ARTICLE IN PRESS Developmental and Comparative Immunology ■■ (2015) ■■–■■
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Developmental and Comparative Immunology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d c i
Short communication
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Molecular cloning and expression studies of the adapter molecule myeloid differentiation factor 88 (MyD88) in turbot (Scophthalmus maximus)
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Jing-Yun Lin a, Guo-Bin Hu a,b,*, Chang-Hong Yu c, Song Li a, Qiu-Ming Liu a, Q2 Shi-Cui Zhang a,b a
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College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China c College of Medicine, Qingdao University, Qingdao 266071, China b
A R T I C L E
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Article history: Received 12 March 2015 Revised 22 May 2015 Accepted 23 May 2015 Available online Keywords: MyD88 Scophthalmus maximus LPS CpG-ODN TRBIV Gene expression
A B S T R A C T
Myeloid differentiation factor 88 (MyD88) is an adapter protein involved in the interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR)-mediated activation of nuclear factor-kappaB (NF-κB). In this study, a full length cDNA of MyD88 was cloned from turbot, Scophthalmus maximus. It is 1619 bp in length and contains an 858-bp open reading frame that encodes a peptide of 285 amino acid residues. The putative turbot (Sm)MyD88 protein possesses a N-terminal death domain and a C-terminal Toll/IL-1 receptor (TIR) domain known to be important for the functions of MyD88 in mammals. Phylogenetic analysis grouped SmMyD88 with other fish MyD88s. SmMyD88 mRNA was ubiquitously expressed in all examined tissues of healthy turbots, with higher levels observed in immune-relevant organs. To explore the role of SmMyD88, its gene expression profile in response to stimulation of lipopolysaccharide (LPS), CpG oligodeoxynucleotide (CpG-ODN) or turbot reddish body iridovirus (TRBIV) was studied in the head kidney, spleen, gills and muscle over a 7-day time course. The results showed an up-regulation of SmMyD88 transcript levels by the three immunostimulants in all four examined tissues, with the induction by CpG-ODN strongest and initiated earliest and inducibility in the muscle very weak. Additionally, TRBIV challenge resulted in a quite high level of SmMyD88 expression in the spleen, whereas the two synthetic immunostimulants induced the higher levels in the head kidney. These data provide insights into the roles of SmMyD88 in the TLR/IL-1R signaling pathway of the innate immune system in turbot. © 2015 Published by Elsevier Ltd.
46 1. Introduction
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The innate immunity in fish, as in all vertebrates, is the first line of defense and provides crucial signals for activation of adaptive immune responses (Akira et al., 2001). Detection and clearance of invading pathogens by the innate immune system are associated with plenty of signaling pathways that are evolutionarily conserved throughout vertebrates. It works through the way of being triggered when pathogen-associated molecular patterns (PAMPs) come into contact with host-expressed pattern recognition receptors (PRRs) (Medzhitov and Janeway, 2000). One of the wellcharacterized PRRs is the family of Toll-like receptors (TLRs) that detects microbial PAMPs such as bacterial lipopolysaccharides (LPS), peptidoglycans (PGN) and flagellin, viral RNA, unmethylated CpG DNA of viruses, bacteria and protozoa, β-glycan of fungi and
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* Corresponding author. College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China. Tel.: +86 532 82032583; fax: +86 532 82032583. E-mail address: [email protected] (G.-B. Hu).
