its continuous demand and habitat destructions are the main reasons

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behind its inclusion in the list of endangered species [38]. In addition, it is highly vulnerable

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to invading pathogens, which lead to mortalities [39, 40]. Thus, it is crucial to investigate the

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immune mechanisms present in seahorses at the molecular level to formulate therapeutic

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strategies.

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In this study, we identified a CXC chemokine gene from H. abdominalis (ShCXCL)

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and analyzed its molecular features, transcriptional profile under different immune stimuli

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and functions, including nitric oxide (NO) production and chemotaxis, using recombinant

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protein.

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2. Materials and methods

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2.1. Construction of seahorse cDNA database and identification of CXCL chemokine We have established a big-belly seahorse cDNA database using the 454 GS-FLX

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sequencing technique as described in our previous report [41]. The full-length cDNA

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sequence of the big-belly seahorse CXC chemokine was identified from the cDNA database,

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using the BLAST (basic local alignment search tool) algorithm at NCBI website

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(http://blast.ncbi.nlm.nih.gov/Blast.cgi), and designated it as ShCXCL.

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2.2. In silico analysis of ShCXCL sequence

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The ShCXCL cDNA sequence was analyzed using the BLAST algorithm at NCBI

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website (http://blast.ncbi.nlm.nih.gov/Blast.cgi) in order to identify its orthologs. The open

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reading frame (ORF) and its amino acid sequence were determined using the DNAssit (2.2)

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software. Using the derived amino acid sequence, the protein domains and the

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physicochemical properties of ShCXCL were predicted using the ExPASy Prosite database

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(http://prosite.expasy.org). The pairwise and multiple sequence alignments were carried out

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using the EMBOSS Needle (http://www.Ebi.ac.uk/Tools/emboss/align) and ClusterW2

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(http://www.Ebi.ac.uk/Tools/clustalw2) software, respectively. The phylogenetic analysis of

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ShCXCL along with its orthologs was performed by the neighbor-joining (NJ) method in the

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MEGA software version 5 [42].

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2.3. Cloning and construction of recombinant plasmid (pMAL-c2X/ShCXCL) The ORF of ShCXCL (without signal peptide) was amplified by polymerase chain reaction (PCR) using gene specific primers with EcoRI and HindIII restriction sites (Table 1).

ACCEPTED MANUSCRIPT The PCR was performed in a 50-µL reaction mixture containing 4 U of Ex Taq™ polymerase

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(TaKaRa, Japan), 5 µL of 10X Ex Taq™ buffer, 4 µL of 2.5 mM dNTPs, 10 pmol/µL of each

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primer and 50 ng of kidney cDNA synthesized from healthy fish. The PCR was carried out as

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follows: initial denaturation at 94 °C for 3 min, 35 cycles of denaturation at 94 °C for 30 s,

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annealing at 58 °C for 30 s and extension at 72 °C for 30 s, followed by a final extension step

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at 72 °C for 4 min. The PCR product was gel-purified using the AccuPrep Gel Purification

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Kit (Bioneer, Korea). The purified PCR product and pMAL-c2X vector (New England

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Biolabs) were digested with EcoRI and HindIII restriction enzymes, followed by the gel

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purification of the digested fragments. The digested vector and PCR product were ligated

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using the DNA Ligation Kit Mighty mix (TaKaRa, Japan) in a total volume of 10 µL at 16 °C

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for 30 min. The ligated pMAL-c2X/ShCXCL construct was then transformed into

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Escherichia coli DH5α cells in 1:10 ratio, and the purified plasmid containing ShCXCL gene

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was confirmed by sequencing.

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2.4. Overexpression and purification of recombinant ShCXCL fusion protein

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The recombinant plasmid pMAL-c2X/ShCXCL was transformed into E. coli BL21

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(DE3) cells for protein expression. Subsequently, a single colony was inoculated in 500 mL

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of LB broth containing 100 µg/mL ampicillin and 1% glucose, followed by incubation at

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37 °C with continuous shaking until the optical density of the culture reaches 0.5 at 600 nm.

