Cloning, promoter analysis and expression in response to bacterial exposure of sea bass (Dicentrarchus labrax L.) interleukin-12 p40 and p35 subunits

Molecular Immunology 44 (2007) 2277–2291

Cloning, promoter analysis and expression in response to bacterial exposure of sea bass (Dicentrarchus labrax L.) interleukin-12 p40 and p35 subunits Diana S. Nascimento a,b,∗ , Ana do Vale a , Ana M. Tom´as a,c , Jun Zou b , Christopher J. Secombes b , Nuno M.S. dos Santos a a

IBMC–Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal b Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, UK c Abel Salazar Institute for Biomedical Research, University of Porto, Porto, Portugal Received 12 May 2006; received in revised form 27 October 2006; accepted 6 November 2006 Available online 29 December 2006

Abstract Interleukin-12 (IL-12) is a heterodimeric cytokine pivotal in resistance to microbial and viral infections. In the search for immunoregulatory genes in sea bass the genes for the two IL-12 subunits p40 and p35 were cloned and sequenced. Molecular characterization of these two genes was performed at both the cDNA and genomic levels. Sea bass IL-12 p40 and p35 conserve most cysteines involved in the intra-chain disulfide bonds of human IL-12 subunits as well as the important structural residues for human IL-12 heterodimerization. The gene organization of sea bass IL-12 p40 is similar to the human orthologue, whilst the sea bass IL-12 p35 gene structure, as reported for pufferfish, differs from the human one in containing an additional exon and lacking a second copy of a duplicated exon present in the mammalian genes. The promoter analysis of both sea bass and pufferfish IL-12 genes showed the presence of the main cis-acting elements involved in the transcriptional regulation of human and mouse orthologues. The involvement of IL-12 in sea bass anti-bacterial immune responses was demonstrated by investigating the expression profiles of IL-1␤, IL-12 p40 and p35 in the head-kidney and spleen following intraperitoneal injection of UV-killed and live Photobacterium damselae ssp. piscicida (Phdp). Finally, the importance of nuclear factor (NF)-␬B on UV-killed Phdp-induced IL-12 p40 and p35 gene transcription was shown by the use of pyrrolidine dithiocarbamate (PDTC). © 2006 Elsevier Ltd. All rights reserved. Keywords: Sea bass; Dicentrarchus labrax; IL-12; IL-12 p35; IL-12 p40; Cytokines; Gene expression; Promoter

1. Introduction Interleukin-12 (IL-12) is a key pro-inflammatory cytokine that bridges both innate and adaptive immunity. In mammals, the central IL-12-producing cells are activated phagocytes and dendritic cells but B cells and accessory cells are also capable of producing this cytokine (reviewed by Ma and Trinchieri, 2001; Trinchieri, 2003). IL-12 acts mostly on natural killer (NK) and T cells by binding to a receptor composed of two chains: IL-12 receptor (IL-12R)␤1 and IL-12R␤2 (Presky et al., 1996). The essential pro-inflammatory role of IL-12 is related to the efficient

∗

Corresponding author. Tel.: +351 226074900; fax: +351 226099157. E-mail addresses: [email protected], [email protected] (D.S. Nascimento). 0161-5890/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2006.11.006

induction of interferon (IFN)-␥ production in these cells (Chan et al., 1992; Kubin et al., 1994; Murphy et al., 1994). IL-12 can also enhance the proliferation and cytolytic activity of NK cells and T cells and promotes T-helper type 1 cell (Th1) differentiation, thus driving cell-mediated immunity (reviewed by Trinchieri, 2003). IL-12 is active only as a heterodimer composed of two covalently linked subunits: a 40 kDa chain known as IL-12 p40 (or IL-12␤) and a 35 kDa chain called IL-12 p35 (or IL-12␣) (Kobayashi et al., 1989). The IL-12 p35 primary sequence resembles other class I helical cytokines, such as IL-6 and ganulocyte colony-stimulating factor (G-CSF), whereas IL-12 p40 has homology to the extracellular domains of the hematopoietic cytokine receptor family members, particularly the IL-6 receptor ␣-chain (IL-6R␣) and the ciliary neurotrophic factor receptor (CNTFR) (Merberg et al., 1992). Recently, other

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heterodimeric cytokines have been identified with a structure similar to that of IL-12 (Oppmann et al., 2000; Pflanz et al., 2002), as is the case of IL-23, which is a heterodimeric cytokine composed of the p40 subunit of IL-12 covalently linked to p19, an IL-12 p35-related molecule (Oppmann et al., 2000). IL-12 p40 and p35 subunits must be co-expressed in the same cell to generate the bioactive IL-12 (Gubler et al., 1991), even though the encoding genes of these molecules are located in different chromosomes (Sieburth et al., 1992; Tone et al., 1996) resulting in an independent regulation of transcription (Liu et al., 2005). In mammals, IL-12 p40 expression is restricted to cells that produce the bioactive IL-12 while IL-12 p35 is more ubiquitously expressed (D’Andrea et al., 1992; Schoenhaut et al., 1992). Diverse microbial and viral pathogens and molecules can stimulate IL-12 production (Romani et al., 1997). The sequences of IL-12 p35 and p40 promoter regions have diverse transcription factor binding sites (Ma et al., 1996; Murphy et al., 1995). Indeed, the molecular mechanisms involving the IFN-␥ and LPS priming of IL-12 p40 and p35 gene expression have been studied in detail (reviewed by Liu et al., 2005). Nuclear factor (NF)-␬B (Kollet and Petro, 2006; Murphy et al., 1995), several interferon regulatory factor (IRF) family members (Kollet and Petro, 2006; Maruyama et al., 2003; Masumi et al., 2002) and interferon consensus binding protein (ICSBP) (Masumi et al., 2002; Wang et al., 2000) are among the nuclear factors involved in the complex regulation of these genes. Recently, the IL-12 p40 and p35 subunits have been characterized in two fish species, pufferfish (Yoshiura et al., 2003) and carp (Huising et al., 2006), showing that IL-12 is conserved across vertebrates. Interestingly, two other IL-12 p40-like genes were identified in carp suggesting an expansion of the vertebrate heterodimeric cytokine family (Huising et al., 2006). In this paper, the IL-12 p40 and p35 subunits and their respective promoters were characterized in sea bass, an important species for the Mediterranean fish-farming industry. Moreover, the in vivo mRNA expression kinetics of these genes was determined in the head-kidney and spleen during a pro-inflammatory response following the intraperitoneal (ip) injection of UV-killed Photobacterium damselae ssp. piscicida (Phdp) strain PP3, as well as during a septicaemic infection with that pathogen, to investigate the role of IL-12 in sea bass anti-bacterial immune responses. An important role of NF-␬B in the transcriptional activation of sea bass IL-12 p40 and p35 genes by UV-killed bacteria was also demonstrated. 2. Materials and methods 2.1. Fish Sea bass, Dicentrarchus labrax, were kept in a recirculating, UV-treated salt-water (30–35%) system at 20 ± 1 ◦ C, and fed at a ratio of 2% body weight per day. For collection of organs fish were killed after being anaesthetised in 2-phenoxyethanol (Panreac).

