CXCL8 of Scophthalmus maximus: Expression, biological activity and immunoregulatory effect

Developmental and Comparative Immunology 35 (2011) 1030–1037

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CXCL8 of Scophthalmus maximus: Expression, biological activity and immunoregulatory effect Yong-Hua Hu a , Ling Chen a,b , Li Sun a,∗ a b

Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, PR China Graduate University of the Chinese Academy of Sciences, Beijing 100049, PR China

a r t i c l e

i n f o

Article history: Received 13 January 2011 Received in revised form 31 March 2011 Accepted 1 April 2011 Available online 20 April 2011 Keywords: CXCL8 Interleukin-8 Scophthalmus maximus Inflammation

a b s t r a c t CXCL8, or interleukin-8, is a CXC chemokine that promotes neutrophil migration in response to inflammatory stimuli. In this study, we identified and analyzed a CXCL8 orthologue, SmCXCL8, from turbot (Scophthalmus maximus). The deduced amino acid sequence of SmCXCL8 is 99-residue in length and shares 52–83% overall identities with the lineage 1 CXCL8 of a number of teleost. SmCXCL8 possesses a CXC chemokine domain that contains the conserved CXC motif preceded by the tripeptide sequence EMH. Purified recombinant SmCXCL8 (rSmCXCL8) induced chemotaxis in peripheral blood neutrophils and, to lesser extents, head kidney (HK) lymphocytes and macrophages in a dose-dependent manner. Mutation of the EMH motif by alanine substitution reduced the chemoattractive effect of rSmCXCL8. Expression of SmCXCL8 as determined by quantitative real time RT-PCR (qRT-PCR) was detected mainly in immune organs under normal physiological conditions and was upregulated by experiment challenges with bacterial pathogen and poly(I:C). In addition, SmCXCL8 expression was also induced to significant extents following vaccination of turbot with a subunit vaccine. When rSmCXCL8 was added to the cell cultures of peripheral blood leukocytes and HK lymphocytes and macrophages, it stimulated the proliferation of these cells and enhanced cellular resistance against intracellular bacterial survival. qRT-PCR analysis showed that rSmCXCL8 induced the expression of TNF-␣ and suppressor of cytokine signaling 3 in HK lymphocytes in different time frames. On the other hand, SmCXCL8 expression was also upregulated by TNF-␣. Taken together, these results indicate that SmCXCL8 is a functional CXC chemokine with immunomodulatory effect and plays a role in inflammatory response induced by bacterial infection. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Chemokines are a family of small extracellular proteins that are defined by homology in primary and higher-order structure (Murphy, 2008). Based on their expression and functional properties, chemokines are categorized into two groups: homeostatic chemokines and inflammatory chemokines (Moser and Loetscher, 2001). Homeostatic chemokines are expressed constitutively and mediate migration of hematopoietic precursor cells and other types of cells, while inflammatory chemokines are expressed after cell activation and function in the regulation of leukocyte trafficking (Fernandez and Lolis, 2002; Laing and Secombes, 2004). Chemokines exert their function by binding to specific receptors that belong to a large family of seven-transmembrane-domain G-protein-coupled receptors, which mediate signal transduction through heterotrimeric G-proteins (Murphy, 1994). Owing to their immunoregulatory capacity, chemokines play important roles in

∗ Corresponding author. Tel.: +86 532 82898829; fax: +86 532 82898829. E-mail address: [email protected] (L. Sun). 0145-305X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2011.04.002

inflammation and host immune response against pathogen infection. A common feature of all chemokines is the presence of at least two cysteine residues that form conserved intramolecular disulfide bridge. According to the number and location of the conserved cysteine residues, chemokines are classified into four subfamilies: CXC, CC, C, and CX3 C (Murphy et al., 2000; Zlotnik and Yoshie, 2000). In CXC chemokines, the two N-terminal cysteine residues are separated by one nonconserved amino acid (X). CXC chemokines are further divided into two subgroups based on the presence or absence of a conserved glutamic acid–leucine–arginine (ELR) motif immediately preceding the CXC motif. CXC chemokines with ELR motif are known to be chemotatic for neutrophils and possess angiogenic property, while CXC chemokines lacking the ELR motif are angiostatic and chemotatic for lymphocytes (Bizzarri et al., 2006; Strieter et al., 1995, 2006). CXCL8, or interleukin 8, is a member of the CXC chemokine family. Mammalian CXCL8, which is ELR positive, is a potent proinflammatory factor and functions primarily as an inducer of chemotaxis in neutrophils in response to an inflammatory stimulus. In addition to neutrophils, other cells, such as endothelial