lipoproteins of various pathogens (Akira et al., 2006; Mogensen, 2009). The signaling pathways mediated by TLRs are broadly classified into the myeloid differentiation factor 88 (MyD88)-dependent and -independent ones. The former uses MyD88 as an adapter molecule to activate the signaling cascades and produces inflammatory mediators. Although MyD88 was first found in mice in 1990 as a myeloid differentiation primary response gene induced during terminal differentiation of M1D + myeloid precursor cells in response to interleukin (IL)-6 treatment (Lord et al., 1990), its function as a key adapter molecule in the interleukin-1 receptor (IL-1R)/TLR-mediated signaling remains unknown until 1997 (Wesche et al., 1997). MyD88 has a bipartite structure composed of an N-terminal death domain and a C-terminal Toll/IL-1 receptor (TIR) domain with a short intervening linker segment. Upon activation by PAMPs, all of TLRs except TLR3 recruit MyD88 through the TIR domain; the death domain interacts with the corresponding domain in IL-1R-associated kinases (IRAKs), leading to recruitment of downstream tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6); these events eventually result in activation of NF-κB and interferon
http://dx.doi.org/10.1016/j.dci.2015.05.013 0145-305X/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: Jing-Yun Lin, et al., Molecular cloning and expression studies of the adapter molecule myeloid differentiation factor 88 (MyD88) in turbot (Scophthalmus maximus), Developmental and Comparative Immunology (2015), doi: 10.1016/j.dci.2015.05.013
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regulator factors (IRFs) and induction of pro-inflammatory cytokines or antiviral genes that play an important role in combating pathogens (Deepika et al., 2014). To date, MyD88 has been identified in mammals, birds, reptiles, amphibians, fishes and invertebrates (Bonnert et al., 1997; Deepika et al., 2014; Li et al., 2011; Prothmann et al., 2000; Wheaton et al., 2007). The fishes with MyD88 identified include zebrafish (van der Sar et al., 2006), Japanese flounder (Takano et al., 2006), half-smooth tongue sole (Yu et al., 2009), large yellow croaker (Yao et al., 2009), Atlantic salmon (Skjæveland et al., 2009), rainbow trout (Rebl et al., 2009), rock bream (Whang et al., 2011), common carp (Kongchum et al., 2011), grass carp (Su et al., 2011), orange-spotted grouper (Yan et al., 2012), miiuy croaker (Tang et al., 2012), etc. Fish MyD88 exhibits structural and functional homologies with its mammalian counterpart. Turbot, Scophthalmus maximus, is an important commercial marine species cultured widely in the world. However, the knowledge about turbot (Sm)MyD88 is scarce. The aim of this study is to improve understanding of the innate immune system in turbot by studies of a MyD88 gene, which will help in development of strategies of microbial infectious disease control for this species. Herein, we report the cDNA sequence, mRNA tissue distribution and transcriptional modulation of SmMyD88. The last study was performed in vivo upon stimulation of turbots with LPS, synthetic CpG oligodeoxynucleotide (CpG-ODN) or turbot reddish body iridovirus (TRBIV). We demonstrated the involvement of SmMyD88 in immune responses of turbots to these three immunostimulants. 2. Materials and methods 2.1. Fish, stimulants and immunostimulation experiments Turbot (S. maximus) juveniles (68.4 ± 4.5 g, n = 170) were purchased from a local fish farm. Fish were kept in aerated seawater tanks at 16 °C for 1 week before use. LPS (L2880, Escherichia coli 055:B5; Sigma, St. Louis, MO, USA) was diluted in phosphate buffer saline (PBS, pH 7.4) to make stock. CpG-ODN 2395 (class C, 5′-T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G-3′, phorothioate modifications are marked with *; Coley Pharmaceutical Group, Ottawa, CA) was dissolved in TE buffer (10 mM Tris, 1 nM EDTA, pH 8.0). TRBIV was isolated from tumor-carrying turbots as previously described (Hu et al., 2012). The viral titers were measured by a 50% tissue culture infective dose (TCID50) assay according to the method of Reed and Muench (1938). Three groups of turbots were intraperitoneally (i.p.) injected with LPS (2.5 mg/ml, 112 μl per fish), CpG-ODN 2395 (0.15 mg/ml, 95 μl per fish) or TRBIV (2 × 106 TCID50/ml, 120 μl per fish), respectively. Control fish for LPSor TRBIV-treated group were injected with PBS, while those for CpGODN 2395-treated group were injected with TE buffer. The volume of PBS or TE buffer used was same with that of corresponding stimulant. The spleen, head kidney, gills and muscle of injected fish were collected for gene expression assay at various time points post injection (0, 3, 6 and 12 hours and 1, 2, 3, 4, 5 and 7 days after LPS or CpG-ODN 2395 injections, or 0, 3 and 6 hours and 1, 2, 3, 4, 5 and 7 days after TRBIV injection), while the untreated healthy fish were used for tissue distribution analysis. 