2.2. cDNA cloning and sequencing Cloning of sea bass IL-12 (sbIL-12) p40 subunit was as follows. Survivors from a nodavirus vaccination trial were re-infected ip with live nodavirus (107 tissue culture infectious dose infecting 50% of inoculated cultures—TCID50). At the third day post-challenge head-kidneys from three fish were collected and total RNA was isolated by guanidinium thiocyanate–phenol–chloroform extraction (Chomczynski and Sacchi, 1987). A SuperscriptTM II first-strand synthesis system (Invitrogen) was used to synthesize first-strand cDNA with the primer APv (Fig. 1). Degenerate primers were designed based on conserved regions from the multiple alignment (CLUSTAL W) (Higgins, 1994) of fish IL-12 p40 amino acid sequences available in GenBank. cDNA was amplified by two successive polymerase chain reaction (PCR) rounds with primers IL12BFW1/IL12BRV1 (Fig. 1). The reaction was adjusted to a final volume of 50 ␮l containing 200 ␮M of each dNTP, 1.5 mM MgCl2 , 5 ␮L of 10× PCR buffer (Invitrogen), 0.4 ␮M of each primer, 2 ␮L of cDNA and 1.25 U of Taq polymerase (Invitrogen). Cycling conditions were: 94 ◦ C for 2 min; 30 cycles of 94 ◦ C for 45 s, 45 ◦ C for 1 min, 72 ◦ C for 2 min; and 72 ◦ C for 10 min. A semi-nested PCR was then performed as described above with primers IL12BFW1/IL12BRV2 (Fig. 1) using an annealing temperature of 48 ◦ C, and extension time of 30 s. A band of ∼370 bp was purified (QIAIXII, Qiagen) and reamplified using the same conditions. The final PCR product was purified and sequenced using primers IL12BFW1 and IL12BRV2 (Fig. 1) according to Big Dye terminator 2.0 (Applied Biosystems) in an automated sequencer. The 3 untranslated region (UTR) of the sbIL-12 p40 gene was cloned by rapid amplification of cDNA ends (RACE) PCR. Briefly, cDNA was amplified with degenerate primer IL12BFW1 and APv (Fig. 1) using the same PCR conditions as above, followed by a nested PCR with the primers DLIL12BFW1/AUAP (Fig. 1). Cycling conditions were: 94 ◦ C for 2 min; 30 cycles of 94 ◦ C for 45 s, 52 ◦ C for 1 min, 72 ◦ C for 45 s; and 72 ◦ C for 5 min. The PCR band of ∼750 bp was purified (QIAquick Gel extraction kit, Qiagen), ligated and cloned into E. coli XL1-blue cells following the pGEM-T easy Vector System (Promega) specifications. Positive transformants were identified by blue-white colour selection in 100 ␮g/mL ampicilin (Sigma) Luria Broth Base plates. Plasmid DNA was isolated (QIAprep Spin Miniprep kit, Qiagen) and sequenced using vector primers T7 and SP6 (Fig. 1) as above. Specific primers, DLIL12BRV1 and DLIL12BRV2 (Fig. 1), were designed based on the obtained sequence and used to amplify the 5 UTR by 5 RACE-PCR. The PCR band of ∼480 bp was purified, cloned and sequenced as above. This fragment did not reach the N-terminal methionine, so the same procedure was repeated using primers DLIL12BRV1 and DLIL12BRV5 (Fig. 1). The primer combination DLIL12BFW4/DLIL12BRV6 (Fig. 1) was used to amplify the full-length sbIL-12 p40 cDNA. The reaction was adjusted to a final volume of 50 ␮l containing 200 ␮M of each dNTP, 1.5 mM MgCl2 , 5 ␮L of 10× PCR buffer (Bioline), 0.4 ␮M of each primer, 2 ␮L of template and 1.25 U of Taq polymerase (Bioline) and 0.15 U of Pfu (Promega). Cycling conditions were: 94 ◦ C for 2 min; 30 cycles of 94 ◦ C

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Fig. 1. Schematic representation of the primer sites and their sequences used in this work to isolate sbIL-12 p40 and p35 genes. Letter nucleotide code: D = A, G, T; H = A, C, T; R = A, G; Y = C, T; S = G, C; W = A, T; N = A, T, C, G; I = deoxyinosine. Universal and degenerate primers used are shown below the figure and in parentheses are the sea bass nucleotide sequences corresponding to the region where the degenerate primers were designed.

for 30 s, 55 ◦ C for 1 min, 72 ◦ C for 1.5 min; and 72 ◦ C for 5 min. The PCR product was purified, cloned and three independent clones were sequenced at MWG (http://www.mwg-biotech. com/). Cloning of sbIL-12 p35 was as follows. Degenerate primers were designed based on conserved regions from the multiple alignment (CLUSTAL W) of SCHIP-1 amino acid sequences, a gene known to be in tandem with human and pufferfish IL-12 (pfIL-12) p35 (Yoshiura et al., 2003). Genomic DNA was amplified by PCR with primers SCHIPFW2/SCHIPRV1 (Fig. 1) as described above except for the annealing temperature (50 ◦ C) and extension time (20 s). A semi-nested PCR, followed by a reamplification, were performed using SCHIPFW2/SCHIPRV2 with the same conditions. The obtained product of ∼80 bp was purified, cloned and sequenced as previously described. Sea bass SCHIP-1 specific primers were designed based on the obtained sequence (Fig. 1) and used to walk downstream of the SCHIP-1 gene within the sea bass genome in order to isolate sbIL-12 p35.

