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Fish & Shellsh Immunology 30 (2011) 118e127

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Cloning of two rainbow trout nucleotide-binding oligomerization

domain containing 2 (NOD2) splice variants and functional
characterization of the NOD2 effector domains
Mingxian Chang a, b, Tiehui Wang a, Pin Nie b, Jun Zou a, Christopher J. Secombes a, *
Scottish Fish Immunology Research Centre, University of Aberdeen, Aberdeen AB24 2TZ, UK
State Key Laboratory of Freshwater Ecology and Biotechnology, and Laboratory of Fish Diseases, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan,
Hubei Province, 430072, P. R. China

a r t i c l e i n f o a b s t r a c t

Article history: Nucleotide-binding oligomerization domain 2 (NOD2) is a cytoplasmic pattern recognition receptor
Received 28 July 2010 (PRR), which is involved in innate antibacterial and antiviral responses. Here, two NOD2 splice variants,
Received in revised form trNOD2a and trNOD2b, are reported in rainbow trout Oncorhynchus mykiss, that share 63% and 61%
20 September 2010
similarity with human NOD2, respectively. These two trout NOD2 splice variants were shown to be
Accepted 20 September 2010
Available online 1 October 2010
constitutively expressed in thymus, gills, skin, muscle, liver, spleen, head kidney, intestine, heart, and
brain, with the expression of trout NOD2 (trNOD2) mainly contributed by trNOD2a in all the examined
tissues. PolyI:C transfection up-regulated the expression of trNOD2a and trNOD2b in RTG-2 cells. The
Rainbow trout
expression of trNOD2a/b was modulated by the inammatory stimulant interferon-g (IFN-g) or inter-
NOD2 leukin-1b (IL-1b). Overexpression of trout NOD2 effector domains resulted in induced expression of
Proinammatory cytokine proinammatory cytokines including IL-1b, tumor necrosis factor-a (TNF-a), IL-6 and IL-8, the antibac-
Apoptosis terial peptide cathelicidin-2, a variety of caspases including caspase-2, -6, -7, -8, -9, and type I and type II
IFN. These results suggest that sh NOD2 functions in inammatory events, possibly via NF-kB activation,
regulation of apoptosis, and triggering of antibacterial and antiviral defences.
2010 Elsevier Ltd. All rights reserved.

1. Introduction receptors (NLRs). RLRs are helicases that sense primarily viruses [3].
In contrast, NLRs are mainly involved in antibacterial immune
The innate immune system, the rst line of defence against responses [4].
infection, relies on host germline-encoded pathogen recognition In mammals, NLRs are multi-domain proteins composed of an
receptors (PRRs) to detect pathogen-associated molecular patterns amino-terminal effector-binding domain (EBDs) such as a caspase
(PAMPs). After sensing the presence of a PAMP, host innate immune recruitment domain (CARD), pyrin domain (PYD), acidic domain, or
cells initiate a broad spectrum of defence mechanisms that result in baculovirus inhibitor repeats (BIRs), a central nucleotide-binding
the development of inammation and host resistance to infection domain (NOD, also designated a NACHT domain) and carboxy-
[1]. PRRs comprise an array of sensors present in the extracellular, terminal leucine-rich repeats (LRRs) [5]. The N-terminal domain of
membrane, and cytoplasmic compartments. When pathogens the NLRs mediates signaling through its interaction with down-
bypass the membrane-associated PRRs by entering the cytosol of stream factors. The NOD domain is closely related to the oligo-
cells directly, or are actively transported into host cells by type III or merization module, which has been shown to be required for the
type IV secretion systems from microbes residing extracellularly, activation of downstream effector molecules [6]. The LRRs are
cytoplasmic PRRs are required to detect PAMPs [2]. Cytoplasmic essential for pathogen detection and recognition [7].
PRRs can be grouped into three families: interferon (IFN)-inducible The NOD2 gene belongs to the subfamily of NLRs characterized
proteins, the retinoic acid-inducible gene I (RIG-I)-like receptors by CARD-containing effector-binding domains. Vertebrate NOD2 is
(RLRs), and nucleotide-binding oligomerization domain (NOD)-like composed of two adjacent N-terminal CARDs, a centrally located
NOD domain and multiple C-terminal LRRs [8e10]. In mammals,
NOD2 induces multiple effector signaling pathways to resist
* Corresponding author. Tel.: 44 1224 272872; fax: 44 1224 272396. microbial pathogens. The best characteristic of these is the activa-
E-mail address: (C.J. Secombes). tion of NF-kB and MAPKs through interactions with the CARD

