Molecular cloning and characterization of Toll-like receptor 9 in Japanese flounder, Paralichthys olivaceus

Tomokazu Takanoa, Hidehiro Kondoa, Ikuo Hironoa, Makoto Endoa, Tatsuo SaitoTakib and Takashi Aokia,
a ,

Laboratory of Genome Science, Graduate School of Marine Science and Technology,

Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato, Tokyo 1088477, Japan
b

Department of Microbiology, School of Allied Health Science, Kitasato University,

Kitasato 1-15-1, Sagamihara-city, Kanagawa 228-8555, Japan

Received 4 August 2006; revised 5 October 2006; accepted 10 October 2006. Available online 21 November 2006.

Abstract
Toll-like receptor (TLR) 9 cDNA and gene were cloned from Japanese flounder, Paralichthys olivaceus. The Japanese flounder TLR9 cDNA encodes 1065 amino acids. The leucine-rich domain (LRD) and the Toll/interleukin-1 receptor (TIR) domain found in other vertebrate TLR9s were conserved in Japanese flounder TLR9. The gene is composed of three exons and two introns. The Japanese flounder tumor necrosis factor (TNF) gene promoter was activated in Japanese flounder TLR9-transformed hirame natural embryo (HINAE) cells upon stimulation with synthesized CpG oligodeoxynucleotide (ODN), but not by stimulation with GpC ODN. The Japanese flounder TLR9 gene was highly expressed in epithelial and lymphoid organs, such as the gills, intestines, kidney, spleen and stomach in an apparently healthy fish. The mRNA

copy numbers of Japanese flounder TLR9 and its adapter protein, the myeloid differentiation factor 88 (MYD88) were increased in some organs including blood, gill, kidney and spleen after Edwardsiella tarda challenge. Immunohistochemical analysis revealed that TLR9 and MYD88 were expressed in the same cells of kidney. Few TLR9expressing cells were found in gill, kidney and spleen in healthy Japanese flounder, but many were found in these organs after E. tarda challenge and were coincident with lesions that had been colonized by the bacteria. Keywords: Innate immunity; Japanese flounder; Luciferase; Myeloid differentiation factor 88; Promoter; Reporter assay; Toll-like receptor; Tumor necrosis factor

Article Outline
1. Introduction 2. Materials and methods 2.1. Cloning of Japanese flounder TLR9 cDNA and gene 2.2. Construction of plasmid vector for reporter assay 2.3. Transfection and luciferase assay 2.4. RT-PCR analysis 2.5. Experimental challenge of E. tarda 2.6. Real-time RT-PCR analysis 2.7. Polyclonal antibody preparation against Japanese flounder TLR9 2.8. Immunohistochemical analysis 3. Results 3.1. Japanese flounder TLR9 cDNA and gene 3.2. Activation of Japanese flounder TNF gene promoter by CpG ODN 3.3. Gene expression of Japanese flounder TLR9 and MYD88 3.4. Immunohistochemical analysis of Japanese flounder TLR9 4. Discussion Acknowledgements References

1. Introduction
Toll-like receptors (TLRs), which are type I transmembrane receptors, are crucial molecules for recognizing pathogen-associated molecular patterns (PAMPs) (Takeda and Akira, 2005). After TLRs recognize the PAMPs, the intracellular domain of the Toll/interleukin-1 receptor (TIR) domains binds to the TIR domain of adapter molecules