Four genome walker libraries were constructed according to the GenomeWalker Kit (BD Biosciences Clontech). Two genomewalking experiments were needed to reach the IL-12 p35 gene. Briefly, first round PCR was performed using the adaptor primer 1 (AP1) and the gene specific primer, DLSCHIP1FW1 (for the first genome walking experiment) or DLSCHIP1FW3 (for the second genome walking experiment) (Fig. 1). Nested PCR was carried out with the adaptor primer 2 (AP2) and the gene-specific primer, DLSCHIP1FW2 (for the first genome walking experiment) or DLSCHIP1FW4 (for the second genome walking experiment) (Fig. 1). PCR reactions were carried out according to the manufacturer’s instructions. Specific primers were designed based on the obtained sequence (Fig. 1) and used to amplify the full-length cDNA in a PCR with DLIL12aFW4 and APv, followed by a semi-nested PCR with DLIL12aFW5 and Apv, using the same PCR conditions as above. The PCR product was purified, cloned and four independent clones sequenced at MWG (http://www.mwg-biotech.com/).

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2.3. Genomic DNA cloning and sequencing Genomic DNA was isolated from sea bass erythrocytes as described by Stet et al. (1993). To obtain the full sbIL-12 p40 gene the DNA was amplified by PCR with the following primer combinations: DLIL2BFW3/DLIL12BRV5, DLIL12BFW3/DLIL12BRV1, DLIL12BFW4/DLIL12BRV2, DLIL12BFW1/DLIL12BRV4, DLIL12BFW1/DIL12BRV6 (Fig. 1). Wax-mediated hot start PCR was performed in a 50 ␮l PCR reaction (200 ␮M of each dNTP, 2 mM MgCl2 , 5 ␮L 10× PCR buffer (Bioline), 0.4 ␮M of each primer and 1.25 U of Taq DNA polymerase (Bioline) and 0.15 U of Pfu DNA polymerase (Promega)). The cycling conditions were: 94 ◦ C for 2 min; 30 cycles of 94 ◦ C for 30 s, 55 ◦ C for 1 min, 72 ◦ C for 1.5 min; and 72 ◦ C for 5 min. The obtained PCR product was cloned as previously described and two clones for each product were sequenced at MWG (http://www.mwg-biotech.com/). To obtain the full sbIL-12 p35 gene, DNA was amplified by PCR using primers DLIL12AFW6/DLIL12ARV3 as described above. The PCR product was purified, cloned and four independent clones were sequenced at MWG (http://www.mwgbiotech.com/). 2.4. Sea bass IL-12 p40 and p35 promoter sequencing The sbIL-12 p40 promoter region was achieved by genomewalking as detailed above. Two genome-walking experiments were performed to clone the IL-12 p40 gene. Briefly, first round PCR was performed using AP1 and the gene specific primer, DLIL12BRV7 (for the first genome walking experiment) or DLIL12BRV10 (for the second genome walking experiment) (Fig. 1). Nested PCR was carried out with AP2 and the genespecific primer, DLIL12BRV8 (for the first genome walking experiment) or DLIL12BRV11 (for the second genome walking experiment) (Fig. 1). PCR reactions were carried out according to the manufacturer’s instructions. Four independent clones were sequenced at http://www.mwg-biotech.com/. The sbIL-12 p35 promoter was included in the DNA fragment cloned in the second IL-12 p35 genome-walking experiment described above. This PCR product was cloned and three independent clones were sequenced at http://www.mwgbiotech.com/. 2.5. Expression analysis by RT-PCR Sea bass (10 g) were injected ip with a 100 ␮L suspension of 3 × 107 UV-killed Phdp (Japanese strain PP3) in tryptic soy broth containing 1% NaCl (TSB-1). Control fish received 100 ␮l of TSB-1. Head-kidneys and spleens were collected at 3, 6 and 24 h after injection. Pyrrolidine dithiocarbamate (PDTC) was used to inhibit NF-␬B (Schreck et al., 1992). Animals in the UV-killed PP3 plus PDTC (Sigma) group were injected ip with 200 mg PDTC per kg body weight in 100 ␮l inocula 1 h before administration of the UV-killed bacteria (same dose as above) and organs were collected 3 h after the challenge. Control animals were injected with PDTC, as above, without inoculat-

ing with UV-killed bacteria. The samples were processed as independent pools of tissue from three fish. Total RNA was extracted according to the VersageneTM RNA tissue kit (Gentra Systems) protocol. RNA was reverse transcribed following the instructions of the BioScript RNase H (Bioline) protocol. The cDNAs were differentially diluted in order to obtain normalised ␤-actin detectable and non-saturated PCR products. PCR primers used were DL␤actinFW/DL␤actinRV for ␤-actin, DLIL1BFW/DLIL1BRV for IL-1␤ (used as a positive control in this experiments), DLIL12BFW6/RV4 for IL-12 p40 and DLIL12AFW7/RV5 for IL-12 p35 (Fig. 1). ␤-Actin PCRs were performed with 2 ␮L of cDNA with the following cycle conditions: 94 ◦ C for 2 min; 30 cycles of 94 ◦ C for 30 s, 65 ◦ C for 1 min, 72 ◦ C for 30 s; and 72 ◦ C for 5 min. The other transcripts were amplified using 2 ␮L of cDNA with the following PCR cycling conditions: 94 ◦ C for 2 min; 35 cycles of 94 ◦ C for 30 s, 59 ◦ C for 1 min, 72 ◦ C for 1 min; and 72 ◦ C for 5 min. PCR products were separated in a 1% agarose gel, stained by soaking with ethidium bromide and analysed by densitometry using the ImageQuant® Molecular Dynamics® . The densitometry results for each pool were normalized (dividing by the respective ␤actin value). The average values for each time point are shown as a bar graph. Sea bass (10 g) were infected by ip injection of 4.5 × 103 colony forming units/fish of Phdp strain PP3 in 100 ␮l of TSB-1. This inoculum was found to kill 10 g sea bass within 48 h. Control fish received 100 ␮l of TSB-1. Head-kidneys and spleens were collected at 3, 6 and 24 h after injection. The samples were processed as pools of three organs. Total RNA was extracted according to the NucleoSpin® RNA II Kit (Macherey-Nagel) and the VersageneTM RNA tissue kit (Gentra Systems) protocol. RNA was reverse transcribed following the instructions of the BioScript RNase H Minus kit (Bioline) protocol. The same procedure as above was followed to normalise the cDNAs and amplify IL-1␤, IL-12 p40 and IL-12 p35 transcripts. PCR products were separated, stained and analysed as above. 2.6. Sequence analysis The IL-12 p40 and p35 amino acid sequences were deduced using the Expasy translate tool (http://www.expasy.org/). Full nucleotide and protein sequences from sbIL-12 p40 and p35 subunits were compared to their counterpart sequences currently available in GenBank, retrieved using the BLAST program (http://www.ncbi.nlm.nih.gov/). A multiple alignment was made using CLUSTAL W (Higgins, 1994) and formatted with Bioedit programs (Hall, 1999). Putative domains and possible N-glycosylation sites were identified by PROSITE (http://www.expasy.org/prosite) (Falquet et al., 2002) and the InterProScan (Zdobnov and Apweiler, 2001). Signal peptide prediction was performed using SignalP 3.0 (Bendtsen et al., 2004) and protein molecular weights were calculated by Expasy’s compute pI/Mw tool (http://www.expasy.org/). Neighbour-joining phylogenetic trees were constructed using the MEGA 3 Program (Kumar et al., 2004), with p-distance parameter and complete deletion of gaps. The phylogenetic trees were tested for reliability using 2000 bootstrap replications. The percent-