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cells, basophils, and resting T lymphocytes, are also responsive to CXCL8 (Laing and Secombes, 2004). In fish, CXCL8-like sequence was first found in lamprey (Lampetra fluviatilis) (Najakshin et al., 1999) and subsequently identified in a large number of teleost species (Chen et al., 2005; Corripio-Miyar et al., 2007; Covello et al., 2009; Fujiki et al., 2003; Huising et al., 2003; Laing et al., 2002; Lee et al., 2001; Nomiyama et al., 2008; Oehlers et al., 2010; Saha et al., 2007; Seppola et al., 2008; Cai et al., 2008), however, only a few piscine CXCL8 orthologues, i.e. those from rainbow trout (Oncorhynchus mykiss), black sea bream (Acanthopagrus schlegeli), and common carp (Cyprinus carpio), have been shown to possess chemotactic activity (Cai et al., 2009; Harun et al., 2008; Montero et al., 2008). Unlike mammalian CXCL8, which are ELR positive, fish CXCL8, with the exceptions of those from haddock (Melanogrammus aeglefinus) and Atlantic cod (Gadus morhua), in general lack a complete ELR. Recent studies indicate that teleosts have at least two CXCL8-like lineages, with lineage 1 present among multiple fish species while lineage 2 having so far been found only in carp and zebrafish (Abdelkhalek et al., 2009; van der Aa et al., 2010). In this study, we cloned and analyzed a CXCL8 orthologue from turbot (Scophthalmus maximus), an important economic fish species cultured widely in north China and other Asian areas. We found that turbot CXCL8 (SmCXCL8) is upregulated at transcription level by microbial challenge and antigen stimulation and that purified recombinant SmCXCL8 induced chemotaxis in target cells and enhanced cellular resistance against intracellular bacterial survival. In addition, we also observed a regulatory effect of SmCXCL8 on the expression of tumor necrosis factor-alpha (TNF-␣) and suppressor of cytokine signaling 3 (SOCS3). 2. Materials and methods 2.1. Fish Turbot (S. maximus) were purchased from a commercial fish farm in Shandong Province, China and maintained at 19 ◦ C in aerated seawater. Fish were acclimatized in the laboratory for two weeks before experimental manipulation. Fish were anaesthetized with tricaine methanesulfonate (Sigma, St. Louis, MO, USA) prior to experiments involving blood collection and sacrificed with an overdose of tricaine methanesulfonate before tissue collection. 2.2. Cloning of SmCXCL8 Plasmid DNA was isolated from clones of a mixed cDNA library of turbot head kidney, spleen, and liver (Zheng et al., 2010) and subjected to DNA sequencing. One of the clones was found to contain the full length cDNA of SmCXCL8 with 5 - and 3 untranslated regions (UTRs). The nucleotide sequence of SmCXCL8 has been deposited in GenBank database under the accession number HQ872499.