including myeloid differentiation factor 88 (MYD88) (Akira and Takeda, 2004). TLR signalling via MYD88-dependent or -independent pathways results in the activation of the appropriate immune response (Akira and Takeda, 2004). The mammalian TLR9 recognizes unmethylated CpG DNA of bacterial genomic DNA and activates the nuclear factor (NF)-κB via a MYD88-dependent pathway (Akira and Takeda, 2004). Synthetic CpG oligodeoxynucleotides (ODNs) mimic the bacterial CpG DNA and induce the production of cytokines (Hemmi et al., 2000). The TLR9 gene is expressed primarily on antigen-presenting cells (APCs), such as dendritic cells (DCs) (Hornung et al., 2002). In human, TLR9 is retained in the endoplasmic reticulum and is trafficked to endocytic vesicles for sensing bacterial DNA (Leifer et al., 2004). Synthetic CpG ODNs also activate innate immune responses of fish. For example, cytotoxicity of non-specific cytotoxic cells in channel catfish (Ictalurus punctatus) (Oumouna et al., 2002) and respiratory burst in leukocytes of Japanese flounder (Paralichthys olivaceus) were modulated by stimulation with CpG ODN (Lee et al., 2003). The expression of cytokine genes in common carp (Cyprinus carpio L.) leukocytes (Tassakka et al., 2006), and production of type I IFN-like antiviral activity from Atlantic salmon (Salmo salar L.) head kidney (HK) leukocytes (Jørgensen et al., 2003) were also induced by CpG ODNs. TLR genes have been found in several fish species from genomic DNA and EST analysis (Oshiumi et al., 2003, Stafford et al., 2003, Jault et al., 2004, Meijer et al., 2004, Hirono et al., 2004, Tsujita et al., 2004, Bilodeau and Waldbieser, 2005 and Tsoi et al., 2006). Fish TLR9 genes have also been identified in puffer fish (Takifugu rubripes) and zebrafish (Danio rerio) by in silico screening (Oshiumi et al., 2003, Jault et al., 2004 and Meijer et al., 2004). Further, we identified Japanese flounder MYD88 gene and demonstrated the assemblage of MYD88-expressing cells at the lesion that was formed by Edwardsiella tarda infection (Takano et al., 2006). However, the role of TLR9 and the functional involvement of TLR9 and MYD88 in fish are unclear. In this study, a cDNA and gene encoding the Japanese flounder TLR9 were cloned. We found that activation of the Japanese flounder tumor necrosis factor (TNF) gene promoter

in Japanese flounder TLR9 gene-expressing cells was ligand-dependent. We also investigated the dynamics of TLR9-expressing cells in several tissues after experimental challenge with E. tarda.

2. Materials and methods
2.1. Cloning of Japanese flounder TLR9 cDNA and gene Total RNA was isolated from kidney of Japanese flounder using TRIzol (Invitrogen, USA). cDNA was synthesized from 1 μg of total RNA using SuperScript II reverse transcriptase (Invitrogen, USA) according to the manufacturer's protocol. Degenerate primers for Japanese flounder TLR9 designated as TLR9-f and TLR9-r (Table 1) were designed from conserved regions of published nucleotide sequences of TLR9 genes from human, Homo sapiens (BC032713), mouse, Mus musculus (AF314224) and puffer fish, Takifugu rubripes (AC156439). PCR was performed using 1 μl of the synthesized cDNA as template. The PCR conditions were: an initial denaturation step of 2 min at 95 °C, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 48 °C for 30 s and 1 min of extension at 72 °C and a final elongation step at 72 °C for 5 min to complete the reaction. The nucleotide sequences of the amplified DNA fragments were determined by an automated DNA sequencer LC4200 (Li-Cor, USA). The full-length TLR9 cDNA was determined by 3′- and 5′-RACE using the SMART RACE cDNA amplification kit (BD Biosciences, USA) according to the manufacturer's protocol with gene-specific primers (Table 1), and the nucleotide sequence of the full-length cDNA was determined as described. The structure of the deduced amino acid sequence of the TLR9 was characterized using the SMART program (http://smart.embl-heidelberg.de/). Table 1. PCR primers used in this study

Primer name Degenerate primer TLR9-f TLR9-r

Sequence of oligonucleotides (references or accession numbers and position of the primer in the sequence)

5′-CTCTNNGGCTGGGANNTNTGGTA-3′ 5′-TGTCGAANACCACNAANGCAT-3′

TLR9 gene-specific primers for RACE-PCR jfTLR9(3′-RACE) jfTLR9(5′-RACE) 5′-TTTGGGCAGGGCATAAAGGA-3′ (AB234023, 2710–2729) 5′-TCCTTTATGCCCTGCCCAAA-3′ (AB234023, 2710–2729)

TLR9 gene-specific primers for RT-PCR jfTLR9RT-f jfTLR9RT-r Jfβ-actinRT-f Jfβ-actinRT-r 5′-GCTCCTGGATGAAAAGGTGGA-3′ (AB234023, 3038–3058) 5′-TAATTTGAGGTTATCTGACGACAATGC-3′ (AB234023, 3201–3227) 5′-TTTCCCTCCATTGTTGGTCG-3′ (Takano et al., 2004) 5′-GCGACTCTCAGCTCGTTGTA-3′ (Takano et al., 2004)