1050-4648/$ e see front matter 2010 Elsevier Ltd. All rights reserved.
M. Chang et al. / Fish & Shellsh Immunology 30 (2011) 118e127 119

domain of receptor-interacting protein 2 (RIP2) [11]. NOD2 role of NOD2 in NF-kB activation, regulation of apoptosis, and
signaling by activating NF-kB and MAPKs leads to the induction of triggering of antibacterial and antiviral defences is conserved in
proinammatory cytokines and chemokines [12], and anti-micro- teleost sh.
bial peptides [13], which mediate the antimicrobial response.
Besides the function in antibacterial defence, new studies show 2. Materials and methods
that NOD2 also has an important role in recognizing viruses and
inducing type I IFN production through association with the 2.1. Cloning and sequencing of trout NOD2
mitochondrial antiviral signaling protein (MAVS) [14].
In teleost sh, orthologs of human NOD2 have been reported in The expressed sequence tag (EST) sequences in the Computa-
zebrash [15,16], channel catsh [17], and grass carp [10]. In the tional Biology and Functional Genomic Laboratory (http://compbio.
present study, our main aims were to assess whether sh NOD2 were searched using
can undergo alternative splicing, as well as whether NOD2 the grass carp NOD2 protein sequence [10] as a bait sequence for
domains have different effects in inammatory events. The results TBLASTX analysis. Several candidate ESTs were found with high
we present clearly show that a region of the NOD2 RNA transcript sequence homology to known NODs, among which EST sequences
that encodes the N-terminal CARD domains is alternatively TC74620 (Salmo salar) and CX244678 (Oncorhynchus mykiss)
spliced, and that overexpression of the trout NOD2 different aligned well with the C terminal and middle region of NOD2
domains has a similar role in the production of proinammatory respectively. Based on the sequence of a partial trout NOD2
cytokines, an antibacterial peptide, a variety of caspases, and type I (CX244678), nested primers trNOD2GSP1 and trNOD2GSP2
and type II IFN. The present study provides evidence that the (Table 1) were designed and used in 50 RACE using a GeneRacer

Table 1
Primer sequences used in this study.

The rst run PCR for 50 RACE GeneRacer 50 Primer CGACTGGAGCACGAGGACACTGA

The nested PCR for 50 RACE GeneRacer 50 Nested Primer GGACACTGACATGGACTGAAGGAGTA
The rst run PCR for 30 coding region trNOD2Fout GGACCGCTCCACCATCCCTAA
The nested PCR for 30 coding region trNOD2Fin GCAAGACAGGCAGCAACAGC
120 M. Chang et al. / Fish & Shellsh Immunology 30 (2011) 118e127