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2.4. Plasmid construction To construct pEtSmCXCL8, which expresses SmCXCL8 linked to a protein tag (Trx-tag) derived from the backbone plasmid pET32a (Novagen, San Diego, CA, USA), the coding sequence of SmCXCL8 without signal sequence was amplified by PCR with primers F2 (5 GATATCATGGTGAGTCTGAGAAGCCTG-3 ; underlined sequence, EcoRV site) and R1 (5 -GCGATATCGCGTCTTCTGTTGGACA-3 ; underlined sequence, EcoRV site); the PCR products were ligated with the T–A cloning vector pBS-T (Tiangen, Beijing, China), and the recombinant plasmid was digested with EcoRV to retrieve the SmCXCL8 fragment, which was inserted into pET32a at the EcoRV site. To construct pEtSmCXCL8M1, which expresses the mutant SmCXCL8 bearing three alanine substitutions at 32 EMH34 , the coding sequence of the mutant SmCXCL8 was created by PCR with primers F3 (5 -GATATC ATGGTGAGTCTGAGAAGCCTGGGAGTGGCGGCAGCCTGTCGCT-3 ; underlined sequence, EcoRV site) and R1, and the PCR products were inserted into pET32a at the EcoRV site as described above. To construct pEtSmCXCL8M2, which expresses the mutant SmCXCL8 bearing LR substitutions at 33 MH34 , the coding sequence of the mutant SmCXCL8 was created by PCR with primers F4 (5 -GATATC ATGGTGAGTCTGAGAAGCCTGGGAGTGGAGCTGCGCTGTCGCT-3 ; underlined sequence, EcoRV site) and R1, and the PCR products were inserted into pET32a at the EcoRV site as described above. 2.5. Purification of recombinant protein Escherichia coli BL21(DE3) (Tiangen, Beijing, China) was transformed separately with pEtSmCXCL8, pEtSmCXCL8M1, pEtSmCXCL8M2, and the control plasmid pET32a, resulting in the transformants BL21(DE3)/pEtSmCXCL8, BL21(DE3)/pEtSmCXCL8M1, BL21(DE3)/pEtSmCXCL8M2, and BL21(DE3)/pEt32a respectively. The transformants were cultured in Luria-Bertani broth (LB) medium at 37 ◦ C to mid-log phase, and isopropyl-␤-d-thiogalactopyranoside was then added to the culture to a final concentration of 1 mM. After growth at 37 ◦ C for an additional 3 h, the proteins were purified under native conditions using nickel–nitrilotriacetic acid (Ni–NTA) columns (GE Healthcare, USA) as recommended by the manufacturer. The tag protein Trx-tag, which is expressed by the control plasmid pEt32a, was similarly purified from BL21(DE3)/pEt32a. The purified proteins were dialyzed for 24 h against phosphate-buffered saline (PBS) and concentrated using Amicon Ultra Centrifugal Filter Devices (Millipore, Billerica, MA, USA). The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized after staining with Coomassie brilliant blue R-250. The concentration of the purified protein was determined using the Bradford method (Bradford, 1976) with bovine serum albumin as a standard. 2.6. Quantitative real time reverse transcriptase-PCR (qRT-PCR) analysis of SmCXCL8 expression under different conditions

2.3. Sequence analysis The cDNA and amino acid sequences of SmCXCL8 were analyzed using the BLAST program at the National Center for Biotechnology Information (NCBI) and the Expert Protein Analysis System. Domain search was performed with the simple modular architecture research tool (SMART) version 4.0 and the conserved domain search program of NCBI. The molecular mass and theoretical isoelectric point were predicted using EditSeq in DNASTAR software package (DNASTAR Inc. Madison, WI, USA). Subcellular localization was predicted with the WoLF PSORT server. Multiple sequence alignment was created with the ClustalX program. Signal peptide search was performed with SignalP 3.0.

2.6.1. SmCXCL8 expression in fish tissues under normal physiological conditions Brain, heart, gill, kidney, spleen, liver, muscle, and blood were taken aseptically from four fish and used for total RNA extraction with the RNAprep Tissue Kit (Tiangen, Beijing, China). One microgram of total RNA was used for cDNA synthesis with the Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). qRT-PCR was carried out in an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) using the SYBR ExScript qRT-PCR Kit (Takara, Dalian, China) as described previously (Hu et al., 2010). Melting curve analysis of amplification products was performed at the end of each PCR to confirm that only one PCR product was amplified and

