Molecular cloning and characterization of Toll-like

receptor 9 in Japanese flounder, Paralichthys

olivaceus
Tomokazu Takanoa, Hidehiro Kondoa, Ikuo Hironoa, Makoto Endoa, Tatsuo Saito-
Takib 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 108-
8477, 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 TLR9-
expressing 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

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

Degenerate primer

TLR9-f 5′-CTCTNNGGCTGGGANNTNTGGTA-3′

TLR9-r 5′-TGTCGAANACCACNAANGCAT-3′

TLR9 gene-specific primers for RACE-PCR

jfTLR9(3′-RACE) 5′-TTTGGGCAGGGCATAAAGGA-3′ (AB234023, 2710–2729)

jfTLR9(5′-RACE) 5′-TCCTTTATGCCCTGCCCAAA-3′ (AB234023, 2710–2729)

TLR9 gene-specific primers for RT-PCR

jfTLR9RT-f 5′-GCTCCTGGATGAAAAGGTGGA-3′ (AB234023, 3038–3058)

5′-TAATTTGAGGTTATCTGACGACAATGC-3′ (AB234023,
jfTLR9RT-r
3201–3227)

Jfβ-actinRT-f 5′-TTTCCCTCCATTGTTGGTCG-3′ (Takano et al., 2004)

Jfβ-actinRT-r 5′-GCGACTCTCAGCTCGTTGTA-3′ (Takano et al., 2004)

Primers for construction of TLR9 gene expression vector

5′-TTTAAGCTTTCATCTGCCTCCTGATTAGAATTAT-3′
jfTLR9(HindIII)-f
(AB234023, 33–58)

5′-TTTGGATCCTCAGACAAAACTCTCACTCATGTTGTTGTC-
jfTLR9(BamHI)-r
3′ (AB234023, 3231–3261)

Primers for TNT ptomoter

5′-GGTACCCGACCTGAAAGAGGCAGTAA-3′ (Yazawa et al.,

jfTNF-LUC(KpnI)-f
2005)

5′-AGATCTCCAGTCGGTGGACGACTTCT-3′ (Yazawa et al.,

jfTNF-LUC(Bg/II)-r
2005)
 Sequence of oligonucleotides (references or accession numbers
Primer name
and position of the primer in the sequence)

Primers for real time PCR (for construction of standard curve)

TLR91ong-f 5′-GCTCCTGGATGAAAAGGTGGA-3′ (AB234023, 3038–3058)

5′-TAATTTGAGGTTATCTGACGACAATGC-3′ (AB234023,
TLR91ong-r
3201–3227)

MyD881ong-f 5′-GTCCTGGAGCCCAACAGAAA-3′ (AB241074, 751–770)

MyD881ong-r 5′-GACGGTATATGAAGCCACAGTTAG-3′ (AB241074, 927–950)

EF (elongation factor
5′-ACGTTCTTGATGTTGAAGCCGA-3′ (AU090803, 760–781)
1 α) long-f

EFlong-r 5′-CCCTGCAGGACGTCTACAAA-3′ (AU090803, 930–959)

RP (ribosomal 5′-GGAATTCAACCCAACTTTCTGACGT-3′ (AU050650, 196–

protein L10) long-f 230)

RPlong-r 5′-TGGTGCGATCAGGTAGCTAC-3′ (AU050650, 376–395)

For detection of mRNAs

TLR9short-f 5′-CGTGCTGGTTCTGTTGGATGAG-3′ (AB234023, 3065–3086)

TLR9short-r 5′-AACCTCTTCCTCAGCTGGAGG-3′ (AB234023, 3107–3127)

MyD88short-f 5′-CCAGTGGTGTACAAGCCGATG-3′ (AB241074, 780–800)

MyD88short-r 5′-ACACGGTGAGGAAGCGCAAG-3′ (AB241074, 821–840)

EFshort-f 5′-CTCGGGCATAGACTCGTGGT-3′ (AU090803, 801–820)