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At this point, isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture at a final

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concentration of 0.25 mM, followed by incubation at 20 °C for 12 h. Thereafter, cells were

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harvested by centrifugation (3,000 rpm for 30 min at 4 °C). The harvested bacterial pellet was

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resuspended in 20 mL of column buffer (20 mM Tris-HCl, pH 7.4 and 200 mM NaCl), and

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stored at −20 °C until purification. The bacterial suspension was thawed on ice and then cold

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ACCEPTED MANUSCRIPT sonicated to lyse the cells. Subsequently, the supernatant from centrifugation (13,000 rpm for

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30 min at 4 ºC) was subjected to amylose-resin affinity chromatography-pMAL™ Protein

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Fusion and Purification System protocol (New England BioLabs, USA). The concentration of

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the purified proteins was assessed by the Bradford method using bovine serum albumin as the

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standard [43]. The purity and size of the expected fusion protein (rShCXCL) was determined

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by 12% SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The

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maltose binding protein (MBP) was also purified for the control experiment.

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2.5. Cell culture and NO production assay

The murine macrophage cell line RAW 264.7 was grown in DMEM supplemented

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with 10% FBS, penicillin (100 U/mL) and streptomycin (100 U/mL) at 37 °C in an incubator

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with 5% CO2. Cells were seeded in a 96-well plate (1 × 105 cells/mL), 24 h prior to the

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treatments. Cells were treated with MBP (1 ng/µL), rShCXCL (1 ng/µL), or

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lipopolysaccharide (LPS, 1 ng/µL) followed by incubation at 37 °C for 24 h. Subsequently,

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50 µL of the supernatant (only media) was transferred to a new 96-well plate and 50 µL of

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Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5%

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phosphoric acid) was added, and kept at room temperature for 10 min. Subsequently, the

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absorbance was measured at 540 nm in a microplate reader (Multiskan GO, Thermo

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Scientific, USA). The assay was conducted in triplicate. The significant difference was

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estimated

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(http://graphpad.com/quickcalcs/ttest1.cfm).

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2.6. Chemotaxis assay

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ACCEPTED MANUSCRIPT In order to perform the chemotactic assay, the peripheral blood leukocytes (PBLs)

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were isolated from olive flounder whole blood using Optiprep density gradient medium

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(Sigma) as described in the manufacturer’s protocol. The assay was carried out in Transwell®

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24-well plates (Corning®, USA). Briefly, the recombinant ShCXCL and MBP proteins were

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diluted in L-15 medium (Sigma, USA) to a concentration of 1 µg/mL and 10 µg/mL,

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respectively. Subsequently, 800 µL of the diluted protein was applied to the lower chamber of

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the transwell plate. The upper chamber containing a polycarbonate membrane of 4-µm pore

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size was placed on top of the lower chamber of the transwell plate. Thereafter, 200 µL of

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PBLs were added to the upper chamber and the plate was incubated at 25 °C for 90 min. The

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lower chamber containing the migrated cells and media were transferred in to a 1.5 mL tube,

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and then centrifuged at 6,000 rpm for 1 min. The supernatant was removed and the resulting

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pellet was resuspended in phosphate buffered saline (PBS). The cells were counted using a

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hemocytometer. The chemotactic index was presented as a fold increase in the number of

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migrated cells induced by the purified recombinant protein compared to that in the elution

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buffer. The assay was performed in triplicate. The significant difference was estimated by

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pairwise

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(http://graphpad.com/quickcalcs/ttest1.cfm).