Primers for construction of TLR9 gene expression vector jfTLR9(HindIII)-f jfTLR9(BamHI)-r 5′-TTTAAGCTTTCATCTGCCTCCTGATTAGAATTAT-3′ (AB234023, 33–58) 5′-TTTGGATCCTCAGACAAAACTCTCACTCATGTTGTTGTC3′ (AB234023, 3231–3261)

Primers for TNT ptomoter jfTNF-LUC(KpnI)-f jfTNF-LUC(Bg/II)-r 5′-GGTACCCGACCTGAAAGAGGCAGTAA-3′ (Yazawa et al., 2005) 5′-AGATCTCCAGTCGGTGGACGACTTCT-3′ (Yazawa et al., 2005)

Primer name

Sequence of oligonucleotides (references or accession numbers and position of the primer in the sequence)

Primers for real time PCR (for construction of standard curve) TLR91ong-f TLR91ong-r MyD881ong-f MyD881ong-r 5′-GCTCCTGGATGAAAAGGTGGA-3′ (AB234023, 3038–3058) 5′-TAATTTGAGGTTATCTGACGACAATGC-3′ (AB234023, 3201–3227) 5′-GTCCTGGAGCCCAACAGAAA-3′ (AB241074, 751–770) 5′-GACGGTATATGAAGCCACAGTTAG-3′ (AB241074, 927–950)

EF (elongation factor 5′-ACGTTCTTGATGTTGAAGCCGA-3′ (AU090803, 760–781) 1 α) long-f EFlong-r RP (ribosomal protein L10) long-f RPlong-r 5′-CCCTGCAGGACGTCTACAAA-3′ (AU090803, 930–959) 5′-GGAATTCAACCCAACTTTCTGACGT-3′ (AU050650, 196– 230) 5′-TGGTGCGATCAGGTAGCTAC-3′ (AU050650, 376–395)

For detection of mRNAs TLR9short-f TLR9short-r MyD88short-f MyD88short-r EFshort-f EFshort-r RPshort-f RPshort-r 5′-CGTGCTGGTTCTGTTGGATGAG-3′ (AB234023, 3065–3086) 5′-AACCTCTTCCTCAGCTGGAGG-3′ (AB234023, 3107–3127) 5′-CCAGTGGTGTACAAGCCGATG-3′ (AB241074, 780–800) 5′-ACACGGTGAGGAAGCGCAAG-3′ (AB241074, 821–840) 5′-CTCGGGCATAGACTCGTGGT-3′ (AU090803, 801–820) 5′-CATGGTCGTGACCTTCGCTC-3′ (AU090803, 841–860) 5′-GCTCCTCTGGTGCAGTTTGTGA-3′ (AU050650, 236–257) 5′-TGGTGTTTGCTGGCGTCACTCT-3′ (AU050650, 324–345)

Primers for recombinant TLR9 protein production

Primer name

Sequence of oligonucleotides (references or accession numbers and position of the primer in the sequence)

jfTLR9recombinant-f 5′-CCCATATGCACCACCACCACCACCACGAGATG GTTTTCAGAGAGCT-3′ (AB234023, 612–631) jfTLR9recombinant-r 5′-GGGGAATTCTTAGTCAGAGAGGTCCAAACTCT-3′ (AB234023, 679–698)

The DNA fragment of Japanese flounder TLR9 was used as a DNA probe for screening of BAC clones from a Japanese flounder genomic BAC library (Katagiri et al., 2000 and Hirono et al., 2000). 2.2. Construction of plasmid vector for reporter assay The ORF of Japanese flounder TLR9 was amplified from the head kidney cDNA by the PCR primer set jfTLR9(HindIII)-f and jfTLR9(BamHI)-r (Table 1). The amplified TLR9 DNA fragments were cloned into pcDNA3.1(+) (Invitrogen, USA), which was designated as pcDNA-ParoliTLR9. The 5′-flanking region of the Japanese flounder TNF gene containing 1984 bp (−1782 to +202 from the transcription initiation site) (Yazawa et al., 2005) was also amplified using a set of PCR primers (Table 1) from a Japanese flounder BAC clone. The fragment was inserted between KpnI and BglII site of pGL3-Basic (Promega, USA), and designated pGL-ParoliTNF. This plasmid was used as a reporter for TLR9 signal activation. The phRL-SV40 vector (Promega, USA) was used as an internal control. 2.3. Transfection and luciferase assay Hirame natural embryo (HINAE) cells (Kasai and Yoshimizu, 2001) were cultured at 20 °C in Leibovitz's L-15 medium (Gibco-BRL, USA), supplemented with 20% fetal bovine serum (FBS) (JRH biosciences, USA), 100 IU/ml penicillin G and 100 μg/ml streptomycin (Gibco-BRL, USA).