Kit (Invitrogen). The cDNA template used in the PCR was generated 2.5. Modulation of trout NOD2 gene expression by IL-1b and IFN-g
from RTS-11 cells [18] stimulated with 50 ng/mL IFN1 recombinant in RTG-2 cells
protein [19] for 4 h. The annealing temperature of the rst round
and nested PCR was 66  C and 68  C, respectively. Specic primers The cells were passaged into fresh culture asks 2 days before
trNOD2Fout/trNOD2Fin (based on the trout NOD2 sequence) and being stimulated for 6 h with either 20 ng/ml recombinant trout
trNOD2Rout/trNOD2Rin (based on the salmon NOD2 30 non-coding IL-1b (rIL-1b) protein [22], or 20 ng/ml recombinant trout inter-
sequence from TC74620) (Table 1) were designed to obtain the feron-g protein (rIFN-g) [23] or left untreated as control. The RNA
30 end of the coding region. The annealing temperature of the rst preparation and real-time PCR analysis were as described above.
and nested PCR was 55  C. The primer pairs for analysis of trout NOD2 expression were
To conrm the two transcripts of trout NOD2 resulted from trNOD2F/trNOD2R, trNOD2aF/trNOD2abR and trNOD2bF/trNO-
alternative splicing of the same gene, two primers (trNOD2F1 and D2abR (Table 1).
trNOD2R1) were designed across a divergent region of the cDNA
sequences and used for PCR amplication from a set of genomic 2.6. Plasmid construction for overexpression of NOD2 domains
DNA and cDNA samples from three trout cell lines (RTGill, RTG-2
and RTS-11 cells). The annealing temperature of this PCR For overexpression studies of different effector domains, an
was 65  C. expression vector ptGFP1 was modied from the pTurboGFP-N
vector and contained two sets of the CMV promoter and SV40 30
UTR to drive expression of the target gene and GFP as separate
2.2. Sequence analysis proteins rather than as a fusion protein. The trNOD2a CARD,
trNOD2b CARD, NACHT and LRR domain was amplied with primer
Protein prediction was performed using software at the ExPASy pairs trNOD2aF/trNOD2aR, trNOD2bF/trNOD2bR, NACHTF/NACHTR
Molecular Biology Server ( The putative and LRRF/LRRR (Table 1) respectively, and inserted into the Hind III
ORFs were analyzed for the presence of signal peptides using the and Sac II sites of the ptGFP1 vector to make constructs ptGFP1-
algorithms Signal P 3.0 ( trNOD2a CARD, ptGFP1-trNOD2b CARD, ptGFP1-NACHT and
Multiple alignments were generated by the CLUSTAL 1.8 program ptGFP1-LRR.
within DNASTAR. A phylogenetic tree was constructed based on the
deduced amino acid sequences using the Neighbour-Joining (NJ)
algorithm within MEGA version 4 ( 2.7. Database mining of trout caspase genes
mega.html). Data were analyzed using Poisson correction, and
gaps were removed by pairwise deletion. Reliability of the tree was In trout, only caspase-6 has been reported previously [24],
assessed by 1000 bootstrap repetitions. although in salmon caspase-3, -6, and -7 have also been reported
[25]. To identify other caspase genes in trout, a tblastn search using
salmon caspase protein sequences as baits was performed against
2.3. Expression of trout NOD2 splice variants in vivo the rainbow trout ESTs in the Computational Biology and Func-
tional Genomic Laboratory database. The translated proteins from
The selection of tissues, RNA preparation, cDNA synthesis and predicted transcripts were veried by BLASTP in the NCBI non-
real-time PCR analysis of gene expression was described previously redundant protein sequence database. To help dene the homology
[20]. Briey, six healthy rainbow trout (average of trout caspase molecules among caspase subfamilies, a phyloge-
weight  SEM 106  5 g) were killed and ten tissues (thymus, netic tree was constructed using the MEGA4 program.
gills, skin, muscle, liver, spleen, head kidney, intestine, heart, and
brain) were collected. Total RNA was prepared from each tissue and 2.8. Modulation of the inammasome and apoptosis signaling
converted to cDNA, and the 60 (6  10) cDNA samples generated pathways in RTG-2 cells
were used in the gene expression analysis. The primer pairs used to
amplify the trout NOD2 splice variants were trNOD2aF/trNOD2abR RTG-2 cells were used for the overexpression studies because of
and trNOD2bF/trNOD2abR (Table 1). several trout cell lines available to us (e.g. RTGill, RTG-2 and RTS-11
cells), only RTG-2 cells had an optimised transfection protocol
already established. RTG-2 cells were maintained in L-15 medium
2.4. Expression of trout NOD2 splice variants in RTG-2 cells supplemented with 10% foetal bovine serum (FBS) (Sigma), 100 U/
transfected with polyI:C ml penicillin (P) and 100 mg/ml streptomycin (S) at 20  C and
passaged 2 days before use. 5  106 RTG-2 cells were transfected
RTG-2 cells (5  106 cells in 100 ml nucleofector solution) were with 2 mg ptGFP1, ptGFP1-trNOD2a CARD, ptGFP1-trNOD2b CARD,
electroporated with 5 ml polyI:C (1 mg/ml) or 5 ml PBS using an ptGFP1-NACHT or ptGFP1-LRR. The cells were washed immediately
Amaxa Nucleofector II transfection system (Lonza) under Pro- with culture medium and incubated at 20  C for 6 h. The RNA
gramme T20, washed immediately with 1 ml HBSS buffer and preparation and real-time PCR analysis was as described above. The
cultured at 20  C for 24 h. The cells were collected for RNA genes studied by real time PCR were trNOD2 (this paper), IRF3 and
extraction and real time PCR analysis as described previously [21]. IRF7 [26], IFN1 and IFN2 [19], IFN-g1 [23], IFN-g2 [27], IL-1b1 [28],
Namely after total RNA was extracted using the RNASTAT reagent IL-6 [29], IL-8 [30], TNFa [31], CATH2 [32], caspase-1 (TC Number of
(AMS Biotechnology (Europe) Ltd) and treated with RNase free rainbow trout EST, TC172333), -2 (TC Number of rainbow trout EST,
DNase I (Fermentas Life Sciences, Germany), the RNA was reverse TC170354), -3 (TC Number of rainbow trout EST, TC148559), -6
transcribed into cDNA using a rst strand cDNA synthesis kit (Fer- [24], -7 (TC Number of rainbow trout EST, TC154414), -8 (TC
mentas Life Sciences, Germany). Real time PCR analysis was per- Number of rainbow trout EST, TC133215), and -9 (GeneBank
formed on a Roche LightCycler 480 using the following accession no. NP_001118119). The primer sequences of IFN1 and
conditions: 1 cycle of 95  C/10 min, 45 cycles of 95  C/20 s, 60  C/ IFN2 were reported by Zou et al. [19], IFN-g1 by Daz-Rosales et al.
20 s and 72  C/30 s. Each sample was run in triplicate in a 96 well [33], IL-1b1 and IL-8 by Wang et al. [34]. The primer sequences of
plate and the mean value recorded. other genes are listed in Table 1.
M. Chang et al. / Fish & Shellsh Immunology 30 (2011) 118e127 121