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2.7. Experimental fish and tissue collection Healthy seahorses were purchased from Korea Marine Ornamental Fish Breeding

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Center, Jeju Island, Republic of Korea and were kept in a 300-L laboratory tank filled with

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sand-filtered seawater (salinity 34 ± 0.6‰; 18 ± 2 °C) for 1 week prior to the experiments for

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acclimatization. No food was provided during the experiment. To analyze the tissue

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distribution of ShCXCL under normal condition, six seahorses (3 males and 3 females) with

ACCEPTED MANUSCRIPT the average body weight of 8 g were selected and the blood from their tails was collected in

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heparinized tubes. The peripheral blood cells were collected by immediate centrifugation at

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3,000 × g for 10 min at 4 °C. Other tissues, including the heart, gill, liver, spleen, kidney,

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intestine, stomach, skin, muscle, pouch and brain, were excised and snap-frozen in liquid

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nitrogen and stored at −80 °C until RNA extraction.

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2.8. Immune challenge experiment

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The 175 healthy seahorses with an average body weight of 3 g were divided in to five

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groups and were injected intraperitoneally with 100 µL of PBS containing LPS (1.25 µg/µL),

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poly I:C (1.5 µg/µL), Edwardsiella tarda (5 ｘ 103 CFU/µL), or Streptococcus iniae (105

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CFU/µL), or PBS alone for each fish in the respective group. The blood and kidneys were

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sampled from five individuals at 0, 3, 6, 12, 24, 48 and 72 h post-injection (p.i.).

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The total RNA from each sample was extracted using RNAiso Plus (TaKaRa. Japan),

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followed by purification with spin columns (Qiagen, USA). The concentration of RNA was

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determined spectrophotometrically at 260 nm using a µDrop Plate (Thermo Scientific, USA),

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and its quality was assessed by 1.5% agarose gel electrophoresis. Fist-strand cDNA was

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synthesized in a total volume of 20 µL containing 2.5 µg of RNA using PrimeScriptTM II 1st

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strand cDNA Synthesis Kit (TaKaRa, Japan). The synthesized cDNA was diluted 40-fold in

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nuclease free water and stored at −80 °C until use.

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2.10. Quantitative real-time PCR (qPCR) The mRNA expression of ShCXCL in healthy and immune-challenged tissues were

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determined by qPCR in a reaction mixture of 10 µL, containing 3 µL of cDNA, 0.5 µL of

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each primer (10 pmol/µL), 5 µL SYBR green master mix (TaKaRa, Japan) and 1 µL of

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nuclease free ddH2O. The qPCR was carried out as follows: initial denaturation at 94 °C for

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30 s, followed by 45 cycles of denaturation at 95 °C for 5 s, annealing at 58 °C for 10 s and

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extension at 72 °C for 20 s and a final dissociation step (95 °C for 15 s, 60°C for 30 s and

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95°C for 15 s). Relative mRNA expression was calculated by the 2−∆∆Ct method [44]. The

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seahorse 40S ribosomal protein S7 was used as the housekeeping gene in all calculations. All

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data were presented as mean relative mRNA expression ± standard deviation (SD). To

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determine the statistical significance (P < 0.05) of ShCXCL transcription in healthy and

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immune-challenged tissues, the obtained data was subjected to a one-way analysis of

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variance (ANOVA) followed by Duncan’s multiple range test using the SPSS 16 program.

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3. Results and discussion

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3.1. Characterization and in silico analysis of ShCXCL

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The ShCXCL cDNA was identified from the previously established big-belly

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seahorse cDNA library [41], and submitted to the NCBI database (GenBank accession No:

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KX966279). The ShCXCL cDNA consisted of a 342-bp ORF encoding a putative peptide of

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113 aa with a molecular mass of 13 kDa. A signal peptide and a family-specific small

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cytokine domain (SCY) were identified from M1–G25 and P29–W92, respectively (Fig. 1).