2.2. RNA extraction Fish were sacrificed and various tissues (including brain, gills, stomach, intestine, heart, head kidney, kidney, liver, spleen, gonad, muscle and skin) were excised, immediately snap-frozen in liquid nitrogen and stored at −80 °C until used. Total RNA was extracted from each tissue using Isogen reagent (Nippon Gene, Tokyo, Japan). RNA samples were incubated with DNase I to remove genomic DNA contamination using Turbo DNA-free Kit (Ambion, Austin, TX, USA). The RNA concentration was determined by measuring the absorbance
at 260 nm, and its quality was monitored by A260 nm/A280 nm ratios >1.8. 2.3. Cloning of SmMyD88 cDNA The SMART cDNAs were generated from 1 μg of total RNA extracted from head kidney of a turbot using a cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA). Based on the conserved sequences of known fish MyD88s, degenerate primers (Supplementary Table S1) were designed. A 539-bp partial cDNA of SmMyD88 was obtained by homology cloning, while the 5′- and 3′-end fragments, with lengths of 305 bp and 1008 bp, respectively, were obtained by a rapid amplifications of cDNA ends (RACE). The full length cDNA sequence was assembled and its continuity was confirmed by sequencing the cloned PCR product amplified with a pair of terminal primers. PCRs across this group were carried out with Ex Taq DNA polymerase (TaKaRa, Dalian, Liaoning, China) under the following condition: initial denaturation at 94 °C for 4 min, then 25–40 cycles of 94 °C for 40 s, 48.9–61 °C for 30 s and 72 °C for 40 s–2 min, and final extension at 72 °C for 7 min. The PCR products were isolated using an E.Z.N.A Gel Extraction Kit (Omega Bio-tek, Doraville, GA, USA), cloned into pMD18-T vector (TaKaRa) and sequenced with an ABI PRISM 3100 DNA sequencer (Applied Biosystem, Foster City, CA). 2.4. Sequence analysis Sequence result of SmMyD88 was compared with the GenBank/ EMBL database by using the BLASTX and BLASTP search programs (http://blast.genome.ad.Jp). The nucleotide sequence was trans- Q3 lated to protein sequence using Translate Tool DNAman. The multiple alignment of protein sequences was produced by the Clustal W (www.ddbj.nig.ac.jp/E-mail/clustalw-e.html). The phylogenetic tree was created using the neighbor-joining (NJ) method by MEGA version 5.0. Bootstrap values were calculated with 1000 replications to estimate the robustness of internal branches. 2.5. Quantitative real time PCR (qPCR) qPCR analysis was employed to study SmMyD88 mRNA tissue distribution and gene expression in response to TRBIV, CpG-ODN 2395 or LPS stimulation in specific organs. Five individuals were studied for tissue distribution and, also, five individuals for each time point of gene expression assay. One microgram of total RNA from each tissue was reverse-transcribed into cDNA by random primers using Superscript First Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). qPCR was conducted in 20 μl volume containing 1 × SYBR Green Real Time PCR Master Mix (Toyobo, Osaka, Japan), 0.2 μM each of gene-specific forward and reverse primers (Supplementary Table S1) and 1.0 μl diluted cDNA (50 ng/μl). PCR conditions were 94 °C for 4 min, followed by 40 cycles of 94 °C for 40 s, 61.5 °C for 30 s, 72 °C for 20 s, and final elongation at 72 °C for 7 min. Turbot 18S rRNA (GenBank accession number: EF126038) was used as endogenous control. All samples were amplified in triplicates. Fluorescent detection was performed after each extension step. A dissociation protocol was performed after thermo cycling to verify that a single amplicon of expected size was amplified. The expression levels of the target gene were normalized to 18S rRNA, and further expressed as fold change relative to the expression level in control according to the 2−ΔΔCT method (Livak and Schmittgen, 2001) in the gene expression assay upon immune stimulation. 2.6. Statistical analysis Statistical analysis was performed using SPSS13.0 software (SPSS Inc., Chicago, IL, USA). Differences in the data were compared by oneway analysis of variance (ANOVA) followed by Duncan’s post hoc