EFshort-r 5′-CATGGTCGTGACCTTCGCTC-3′ (AU090803, 841–860)

RPshort-f 5′-GCTCCTCTGGTGCAGTTTGTGA-3′ (AU050650, 236–257)

RPshort-r 5′-TGGTGTTTGCTGGCGTCACTCT-3′ (AU050650, 324–345)

Primers for recombinant TLR9 protein production

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

jfTLR9recombinant-f 5′-CCCATATGCACCACCACCACCACCACGAGATG

GTTTTCAGAGAGCT-3′ (AB234023, 612–631)

5′-GGGGAATTCTTAGTCAGAGAGGTCCAAACTCT-3′
jfTLR9recombinant-r
(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-d-
thiogalactopyranoside 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 nickel-
nitrilotriacetic 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
Animal (accession Human Puffer fish Zebrafish
flounder
no.) (NP_059138) (AAW69377) (XP_691003)
(AB234023)

ORF (bp) 3198 3099 3138 3198

Amino acids (aa) 1065 1032 1045 1065

Percent identity of
amino acid
– 35.0 (47.2)a 60.7 (78.8)a 49.5 (68.1)a
sequence between
jfTLR9

Number of LRR 12 18 14 14
a
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 ODN-
1668 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 non-
stimulated 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 0.5 1 3 7

TLR9 Blood 1.0 2.9 ± 0.80a 3.0 ± 0.20a 3.1 ± 0.51a 3.9 ± 0.38a

Gill 1.0 1.4 ± 0.21 2.4 ± 0.08a 1.4 ± 0.39 3.3 ± 0.32a

Kidney 1.0 1.5 ± 0.06 0.9 ± 0.08 1.6 ± 0.13 2.6 ± 0.36

Spleen 1.0 2.9 ± 0.16 2.2 ± 0.52 3.3 ± 0.29 11.9 ± 1.11a

MyD88 Blood 1.0 56.4 ± 12.9a 28.7 ± 8.39a 1.4 ± 0.13 8.1 ± 1.85

Gill 1.0 1.5 ± 0.05 1.9 ± 0.21a 1.1 ± 0.04 1.9 ± 0.19a

Kidney 1.0 3.3 ± 0.08a 2.8 ± 0.31a 4.0 ± 0.28a 13.5 ± 0.29a

Spleen 1.0 6.4 ± 0.90a 1.1 ± 0.08 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 DAPI-
stained 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 TLR9-
expressing cells are visible in the kidney of a healthy fish (D). An assemblage of TLR9-
expressing cells (E) in an E. tarda-infected site (F) at 7 dpi was observed. A few TLR9-
expressing cells were observed in the spleen of a healthy fish (G). An increase of TLR9-
expressing 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 species-
specific.

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 ODN-
1668 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 TLR9-
expressing 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 TLR9-
expressing 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 TLR9-
expressing 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.

References

Akira and Takeda, 2004 S. Akira and K. Takeda, Toll-like receptor signalling, Nat. Rev.
Immunol. 4 (2004), pp. 499–511. Full Text via CrossRef | View Record in Scopus | Cited
By in Scopus (1755)
Bell et al., 2003 J.K. Bell, G.E. Mullen, C.A. Leifer, A. Mazzoni, D.R. Davies and D.M.
Segal, Leucine-rich repeats and pathogen recognition in Toll-like receptors, Trends.

Immunol. 24 (2003), pp. 528–533. Article | PDF (541 K) | View Record in Scopus |
Cited By in Scopus (195)
Bilodeau and Waldbieser, 2005 A.L. Bilodeau and G.C. Waldbieser, Activation of TLR3
and TLR5 in channel catfish exposed to virulent Edwardsiella ictaluri, Dev. Comp.