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detected. The expression level of SmCXCL8 was analyzed using comparative threshold cycle method (2−CT ) with RNA polymerase II subunit D (RPSD) as the control (Dang and Sun, 2011). All data are given in terms of mRNA levels relative to that of RPSD and expressed as means plus or minus standard errors of the means (SE). 2.6.2. SmCXCL8 expression in response to microbial challenge The fish bacterial pathogen Edwardsiella tarda TX1 (Zhang et al., 2009) was cultured in LB medium and resuspended in PBS to 1 × 107 colony forming units (CFU)/ml. Polyinosinic–polycytidylic acid {poly(I:C)} (Sigma, St. Louis, MO, USA) was suspended in PBS to 0.5 mg/ml. Turbot (∼12.6 g) were divided randomly into groups of five fish and injected i.p. with 100 ␮l of E. tarda, poly(I:C), and PBS, respectively. Fish were sacrificed at 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h post-challenge, and head kidney (HK) was excised under aseptic conditions and used for qRT-PCR analysis of SmCXCL8 expression as described above. 2.6.3. SmCXCL8 expression following vaccination Purified recombinant Eta21, a protective E. tarda antigen (Jiao et al., 2009), was prepared as described previously (Jiao et al., 2009) and resuspended in PBS to 150 ␮g/ml. Turbot (∼12.6 g) were divided randomly into two groups and injected intraperitoneally with 100 ␮l of PBS (control) or Eta21. Fish were sacrificed at 4 h, 12 h, 1 d, 2 d, 3 d, 7 d, 14 d, and 21 d post-vaccination. SmCXCL8 expression in HK was determined by qRT-PCR as described above. 2.7. Preparation of peripheral blood leukocytes (PBL), blood neutrophils, head kidney (HK) lymphocytes, and HK macrophages To prepare PBL, blood was collected from the caudal veins of turbot and diluted 1:5 with RPMI 1640 culture medium (Gibco, Invitrogen Corp., Carlsbad, CA, USA). The diluted blood was placed on top of 54% Percoll (Solarbio, Beijing, China) and centrifuged at 400 × g for 30 min. The layer of PBL was recovered and washed three times with PBS. A portion of the cells was examined under microscope (Supplementary data Figure 1). The cells were resuspended in RPMI 1640 containing 0.1% calf serum (Thermo Scientific HyClone, Beijing, China), 1% penicillin and streptomycin (Sangon, Shanghai, China), and 25 U/ml heparin (Sigma, St. Louis, MO, USA). Blood neutrophils were prepared according to the method of Haslett et al. (1985) with modification as follows. Two milliliters of blood was centrifuged at 500 × g for 20 min, and the thrombocyterich layer and the leukocyte layer were carefully aspirated. The thrombocyte-rich layer was centrifuged at 2500 × g for 15 min to produce thrombocyte-poor plasma (TPP). The leukocyte layer was diluted with 1.6 ml PBS in a polyethylene tube, followed by adding 0.4 ml 6% dextran. The tube was inverted a few times to mix the content and then incubated at room temperature for 60 min. After incubation, the supernatant was taken and centrifuged at 500 × g for 5 min. The pellet was resuspended in 0.4 ml TPP and transferred to a fresh polyethylene tube, where it was underlayered sequentially with 0.8 ml 60% Percoll and 0.8 ml 75% Percoll in PPP. The gradients were centrifuged at 500 × g for 30 min, and the intermediate neutrophil layer was taken out and washed three times with PBS. The cells were resuspended in RPMI 1640 containing 10% fetal bovine serum (FBS) and adjusted to a concentration of 1 × 106 cells/ml. Turbot HK macrophages were prepared as reported previously (Chung and Secombes, 1988). In brief, HK was removed, washed three times with PBS, and passed through a sterile metal mesh. The cells were resuspended in L-15 medium (Thermo Scientific HyClone, Beijing, China) and placed onto a 34/51% Percoll gradient, followed by centrifugation at 400 × g for 30 min. After centrifugation, the cells at the 34/51% interface were recovered and washed three times with PBS. A portion of the cells was examined under