2.9. Statistical analysis

The relative expression of target genes was normalized to the

expression of elongation factor (EF) -1a and expressed as arbitrary
units or fold change relative to the corresponding control group.
The mean of at least three independent experiments was used for
statistical analysis using the paired student t test. P < 0.05 was
considered to be signicant.

3. Results

3.1. Sequence analysis of NOD2 transcripts in rainbow trout

By 50 -RACE, three main products were amplied from rainbow

trout. The sizes of these products were approximately 1.9 kb, 1.5 kb
and 1 kb. Five clones for every product were sequenced. The
products of 1.9 kb and 1.5 kb were found to be NOD2 transcripts,
whereas non-specic amplication resulted in the 1 kb product.
The open reading frame of the two NOD2 transcripts was conrmed
by RT-PCR. The shorter transcript of rainbow trout NOD2 (named
trNOD2a, GenBank accession No, HM133906) is 3121 bp, and
encodes a protein of 996 aa. Another transcript of 3511 bp (named
trNOD2b, GenBank accession No, HM133907) was also sequenced,
and had a longer 50 UTR and a 65 bp deletion including the normal
start codon ATG, resulting in the predicted translation starting from
the next downstream ATG. As a result, trNOD2b is predicted to be
38 aa shorter than trNOD2a (Fig. 1). Pfam HMM analysis predicted
two CARD domains at the N terminus and a NACHT domain in the
middle both for trNOD2a and trNOD2b, whereas the rst CARD
domain of trNOD2b had insignicant Pfam-A matches by Pfam
HMM analysis due to the high E-value (0.017) and low score (14.6).
The conserved domains analysis in the NCBI database also pre-
dicted a multi Leucine-rich repeats (LRRs) domain in aa 766-987/
728-949 for trNOD2a/trNOD2b, which was not predicted by Pfam
HMM analysis.
The full ORF of trNOD2a has a similarity of 81.1% with the
reported grass carp NOD2, and 81.2% with zebrash NOD2. The
N-terminal two CARD domains, central NACHT domain and
C-terminal LRR of trNOD2a have similarities of 71.3%, 74.1%, 88.4%
and 89.3% with the corresponding domains of grass carp NOD2, and
of 56%, 56.3%, 75.6% and 66.2% with the corresponding domains of
human NOD2. The rst CARD domain of trNOD2b has a similarity of
45.2% and 30.8% with grass carp and human NOD2, respectively.

3.2. Rainbow trout NOD2 transcripts are splice variants

from the same gene

A single product was amplied from genomic DNA from six sh

studied but two products were amplied from different cDNA
samples, as seen with the cell line samples used (Fig. 2A). The larger
product was strong and of the expected size for trNOD2a (343 bp)
whilst the smaller product was weaker and of the expected size
from trNOD2b (278 bp).
The product amplied from genomic DNA was sequenced, and
the sequence results showed that all the products from genomic
DNA were identical and 805 bp in length. By comparison with the
cDNA sequences, it was clear that 462 bp of the genomic sequence
was spliced out in NOD2a, whilst a further 65 bp downstream was
also spliced out in NOD2b (Fig. 2B).