Immunol. 29 (2005), pp. 713–721. Article | PDF (152 K) | View Record in Scopus |
Cited By in Scopus (26)
Dalpke et al., 2002 A.H. Dalpke, M.K. Schafer, M. Frey, S. Zimmermann, J. Tebbe, E.
Weihe and K. Heeg, Immunostimulatory CpG-DNA activates murine microglia, J.
Immunol. 168 (2002), pp. 4854–4863. View Record in Scopus | Cited By in Scopus (72)
Griebel et al., 2005 P.J. Griebel, R. Brownlie, A. Manuja, A. Nichani, N. Mookherjee, Y.
Popowych, G. Mutwiri, R. Hecker and L.A. Babiuk, Bovine Toll-like receptor 9: a
comparative analysis of molecular structure, function and expression, Vet. Immunol.

Immunopathol. 108 (2005), pp. 11–16. Article | PDF (139 K) | View Record in Scopus
| Cited By in Scopus (17)
Hemmi et al., 2000 H. Hemmi, O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M.
Matsumoto, K. Hoshino, H. Wagner, K. Takeda and S. Akira, A Toll-like receptor
recognizes bacterial DNA, Nature 408 (2000), pp. 740–745. View Record in Scopus |
Cited By in Scopus (2584)
Hirono et al., 2000 I. Hirono, B.H. Nam, T. Kurobe and T. Aoki, Molecular cloning,
characterization, and expression of TNF cDNA and gene from Japanese flounder
Paralichthys olivaceus, J. Immunol. 165 (2000), pp. 4423–4427. View Record in Scopus |
Cited By in Scopus (96)
Hirono et al., 2004 I. Hirono, M. Takami, M. Miyata, T. Miyazaki, H.J. Han, T. Takano,
M. Endo and T. Aoki, Characterization of gene structure and expression of two Toll-like
receptors from Japanese flounder, Paralichthys olivaceus, Immunogenetics 56 (2004), pp.
38–46. View Record in Scopus | Cited By in Scopus (30)
Hornung et al., 2002 V. Hornung, S. Rothenfusser, S. Britsch, A. Krug, B. Jahrsdorfer, T.
Giese, S. Endres and G. Hartmann, Quantitative expression of Toll-like receptor 1–
10 mRNA in cellular subsets of human peripheral blood mononuclear cells and
sensitivity to CpG oligodeoxynucleotides, J. Immunol. 168 (2002), pp. 4531–4537. View
Record in Scopus | Cited By in Scopus (658)
Iwasaki and Medzhitov, 2004 A. Iwasaki and R. Medzhitov, Toll-like receptor control of
the adaptive immune responses, Nat. Immunol. 5 (2004), pp. 987–995. Full Text via
CrossRef | View Record in Scopus | Cited By in Scopus (1188)
Jault et al., 2004 C. Jault, L. Pichon and J. Chluba, Toll-like receptor gene family and

TIR-domain adapters in Danio rerio, Mol. Immunol. 40 (2004), pp. 759–771. Article |
PDF (725 K) | View Record in Scopus | Cited By in Scopus (97)
Jørgensen et al., 2003 J.B. Jørgensen, L.H. Johansen, K. Steiro and A. Johansen, CpG
DNA induces protective antiviral immune responses in Atlantic salmon (Salmo salar L.),
J. Virol. 77 (2003), pp. 11471–11479. Full Text via CrossRef | View Record in Scopus |
Cited By in Scopus (39)
Kasai and Yoshimizu, 2001 H. Kasai and M. Yoshimizu, Establishment of two Japanese
flounder embryo cell lines, Bull. Fish. Sci. Hokkaido Univ. 52 (2001), pp. 67–70.
Katagiri et al., 2000 T. Katagiri, S. Asakawa, I. Hirono, T. Aoki and N. Shimizu,
Genomic bacterial artificial chromosome library of the Japanese flounder Paralichthys
olivaceus, Mar. Biotechnol. 2 (2000), pp. 571–576. Full Text via CrossRef | View
Record in Scopus | Cited By in Scopus (33)
Lee et al., 2003 C.H. Lee, H.D. Jeong, J.K. Chung, H.H. Lee and K.H. Kim, CpG motif
in synthetic ODN primes respiratory burst of olive flounder Paralichthys olivaceus
phagocytes and enhances protection against Edwardsiella tarda, Dis. Aquat. Organ. 56
(2003), pp. 43–48. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus
(18)
Leifer et al., 2004 C.A. Leifer, M.N. Kennedy, A. Mazzoni, C. Lee, M.J. Kruhlak and
D.M. Segal, TLR9 is localized in the endoplasmic reticulum prior to stimulation, J.
Immunol. 173 (2004), pp. 1179–1183. View Record in Scopus | Cited By in Scopus (105)
Lovy et al., 2006 J. Lovy, G.M. Wright and D.J. Speare, Morphological presentation of a
dendritic-like cell within the gills of chinook salmon infected with Loma salmonae, Dev.