Please cite this article in press as: Jing-Yun Lin, et al., Molecular cloning and expression studies of the adapter molecule myeloid differentiation factor 88 (MyD88) in turbot (Scophthalmus maximus), Developmental and Comparative Immunology (2015), doi: 10.1016/j.dci.2015.05.013
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test for multiple comparisons. Differences were considered significant at P < 0.05. 3. Results and discussion MyD88 in mammals is one of the key adapter proteins to IL-1R/TLR signal transduction that triggers downstream cascades involved in innate immunity (Krishnan et al., 2007). After activation by PAMPs, almost all TLRs and IL-1Rs directly or indirectly recruit MyD88 to propagate the downstream signaling that orchestrate the immuno-inflammatory responses (Yamamoto et al., 2003). In this study, a MyD88 ortholog in turbot (SmMyD88) was identified and characterized. The full-length cDNA sequence of SmMyD88 is composed of 1619 bp, containing an 858-bp open reading frame that encodes a putative protein of 285 amino acid residues, a 31-bp 5′-untranslated region (UTR) and a 733-bp 3′-UTR (Supplementary Fig. S1). In the 3′-UTR, four putative polyadenylation consensus signals (958ATTAAA963, 1087ATTAAA1092, 1572AATAAA1577 and 1578AATAAA1583) and a RNA instability motif (1214ATTTA1218) were found. The deduced protein shows a relatively high sequence homology to other known MyD88s with the highest identity (97.9%) to MyD88 from its close relative Japanese flounder (Supplementary Table S2). Protein domain prediction by Simple Modular Architecture Reach Tool (SMART) (http://smart.embl-heidelberg.de/) revealed that SmMyD88 possesses a death domain at position 12–103 of the amino terminus and a TIR domain at position 149–285 of the carboxyl terminus. The multiple alignment showed a significant conservation for the two domains in fish species and mammals, with the TIR domain being more conservative that harbors three highly conserved regions, Boxes 1, 2 and 3 (Fig. 1). The death domain is characterized by involvement in the signaling leading to apoptosis and inflammatory responses (Hofmann and Tschopp, 1995; Itoh and Nagata, 1993).
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In most reported death domain-containing proteins, it is typically located at the extreme carboxyl terminus (Weber and Vincenz, 2001). In contrast, it is located at the N-terminus in MyD88 and mediates downstream interactions with the IRAK family (Medzhitov et al., 1998). The TIR domain interacts with its cognate domains located in the cytoplasmic tails of activated TLRs or IL-1R. This process is mediated primarily through Box 1 (Xu et al., 2000), while Box 2 is important for the formation of loop structure that probably regulates downstream elements (Takano et al., 2006) and Box 3 is involved in controlling trafficking and localization of the receptor in IL-1R (Slack et al., 2000), but whether it has such a function in MyD88 remains unclear. Taken together, the structural characteristics of SmMyD88 suggest that it may share similar functions with its mammalian orthologs. To determine the evolutionary position of SmMyD88, a phylogenetic tree containing 22 MyD88 protein sequences from various vertebrates was constructed (Supplementary Fig. S2). The MyD88 proteins were separated into three clades, mammalian MyD88, amphibian MyD88 and fish MyD88. SmMyD88 was grouped into the clade of fish MyD88. It exhibited the closest relationship to MyD88s from two other flatfishes, Japanese flounder and tongue sole, followed by other percomorpha fishes. A less close relationship was seen to basal teleosts like zebrafish and a chondrostei species Chinese sturgeon and a remote relationship to mammals. This result matches the evolutionary relationships among various species. qPCR analysis showed that SmMyD88 mRNA was constitutively expressed in all examined tissues of healthy turbots (Supplementary Fig. S3). The strong expression was detected in the heart, head kidney, kidney, spleen and liver, while a weak expression in the intestine and gonad. This result is in general consistent with the reports for MyD88s from mammals and other teleosts which also have a broad tissue expression spectrum (Burns et al., 1998; Janssens et al., 2002). MyD88 is a universal intracellular immune
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Fig. 1. Multiple alignment of SmMyD88 and other known MyD88 proteins. The death domain and TIR domain are indicated by lines above the aligned sequences. The high highly conserved regions Box 1, Box 2 and Box 3 in the TIR domain are boxed. The accession numbers of the sequences are presented in Supplementary Table S2.