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ages of similarity and identity were calculated by the MatGat (Campanella et al., 2003) program using default parameters. The 5 flanking regions (promoter region, exon I and intron I) of the sea bass and pfIL-12 genes were searched for transcription factor binding motifs deposited in the TFD and TRANSFAC databases (Prestridge, 1991). The cis-acting elements involved in the transcriptional regulation of both genes in humans and mouse were also examined using the Genamics Expression program. 3. Results 3.1. Cloning and characterization of sea bass IL-12 p40 The sbIL-12 p40 cDNA (GenBank accession no DQ388039) comprises a 182 bp 5 untranslated region (UTR), a 1050 bp open reading frame (ORF) encoding a predicted protein of 349 amino acids and a 3 UTR of 148 bp with a consensus polyadenylation signal (AATAAA) 22 bp upstream of the polyA tail (not shown). One putative mRNA destabilizing motif TATTTAT was found within the sbIL-12 p40 3 UTR. Analysis of the sbIL-12 p40 revealed an 18-aa signal-peptide (Fig. 2a) which would generate a mature protein of calculated molecular mass of 38.3 kDa encoding 15 cysteines. Sea bass C46 , C94 , C129 , C140 , C199 and C205 are conserved across all species while C188 , C283 , C286 , C290 , C317 and C325 are conserved within the fish IL-12 p40 sequences. Seven potential N-glycosylation sites were predicted for sbIL-12 p40. The N200 involved in the Nlinked sugar modification of human IL-12 (hIL-12) p40 (Yoon et al., 2000) is preserved within sea bass (N229 YS), spotted green pufferfish and chicken IL-12 p40 but absent from cyprinid and pufferfish IL-12 p40 sequences. Sea bass N91 YT and N251 NT are conserved among fish and chicken IL-12 p40 molecules. The residues D312 and E203 shown in the hIL-12 crystal structure (Yoon et al., 2000) to be involved in two important charged interactions with hIL-12 p35, are conserved in sbIL-12 p40 (D322 and E209 ). Furthermore, hIL-12 p40 forms a key hydrophobic binding pocket (Y136 , P200 , Y268 , Y314 , Y315 ) fully conserved in sea bass or substituted by other hydrophobic amino acids (Y134 , P206 , Y269 , L324 , C325 ) (Fig. 2a). SbIL-12 p40, as a member of the long hematopoietin receptor family (Merberg et al., 1992), contains an N-terminal immunoglobulin (Ig) domain and two fibronectin type-III domains (Fig. 2a). The first fibronectin domain conserves four typical cysteines (C129 , C140 , C168 , C199 ) and the second contains the well-conserved WSXWS motif. The family signature of the long hematopoietin receptor soluble ␣-chains is also partially conserved on sbIL-12 p40 (Fig. 2a). Pair-wise alignments determined that sbIL-12 p40 amino acid similarity and identity with mammalian and chicken counterparts is 47.0–49.9% and 27.6–28.2%, respectively (Table 1a). Among fish IL-12 p40, pfIL-12 p40 is the most similar to sea bass with 72.7% similarity and 57.1% identity. Regarding the three carp IL-12 p40-like sequences, which have been shown to have different expression profiles (Huising et al., 2006), the p40-like-c is the sequence with least amino acid similarity (41.8%) and identity (24.4%) with sbIL-12 p40. A phylogenetic tree was constructed using IL6-R␣, Epstein-Barr virus

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Table 1 Similarity and identity between sea bass and other species IL-12 p40 (a) and p35 (b) protein sequences Sea bass IL-12 p40

Pufferfish Spotted green pufferfish CRFA15 Zebrafish Carp p40-like a Carp p40-like b Spotted green pufferfish CRFA14 Man Sheep Rat Water buffalo Horse Mouse Pig Cat Chicken Carp p40-like c

Similarity (%)

Identity (%)

72.7 72.1 60.5 59.9 53.3 51.6 49.9 49.9 49.6 49.0 48.4 48.1 47.9 47.9 47.0 41.8

57.1 56.0 39.9 38.4 30.3 29.5 28.2 26.9 29.6 26.4 28.3 27.8 27.4 27.2 27.6 24.4

Sea bass IL-12 p35

Pufferfish Spotted green pufferfish Carp Guinea pig Man Sheep Cat Chicken Horse Mouse Rat

Similarity (%)

Identity (%)

73.4 70.9 59.3 49.3 47.5 47.1 45.9 42.9 42.3 42.0 41.9

52.0 52.8 36.7 27.4 24.6 25.0 24.1 24.3 24.3 22.7 24.3

induced protein 3 (EBI3) and CNTFR as out-groups (Fig. 3a). Pufferfish and zebrafish homologues of the carp IL-12 p40like b and c molecules were retrieved from each genome and added to the phylogenetic analyses. SbIL-12 p40 clustered with the vertebrate IL-12 p40 molecules and apart from the other hematopoietin receptor family members. SbIL-12 p40 was most related to the pufferfish and spotted green pufferfish CRFA15 molecules. The sbIL-12 p40 clustered first with the IL-12 p40like a and b sequences before clustering with the mammalian and chicken IL-12 p40 molecules, which, as proposed by Huising et al. (2006), suggests a gene duplication after the divergence between teleosts and mammals ∼450 million years ago (Kumar and Hedges, 1998). The group of IL-12 p40-like c molecules forms a cluster distinct from the other IL-12 p40 sequences. This tree topology corroborate the similarity and identity results (Table 1a), in which the carp IL-12 p40-like c showed the lowest amino acid homology with sea bass of the other vertebrate IL-12 p40 sequences. The sbIL-12 p40 gene (GenBank accession no. DQ388040) has 3225 bp and contains an eight exon–seven intron structure (Fig. 4a). Exon–intron junction positions are conserved relative to the hIL-12 p40 except for the exon VIII that in sea bass, as in the pufferfish, includes ORF. Typical ‘gt· · ·ag’

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intron splice motifs were also identified in the flanks of each intron.

exon VI) than hIL-12 p35. Typical ‘gt· · ·ag’ intron splice motifs were also identified in the flanks of each intron.