3.3. Expression of trout NOD2 splice variants in vivo Fig. 1. Multiple alignment of sh NOD2 protein sequences. Identical amino acids are
indicated with asterisks, and conservative substitutions are indicated with . or :.
The CARD, NACHT and LRR domains are underlined, double-underlined and bold
The expression of trNOD2a and trNOD2b splice variants was underlined, respectively. The amino acids labeled in grey are spliced out in trNOD2b.
examined in ten tissues, including gill, thymus, brain, skin, muscle,
liver, spleen, head kidney, intestine, and heart, from six healthy sh
122 M. Chang et al. / Fish & Shellsh Immunology 30 (2011) 118e127

Fig. 2. Conrmation of two trout NOD2 splice variants. A: the amplication of trout NOD2 in cDNA from RTGill, RTG and RTS-11 cell lines, and genomic DNA from 6 sh. B: the
alignment of trout NOD2 genomic DNA with the cDNA of trNOD2a and trNOD2b. 462 bp of the genomic sequence (in lower case) was spliced out in NOD2a, whilst a further 65 bp
downstream (highlighted in grey) was also spliced out in NOD2b. The primers used for amplication are underlined. The consensus GT/AG splice sequences are boxed. The start
codons of trNOD2a and trNOD2b are highlighted in bold, with the latter 38 aa shorter than the former.

using real-time PCR. The expression of the two splice variants was respectively. A high level expression of trNOD2a was also detected
detectable in all the tissues examined (Fig. 3). The highest level of in skin and brain. It was apparent that the expression of trNOD2
trNOD2a and trNOD2b was detected in muscle, whereas the lowest was mainly contributed by trNOD2a in all the examined tissues
level of trNOD2a and trNOD2b was observed in heart and liver, (>95%).
M. Chang et al. / Fish & Shellsh Immunology 30 (2011) 118e127 123

(at 20 ng/ml) for 6 h. Stimulation with rIL-1b or rIFN-g has

a differential effect on trNOD2a and trNOD2b. The expression of
trNOD2a was signicantly increased by stimulation with rIFN-g,
and of trNOD2b by rIL-1b. The total expression of trout NOD2 was
also detected by real time PCR. Similar to trNOD2a, stimulation with
rIFN-g resulted in increased overall gene expression of trNOD2,
whereas there was no effect of rIL-1b (Fig. 5).

3.6. Identication of trout inammatory and apoptosis caspases

Fig. 3. In vivo expression of the two trout NOD2 splice variants. The expression of the
trout NOD2 splice variants was determined by real time PCR in tissues of six sh. The In addition to trout caspase-6, multiple caspase EST sequences
transcript level was rst calculated using a serial dilution of references in the same run. including caspase-1, -2, -3, -7, -8 and -9 were found in the trout
The relative expression level for each gene was then expressed as arbitrary units database. The EST sequences of caspase-8 and -9 encode for the
normalized against the expression level of EF-1a. The results presented are the complete open reading frame. Although the other EST sequences of
average S.E.M. of six sh.
caspase-1, -2, -3 and -7 were partial, these EST sequences when
translated were found to encode for the conserved caspase domain
3.4. Expression of trout NOD2 splice variants in RTG-2 cells for caspase-2, -3 and -7, or the conserved CARD domain for caspase-
transfected with polyI:C 1. In order to conrm these caspase molecules were true orthologs
of known mammalian caspases, a phylogenetic tree based on
It is known that NOD2 is an intracellular pattern recognition a multiple alignment of selected vertebrate caspase members was
receptor. To study the effect of polyI:C, a synthetic double stranded constructed using the MEGA4 program. Each trout caspase mole-
RNA mimicking viral dsRNA, on trout NOD2 expression, RTG-2 cells cules formed a sub-branch with corresponding caspase molecule
were electroporated with 5 ml polyI:C or 5 ml PBS as a control. from Atlantic salmon and/or zebrash and, with the exception of
trNOD2a and trNOD2b were both signicantly induced by this caspase 1, formed clear clades with other vertebrate caspase
stimulation, with a 17.4- and 21.8-fold increase of expression level molecules (Fig. 6). The high bootstrap value of all sub-branches
respectively (Fig. 4). (>80%) suggests this is a reliable phylogenetic tree.