HINAE cells (1 × 105 cell) seeded onto a 24-well cell culture microplate (Corning, USA) were transiently transfected with 1 μg of pGL-ParoliTNF, 0.02 μg of phRL-SV40 and 1 μg of pGL3-Basic or pcDNA-ParoliTLR9 with Lipofectamine™ 2000 (Invitrogen, USA) according to the manufacturer's protocol. The transformants were washed with PBS at 36 h after transfection, then added 0.2 ml of culture medium containing synthesized phosphodiesters (Hokkaido System Science, Japan) of CpG ODN-1668: 5′-TCCATGACGTTCCTGATGCT-3′ (1, 10 μg/ml) or GpC ODN-1720; 5′-TCCATGAGCTTCCTGATGCT-3′ (1, 10 μg/ml) and cultured for another 36 h. Luciferase luminescence assays were performed using the Dual-Luciferase® reporter assay system (Promega, USA) with a LUMI-COUNTER 2500 (Microtec Nition, Japan). Relative light units were calculated using phRL-SV40 as standard. The test was done in triplicate and relative light units were expressed with standards error of the mean. Significant differences between relative light units were analyzed by Student's t-test. 2.4. RT-PCR analysis Peripheral blood, brain, gill, heart, intestine, kidney, liver, muscle, skin, spleen and stomach were extirpated from a Japanese flounder juvenile (body weight of 8.4 g), and total RNAs were extracted using TRIzol (Invitrogen, USA). One micrograms of total RNAs were used for cDNA synthesis in a 20 μl reaction mixture as described above. One microlitre of the reverse-transcribed product was used in a 50 μl PCR reaction mixture. PCR was performed with gene-specific primers for TLR9 and beta-actin (ACTB) (Takano et al., 2004) (Table 1). The PCR conditions were: an initial denaturation step of 2 min at 95 °C, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s and 1 min of extension at 72 °C, a final elongation step at 72 °C for 5 min completed the reaction. The amplified PCR product (5 μl) was electrophoresed on a 0.8% agarose gel. 2.5. Experimental challenge of E. tarda

Japanese flounder juveniles (average weight of 8.9 g) were used for the experimental challenge. E. tarda strain NE9505 was grown in tryptic soy broth (BD Biosciences, USA) with 2% sodium chloride at 25 °C. The cultured E. tarda was diluted to 2.3 × 107 cfu/ml with artificial seawater in a 15-l tank. Then, Japanese flounder juveniles were placed in the tank for 10 min at 25 °C. After exposure to E. tarda, the fish were transferred to another 200-l tank that is provided with UV treated artificial seawater in a recirculating system at 25 °C. The challenged fish were used for real-time RT-PCR and immunohistochemical analysis of TLR9. 2.6. Real-time RT-PCR analysis Gene expression patterns of Japanese flounder TLR9 and its adapter protein, MYD88 after E. tarda infection were determined. To confirm the chronological alternation of mRNA copy numbers, the blood, gills, kidney and spleen were surgically excised and pooled from three fish at 0, 0.5, 1, 3 and 7 days after E. tarda challenge. The mRNA copy numbers in each tissue were quantified by real-time RT-PCR analysis as previously described (Takano et al., 2004), and this test was conducted three times. The mRNA copy numbers of Japanese flounder elongation factor 1 α (AU090803) were used as internal control in blood, gill and spleen, and ribosomal protein L10 (AU050650) was used in kidney. The gene-specific primers used in the real-time RT-PCR analysis are shown in Table 1. The differences between fold inductions were tested by ANOVA and analyzed with Tukey's method. 2.7. Polyclonal antibody preparation against Japanese flounder TLR9 A cDNA fragment of Japanese flounder TLR9 was amplified using the primer set of jfTLR9recombinant-f and jfTLR9recombinant-r (Table 1). The amplified fragments were cloned into the NdeI and EcoRI restriction sites of pET-32a(+) DNA (Novagen, Germany) and transformed into competent Escherichia coli BL21 cells for producing the recombinant TLR9. The transformants were cultured overnight in 3 ml of Luria-Bertani (LB) broth (1% Bacto Tryptone; 0.5% Yeast Extract; 1% NaCl; 100 μg/ml ampicillin). The inoculum was transferred to 200 ml of LB broth and incubated at 37 °C with