microscope (Supplementary data Figure 2A). The cells were resuspended in L-15 medium containing 0.1% calf serum (Thermo Scientific HyClone, Beijing, China), 100 U/ml of penicillin and streptomycin (Sangon, Shanghai, China), and 20 U/ml heparin (Sigma, St. Louis, MO, USA). The cells were added to 96-well microplates (2 × 105 cells/well) and incubated at 25 ◦ C for 2 h. Non-adherent cells were washed off after the incubation. To prepare HK lymphocytes, turbot HK was removed under aseptic conditions and washed three times with PBS containing 100 U of penicillin and streptomycin. The tissue was passed through a metal mesh, and the cell suspension was overlayered on a 1.070 and 1.077 discontinuous density of Percoll solution (Solarbio, Beijing, China). After centrifugation at 300 × g for 30 min at 4 ◦ C, the interface fraction was collected and washed three times with PBS. The cells were examined under a microscope to confirm lymphocyte identity (Supplementary data Figure 2B). The lymphocytes were resuspended in L-15, and the viability of the cells was examined by trypan blue dye exclusion method. The cells were adjusted to 5 × 105 viable cells/ml in L-15 and distributed into 96-well tissue culture plates. All cell cultures were conducted at 22 ◦ C. 2.8. Chemotaxis assay Chemotaxis assay was carried out in 24-well Costar Transwell (Corning Costar Co., Cambridge, MA, USA) according to the method of Montero et al. (2008). Briefly, purified wild type and mutant SmCXCL8 and Trx-tag were diluted in RPMI 1640 medium to various concentrations, and 0.6 ml of each dilution was applied to a lower chamber of Transwell. The upper chamber containing a polycarbonate membrane of 3-␮m pore size was placed on top of the lower chamber. One hundred microliters of target cells (105 ) were added to the upper chamber, and the plate was incubated at 22 ◦ C for 40 min. The number of cells migrated into the lower chamber was counted with a microscope. To distinguish between chemotaxis and chemokinesis, the above assay was also performed with the same concentration of rSmCXCL8 present in both the upper and lower chambers of Transwell. Each assay was performed independently for three times. 2.9. Cell proliferation PBL, HK lymphocytes, or HK macrophages prepared above were added to a 96-well culture plate (∼5 × 105 cells/well). One hundred microliters of RPMI 1640 (control) or rSmCXCL8 diluted in RPMI 1640 to various concentrations was added to the plate. The plate was incubated at 22 ◦ C for 3 d and added with 20 ␮l of 5 mg/ml MTT {3-(4,5)-dimethylthiahiazo (-z-yl)3,5-di-phenytetrazoliumromide} (Sangon, Shanghai, China). After incubation at 22 ◦ C for 4 h, 200 ␮l dimethyl sulfoxide was added to the plate to dissolve the reduced formazan. The plate was then read at 540 nm with a microplate reader. Results were expressed as proliferation index, which was calculated as follows: (A540 of rSmCXCL8-treated cells − A540 of control cells)/A540 of control cells. The assay was performed independently for three times. 2.10. Effect of rSmCXCL8 on bacterial infection E. tarda TX1 was cultured to mid-logarithmic phase, washed, and resuspended in PBS to 5 × 106 CFU/ml. HK lymphocytes prepared above in a 96-well culture plate (105 cells/well) were washed with PBS; the cells were added with rSmCXCL8 (100 ng/ml) or Trx-tag (100 ng/ml) (control) and incubated at 22 ◦ C for 0.5 h. After the incubation, the cells were mixed with E. tarda (100 ␮l/well). The plate was incubated at 22 ◦ C for 2 h and washed three times with PBS. The cells were lysed by adding 50 ␮l of 0.2% Tween 20 to each well. The lysate was plated on LB agar plates. The plates were incubated

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Fig. 1. Alignment of the deduced amino acid sequences of CXCL8 homologues. Dots denote gaps introduced for maximum matching. The residues that are conserved among all CXCL8 homologues are in black; the residues that are conserved among fish CXCL8 are in grey; the ELR motif was doubly underlined. Numbers in brackets indicate overall sequence identities between SmCXCL8 and the compared CXCL8 sequences.

at 28 ◦ C for 32 h, and the colonies that emerged on the plates were counted. The assay was performed three times independently. 2.11. qRT-PCR analysis of SmCXCL8, TNF-˛, and SOCS3 expression in HK lymphocytes To examine the effect of SmCXCL8 on the expression of TNF-˛ and SOCS3, rSmCXCL8 (100 ng/ml) or Trx-tag (100 ng/ml) (control) was added to HK lymphocytes in a 96-well culture plate as described above. The plate was incubated at 22 ◦ C for 1 h, 2 h, 4 h, and 8 h respectively. Total RNA was prepared from the cells with Total RNA Kit I of Omega Bio-tek (Norcross, GA, USA) and used for qRT-PCR analysis of TNF-˛ and SOCS3 expression as described above. To examine the effect of TNF-␣ on SmCXCL8 expression, recombinant turbot TNF-␣ was prepared as described previously (Zhang et al., 2011) and added to HK lymphocyte culture to 100 ng/ml. Cells were collected at various hours and determined for SmCXCL8 expression by qRT-PCR as described above.

residue in length with a predicted molecular mass of 11.0 kDa and a theoretical isoelectric point of 8.21. SmCXCL8 is predicted to localize in the extracellular and contains a putative signal peptide formed by residues 1–23. In silico analysis identified in SmCXCL8 a CXC chemokine domain (residues 32–93) that contains the conserved CXC motif in the form of 35 CRC37 , which is preceded by the tripeptide sequence EMH. In addition to the cysteine residues that form the CXC motif, two more cysteine residues (C61 and C78) that are known to be essential to the tertiary structure of CXCL8 (Rajarathnam et al., 1999) were also found in SmCXCL8. BLAST analysis indicated that SmCXCL8 shares 52–83% overall sequence identities with the lineage 1 CXCL8 of a number of teleosts, including Latris lineate, Lateolabrax japonicus, Dicentrarchus labrax, Anoplopoma fimbria, Paralichthys olivaceus, and M. aeglefinus (GenBank accession nos: ACQ99511, ACK57558, CAM32186, ACQ57874, BAB86884, and CAD97422, respectively). In contrast, SmCXCL8 shares 37% overall sequence identity with human CXCL8 and only 27% overall sequence identity with the lineage 2 CXCL8 of carp and zebrafish (Fig. 1).