3.5. The effect of proinammatory cytokines on trout NOD2 3.7. Overexpression of trout NOD2 domains induced expression
of trNOD2
To study the effect of proinammatory cytokines on trout NOD2
gene expression, RTG-2 cells were stimulated with rIL-1b or rIFN-g To determine whether the different trout NOD2 domains play
any role in inammation and/or apoptosis, RTG-2 cells were
transfected with ptGFP1-trNOD2a CARD, ptGFP1-trNOD2b CARD,
ptGFP1-NACHT, ptGFP1-LRR or ptGFP1 as a control. Overexpression
of trout NOD2 was apparent, with a signicant increase of the
transcript levels compared with ptGFP1 transfected cells; 35- fold
for the trNOD2a CARD domain, 82- fold for the trNOD2b CARD
domain, 8- fold for the NACHT domain, and 30- fold for the LRR
domain, respectively (Fig. 7).

3.8. Modulation of inammatory cytokine and AMP expression

by trout NOD2 effector domains

In mammals, stimulation of NOD2 results in the activation of

caspase-1 and the secretion of proinammatory cytokines. In the
present study, overexpression of trout NOD2 effector domains

Fig. 4. The effect of transfected polyI:C on expression of the two trout NOD2 splice
variants in RTG-2 cells. The RTG-2 cells (5  106 cells) were electroporated with 5 mg Fig. 5. The effect of proinammatory cytokines on trout NOD2 expression. RTG-2 cells
polyI:C, washed and cultured for 24 h. The cells were collected and used for RNA were stimulated for 6 h with 20 ng/ml trout rIL-1b or rIFN-g. The cells were collected
extraction and real time PCR analysis. The relative expression of target genes was and used for RNA extraction and real time PCR analysis. The relative expression of
normalized to the expression of EF-1a and expressed as fold change relative to the target genes was normalized to the expression of EF-1a and expressed as fold change
appropriate control group. The mean of three independent experiments is shown and relative to the control group. The mean of three independent experiments is shown
bars indicate the SEMs. Asterisks indicate where the increase of gene expression is and bars indicate the SEMs. Asterisks indicate where the increase of gene expression is
signicant (P < 0.05). signicant (P < 0.05).
124 M. Chang et al. / Fish & Shellsh Immunology 30 (2011) 118e127

Fig. 7. Construction and effect of different trout NOD2 domains. A: The domains used
to construct ptGFP1-trNOD2a CARD, ptGFP1-trNOD2b CARD, ptGFP1-NACHT and
ptGFP1-LRR plasmids. B: Effect of overexpression of different trout NOD2 domains on
expression of trNOD2. RTG-2 cells (5  106 cells) were electroporated with 2 mg ptGFP1,
ptGFP1-trNOD2a CARD, ptGFP1-trNOD2b CARD, ptGFP1-NACHT or ptGFP1-LRR
plasmid. After 6 h, the cells were collected and used for RNA extraction and real time
PCR analysis. The relative expression of target genes was normalized to the expression
of EF-1a and expressed as fold change relative to the appropriate control group. The
mean of three independent experiments is shown and bars indicate the SEMs. Aster-
isks indicate where the increase of gene expression is signicant (P < 0.05).

Fig. 6. Phylogenetic relationship of vertebrate caspases genes. The amino acid

sequences were aligned using the CLUSTAL program within DNASTAR and the phylo-
genetic tree was constructed using the Neighbour-Joining algorithm within MEGA
version 4. The tree was boosted 1000 times and the percentages are shown. The
sequences used for tree construction are as follows: Atlantic salmon caspase 1,
ACI69403; rainbow trout caspase 1, TC172333; rabbit caspase 1, XP_002708463;
Norway rat caspase 1, NP_036894; human caspase 1, BAD97223; Atlantic salmon
caspase 3, ACN11423; rainbow trout caspase 3, TC148559; Fugu caspase 3, AAM43816;
zebrash caspase 3, CAX14649; chicken caspase 3, NP_990056; mouse caspase 3,
AAH38825; human caspase 3, CAC88866; Atlantic salmon caspase 7, AAY28975;
rainbow trout caspase 7, TC154414; zebrash caspase 7, NP_001018443; African clawed
frog caspase 7, NP_001081408; mouse caspase 7, NP_031637; human caspase 7,
AAH15799; rainbow trout caspase 6, NP_001117743; Atlantic salmon caspase 6A,
AAY28971; Atlantic salmon caspase 6B, AAY28974; zebrash caspase 6,
NP_001018333; African clawed frog caspase 6, NP_001081406; mouse caspase 6,
AAH02022; human caspase 6, AAH04460; rainbow trout caspase 9, NP_001118119;
zebrash caspase 9, NP_001007405; mouse caspase 9, NP_056548; African clawed frog
caspase 9, NP_001079035; human caspase 9, AAO21133; rainbow trout caspase 8,
TC133215; Atlantic salmon caspase 8, ACN10768; zebrash caspase 8, NP_571585;
mouse caspase 8, CAA07677; human caspase 8, AAD24962; chicken caspase 8,
NP_989923; rainbow trout caspase 2, TC170354; zebrash caspase 2, NP_001036160;
chicken caspase 2, NP_001161173; mouse caspase 2, NP_031636; human caspase 2,