shaking. When OD600 reached 0.6, the cultures were induced with 1 mM of isopropyl-β-dthiogalactopyranoside for 4 h. The bacterial culture was centrifuged and the cell pellet was resuspended in phosphate buffered saline (PBS) (5.2 mM Na2HPO4; 1.7 mM KH2PO4; 150 mM NaCl; pH 7.4), and kept at −80 °C for 3 h. The cells were thawed, sonicated then centrifuged at 5000 × g. The inclusion bodies were solubilized in a denaturation buffer containing 8 M urea, 50 mM sodium dodecyl sulfate and 10 mM Tris–HCl (pH 8.0). The solubilized recombinant TLR9 protein was mixed with nickelnitrilotriacetic acid (Ni-NTA) agarose (QIAGEN, Germany). The solution was transferred onto a polypropylene column and recombinant protein was eluted using an imidazole gradient (300–500 mM). The eluate was dialyzed by PBS and the purified recombinant protein was analyzed by 20% SDS PAGE with Coomassie brilliant blue (CBB) staining. The anti-TLR9 polyclonal antibody (PAb ParoliTLR9) from mouse was obtained following protocols described previously (Ooi et al., 2005). 2.8. Immunohistochemical analysis The kidney was surgically excised from a Japanese flounder at 7 days post-infection (dpi). The tissues were fixed in 4% phosphate buffered paraformaldehyde at room temperature for 12 h, embedded in paraffin and sectioned at 4 μm. PAb ParoliTLR9 and PAb ParoliMYD88 (Takano et al., 2006) were labeled with Zenon Alexa Fluor 488 mouse IgG1 labeling reagent (green) (Invitrogen, USA) and Zenon Alexa Fluor 594 mouse IgG1 labeling reagent (red) (Invitrogen, USA), respectively. The immunostained cells were observed by fluorescence microscopy using appropriate filters. Gills, kidney and spleen were surgically excised from an apparently healthy Japanese flounder juvenile and from an infected fish at 3 and/or 7 dpi. Sections were immunostained by PAb ParoliTLR9 or anti-E. tarda polyclonal serum (Verjan et al., 2005) according to the protocols previously described (Takano et al., 2006).

3. Results
3.1. Japanese flounder TLR9 cDNA and gene

The coding region of Japanese flounder TLR9 (AB234023) was 3198 bp and encoded 1065 amino acid residues. The TLR9 contains a leucine rich domain (LRD), which is the functional extracellular domain (Fig. 1). Part of the CpG-DNA-binding domain is similar to a sequence in mouse TLR9 LRD and contains two essential amino acid residues for interacting with CpG-DNA (Rutz et al., 2004.). These amino acid residues are also conserved in the LRDs of the TLR9s of Japanese flounder (Asp562 and Tyr564), puffer fish (Asp544 and Tyr546) and human (Asp534 and Tyr536) TLR9 (Fig. 1). The Japanese flounder TLR9 also has two CXXC motifs separated by six amino acid residues (Fig. 1). This region is expected to bind directly to unmethylated CpG dinucleotide sequences (Bell et al., 2003).

Full-size image (180K) Fig. 1. Amino acid sequence alignment of Japanese flounder, Paralichthys olivaceus TLR9 (AB234023) to human, Homo sapiens TLR9 (NP_059138), puffer fish, Fugu rubripes TLR9 (AAW69377) and zebrafish, Danio rerio TLR9 (XM_685911). Signal peptides are indicated with small letters. The LRR regions are indicated with underline and the first amino acid residue in LRRs are indicated with a gray letter. LRRCT is represented with a broken underline. Underlined residues above double line correspond to the transmembrane domains, and the TIR domains are boxed. Sequence gaps and identical symbols are indicated with dots and hyphens, respectively. The region containing two CXXC-containing motifs that are expected to be important for binding to the unmethylated CpG DNAs, are indicated by a gray box. Asterisks indicate the conserved amino acid residues that exist in the CpG-DNA-binding domain. The amino acid residue that is encoded by the first codon of each exon is framed.