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Four conserved cysteine residues, which are important for maintaining the proper structure

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and function of the ShCXCL protein [1], were identified in this SCY domain as C32, C34,

ACCEPTED MANUSCRIPT C57and C77. The ShCXCL protein lacked the N-terminal ELR motif; thus, it belongs to the

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ELR− group. In mammals, the CXC chemokines having an ELR motif played a vital role in

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angiogenesis and neutrophil induction, whereas the CXC chemokines lacking an ELR motif

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functioned as a chemoattractant for lymphocytes and monocytes [45]. However, both groups

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responded to inflammatory signals [46]. Previous studies on the fish-specific CXC

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chemokines reported that some of the functions were independent of the presence of an ELR

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motif; they showed chemotaxis towards leukocytes [46, 47] and leukocyte proliferation [47].

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Such evidence indicates that ShCXCL might also possess a chemotaxis activity.

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The multiple sequence alignment of ShCXCL with its homologs (Fig. 2) illustrated

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that seven cysteine residues, including four important residues for disulfide bond formation in

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vertebrates, were exclusively conserved [23, 47, 48]. These four residues are important for

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the tertiary structure and classification of chemokines [49]. Furthermore, the constructed

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phylogenetic tree (Fig. 3) revealed that the ShCXCL protein was clustered in the fish-specific

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CXCL group, and it shared close relationship with Cynoglossus semilaevis CXC chemokine,

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which also belonged to the ELR− group. The CXCL chemokines, which belong to the fish

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species, were distinctly clustered into a separate subgroup, showing a closer evolutionary

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relationship with the CXCL11 subgroup. In fact, it is very hard to classify ShCXCL into a

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distinct family of chemokines as in mammals, since it belongs to the fish-specific group,

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which is still under investigation.

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3.2. Recombinant protein (rShCXCL)

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The recombinant MBP-fusion protein (rShCXCL) was purified by affinity

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chromatography. The precise band corresponding to the expected size of the recombinant

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protein (51.7 kDa) was observed in both, the soluble fraction and the purified eluent (Fig.4).

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The size of the recombinant MBP was 42.5 kDa.

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3.3. NO production assay

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Murine macrophage RAW 264.7 cell line was used to explore the pro-inflammatory

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(NO production) activity of ShCXCL as this cell line was used elsewhere for the same

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experiment [50]. The LPS- and rShCXCL-treated cells showed significant NO production

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compared to the untreated and MBP-treated controls (Fig. 5). In comparison with untreated

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control cells, LPS and rShCXCL triggered the NO production by 3-fold and 2.3-fold,

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respectively. The LPS-induced NO production has been reported by previous studies [51].

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Previous studies reported that some chemokines have the ability to induce NO production

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[52-54]. The activation of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and

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interleukin-8 (IL-8) lead to the induction of cyclooxygenase-2 (COX-2) and inducible nitric

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oxide synthase (iNOS) genes [54, 55], which further induce the production of superoxide and

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inflammation via NO production [56]. The present results showed a significant involvement

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of rShCXCL in NO production in macrophages, as a first record of the fish-specific

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chemokine, and further indicates its role in inflammation and host immune defense in

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seahorse [53, 57, 58]. Based on the current results, ShCXCL could be considered a pro-

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inflammatory chemokine.

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3.3. Chemotactic activity of rShCXCL

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To investigate the chemotactic activity of rShCXCL, the PBLs isolated from the

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olive flounder and the rShCXCL were placed on the upper and lower chambers of the

ACCEPTED MANUSCRIPT Transwell (Corning®, USA), respectively. Here, we have used the blood from olive founder

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instead of seahorse blood because of the practical difficulties and the reason that olive

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flounder chemokine sequence is much closer to the sequence of ShCXCL. The migration of

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PBLs towards the lower chamber was observed in a dose-dependent manner and illustrated as

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the chemotactic index as shown in Fig. 6. The results indicated that, like other CXC

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chemokines, rShCXCL also has a potential chemotactic activity, which increased with

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increasing concentration of rShCXCL. Similarly, a few number of fish chemokines, including

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CXC chemokine from Oplegnathus fasciatus [47], two CC chemokines (Paol-SCYA104 [59]

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and JFCCL3 [60]) from P. olivaceus, a CK-1 from Oncorhynchus mykiss [61] and a CCL21

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from C. semilaevis [62] also showed the apparent chemotactic activity towards leukocytes.