3.2. Cloning and characterization of sea bass IL-12 p35

3.3. Comparative analysis of sea bass and pufferfish IL-12 p40 and p35 promoters

The sbIL-12 p35 cDNA (GenBank accession no. DQ388037) comprises a 5 UTR of 39bp (deduced according to the TATA box position), a 600 bp ORF encoding a predicted protein of 199 amino acids, and a 3 UTR of 337 bp with a consensus polyadenylation signal (AATAAA) 17 bp upstream of the polyA tail (not shown). Three putative mRNA destabilizing motifs TATTTAT were identified within the sbIL-12 p35 3 UTR. Overlapping with the first TATTTAT was found a TTATTTATA sequence, which has been described as a fully functional mRNA destabilizing motif (Lagnado et al., 1994). Analysis of sbIL-12 p35 revealed a 27-aa signal peptide (Fig. 2b), which would generate a mature protein of calculated molecular mass of 18.9 kDa encoding seven cysteines. Sea bass C40 , C66 , C83 , C90 , C103 and C172 are conserved across all species and C143 is present in all fish IL-12 p35. According to the alignment, the human C96 that is involved in the interchain disulphide bond of the IL-12 p70 complex (Yoon et al., 2000) is not conserved in any fish IL-12 p35. Two potential N-glycosylation sites were predicted for sbIL-12 p35. N50 IT is conserved across fish IL-12 p35 sequences. Key IL-12 p35 residues, involved in the human heterodimer formation, are conserved in sbIL-12 p35 (R181 , T184 , R187 and Y191 ) as well as other relevant structural amino acids, which are identical or conservatively substituted in sbIL-12 p35 (Fig. 2b). Pair-wise alignments showed that amino acid similarity and identity between sbIL-12 p35 and its mammalian and chicken counterparts are 41.9–49.3% and 22.7–27.4%, respectively (Table 1b). Among fish IL-12 p35, pfIL-12 p35 is the most similar to sea bass with 73.4% similarity and 52.0% identity (Table 1b). IL-12 p35 has homology to other single-chain cytokines such as IL-6 (Merberg et al., 1992), thus the phylogenetic tree was constructed using IL-6 molecules as outgroup. The tree clustered the sea bass molecule with the other vertebrate IL12 p35 sequences supported by a 100% bootstrap value. Within the fish IL-12 p35 group, pufferfish and spotted green pufferfish were more closely related to sbIL-12 p35 than carp (Fig. 3b). The sbIL-12 p35 gene (GenBank accession no. DQ388037) is 2408 bp and includes seven exons and six introns, with similar exon–intron junction positions to pfIL-12 p35 (Fig. 4b). HIL-12 p35 seems to have two similar exons (exon IV and exon V) while only one is present in the sea bass (exon IV) and pufferfish IL-12 p35 gene. Sea bass and pufferfish have one extra exon (sea bass

SbIL-12 p40 and p35 5 flanking regions were cloned and sequenced and pufferfish orthologues were downloaded from the Ensembl database (http://www.ensembl.org/). The molecular mechanisms involved in the transcription of human and mouse IL-12 genes have been clarified. Although the nucleotide similarity between the promoter of sea bass and pfIL12 subunits is very low (not shown), the binding sites for the main transcription factors involved in the transcriptional regulation of both genes were identified. Putative promoter regulatory elements found within sea bass and pfIL-12 genes are summarized in Table 2 and Fig. 5. Concerning sea bass and pfIL-12 p40, no consensus sequence for the TATA-box was found in the expected region upstream of the transcription initiation site (TSS). Among the potential cisacting elements identified in sbIL-12 p40 were ten sites for the NF-IL6 (CCAAT/enhancer binding protein beta (C/EBP␤)), six interferon-␥ activation sites (GAS), four sites for activation protein-1 (AP-1), three interferon regulatory factor elements (IRF-E), three sites for the PU.1, two sites for Kruppel-like factor (KLF) and one site for the ets-family of DNA binding proteins. With one nucleotide mismatch to the consensus sequence were also found: five NF-␬B and eleven Sp-1 binding-sites. Similar sites were found within the pfIL-12 p35 promoter: eight sites for the NF-IL6, six GAS, two IRF-Es, two PU.1 sites, one site for KLF, one ets site and, with one nucleotide mismatch to the consensus sequence, four NF-␬B sites. No Sp-1 and AP-1 cis-acting elements were identified in the pfIL-12 p40 promoter. Within sea bass and pfIL-12 p35, a consensus sequence for the TATA-box was found in the expected region upstream of the TSS. Among the potential cis-acting elements identified in sbIL-12 p35 were five sites for the NF-IL6, six GAS, four sites for AP-1, one IRF-E, one site for the NF-␬B and one octamer sequence motif (Oct). Similar sites were found within the pfIL12 p35 promoter, with two sites for NF-IL6, six GAS elements and one site for AP-1. Two IRF-E and four NF-␬B sites with a single nucleotide mismatch from the consensus sequence were also found. Furthermore, eighteen Sp-1 binding sites with one nucleotide mismatch, a AP-2 binding site, a site for the PU.1 and a cAMP response element (CRE) were also present within the pfIL-12 p35 promoter. Fourteen sites for KLF were also found

Fig. 2. Multiple sequence alignment of IL-12 p40 (a) and p35 (b) amino acid sequences. The accession numbers for the sequences are: pfIL-12 p40, AB096268; spotted green pufferfish class I helical cytokine receptor number 15 (CRFA15), AY374487; zebrafish IL-12 p40, NM 001007108; carp IL-12 p40 a, CAF18555; chicken IL-12 p40, CAD91902; man IL-12 p40, P29460; pfIL-12 p35, AB096267; spotted green pfIL-12 p35, AAR25700; carp IL-12 p35, AJ580354; chicken IL-12 p35, NM 213588; man IL-12 p35, AAD16432. SbIL-12 p40 and p35 are shown in bold print. Dashes indicate gaps introduced to optimize the similarity between sequences. Identical (*) and similar amino acids (: or .) conserved among species are indicated. The predicted signal peptide is shaded in grey and boxed. Cysteines are shaded in grey and numbered according to the full-length sbIL-12 p40 and p35 amino acid sequence. Potential N-glycosylation sites are shaded in black. Open arrows indicate the cysteine residues involved in the hIL-12 inter-chain disulphide bridge. Cysteine pairs forming intra-chain disulphide bridges in hIL-12 are indicated by closed numbered arrows. D1, D2 and D3 structural domains of hIL-12 p40 are indicated below the alignment. The IL-12 p40 key cysteine residues of the fibronectin domain, and conserved residues of the long hematopoietin receptor family signature are boxed. Structurally critical (♦) and important amino acids () in hIL-12 p70 are shown above the alignments. Exons of sea bass and hIL-12 p35 are indicated above and below the alignment, respectively.