A TIR domain that is functionally important for TLR signal transduction was identified in the cytoplasmic domain of Japanese flounder TLR9. The TIR domain of zebrafish TLRs have three boxes that are important for TLR function (Jault et al., 2004). These boxes are conserved in Japanese flounder TLR9 TIR domain (Fig. 1). The amino acid sequence of Japanese flounder TLR9 protein shared sequence homology with human (NP_059138), puffer fish (AAW69377) and zebrafish (XP_691003) at 35.0%, 60.7% and 49.5%, respectively (Table 2). The number of leucine rich repeats (LRRs) in Japanese flounder, human, puffer fish and zebrafish TLR9 are 12, 18, 14 and 14, respectively (Table 2). The genomic sequence of the Japanese flounder TLR9 (AB234024) spanned 3493 bp containing three exons and two introns (Fig. 2). Table 2. Amino acid identities of Japanese flounder TLR9 with other species
Japanese flounder (AB234023) 3198 1065

Animal (accession no.) ORF (bp) Amino acids (aa) Percent identity of amino acid sequence between jfTLR9 Number of LRR
a

Human (NP_059138) 3099 1032

Puffer fish (AAW69377) 3138 1045

Zebrafish (XP_691003) 3198 1065

35.0 (47.2)a

60.7 (78.8)a

49.5 (68.1)a

12

18

14

14

Percent identity of amino acid sequence of the TIR domain of jfTLR9 with other

organisms.

Full-size image (33K) Fig. 2. Schematic diagram of the deduced Japanese flounder, Paralichthys olivaceus TLR9 protein and gene (AB234024: position of the gene from ATG to stop codon in this accession no.; 226–3808) structure. Both diagrams are connected with broken line to delimit the position of splicing frame. The TLR9 gene structure is compared with human, Homo sapiens (AC097637: 197,007–201,309), puffer fish, Fugu rubripes (AC156439: 1105–4315) and zebrafish, Danio rerio (NM_634665: 1,674,474–1,677,671) TLR9 genes. The coding regions in the exons are shown by black box and sizes (bp) are indicated above the box. The sizes (bp) of the introns are represented in parenthesis.

3.2. Activation of Japanese flounder TNF gene promoter by CpG ODN The Japanese flounder TNF gene promoter activity was measured by reporter assay in Japanese flounder TLR9-expressing HINAE cell following stimulation with CpG ODN1668 or GpC ODN-1720. The TNF gene promoter in the pGL3-Basic transfected HINAE cell responds neither to CpG ODN-1668 nor GpC ODN-1720 (Fig. 3). On the other hand, the TNF promoter in TLR9-expressing HINAE cells was activated by CpG ODN-1668 (Fig. 3). The activities of TLR9-expressing HINAE cells stimulated with 1 and 10 μg/ml of CpG ODN-1668 were 1.58- and 1.82-fold greater, respectively, than those of nonstimulated cells.

Full-size image (10K)

Fig. 3. Japanese flounder TNF promoter activities in HINAE cells transiently transfected with pcDNA-ParoliTLR9 and stimulated with 1 and 10 μg/ml of CpG ODN-1668 or GpC ODN-1720. Luminescence of stimulated cells was determined at 36 h post stimulation and measured in relative light units. Error bars indicate the mean ± S.E.M. of each treatment (n = 3). Asterisk indicates a significant increase of relative light units by Student's t-test (p < 0.05).

3.3. Gene expression of Japanese flounder TLR9 and MYD88 The Japanese flounder TLR9 gene was strongly expressed in peripheral blood, gill, intestine, kidney, spleen and stomach, and weakly expressed in brain, heart, liver, muscle and skin (Fig. 4). Exposure of juvenile fishes to E. tarda significantly elevated TLR9 mRNA copy number in the blood, gill, kidney and spleen (Table 3). However, obvious acute-phase increments of TLR9 mRNA were not observed in any of the organs (Table 3). The copy number of MYD88 mRNA rose abruptly more than five-fold at 0.5 dpi in the blood and spleen (Table 3). After acute-phase induction, MYD88 mRNAs in both tissues reverted to original levels until 1 or 3 dpi, respectively. Then the mRNA copy numbers started to increase again until 7 dpi (Table 3). Intense increment of MYD88 mRNAs also occurred in the kidney at 7 dpi (Table 3).

Full-size image (21K) Fig. 4. Relative abundance of Japanese flounder TLR9 transcripts in tissues as measured by RT-PCR. Lane 1, peripheral blood; lane 2, brain; lane 3, gill; lane 4, heart; lane 5, intestine; lane 6, kidney; lane 7, liver; lane 8, muscle; lane 9, skin; lane 10, spleen; lane 11, stomach.