Please cite this article in press as: Jing-Yun Lin, et al., Molecular cloning and expression studies of the adapter molecule myeloid differentiation factor 88 (MyD88) in turbot (Scophthalmus maximus), Developmental and Comparative Immunology (2015), doi: 10.1016/j.dci.2015.05.013
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mediator, functioning as a signal transduction adapter downstream of multiple TLRs that are expressed ubiquitously. This may explain the ubiquitous expression of SmMyD88 in turbot tissues. The higher expression levels were observed in the leukocyte-rich organs, indicating an important role of SmMyD88 in immune system. Interestingly, SmMyD88 was also strongly expressed in non-immune tissues such as the brain, suggesting that its function is not limited to the immune system. Additionally, MyD88 has been reported to participate in the activation of several members of interferon regulatory factor (IRF) family transcription factors, namely, IRF3, -5 and -7 (Yamamoto et al., 2002). Since these IRFs were constitutively expressed also in most tissue types of turbots with higher levels in the leukocyte-rich organs (Hu et al., 2011a, 2011b; Xia et al., 2012), it can be inferred that SmMyD88 has a close relationship with them. To explore the potential role of SmMyD88 in immune responses of turbot, its gene expression profile was studied over a 7-day time course in the gills, head kidney, spleen and muscle following stimulation with LPS, CpG-ODN or TRBIV. The up-regulation of SmMyD88 was observed in all four tested tissues after treatment with each stimulant (Fig. 2), suggesting its general inducibility in the immune and non-immune organs by bacterial or viral infections. The gram-negative endotoxin LPS has been reported to be a powerful stimulator of innate immunity in diverse eukaryotic species (Lemaitre et al., 1996; Ulevitch and Tobias, 1995). Here, its effect on
SmMyD88 expression was investigated (Fig. 2 A1–4). Upon LPS stimulation, SmMyD88 was markedly induced in the head kidney with a peak transcript level of 5.6-fold over control arising at day 1 postinjection and less induced in the gills and spleen with a peak level of 4.2- and 3.2-fold arising at hour 6 and day 1, respectively. The induction in the muscle was weak with a peak level of 1.5-fold arising at day 3, suggesting a weak Toll/IL-1R signal pathway response taking place in this tissue where lymphomyeloid cells are scarce. This expression pattern generally agrees with the reports for MyD88s from Japanese flounder, rock bream and scallop upon LPS treatment (Qiu et al., 2007; Takano et al., 2006; Whang et al., 2011). In mammals, stimulation with LPS leads to a cellular response where TLR4 in membrane recruits MyD88 that acts as an adapter to propagate the downstream signaling after binding to LPS (Netea et al., 2004; Takano et al., 2006). However, fish TLR4 does not recognize LPS (Sepulcre et al., 2009) and some fish species lack a TLR4 ortholog. Further, the results from zebrafish demonstrated that LPS was signaled by a TLR4and MyD88-independent pathway (Sepulcre et al., 2009). Thus, the up-regulation of SmMyD88 is probably mediated independently of SmMyD88 activation, i.e., via a TLR4- and MyD88-independent pathway. Additionally, some studies suggested that fugu TLR23 may participate in LPS recognition and alternative signaling receptors like beta-2 integrins may play a pivotal role in the activation of piscine leukocytes by LPS (Iliev et al., 2005). These findings suggest that
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Fig. 2. Time-course expression profiles of SmMyD88 gene in gills, head kidney, spleen and muscle of turbots injected with 280 ng LPS, 14.25 ng CpG-ODN 2395 or 2.4 × 105 TCID50 TRBIV per fish. A1–4 show fold changes of SmMyD88 expression with LPS stimulation, B1–4 with CpG-ODN 2395 stimulation and C1–4 with TRBIV infection. Values are means ± standard error (S.E.), n = 5. The level of significance of the comparison to the control is indicated by *P < 0.05 and **P < 0.01.