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This information is further supported by the fact that the chemotactic activity might be a

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conserved functional feature of chemokines, although the numerical values are variable. This

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might be due to the fact that the extremely conserved disulfide bonds would preserve the

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three dimensional structural folding throughout the species [1].

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3.5. mRNA expression of ShCXCL with and without immune stimuli

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The mRNA distribution of ShCXCL under normal physiological conditions in

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different tissues is shown in Fig. 7. The mRNA expression in blood was considered as the

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basal value to normalize the fold change expression in the other tissues. The results indicated

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that the ShCXCL transcripts were constitutively expressed in all tissues analyzed, with

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relatively higher expression in spleen, followed by heart and kidney. The present results is

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consistent with the mRNA expression of a CC chemokine of C. semilaevis, which showed the

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highest expression in spleen [63]. Moreover, several other fish-specific chemokines, such as

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CXC12 [23] and a novel CXC [30] from O. fasciatus, a CXCL13-like chemokine from P.

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ACCEPTED MANUSCRIPT olivaceus [28], CXC from S. maximus [64] and CXCL13 from Pseudosciaena crocea [65],

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were highly expressed in the spleen, head kidney and/or kidney tissues. According to the

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previous reports, chemokines play a central role in innate and adaptive immune system [66]

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because of the relatively higher levels of their transcripts in spleen and kidney tissues, which

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are crucial hematopoietic and lymphoid organs in the fish immune system [67]. Hence, the

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results observed in the present study further support the notion of the involvement of

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chemokines in innate and adaptive immunity.

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To understand the immune-defensive role of ShCXCL towards microbial pathogens

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and pathogen-associated molecular patterns (PAMPs), the mRNA expression pattern under

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different immune stimuli in blood and kidney cells were examined (Fig. 8A and 8B). In order

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to reveal the difference of ShCXCL modulatory patterns towards Gram positive and Gram

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negative bacteria, we have used most common pathogens in aquaculture including; E. tarda

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and S. iniae respectively [68]. The expression of ShCXCL mRNA was significantly

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upregulated in response to all immune stimuli; however, the level of expression was

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dependent on both time and stimuli. In blood, live pathogens including, E. tarda and S. iniae,

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significantly induced the expression of ShCXCL at 72 h p.i. compared to the un-induced

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control (0 h). The Poly I:C challenge highly induced the expression of ShCXCL at 12 h p.i,

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whereas LPS stimulated the expression of ShCXCL predominantly at 6 h p.i. In kidney, all

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stimuli significantly upregulated the expression of ShCXCL transcripts at 3 h p.i and the

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pattern was specific to the each stimulus. The poly I:C and E. tarda stimulations showed the

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highest expression at 3 h p.i. and maintained the upregulation at all time points examined,

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except at 72 h p.i of E. tarda. The highest transcription of ShCXCL was detected at 6 h p.i

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with the LPS challenege and at 72 h p.i. with S. iniae infection. In addition, the LPS induction

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upregulated the expression of ShCXCL mRNA till 24 h p.i, and then reached the basal level.

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ACCEPTED MANUSCRIPT Transcriptional changes in different CXC chemokines were reported from several

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fish species under different immune stimuli. A CXC chemokine from S. maximus was

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significantly upregulated in liver and head kidney after the injection of Vibrio anguillarum

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[31], and CXCL12 from O. fasciatus was significantly upregulated in head kidney and spleen

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after the induction of S. iniae and E. tarda [23]. Similarly, a CC chemokine from Channa

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striata (csCC17) was upregulated in blood after the induction (72 h p.i) of Aphanomyces

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invadans [69]. The upregulated pattern of a CXC chemokine upon poly I:C challenge was

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reported from the head kidney and spleen of O. mykiss [70] and from the spleen and kidney

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of P. crocea [71]. The upregulated pattern of CXCL12 expression was reported from O.