Comp. Immunol. 30 (2006), pp. 259–263. Article | PDF (296 K) | View Record in
Scopus | Cited By in Scopus (13)
Meijer et al., 2004 A.H. Meijer, S.F.G. Krens, I. A.M. Rodriguez, S. He, W. Bitter, B.E.
Snaar-Jagalska and H.P. Spaink, Expression analysis of the Toll-like receptor and TIR
domain adaptor families of zebrafish, Mol. Immunol. 40 (2004), pp. 773–783. Article |

PDF (692 K) | View Record in Scopus | Cited By in Scopus (117)

Muzio et al., 1997 M. Muzio, J. Ni, P. Feng and V.M. Dixit, IRAK (Pelle) family
member IRAK-2 and MYD88 as proximal mediators of IL-1 signalling, Science 278
(1997), pp. 1612–1615. Full Text via CrossRef | View Record in Scopus | Cited By in
Scopus (597)
Ooi et al., 2005 E.L. Ooi, T. Ohira, I. Hirono, T. Saito-Taki and T. Aoki, Immunoanalysis
of antiviral Mx protein expression in Japanese flounder (Paralichthys olivaceus) cells,
Dev. Comp. Immunol. 29 (2005), pp. 443–455.
Oshiumi et al., 2003 H. Oshiumi, T. Tsujita, K. Shida, M. Matsumoto, K. Ikeo and T.
Seya, Prediction of the prototype of the human Toll-like receptor gene family from the
pufferfish, Fugu rubripes, genome, Immunogenetics 54 (2003), pp. 791–800. View
Record in Scopus | Cited By in Scopus (72)
Oumouna et al., 2002 M. Oumouna, L. Jaso-Friedmann and D.L. Evans, Activation of
nonspecific cytotoxic cells (NCC) with synthetic oligodeoxynucleotides and bacterial
genomic DNA: binding, specificity and identification of unique immunostimulatory

motifs, Dev. Comp. Immunol. 26 (2002), pp. 257–269. Article | PDF (257 K) | View
Record in Scopus | Cited By in Scopus (24)
Peng, 2005 S.L. Peng, Signaling in B cells via Toll-like receptors, Curr. Opin. Immunol.

17 (2005), pp. 230–236. Article | PDF (2162 K) | View Record in Scopus | Cited By in
Scopus (57)
Press and Evensen, 1999 C.M. Press and Ø. Evensen, The morphology of the immune

system in teleost fishes, Fish. Shellfish. Immunol. 9 (1999), pp. 309–318. Abstract |
PDF (289 K) | View Record in Scopus | Cited By in Scopus (78)
Rutz et al., 2004 M. Rutz, J. Metzger, T. Gellert, P. Luppa, G.B. Lipford, H. Wagner and
S. Bauer, Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-
dependent manner, Eur. J. Immunol. 34 (2004), pp. 2541–2550. Full Text via CrossRef |
View Record in Scopus | Cited By in Scopus (161)
Solem and Stenvik, 2006 S.T. Solem and J. Stenvik, Antibody repertoire development in
teleosts—a review with emphasis on salmonids and Gadus morhua L., Dev. Comp.