2.12. Statistical analysis 3.2. Expression of SmCXCL8 under different conditions All statistical analyses were performed with SPSS 15.0 software (SPSS Inc., Chicago, IL, USA). Data were analyzed with one-way analysis of variance (ANOVA), and statistical significance was defined as P < 0.05. 3. Results 3.1. Characterization of the sequence of SmCXCL8 The full-length cDNA of SmCXCL8 is 913 bp, which contains a 5 untranslated region (UTR) of 198 bp, an open reading frame (ORF) of 300 bp, and a 3 -UTR of 415 bp with a poly-A tail formed by 29 adenines (Supplementary data Figure 3). A putative polyadenylation signal, ATTAAA, was present at 17 bp upstream the poly-A sequence. The deduced amino acid sequence of SmCXCL8 is 99-

3.2.1. SmCXCL8 expression under normal physiological conditions qRT-PCR was carried out to determine the expression levels of SmCXCL8 in the gill, blood, spleen, muscle, kidney, brain, liver, and heart of turbot under normal physiological conditions. The results showed that SmCXCL8 mRNA was detected in all the examined tissues, with relatively low levels found in brain, heart, blood, and muscle and high levels (more than 10-fold of that in brain) found in gill, liver, kidney, and spleen (Fig. 2A). 3.2.2. SmCXCL8 expression after microbial challenge Experimental challenge with the bacterial pathogen E. tarda caused significant induction of SmCXCL8 expression in kidney at 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h post-challenge, with maximum induction occurring at 72 h post-challenge (Fig. 2B). In

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Fig. 2. SmCXCL8 expression under different conditions as determined by quantitative real time RT-PCR. (A) SmCXCL8 expression in turbot tissues under normal physiological conditions; the expression level in brain was set as 1. (B) SmCXCL8 expression in turbot kidney in response to Edwardsiella tarda, poly(I:C) or PBS challenge. (C) SmCXCL8 expression in turbot kidney in response to vaccination with Eta21 or PBS. For panels (B and C) significances between challenged/vaccinated fish and control fish are indicated with asterisks. Vertical bars represent means ± SE (N = 4). * P < 0.05; ** P < 0.01.

contrast, following poly(I:C) challenge, significant SmCXCL8 induction was observed only at 12 h, 24 h, and 48 h post-challenge and to extents that were much lower than those caused by E. tarda challenge. 3.2.3. SmCXCL8 expression following vaccination To examine whether SmCXCL8 expression was affected by antigen stimulation, turbot were vaccinated with an E. tarda subunit vaccine Eta21, which is known to be protective against E. tarda infection (Jiao et al., 2009). qRT-PCR analysis showed that vaccination with Eta21 caused significant induction of SmCXCL8 expression at 4 h to 2 d post-vaccination, with maximum induction occurring at 12 h post-vaccination (Fig. 2C). 3.3. Chemotactic activity of purified recombinant SmCXCL8 (rSmCXCL8) 3.3.1. Chemotactic activity of wild type rSmCXCL8 rSmCXCL8 was purified from E. coli as a recombinant protein fused with a thioredoxin tag (Trx-tag). SDS-PAGE analysis indicated that the purified protein appeared as a single band with a molecular mass matching that predicted for the fusion protein (27 kDa) (Supplementary data Figure 4). In addition, the Trx-tag was also purified from the control vector as a recombinant protein of 20.4 kDa (Supplementary data Figure 4). Chemotactic analysis

Fig. 3. Chemotactic activity of SmCXCL8. Migration of peripheral blood neutrophils (A), head kidney macrophages (B), and lymphocytes (C) induced by different concentrations of rSmCXCL8 was determined using Transwell. Data are presented as means ± SE (N = 3). * P < 0.05; ** P < 0.01.

indicated that rSmCXCL8, but not the purified Trx-tag, induced migration of neutrophils, HK lymphocytes, and HK macrophages in a dose-dependent fashion (Fig. 3). The chemotactic effect of rSmCXCL8 on neutrophils was more pronounced than those on HK lymphocytes and macrophages. When rSmCXCL8 was added to both the upper and lower chambers of Transwell, migration of the cells was significantly (P < 0.01) inhibited, suggesting that migration was due to rSmCXCL8-induced chemotaxis rather than chemokinesis.