resulted in increased gene expression of the proinammatory

cytokines IL-1b, IL-6, IL-8 and TNF-a (P < 0.05), whereas there was
no effect on caspase-1 expression (Fig. 8A). Notably, a 21- and
17-fold increase in IL-1b and TNF-a transcript level was detected in
cells transfected with ptGFP1-trNOD2b CARD.
Mammalian NOD2 signaling leads to the production of antimi- Fig. 8. Modulation of inammasome genes and the antimicrobial peptide cathelicidin-
crobial peptides. In rainbow trout, Chang et al. [32] have previously 2 by trout NOD2 effector domains. A: The effect of overexpression of trout effector
domains on caspase 1 and various proinammatory cytokines. B: The effect of over-
identied two cathelicidin genes, with the expression of cath- expression of trout NOD2 effector domains on the antibacterial peptide cathelicidin-2.
elicidin-2 (cath2) high in RTG-2 cells. Thus, we next examined the The sample preparation and real time PCR analysis were as in Fig. 7. Asterisks indicate
expression of cath2 in cells tranfected with the trout NOD2 effector where the increase of gene expression is signicant (P < 0.05).
M. Chang et al. / Fish & Shellsh Immunology 30 (2011) 118e127 125

domains. Induced expression was apparent with signicant

increases compared with cells transfected with ptGFP1 in all cases.
The induced fold increases seen using trNOD2a CARD, trNOD2b
CARD, NACHT and LRR were 2.4-, 23-, 15-, and 9-fold, respectively
(Fig. 8B).