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Fig. 3. Phylogenetic tree of IL-12 p40 (a) and p35 (b) amino acid sequences. The branches were validated by bootstrap analysis from 2000 repetitions, which are represented by numbers in branch nodes. IL-6R␣ (a), CNTFR (a) and EBI (a) and IL-6 (b) were used as outgroups (dashed branches).

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Fig. 4. Schematic representation of sbIL-12 p40 (a) and p35 (b) gene structure along with human and pfIL-12 counterparts. Boxes are exons and horizontal lines are introns. Coding nucleotides and untranslated regions are differentiated by black and white boxes, respectively. Values above boxes represent the number of nucleotides. Exon numbers are indicated below boxes by roman numerals.

in the pfIL-12 p35 promoter (Fig. 5 and Table 2) in a region very rich in CA dinucleotide repeats (not shown). 3.4. Sea bass IL-12 p40 and p35 gene expression mRNA expression levels of sea bass IL-12 p40, p35 and IL1␤ were compared in the spleen and head-kidney following a UV-killed bacterial pro-inflammatory stimulus (Fig. 6a). In the spleen, the stimulus induced expression of IL-12 p40, IL-12 p35 and IL-1␤ mRNA transcripts in a time-dependent manner. Increases in IL-12 p40 and IL-1␤ mRNA expression were maximal at 3 h after stimulation, and had decreased to resting levels by 24 h. IL-12 p35 mRNA up-regulation was also detectable at 3 h, but declined to basal levels by 6 h. In the head-kidney, IL-12 p40 and IL-1␤ showed the same kinetics of expression as in the spleen although the return to basal levels did differ somewhat from pool to pool, suggesting some variability in this kinetic. In contrast to the spleen results IL-12 p35 mRNA was not upregulated in the head-kidney. The results obtained from the gel images are reinforced by the densitometry analysis graph of the normalized average values for each gene (Fig. 6a). TSB-1 did not stimulate significantly the expression of any of the genes (Fig. 6a). The potential role of NF-␬B in the regulation of the IL-12 subunits and IL-1␤ mRNA was evaluated by the use of pyrrolidine dithiocarbamate (PDTC), an NF-␬B-specific inhibitor (Schreck et al., 1992) (Fig. 6a). In the spleen, IL-12 p35 and p40 mRNA up-regulation at 3 h post-infection with UV-killed PP3 was clearly impaired in PDTC-treated fish while the effect

on IL-1␤ mRNA induction was marginal. In the head-kidney IL12 p40 and, to a lesser extent, IL-1 ␤ expression were decreased by the PDTC treatment when compared to the UV-killed bacteria alone. PDTC treatment by itself had no effect on gene expression (Fig. 6a). SbIL-12 p40, p35 and IL-1␤ expression kinetics were also studied in the spleen and head-kidney of fish challenged ip with live Phdp strain PP3 (Fig. 6b). In the spleen, IL-1␤ mRNA expression was up-regulated at 12 and 24 h after the challenge but no clear stimulation of IL-12 p40 or p35 expression was observed. In the head-kidney IL-1␤ expression was again increased clearly at 12 h post injection but had declined at 24 h. IL-12 p40 was also induced at 12 h post-challenge, returning to resting levels by 24 h (Fig. 6b). No stimulation was detected in the IL-12 p35 mRNA level. The bar graphs based on the densitometry analysis clearly support the visual gel results (Fig. 6b). 4. Discussion In the present study, (i) the sbIL-12 p40 and p35 cDNA and genomic sequences as well as their respective 5 flanking regions were isolated and characterized, (ii) the in vivo expression kinetics of these genes was investigated during a pro-inflammatory response, (iii) the importance of the NF-␬B-dependent pathway in this model was determined, and (iv) the mRNA expression of IL-12 p40 and p35 was studied during an acute infection by the Gram-negative Phdp. That the sbIL-12 p40 and p35 were indeed IL-12 orthologues was clearly supported by the conservation of important structural

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Table 2 Cis-acting elements identified in the promoter, exon I and intron I regions of sea bass and pfIL-12 genes

* NF-␬B with one mismatch were included in Fig. 6 because an NF-␬B-half site, with two mismatches to the NF-␬B consensus sequence, is know to be a key regulatory element of IL-12 p40 expression (Murphy et al., 1995). Elements in black are shown in Fig. 6.

residues, phylogenetic analysis and gene organization. HIL-12 p40 tertiary structure is divided into three structural domains (D1, D2 and D3) that seem to be conserved in sbIL-12 p40 (Yoon et al., 2000). The sbIL-12 p40 retains the N-terminal Iglike region with the two conserved cysteines known to form an intra-chain disulfide bond within the hIL-12 p40 D1 domain. In addition, sbIL-12 p40 conserves the four cysteine residues that form two disulphide bonds in the hIL-12 D2 domain. Within the D3 domain, sbIL-12 p40 conserves the WSXWS motif but, like other fish IL-12 p40 molecules (Huising et al., 2006; Yoshiura et al., 2003), lacks the cysteine pair that forms the disulfide bond in the human orthologue. However, fish IL-12 p40 have several cysteines in this region that are not present in other species and may be available to form an intra-chain disulfide bond. hIL-12 p35 folds as a four-helix bundle structure containing two intra-chain disulfide bonds, which are conserved in all fish orthologues. HIL-12 has a unique p40–p35 interface, defining a novel mode of cytokine-receptor recognition (Yoon et al., 2000). Fish and avian IL-12 p40 and p35, conserve the key structural residues involved in the formation of the hIL-12 heterodimer, giving insight to the presence of a similar interlocking topology in non-mammalian IL-12. In humans, an inter-chain disulfide bond stabilizes the IL-12 p70 complex. The present alignments show