Please cite this article in press as: Jing-Yun Lin, et al., Molecular cloning and expression studies of the adapter molecule myeloid differentiation factor 88 (MyD88) in turbot (Scophthalmus maximus), Developmental and Comparative Immunology (2015), doi: 10.1016/j.dci.2015.05.013
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different receptors might be involved in the activation of signaling pathway by LPS in turbot that needs to be further studied. The innate immune system of vertebrates has evolved an ability to recognize unmethylated CG dinucleotides within certain sequence contexts in bacterial and viral DNA (Krieg, 2002). The synthetic oligodeoxynucleotides (ODNs) are a mimic of these microorganism DNA because of containing CpG motifs to trigger innate immune responses of hosts (Mutwiri et al., 2003). In this study, CpGODN 2395, a class C CpG-ODN with a powerful capacity of inducing IFN-α and B cell proliferation (Krug et al., 2001; Verthelyi et al., 2001; Vollmer et al., 2004), was used to stimulate turbots. Upon CpG-ODN 2395 stimulation, SmMyD88 was markedly induced in the head kidney with a peak level of 7.2-fold at hour 6 post-injection and less induced in the gills, spleen and muscle where the maximum induction were 4.3-, 6.2- and 3.2-fold arising at hours 6 and 3 and day 1, respectively (Fig. 2 B1–4). The up-regulated expression of MyD88 by CpG-ODN 2395 was also found in Atlantic salmon (Skjæveland et al., 2009; Strandskog et al., 2008). TLR9 is the key sensor for CpG signaling. Following the binding of TLR9 to CpG DNA, the MyD88 combines with its cytoplasmic part through TIR–TIR interaction to activate downstream immunological reactions. Previous studies have shown the functional IFN α/β activity following CpG stimulation (Bonnert et al., 1997; Vollmer et al., 2004), suggesting that the stimulation of IFN by CpG might be dependent on TLR9-MyD88 pathway. Therefore, the different inducibilities in the four tissues may be caused by different levels of TLR9 expression there. TRBIV, a double-stranded DNA virus, is regarded as a cause of serious systemic diseases among cultured turbots. In order to explore whether SmMyD88 is involved in antiviral immunity, fish were infected with TRBIV and SmMyD88 expression levels were assessed in different organs post infection. Upon TRBIV challenge, SmMyD88 expression level reached the peak at day 1, 1 and 3 in the gills, head kidney and muscle with 2.4-, 4.3- and 1.4-fold, respectively (Fig. 2 C1, C2 and C4). The induction in the spleen was stronger with a peak level of 6.6-fold arising at day 4 (Fig. 2 C3). These results are in agreement with the findings for MyD88s from grass carp and orangespotted grouper with virus challenge (Su et al., 2011; Yan et al., 2012). In contrast, the two synthetic immunostimulants induced higher levels of SmMyD88 transcripts in the head kidney. TRBIV is expected to possess both DNA (CpG DNA) and RNA (intermediate singleand double-stranded RNA) PAMPs (Hu et al., 2011b). Previous studies have demonstrated the involvement of TLR7/8/9-MyD88 pathways in controlling viral infection and replication through recruitment of IRAKs by MyD88 to activate NF-κB and induce proinflammatory cytokines (Pichlmair and Reise, 2007). Thus, these pathways were likely activated in the response of turbots to TRBIV infection. In summary, we identified and characterized the structure and expression pattern of a MyD88 molecule in turbot in the present study. SmMyD88 had a structural feature same with its mammalian counterpart. It was up-regulated by LPS, CpG-ODN and TRBIV with the induction by CpG-ODN being earliest and strongest and inducibility in the muscle faint. These findings may help a further understanding of the functions and evolution of vertebrate MyD88.
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Acknowledgments This work was supported by grants from the National Basic Research Program of China (2012CB114404), National Natural Science Foundation of China (81100697, 30671604), Fundamental Research Funds for the Central Universities (201362024), Program for New Century Excellent Talents in University of Ministry of Education of China (NCET-11-0467), Promotive Research Fund for Excellent Young and Middle-aged Scientists of Shandong Province
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(2011BSB01433, BS2011YY001) and Shandong Provincial Natural Science Foundation (ZR2013CM045).
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Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.dci.2015.05.013.