Immunol. 30 (2006), pp. 57–76. Article | PDF (276 K) | View Record in Scopus |
Cited By in Scopus (23)
Stafford et al., 2003 J.L. Stafford, K.K. Ellestad, K.E. Magor, M. Belosevic and B.G.
Magor, A Toll-like receptor (TLR) gene that is up-regulated in activated goldfish

macrophages, Dev. Comp. Immunol. 27 (2003), pp. 685–698. Article | PDF (850 K) |
View Record in Scopus | Cited By in Scopus (39)
Takano et al., 2004 T. Takano, A. Iwahori, I. Hirono and T. Aoki, Development of a
DNA vaccine against hirame rhabdovirus and analysis of the expression of immune-
related genes after vaccination, Fish. Shellfish. Immunol. 17 (2004), pp. 367–374. Article

| PDF (127 K) | View Record in Scopus | Cited By in Scopus (22)

Takano et al., 2006 T. Takano, H. Kondo, I. Hirono, T. Saito-Taki, M. Endo and T. Aoki,
Identification and characterization of a myeloid differentiation factor 88 (MYD88) cDNA
and gene in Japanese flounder, Paralichthys olivaceus, Dev. Comp. Immunol. 30 (2006),

pp. 807–816. Article | PDF (2101 K) | View Record in Scopus | Cited By in Scopus
(7)
Takeda and Akira, 2005 K. Takeda and S. Akira, Toll-like receptors in innate immunity,
Int. Immunol. 17 (2005), pp. 1–14. View Record in Scopus | Cited By in Scopus (700)
Tassakka et al., 2006 A.C. Tassakka, R. Savan, H. Watanuki and M. Sakai, The in vitro
effects of CpG oligodeoxynucleotides on the expression of cytokine genes in the common
carp (Cyprinus carpio L.) head kidney cells, Vet. Immunol. Immunopathol. 110 (2006),
pp. 75–85.
Tsoi et al., 2006 S. Tsoi, K.C. Park, H.H. Kay, T.J. O’Brien, E. Podor, G. Sun, S.E.
Douglas, L.L. Brown and S.C. Johnson, Identification of a transcript encoding a soluble
form of Toll-like receptor 5 (TLR5) in Atlantic salmon during Aeromonas salmonicida

infection, Vet. Immunol. Immunopathol. 109 (2006), pp. 183–187. Article | PDF (281
K) | View Record in Scopus | Cited By in Scopus (9)
Tsujita et al., 2004 T. Tsujita, H. Tsukada, M. Nakao, H. Oshiumi, M. Matsumoto and T.
Seya, Sensing bacterial flagellin by membrane and soluble orthologs of Toll-like receptor
5 in rainbow trout (Oncorhynchus mykiss), J. Biol. Chem. 279 (2004), pp. 48588–48597.
View Record in Scopus | Cited By in Scopus (39)
Verjan et al., 2005 N. Verjan, I. Hirono and T. Aoki, Genetic loci of major antigenic
protein genes of Edwardsiella tarda, Appl. Environ. Microbiol. 71 (2005), pp. 5654–
5658. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (9)
Wesche et al., 1997 H. Wesche, W.J. Henzel, W. Shillinglaw, S. Li and Z. Cao, MYD88:
an adapter that recruits IRAK to the IL-1 receptor complex, Immunity 7 (1997), pp. 837–

847. Article | PDF (451 K) | View Record in Scopus | Cited By in Scopus (544)
Yazawa et al., 2005 R. Yazawa, I. Hirono, T. Ohira and T. Aoki, Functional analysis of
tumor necrosis factor gene promoter from Japanese flounder, Paralichthys olivaceus,

using fish cell lines, Dev. Comp. Immunol. 29 (2005), pp. 73–81. Article | PDF (341
K) | View Record in Scopus | Cited By in Scopus (4)