3.3.2. Chemotactic activity of mutant rSmCXCL8 bearing mutations at the EMH motif In order to examine the potential importance of the tripeptide sequence immediately before the CXC motif in SmCXCL8, the 32 EMH34 sequence was mutated to AAA or ELR, and the mutant protein was named SmCXCL8M1 or SmCXCL8M2. The results showed that compared to the wild type, SmCXCL8M1 was dramatically reduced in chemotactic activity (Fig. 3A). In contrast, the chemotactic activity exhibited by SmCXCL8M2 was comparable to that of the wild type protein (data not known).

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Fig. 4. Effect of SmCXCL8 on cell proliferation. Peripheral blood leukocytes (PBL) and head kidney macrophages and lymphocytes were treated with or without different concentration of rSmCXCL8, and proliferation of the cells was determined by MTT assay. Data are presented as means ± SE (N = 3). Significances between rSmCXCL8trerated and untreated cells are indicated with asterisks. * P < 0.05; ** P < 0.01.

3.4. Effect of rSmCXCL8 on the proliferation of PBL, HK lymphocytes, and HK macrophages MTT assay indicated that rSmCXCL8 stimulated the proliferation of PBL, HK lymphocytes, and HK macrophages in a manner that depended on the concentration of rSmCXCL8, with significant stimulations observed when rSmCXCL8 reached 10 ng and 100 ng (Fig. 4). In contrast, purified Trx-tag exhibited no effect on the proliferation of these cells. 3.5. Effect of rSmCXCL8 on intracellular survival of E. tarda To examine whether SmCXCL8 had any effect on bacterial infection, HK lymphocytes pre-treated with or without rSmCXCL8 were infected with E. tarda, and the number of bacterial cells recovered from lymphocytes was determined. The results showed that the intracellular bacterial recovery from rSmCXCL8-treated lymphocytes (736 ± 95 CFU) was significantly (P < 0.05) lower than that from the untreated cells (1820 ± 141 CFU) 3.6. Effect of SmCXCL8 on the expression of TNF-˛ and SOCS3 qRT-PCR analysis showed that when rSmCXCL8 was added to cultured primary HK lymphocytes, it induced significant expression of TNF-˛ and SOCS3 (Fig. 5A and B). However, the induction patterns of TNF-˛ and SOCS3 were different. While maximum induction of TNF-˛ occurred at early hour (1 h) post-rSmCXCL8 treatment, peak level of SOCS3 induction was observed at late hour (8 h) postrSmCXCL8 treatment. Treatment of HK lymphocytes with TNF-␣ induced significant SmCXCL8 expression that increased with time from 1 h to 8 h post-treatment (Fig. 5C). 4. Discussion In this study, we cloned and analyzed a CXCL8 orthologue, SmCXCL8, from turbot. Sequence analysis showed that SmCXCL8 is closely related to the lineage 1 CXCL8 of a number of fish species but remotely related to the lineage 2 CXCL8 of carp, suggesting that SmCXCL8 is a member of the lineage 1 CXCL8. SmCXCL8 possesses the characteristic CXC signature motif of CXC chemokines and, as with most known fish CXCL8 which lack an intact ELR motif, exhibits an EMH, rather than ELR, tripeptide sequence before the CXC motif. In mammalian CXCL8, the ELR motif constitutes the receptor binding site and is required for angiogenesis (Clark-Lewis et al., 1991; Gerber et al., 2000; Hebert et al., 1991; Rosenkilde and Schwartz, 2004). However, it is not clear as to the potential

Fig. 5. Cross regulation of SmCXCL8, TNF-␣, and SOCS3. In (A) and (B), head kidney lymphocytes were treated with or without (control) rSmCXCL8, and TNF-˛ and SOCS3 expression was determined by quantitative real time RT-PCR at various time points post-treatment. In (C), head kidney lymphocytes were treated with or without (control) TNF-␣, and SmCXCL8 expression was determined as in (A) and (B). Data are presented as means ± SE (N = 3). * P < 0.05; ** P < 0.01.