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Developmental and Comparative Immunology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d c i
Short communication
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Molecular cloning and expression studies of the adapter molecule myeloid differentiation factor 88 (MyD88) in turbot (Scophthalmus maximus)
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Jing-Yun Lin a, Guo-Bin Hu a,b,*, Chang-Hong Yu c, Song Li a, Qiu-Ming Liu a, Q2 Shi-Cui Zhang a,b a
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College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China c College of Medicine, Qingdao University, Qingdao 266071, China b
A R T I C L E
I N F O
Article history: Received 12 March 2015 Revised 22 May 2015 Accepted 23 May 2015 Available online Keywords: MyD88 Scophthalmus maximus LPS CpG-ODN TRBIV Gene expression
A B S T R A C T
Myeloid differentiation factor 88 (MyD88) is an adapter protein involved in the interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR)-mediated activation of nuclear factor-kappaB (NF-κB). In this study, a full length cDNA of MyD88 was cloned from turbot, Scophthalmus maximus. It is 1619 bp in length and contains an 858-bp open reading frame that encodes a peptide of 285 amino acid residues. The putative turbot (Sm)MyD88 protein possesses a N-terminal death domain and a C-terminal Toll/IL-1 receptor (TIR) domain known to be important for the functions of MyD88 in mammals. Phylogenetic analysis grouped SmMyD88 with other fish MyD88s. SmMyD88 mRNA was ubiquitously expressed in all examined tissues of healthy turbots, with higher levels observed in immune-relevant organs. To explore the role of SmMyD88, its gene expression profile in response to stimulation of lipopolysaccharide (LPS), CpG oligodeoxynucleotide (CpG-ODN) or turbot reddish body iridovirus (TRBIV) was studied in the head kidney, spleen, gills and muscle over a 7-day time course. The results showed an up-regulation of SmMyD88 transcript levels by the three immunostimulants in all four examined tissues, with the induction by CpG-ODN strongest and initiated earliest and inducibility in the muscle very weak. Additionally, TRBIV challenge resulted in a quite high level of SmMyD88 expression in the spleen, whereas the two synthetic immunostimulants induced the higher levels in the head kidney. These data provide insights into the roles of SmMyD88 in the TLR/IL-1R signaling pathway of the innate immune system in turbot. © 2015 Published by Elsevier Ltd.
46 1. Introduction
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The innate immunity in fish, as in all vertebrates, is the first line of defense and provides crucial signals for activation of adaptive immune responses (Akira et al., 2001). Detection and clearance of invading pathogens by the innate immune system are associated with plenty of signaling pathways that are evolutionarily conserved throughout vertebrates. It works through the way of being triggered when pathogen-associated molecular patterns (PAMPs) come into contact with host-expressed pattern recognition receptors (PRRs) (Medzhitov and Janeway, 2000). One of the wellcharacterized PRRs is the family of Toll-like receptors (TLRs) that detects microbial PAMPs such as bacterial lipopolysaccharides (LPS), peptidoglycans (PGN) and flagellin, viral RNA, unmethylated CpG DNA of viruses, bacteria and protozoa, β-glycan of fungi and
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* Corresponding author. College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China. Tel.: +86 532 82032583; fax: +86 532 82032583. E-mail address: [email protected] (G.-B. Hu).
lipoproteins of various pathogens (Akira et al., 2006; Mogensen, 2009). The signaling pathways mediated by TLRs are broadly classified into the myeloid differentiation factor 88 (MyD88)-dependent and -independent ones. The former uses MyD88 as an adapter molecule to activate the signaling cascades and produces inflammatory mediators. Although MyD88 was first found in mice in 1990 as a myeloid differentiation primary response gene induced during terminal differentiation of M1D + myeloid precursor cells in response to interleukin (IL)-6 treatment (Lord et al., 1990), its function as a key adapter molecule in the interleukin-1 receptor (IL-1R)/TLR-mediated signaling remains unknown until 1997 (Wesche et al., 1997). MyD88 has a bipartite structure composed of an N-terminal death domain and a C-terminal Toll/IL-1 receptor (TIR) domain with a short intervening linker segment. Upon activation by PAMPs, all of TLRs except TLR3 recruit MyD88 through the TIR domain; the death domain interacts with the corresponding domain in IL-1R-associated kinases (IRAKs), leading to recruitment of downstream tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6); these events eventually result in activation of NF-κB and interferon