3.9. Overexpression of trout NOD2 effector domains induces

expression of caspase genes involved in apoptosis

In trout, the sequences of caspase-2, -3, -7, -8, and -9 were

identied by database mining, as described in section 3.6. The cells
transfected with ptGFP1-trNOD2a CARD, ptGFP1-NACHT, or
Fig. 10. Effect of overexpression of trout NOD2 effector domains on expression of IRF3,
ptGFP1-LRR exhibited increased caspase-2, -6, -7, -8, and -9 IRF7 and both type I and II IFN. The sample preparation and real time PCR analysis
expression compared with cells transfected with ptGFP1 (Fig. 9) were as in Fig. 7. Asterisks indicate where the increase of gene expression is signicant
(P < 0.05). Cells transfected with ptGFP1-trNOD2b CARD also had (P < 0.05).
increased expression of caspase-6, -7, -8 and -9 but not caspase-2.
No effect on caspase-3 was seen with any domain.
alternatively spliced, giving two protein products encoded by two
3.10. Overexpression of trout NOD2 effector domains induces mRNA splice variants at the N-terminal [35,36], and one protein
expression of both type I and II IFN product encoded by ten different mRNA splice variants in the LRR
domain [37]. Alternative splice variants also exist in the sh NOD2
Using real-time PCR, the induced mRNA expression of type I and homologs, as seen in the present studies with rainbow trout where
II IFN after overexpression of trout effector domains was analyzed two splice variants were discovered, one (trNOD2b) with the rst
in RTG-2 cells. In each case, the expression of type I and II IFN was CARD domain partially deleted. Many PRRs including NOD1 [38],
increased signicantly in transfected cells (P < 0.05), with the Dectin-1 [39], TLRs [40], PGRPs [41], undergo alternative splicing
exception of IFN-g1 in NACHT transfected cells. In view of these generating multiple forms. It has been argued that each splice
results the transfected cells were also used to examine the variant may have a role in a coordinated defence response [40] or
expression of upstream genes that induce IFN responses. The that the truncated splice variants may be competitive inhibitors in
expression of IRF3 and IRF7, especially IRF7, was signicantly up- downstream signaling induced by the normal form [35].
regulated by the various trNOD2 domains (Fig. 10). However, Similar to human NOD2-S (a truncated splice variant), the
overexpression of the LRR domain had no impact on the expression expression of trNOD2b was rather low under basal conditions
of IRF3 (Fig. 10). compared to trNOD2a, which may suggest that trNOD2a plays
a more dominant role in trout innate immunity. In agreement with
the studies in mammals [42], trNOD2 was up-regulated in trout
4. Discussion
RTG-2 cells by an inammatory stimulant (IFN-g), which conrmed
that proinammatory signals could trigger the induction of NOD2
In this paper we report on the cloning of rainbow trout NOD2, an
in cells. Since NOD2 is able to sense the presence of Gram-negative
intracellular pattern recognition receptor [4], and analyze its
or Gram-positive bacteria in the cytosolic compartment through
expression and function in this species. NOD2 was rst identied in
the detection of MDP, the up-regulation of the two trout splice
2001 by Ogura et al. [8] and functions to activate NF-kB signaling
variants upon polyI:C transfection indicates the two splice variants
through its interaction with the serine-threonine kinase RIP2.
are likely to contribute to the early innate immune defence against
Previous analysis has shown that NOD1/NOD2 are well conserved
invading pathogens.
among vertebrates [15,16], and here we show that the function of
The present work also studied the overexpression of the three
NOD2 in inammation and apoptosis has been conserved during
distinct effector domains of trout NOD2, and showed they are able
vertebrate evolution.
to induce the expression of proinammatory cytokines and the
In mammals, a large variety of alternative splice variants have
antibacterial peptide cathelicidin-2, consistent with reports in
been reported for NOD2. Human NOD2 RNA transcripts that
mammals that show NOD2 is able to mediate antibacterial
encode the N-terminal and leucine-rich repeat (LRR) domains are
responses during inammatory events via NF-kB signaling, to
induce the secretion of proinammatory cytokines and antimi-
crobial peptides [12,43]. However, mammalian NOD2 was also
shown to induce caspase-1 activation [44,45] but the present study
found no effect of trout NOD2 on caspase-1 expression. Whilst
further study is needed to determine whether sh NOD2 could
activate caspase-1 at the post-translational level, it should also be
noted that the role of caspase-1 in the sh inammasome is less
clear with no obvious ICE cute site present in IL-1b for example [28].
Extensive data mining of sh genomes has revealed a range of
orthologs of mammalian caspases in both zebrash and other non-
model sh species such as salmon [25,46]. In rainbow trout little is
known about the structure and function of caspases, except for the
study of Laing et al. [24] which showed the immunoregulatory role
of trout caspase-6. The present study revealed the existence of
Fig. 9. Effect of overexpression of trout NOD2 effector domains on expression of
caspase genes involved in apoptosis. The sample preparation and real time PCR
caspase orthologs in rainbow trout that are potentially involved in
analysis were as in Fig. 7. Asterisks indicate where the increase of gene expression is apoptosis, based on a few functional studies on sh caspases that
signicant (P < 0.05). have shown caspase-2, -3, -6, -7, -8 and -9 can induce apoptosis
126 M. Chang et al. / Fish & Shellsh Immunology 30 (2011) 118e127

[25,47e51]. In humans, members of NLRs can coordinate caspase- [13] Voss E, Wehkamp J, Wehkamp K, Stange EF, Schrder JM, Harder J. NOD2/
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Acknowledgements Molecular characterization of IRF3 and IRF7 in rainbow trout, Oncorhynchus
mykiss: Functional analysis and transcriptional modulation. Mol Immunol
This work was supported by the National Natural Science 2008;46:269e85.
[27] Purcell MK, Laing KJ, Woodson JC, Thorgaard GH, Hansen JD. Characterization
Foundation of China (30830083), the Royal Society of Edinburgh of the interferon genes in homozygous rainbow trout reveals two novel genes,
and the EU Imaquanim project. alternate splicing and differential regulation of duplicated genes. Fish Shell-
sh Immunol 2009;26:293e304.
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