that fish preserve the IL-12 p40 cysteine involved in the interchain disulfide bond but lack the IL-12 p35 cysteine that is present within exon IV of the hIL-12 p35, which may have evolved from a recent exon duplication in the mammalian lineage (Huising et al., 2006). According to the current alignment, fish exon IV and chiken exon III of IL-12 p35 align with the human exon V, leaving a gap in the alignment where exon IV encoded amino acids, including the inter-chain disulfide bond-forming cysteine, are present in the human molecule. The analysis of the hIL-12 p35 C74S mutant showed that the interchain disulphide bond is not required for secretion of the hIL-12 heterodimer, but may have emerged to overcome the IL-12 p35 instability (Yoon et al., 2000). A mutation in the ovine IL-12 p35 cysteine involved in the inter-disulfide bond resulted in a 100-fold reduction in IL-12 activity (De Rose et al., 2000). Conversely, the recombinant chicken IL-12, active only as a p35–p40 heterodimer, shows a high degree of functional similarity with mammalian IL-12, although it works in a species-specific manner (Degen et al., 2004). The homology modelling of carp IL-12 shows that the formation of an inter-chain disulphide bridge is plausible (Huising et al., 2006). However it still remains to be determined whether non-mammalian IL-12 molecules have an inter-chain disulfide bond formed using the IL-12 p35 cysteine residue (sea bass C90 ) available in exon IV of the fish sequences

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Fig. 5. Schematic representation of the cis-acting elements identified in the promoter, exon I and intron I regions of sea bass and pfIL-12 genes. Coding nucleotides and the 5 UTR are differentiated by black and white boxes, respectively. Grey boxes represent the promoter region and horizontal lines are introns. Values above boxes represent the number of nucleotides, starting from the TSS. Asterisks (*) indicate sites with one nucleotide mismatch to the consensus motif whilst dashes (–) indicate motifs present in the reverse complement strand.

(equivalent to exon III in chicken) or whether the p70 complex in these species involves different interactions. The phylogenetic tree shows a greater similarity of the sbIL-12 p40 and p35 with other IL-12 molecules than to their respective IL-6 and IL-6R␣ out groups, clustering sbIL-12 p40 with the other IL-12 p40 molecules and sbIL-12 p35 with other IL-12 p35 sequences. Several carp IL-12 p40 encoding genes have been found recently (Huising et al., 2006) and were designated p40-like a, b and c, in decreasing order of similarity to hIL-12 p40 (Huising et al., 2006). Zebrafish and pufferfish orthologues of carp IL-12 p40-like b and c molecules were retrieved from the genome database in this study and used to reconstruct the phylogenetic position of these molecules. The neighbourjoining tree reveals that sbIL-12 p40 is more related to the fish sequences with higher similarity to mammalian IL-12 p40 and with more conserved structural residues involved in the interlocking topology of IL-12 (Huising et al., 2006). Furthermore, a comparative genomic analysis revealed a conserved synteny between the pufferfish, zebrafish and hIL-12 p40 encoding genes (not shown), suggesting that this cluster of fish IL-12 p40 genes may indeed be a true orthologue of mammalian IL-12 p40. Inter-

estingly, the phylogenetic tree topology is compatible with the hypothesis that carp IL-12 p40-like a- and b-related molecules have evolved from the early whole-genome duplication in the teleost lineage after the divergence from the tetrapods (Jaillon et al., 2004; Woods et al., 2005). Indeed, spotted green pfIL-12 p40 and p40-like b encoding genes are located in chromosomes (1 and 7, respectively) that contain a reasonable number of duplicated genes consistent with their origin from a duplication of an ancestral chromosome (Jaillon et al., 2004; Woods et al., 2005). Furthermore, zebrafish IL-12 p40 and p40-like b-encoding genes are located in chromosomes (14 and 21, respectively) related to the spotted green pufferfish chromosomes 1 and 7. Analysis of the chromosome regions surrounding the genes encoding for zebrafish and pfIL-12 p40 and p-40 like b revealed some preserved synteny with human chromosome 5 near the IL-12 p40 gene. This is congruent with the idea that these fish chromosome segments, which include the IL-12 p40 and p40-like b-encoding genes, may have been ancestrally syntenic. On the other hand, the p40-like c molecules are the most divergent to mammalian and chicken IL-12 and, in contrast to the other fish IL-12 p40, seem to have emerged early in evolution, potentially before the

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Fig. 6. (a) RT-PCR analysis of sbIL-12 p40, p35 and sbIL-1␤ mRNA expression in the spleen and head-kidney of fish injected with UV-killed Phdp strain PP3. Organs were collected from unstimulated fish (C), and from fish injected ip with UV-killed Phdp strain PP3 in TSB-1 or TSB-1 alone at 3, 6, and 24 h after injection. Animals in the UV-killed Phdp plus PDTC group were injected ip with PDTC 1h prior to the bacteria exposure and organs were collected 3 h after the challenge. Animals in the PDTC alone group (without Phdp) were injected ip for an equivalent period. Samples were processed in pools of tissue from three fish with numbers representing independent pools above the gel. Bar graphs below the gel images show the average of the normalized densitometry values for each gene: IL-1␤ (), IL-12 p40 ( ) and p35 (). (b) RT-PCR analysis of sbIL-12 p40, p35 and sbIL-1␤ mRNA expression in the spleen and head-kidney of fish experimentally infected with Phdp strain PP3. Organs were collected from unstimulated fish (C), and from fish injected ip with PP3 in TSB-1 or TSB-1 alone at 3, 6, 12 and 24 h after infection. Samples were processed in pools of tissue from three fish with numbers representing independent pools above the gel. Bar graphs below the gel images show the average of the normalized densitometry values for each gene: IL-1␤ (), IL-12 p40 ( ) and p35 ().

fish-tetrapod split despite the fact that orthologues of the p40-like c molecules have not yet been found in mammalian or chicken genomes. The gene organization of sbIL-12 p40 and p35 was compared to the hIL-12 subunit genes. The sbIL-12 p40 gene contains the same eight exon–seven intron structure of hIL-12 p40 with well-conserved exon–intron junction positions except for the last exon, which in sea bass is within the ORF. Both mouse and pufferfish have the last intron of the IL-12 p40 gene also in between coding regions (Yoshiura et al., 2003). The sbIL-12 p35 gene has seven exons and six introns, as with hIL-12 p35

gene, but the mammalian IL-12 p35 gene seems to have had a duplication event generating an exon not present in chicken and fish. Thus, fish have an extra exon (sea bass exon VI) not seen in other vertebrate IL-12 p35 genes probably due to an intron insertion in the last exon. In mammals, IL-12 is produced in response to both viral and bacterial components (Romani et al., 1997). In pufferfish the expression of the IL-12 p35 gene is responsive to doublestranded RNA but not to LPS while IL-12 p40 mRNA was found to be constitutively expressed and unresponsive to either stimuli (Yoshiura et al., 2003), questioning the importance of IL-12