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fasciatus, when challenged with the rock bream irido virus [23]. Thus, the results of the

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present study confirm that ShCXCL is involved in a potential immune-defensive role against

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viral and bacterial infections in the big-belly seahorse. Moreover, we propose that seahorse

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CXCL could govern the immune-defensive roles that might occur via the recruitment of

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leukocytes and relevant cytokines in the infected area.

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4. Conclusion

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In conclusion, a CXC chemokine, ShCXCL, was successfully identified in the big-belly

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seahorse and was characterized structurally and functionally. Structural insights revealed that

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ShCXCL is a fish-specific chemokine and it belongs to the ELR− group. The transcriptional

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profiling of ShCXCL from a healthy seahorse revealed its abundance in spleen and an

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upregulated expression pattern in spleen and kidney under the effect of both live-bacterial and

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mitogen stimulants, empahsizing its vital role in the immune physiology of seahorse. The

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dose-dependent NO production and chemotactic effect on leukocytes further confirmed its

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crucial role in the defense mechanism of H. abdominalis.

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ACCEPTED MANUSCRIPT Acknowledgments

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This research was a part of the project titled ‘Fish Vaccine Research Center’, funded by the

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Ministry of Oceans and Fisheries, Korea.

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ATGGCTTTGGTTGTCAACAGTTTTCCTCTCCTGCTGTTTGTTGTGGCTGGATTTTGCACA

M--A--L--V--V--N--S--F--P--L--L--L--F--V--V--A--G--F--C--TCAGCTCTATCGAGGTCATGACTTTCCTGGCCGTTGCTCATGTCACAATACAATCAAATTC

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-Q--L--Y--R--G--H--D--F--P--G--R--C--S--C--H--N--T--I--K--F-

60 20

120 40 180

-I--K--G--N--M--S--D--F--Q--V--L--E--K--R--P--G--C--D--K--I-

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GAATTGATTGTCACTATGAACAGGCCAGACAATGCCACTGAAAAGATCTGCATGAACACG

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-E--L--I--V--T--M--N--R--P--D--N--A--T--E--K--I--C--M--N--T-

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GAGGGAAGGATGGCCAGAGCTTTTTTTAGGTGCTGGGAAAGGATAAACAAAGATGAGAAC

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ATCAAAGGCAATATGTCAGATTTCCAAGTGCTTGAAAAGAGACCTGGATGTGATAAAATC

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-E--G--R--M--A--R--A--F--F--R--C--W--E--R--I--N--K--D--E--N-

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CGGAAGATGGAGTGCATCGAAAGAAAAAGAAAGGCAGAGTAA

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R--K--M--E--C--I--E--R--K--R--K--A--E--*

113

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Fig. 1. Nucleotide and amino acid sequence of the seahorse chemokine ShCXCL. In the

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nucleotide sequence, the start codon (ATG) is bold and the stop codon (TAA) is bold and

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marked with an asterisk “*”. In the amino acid sequence, signal peptide is shaded in gray

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color and the SCY domain is underlined. Four conserved cysteine(C) residues are bold and

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boxed.

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Fig. 2. ClustalW multiple sequence alignment of the deduced amino acid sequence of

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ShCXCL and its homologues using the MEGA6 software. The highly conserved (up to

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100%) residues are highlighted in black shadow and the semi-conserved (up to 80%) residues

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are highlighted in gray shadow. The conserved cysteine residues, which are important for

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disulfide bond formation, are indicated by an asterisk “*” on the top of the sequence.