Table 3. The fold induction of Japanese flounder TLR9 and MyD88 gene expression after EdwardsieIla tarda challenge
Gene Organ Days post-challenge 0 TLR9 Blood Gill Kidney Spleen 0.5 1 3.0 ± 0.20a 2.4 ± 0.08a 0.9 ± 0.08 2.2 ± 0.52 3 7

1.0 2.9 ± 0.80a 1.0 1.4 ± 0.21 1.0 1.5 ± 0.06 1.0 2.9 ± 0.16

3.1 ± 0.51a 3.9 ± 0.38a 1.4 ± 0.39 1.6 ± 0.13 3.3 ± 0.29 3.3 ± 0.32a 2.6 ± 0.36 11.9 ± 1.11a

MyD88

Blood Gill Kidney Spleen

1.0 56.4 ± 12.9a 28.7 ± 8.39a 1.4 ± 0.13 1.0 1.5 ± 0.05 1.0 3.3 ± 0.08a 1.0 6.4 ± 0.90a 1.9 ± 0.21a 2.8 ± 0.31a 1.1 ± 0.08 1.1 ± 0.04

8.1 ± 1.85 1.9 ± 0.19a

4.0 ± 0.28a 13.5 ± 0.29a 1.1 ± 0.11 3.2 ± 0.44a

The fold inductions were expressed with standards error of the mean.
a

The fold induction value is significant at the p < 0.05 level by the ANOVA and Tukey's

method.

3.4. Immunohistochemical analysis of Japanese flounder TLR9 Co-localization of Japanese flounder TLR9 and MYD88 was determined in a section of kidney tissues that was obtained from an E. tarda-infected fish at 7 dpi (Fig. 5). A few TLR9-expressing cells were detected in the gills (Fig. 6A), kidney (Fig. 6D) and spleen (Fig. 6G) in a healthy fish. Infiltration of TLR9-expressing cells was observed on the surface of secondary gill lamellae at 3 dpi (Fig. 6B), and assemblages of TLR9-

expressing cells were confirmed at the accreted secondary gill lamellas at 7 dpi (Fig. 6C). A large number of TLR9-expressing cells in the kidney were found at the same locus of a lesion (Fig. 6E) that was formed due to E. tarda proliferation at 7 dpi (Fig. 6F). An increase of TLR9-expressing cells was also observed in the spleen at 7 dpi (Fig. 6H).

Full-size image (43K) Fig. 5. Co-localization of Japanese flounder TLR9 and MYD88 in renal cells in an E. tarda-infected fish. A kidney section of E. tarda-infected fish was immunostained by PAb ParoliTLR9 (A) and PAb ParoliMYD88 (B). Japanese flounder TLR9 and MYD88 can be seen to be co-localized in some cells (C). The blue spots on photo C are DAPIstained nuclei. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Full-size image (283K) Fig. 6. Immunohistochemical detection of Japanese flounder TLR9 in tissues. The kidney and spleen were collected at 0 and 7 days post infection (dpi), and gills were collected at 0, 3 and 7 dpi from an E. tarda-infected Japanese flounder. The TLR9-expressing cells and E. tarda were stained red. A few TLR9-expressing cells were present in the gill of a healthy fish (A). Infiltration of TLR9-expressing cells was observed in the gill at 3 dpi (B) and assemblage of TLR9-expressing cells was observed at 7 dpi (C). A few TLR9expressing cells are visible in the kidney of a healthy fish (D). An assemblage of TLR9expressing cells (E) in an E. tarda-infected site (F) at 7 dpi was observed. A few TLR9expressing cells were observed in the spleen of a healthy fish (G). An increase of TLR9expressing cells was also observed in the spleen at 7 dpi (H). (For interpretation of the

references to colour in this figure legend, the reader is referred to the web version of the article.)