role of the ELR-corresponding region in fish CXCL8. In a recent study of black sea bream CXCL8, it was found that replacement of the ELH sequence before the CXC motif with ELR does not change the biological property of the protein (Cai et al., 2009). In our study, we found that mutation of the EMH sequence to AAA caused severe impairment in the chemotactic capacity of SmCXCL8, suggesting that EMH is required for the optimal activity of SmCXCL8. In contrast, substitution of the EMH motif with ELR had no impact on the biological activity of SmCXCL8, probably because ELR is biochemically similar to EMH. These results suggest the possibility that the immediate region before the CXC motif in SmCXCL8 may be involved in receptor interaction as in the case of human CXCL8. In mammals, ELR-positive CXC chemokines differ in function and target cells from ELR-negative ones. Human CXCL8, which possesses an ELR motif, is known to induce chemotaxis in neutrophils, basophils, and T lymphocytes (Laing and Secombes,

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2004). In fish, purified recombinant rainbow trout CXCL8 has been shown to cause migration of HK neutrophils as well as macrophages and lymphocytes (Harun et al., 2008); likewise, both lineage 1 and lineage 2 carp CXCL8, when in the form of purified recombinant proteins, were chemoattractive to HK phagocytes (van der Aa et al., 2010). Similar to these studies, in our study, we found that rSmCXCL8 was a potent chemotactic mediator that induced migration of peripheral blood neutrophils and, to less extents, HK lymphocytes and macrophages. The relatively weak chemotactic effect of rSmCXCL8 on HK macrophages and lymphocytes suggests a possible weak interaction between rSmCXCL8 and the cognate receptors on these cells. In addition, our study also showed that SmCXCL8 stimulated the proliferation of PBL, HK lymphocytes, and HK macrophages and promoted cellular resistance against bacterial infection. These results suggest that treatment with SmCXCL8 probably activates the target cells and leads to enhanced immune defense against bacterial infection. Gene expression studies have indicated that in a number of teleost fish, CXCL8 expression occurs in a wide arrange of tissues and responds positively to inflammatory stimulations and microbial challenge (Chen et al., 2005; Lee et al., 2001; Oehlers et al., 2010; Seppola et al., 2008; van der Aa et al., 2010). In our study, SmCXCL8 expression was detected in high levels in immune organs under normal physiological conditions and was significantly upregulated following E. tarda and poly(I:C) challenges. It is interesting that the induction effect of E. tarda is much stronger than that of poly(I:C), suggesting that SmCXCL8 is more responsive to bacterial infection. In line with these observations, vaccination of turbot with an E. tarda protein antigen caused significant increase in SmCXCL8 expression, which suggests the possibility that SmCXCL8 induction may constitute part of the protective immunity induced by the subunit vaccine. It is known that in mammals, CXCL8 expression is regulated by, in addition to microbial pathogens, various inflammatory cytokines (Larsen et al., 1989; Strieter et al., 1989; Vaddi et al., 1997). No similar observation has been reported in fish, however, the rainbow trout IL-8 has been shown to stimulate the expression of IL-8, IL-1␤, and TNF-␣ in a monocyte–macrophage like cell line (Montero et al., 2008). In our study, we found that recombinant SmCXCL8 caused significant induction of TNF-˛ and SOCS3 expression at early and late hours post-treatment respectively. On the other hand, TNF-␣ treatment also upregulated SmCXCL8 expression in a time-dependent manner. These results indicate a positive inter-regulation between SmCXCL8 and TNF-␣. Since TNF-␣ is known to stimulate SOCS3 expression in turbot (Zhang et al., 2011), we do not know whether SmCXCL8 induced SOCS3 expression directly or indirectly via TNF-␣. In either case, since SOCS3 is an inhibitor of cytokine signaling, induction of SOCS3 in later hours post-treatment is probably a protective mechanism of the host to prevent over-activation of the cells. In conclusion, the results of this study indicate that SmCXCL8 is a potent neutrophil chemoattractant that requires the EMH sequence before the CXC motif for full activity. Expression of SmCXCL8 is high in immune organs and responds positively to microbial challenge and antigen stimulation. Purified recombinant SmCXCL8 exhibits multiple effects, including stimulating the proliferation of target immune cells, enhancing cellular resistance against intracellular bacterial survival, and modulating TNF-˛ and SOCS3 expression. These results support a role for SmCXCL8 as an inflammatory cytokine. Acknowledgements This work was supported by the grants from the National Natural Science Foundation of China (31025030) and the Knowledge

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