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Fig. 3. Phylogenetic tree of ShCXCL with its homologs. A phylogenetic tree of ShCXCL

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chemokines was constructed by Neighbor-Joining method. The bootstrap confidence was

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calculated from 1000 replications available in the MEGA6 software. The ShCXCL protein of

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Hippocampus abdominalis is labeled with a star.

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Fig. 4. The SDS-PAGE analysis of recombinant seahorse ShCXCL (rShCXCL) protein.

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M: Protein maker (Enzynomics, Korea); 1: total protein of the uninduced E. coli BL21 cells;

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2: total protein of the IPTG-induced soluble fraction; 3: total protein of the IPTG-induced

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pellet; 4: purified rShCXCL fusion protein; 5: Purified MBP protein.

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Fig. 5. Nitric oxide (NO) production assay for rShCXCL protein

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Raw 264.7 cells were treated with MBP (1 ng/µL), ShCXCL (1 ng/µL), or LPS (1 ng/µL).

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After 24 h of incubation, the supernatant from the treated cells were mixed with the Griess

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reagent and the absorbance was measured at OD540. This assay was conducted in triplicates.

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The significant differences from the untreated controls are indicated with an asterisk (*, P <

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0.05)

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Fig. 6. Chemotactic assay for rShCXCL protein.

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The number of migrated leukocytes was determined following induction by two

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concentrations (1 and 10 µg) of rShCXCL protein using Transwell® 24-well plates. After 90

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min of incubation, the cells in the lower chamber was counted using hemocytometer. The

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chemotactic assay was conducted in triplicates. The significant differences from the MBP-

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treated controls are indicated with an asterisk (*, P < 0.05).

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Fig. 7. Tissue distribution of seahorse ShCXCL

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Relative mRNA expression was calculated by the 2−∆∆Ct method using 40S ribosomal protein

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S7 as the housekeeping gene. The values of the relative expression of ShCXCL mRNA were

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represented as relative fold compared with the mean expression of ShCXCL in blood.

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Different letters (a to i) indicate the significant difference P < 0.05.

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ACCEPTED MANUSCRIPT Fig. 8. The mRNA expression of ShCXCL in (A) blood and (B) kidney of the big-belly

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seahorse after in vivo challenge with lipopolysaccharide (LPS),

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polyinosinic:polycytidylic acid (poly I:C), Edwardsiella tarda and Streptococcus iniae.

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Tissues were obtained at different post injection time points (0–72 h) from the immune-

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challenged seahorses for RNA extraction and cDNA synthesis. Transcript levels were

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determined by SYBR green qPCR. The seahorse 40S ribosomal protein S7 was used as the

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housekeeping gene. The results are reported as mean ± standard deviation (SD) of triplicates.

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Data indicated with asterisk (*) are significantly different (P < 0.05) from the untreated (0 h)

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control.

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ACCEPTED MANUSCRIPT Table 1. List of the primers used in this study Orientation

Primer sequences (5′′ → 3′′)

ShCXCL qPCR

Forward

CAGGCCAGACAATGCCACTGAAA

ShCXCL qPCR

Reverse

TGCACTCCATCTTCCGGTTCTCAT

40S ribosomal protein S7 qPCR

Forward

GCGGGAAGCATGTGGTCTTCATT

40S ribosomal protein S7 qPCR

Reverse

ACTCCTGGGTCGCTTCTGCTTATT

ShCXCL cloning

Forward

gagagaattcCATGACTTTCCTGGCCGTTGC

ShCXCL cloning

Reverse

gagaaagcttTTACTCTGCCTTTCTTTTTCTTTCGATGCAC

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Target

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ACCEPTED MANUSCRIPT A CXC chemokine gene (ShCXCL) with CXC family features was identified ShCXCL transcripts were constitutively expressed with highest expression in spleen Modulated transcription of ShCXCL upon challenges revealed its role in host immunity

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rShCXCL strongly induced the NO production in RAW 264.7 cells

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rShCXCL showed an effective chemotactic activity towards leukocytes