4. Discussion
In this study, we cloned Japanese flounder TLR9 cDNA and characterized its functional domains in the amino acid sequence. The TIR domain of Japanese flounder TLR9 has significant identities with the TIR domains of human and fish TLR9 (Table 2). On the other hand, the LRD structure of Japanese flounder TLR9 is different from other TLR9s. Almost all LRRs in Japanese flounder TLR9 are identical between human and puffer fish TLR9, but some of the LRRs do not correspond to human and/or puffer fish TLR9 (Fig. 1). Notably, Japanese flounder TLR9 does not have a LRR in the region that encodes the functional important amino acid residue of Asp562 and Tyr564. The structures of LRRs in TLR are speculated to mediate specific PAMPs recognition (Bell et al., 2003). The difference of TLR9 specificity for unmethylated CpG ODNs has been confirmed in several species (Griebel et al., 2005). Thus, the differences in structure, especially the LRRs of TLR9 extracellular domain may explain why CpG motif recognition is speciesspecific. The TNF promoter activation was observed when TLR9-expressing cells were stimulated with CpG ODN-1668 (Fig. 3). However, the TNF promoter was not activated with GpC ODN-1720 (Fig. 3). CpG ODN-1668 (but not GpC ODN-1720) was found to have a strong immunostimulatory effect in Japanese flounder (Lee et al., 2003), and this action is possibly because of the differential sensitivity of Japanese flounder TLR9 to CpG ODN1668 and GpC ODN-1720. Also in several vertebrates, TNF-α production is up-regulated following stimulation with CpG ODN but not with non-CpG ODN (Tassakka et al., 2006 and Dalpke et al., 2002). Japanese flounder TLR9 is predominantly expressed in blood, gill, kidney and spleen (Fig. 4). After the E. tarda challenge, the TLR9 mRNA copy numbers increased in the

tissues (Table 3), and the number of TLR9-expressing cells markedly increased in tissues of an E. tarda infected fish (Fig. 6B, C, E and H). Hence, the increase of the TLR9 mRNA may be due to the up-regulation of gene expression, proliferation and/or recruitment of TLR9-expressing cells in the tissues. In the early stage of E. tarda infection, TLR9 expression did not clearly increase in any tissues, while MYD88 expression markedly increased in the blood and spleen (Table 3). MYD88 is known to be a major adapter protein not only for TLR9 but also for other IL-1R/TLR superfamily molecules in mammals (Muzio et al., 1997, Wesche et al., 1997 and Akira and Takeda, 2004). We previously demonstrated that Japanese flounder MYD88 gene expression in peripheral blood leukocytes was abruptly induced by lipopolysaccharide (LPS), poly I:C and peptidoglycan (PGN), which are known ligands of mammalian TLRs (Takano et al., 2006). Hence, Japanese flounder MYD88 gene expression seems to be up-regulated more than TLR9 gene expression in leukocytes during the early stage of infection. However, because Japanese flounder TLR9 and MYD88 are expressed in the same cells of the kidney (Fig. 6), MYD88-dependent signalling cascades may be present in TLR9expressing cells. In mammals, TLR9 is predominantly expressed in B cells, plasmacytoid DC, CD8α+ DC and CD11b+ DC (Iwasaki and Medzhitov, 2004 and Peng, 2005). These DCs play crucial roles in antigen recognition in peripheral tissues and in naive T lymphocyte activation in lymphoid tissues (Iwasaki and Medzhitov, 2004). In teleost fish, the spleen is speculated to be a secondary lymphoid tissue (Press and Evensen, 1999). A DC-like cell, which has unique granules and closely resembles mammalian DCs was characterized within the gills of Loma salmonae-infected chinook salmon, Oncorhynchus tshawytscha (Lovy et al., 2006). In this study, numerous TLR9-expressing cells were detected in the spleen (Fig. 6H) and the gill (Fig. 6B and C) of E. tarda-infected fish. Assemblages of TLR9expressing cells were also observed in kidney lesions that formed due to E. tarda infection (Fig. 6D and E). Hence, Japanese flounder TLR9-expressing cells may have a role similar to that of mammalian DCs. The major lymphoid organs in mammals are bone marrow and lymph nodes, whereas in teleost fish they are the kidney and spleen (Solem and Stenvik, 2006). Therefore, it is worthwhile to characterize the nature of Japanese

flounder TLR9-expressing cells for understanding the differences of fish immune system between other vertebrates. In conclusion, Japanese flounder TLR9 functions as a receptor for CpG ODN, and the recognition of CpG ODN leads to the induction of TNF-α gene expression. The TLR9expressing cells were observed in lesions of E. tarda-infected fish. Taken together, these results suggest that Japanese flounder TLR9-expressing cells have roles in sensing bacterial pathogens and in inducing immune responses.

Acknowledgements
This research was supported in part by Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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