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in the antibacterial response in fish. Here we show that in sea bass injected ip with UV-killed Phdp, IL-1␤, IL-12 p40 and p35 mRNA were up-regulated in the spleen and IL-1␤ and IL-12 p40 wre up-regulated in the head-kidney. Surprisingly, IL-12 p35 mRNA expression was not stimulated in the head-kidney after the bacterial injection. The up-regulation of both IL-12 genes in the spleen in response to the UV-killed bacterial challenge suggests that IL-12 may indeed be involved in fish anti-bacterial immune defenses. The haematopoietic nature of the head-kidney (Iwama and Nakanishi, 1996) may account for the difference in IL-12 expression seen in this organ when compared to the spleen. In carp, IL-12 p40-like a and b mRNA expression was responsive to LPS in head-kidney macrophages (Huising et al., 2006). Carp IL-12 p35 was also up-regulated in the same experiment (Huising et al., 2006) in contrast to the present results with sbIL-12 p35 expression in the head-kidney and possibly due to the different experimental approaches used (in vivo versus ex vivo/mixed cell population versus purified cells). In order to understand the differences in the IL-12-responsivness to LPS presented here and the pufferfish work by Yoshiura et al. (2003), the cis-acting elements on the promoter region of IL-12 genes of both species have been studied. The IL-12 p40 promoters of sea bass and pufferfish contain binding sites for several regulatory elements (e.g. NF-␬B, NF-IL6 (c/EBP), EKLF) of human and mouse IL-12 p40 transcription. This suggests that, in fish, IL-12 transcription involves the same molecular mechanisms as in mammals. In both the fish IL-12 p40 promoter regions no TATA-box was identified revealing that in fish the expression of these genes is TATA-box independent. AP-1 has a role, in addition to that of the NF-␬B, in the LPS-induced IL-12 p40 promoter activation in human and mouse (Ma et al., 2004; Zhu et al., 2001). No potential AP-1 binding site was found within the pfIL12 p40 promoter contrasting to the 4 AP-1 sites present in sea bass promoter; whether this influences the LPS-responsiveness of these genes awaits further investigation. The IL-12 p35 promoter regions in both sea bass and pufferfish contain potential binding sites for the main transcription factors regulating the hIL-12 p35 response to LPS (NF-␬B), IFN-␥ (IRF-E, ICSBPRE) and both (Sp-1). The presence of these important cis-acting elements in fish promoters suggests similar regulatory mechanisms of IL-12 p35 expression in fish, and does not help to explain the unresponsiveness of pfIL-12 p35 to LPS (Yoshiura et al., 2003), which contrasts with the sea bass and carp (Huising et al., 2006) expression results. Members of the KLF protein family have been reported as both activators and repressors of gene expression (Lee et al., 1999; Turner and Crossley, 1998; van Vliet et al., 2000). In macrophages, the Erythroid KLF has a dual role regulating the hIL-12 p40 promoter: it activates its transcription in resting cells while it represses it after IFN-␥/LPS stimulation (Luo et al., 2004). Several KLF cis-acting elements were found within pfIL-12 p35 5 flanking region suggesting the involvement of these mediators in the regulation of pfIL-12 p35 transcription. The transcription of hIL-12 p35 seems to initiate from two sites resulting in a longer and shorter mRNA, the latter following a TATA-box-like sequence (Hayes et al., 1995). Multiple transcription initiation sites of murine p35 transcription were also

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identified, resulting in four p35 mRNA isoforms that differ in the number and position of upstream ATGs in the 5 UTR (Babik et al., 1999). The same study showed that p35 expression is controlled by both transcriptional and translational regulation, in which the presence of an upstream ATG in the 5 UTR of p35 mRNA inhibits translation of the p35 subunit. In carp and pfIL-12 p35 only one transcription initiation site was reported (Huising et al., 2006; Yoshiura et al., 2003) showing the lack of alternative initiation sites in fish. However, both sea bass and pfIL-12 p35 genes have an ATG present 11 and 6 bp downstream of the TATA-box (not shown), respectively, which may represent a similar translational control of fish IL-12 p35 expression to that seen in the mouse. The transcription factor NF-␬B is involved in the transcriptional regulation of IL-12 p35 and p40 (Kollet and Petro, 2006; Murphy et al., 1995). The role of NF-␬B in the sbIL-12 p40 and p35 Phdp-induced expression was evaluated by the use of PDTC, a well-known NF-␬B-inhibitor (Schreck et al., 1992). Pre-treatment of the fish with PDTC suppressed the Phdpinduced IL-12 p35 and p40 gene expression, showing that an NK-␬B-dependent pathway mediates the gene activation of these cytokine genes. This is consistent with the finding that a consensus sequence for the NF-␬B-binding site was within the sbIL-12 p35 promoter region and 5 NF-␬B-binding sites with one mismatch were present in the IL-12 p40 promoter region. Phdp is the agent of pasteurellosis, one of the most threatening bacterial diseases in marine aquaculture (Barnes et al., 2005). The pathogenicity of this bacterium is related to AIP56, a apoptogenic exotoxin only detected in virulent strains, that triggers apoptosis in sea bass macrophages and neutrophils (do Vale et al., 2005). The sea bass cytokine production during acute infection with Phdp was also investigated in this study. In the head-kidney, IL-1␤ and IL-12 p40 expression was transiently up-regulated at 12 h post-infection, while no variation was detected in the IL-12 p35 mRNA. The expression profile observed during the Phdp challenge differed from the response to UV-killed bacteria by the lack of IL-12 p40 and p35 gene induction in the spleen, which could be explained by the Phdp induced macrophage and neutrophil apoptosis. In conclusion, the sbIL-12 p40 and p35-encoding genes were cloned and their sequences characterized. Expression studies showed that IL-12 is involved in sea bass anti-bacterial responses and that NF-␬B is implicated in the transcriptional activation of these genes. Analysis of the 5 flanking regions of the sea bass genes revealed a number of regulatory elements consistent with these experimental findings. Acknowledgements This work was partially supported by funding from the FCT, Fundac¸a˜ o para a Ciˆencia e a Tecnologia (POCTI/CVT/ 44925/2002). Diana S. Nascimento and Ana do Vale were supported by Grants SFRH/BD/13054/2003 and SFRH/BPD/ 11538/2002, respectively, from the Portuguese programme POCTI.

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