http://dx.doi.org/10.1016/j.dci.2015.05.013 0145-305X/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: Jing-Yun Lin, et al., Molecular cloning and expression studies of the adapter molecule myeloid differentiation factor 88 (MyD88) in turbot (Scophthalmus maximus), Developmental and Comparative Immunology (2015), doi: 10.1016/j.dci.2015.05.013
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regulator factors (IRFs) and induction of pro-inflammatory cytokines or antiviral genes that play an important role in combating pathogens (Deepika et al., 2014). To date, MyD88 has been identified in mammals, birds, reptiles, amphibians, fishes and invertebrates (Bonnert et al., 1997; Deepika et al., 2014; Li et al., 2011; Prothmann et al., 2000; Wheaton et al., 2007). The fishes with MyD88 identified include zebrafish (van der Sar et al., 2006), Japanese flounder (Takano et al., 2006), half-smooth tongue sole (Yu et al., 2009), large yellow croaker (Yao et al., 2009), Atlantic salmon (Skjæveland et al., 2009), rainbow trout (Rebl et al., 2009), rock bream (Whang et al., 2011), common carp (Kongchum et al., 2011), grass carp (Su et al., 2011), orange-spotted grouper (Yan et al., 2012), miiuy croaker (Tang et al., 2012), etc. Fish MyD88 exhibits structural and functional homologies with its mammalian counterpart. Turbot, Scophthalmus maximus, is an important commercial marine species cultured widely in the world. However, the knowledge about turbot (Sm)MyD88 is scarce. The aim of this study is to improve understanding of the innate immune system in turbot by studies of a MyD88 gene, which will help in development of strategies of microbial infectious disease control for this species. Herein, we report the cDNA sequence, mRNA tissue distribution and transcriptional modulation of SmMyD88. The last study was performed in vivo upon stimulation of turbots with LPS, synthetic CpG oligodeoxynucleotide (CpG-ODN) or turbot reddish body iridovirus (TRBIV). We demonstrated the involvement of SmMyD88 in immune responses of turbots to these three immunostimulants. 2. Materials and methods 2.1. Fish, stimulants and immunostimulation experiments Turbot (S. maximus) juveniles (68.4 ± 4.5 g, n = 170) were purchased from a local fish farm. Fish were kept in aerated seawater tanks at 16 °C for 1 week before use. LPS (L2880, Escherichia coli 055:B5; Sigma, St. Louis, MO, USA) was diluted in phosphate buffer saline (PBS, pH 7.4) to make stock. CpG-ODN 2395 (class C, 5′-T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G-3′, phorothioate modifications are marked with *; Coley Pharmaceutical Group, Ottawa, CA) was dissolved in TE buffer (10 mM Tris, 1 nM EDTA, pH 8.0). TRBIV was isolated from tumor-carrying turbots as previously described (Hu et al., 2012). The viral titers were measured by a 50% tissue culture infective dose (TCID50) assay according to the method of Reed and Muench (1938). Three groups of turbots were intraperitoneally (i.p.) injected with LPS (2.5 mg/ml, 112 μl per fish), CpG-ODN 2395 (0.15 mg/ml, 95 μl per fish) or TRBIV (2 × 106 TCID50/ml, 120 μl per fish), respectively. Control fish for LPSor TRBIV-treated group were injected with PBS, while those for CpGODN 2395-treated group were injected with TE buffer. The volume of PBS or TE buffer used was same with that of corresponding stimulant. The spleen, head kidney, gills and muscle of injected fish were collected for gene expression assay at various time points post injection (0, 3, 6 and 12 hours and 1, 2, 3, 4, 5 and 7 days after LPS or CpG-ODN 2395 injections, or 0, 3 and 6 hours and 1, 2, 3, 4, 5 and 7 days after TRBIV injection), while the untreated healthy fish were used for tissue distribution analysis. 2.2. RNA extraction Fish were sacrificed and various tissues (including brain, gills, stomach, intestine, heart, head kidney, kidney, liver, spleen, gonad, muscle and skin) were excised, immediately snap-frozen in liquid nitrogen and stored at −80 °C until used. Total RNA was extracted from each tissue using Isogen reagent (Nippon Gene, Tokyo, Japan). RNA samples were incubated with DNase I to remove genomic DNA contamination using Turbo DNA-free Kit (Ambion, Austin, TX, USA). The RNA concentration was determined by measuring the absorbance
at 260 nm, and its quality was monitored by A260 nm/A280 nm ratios >1.8. 2.3. Cloning of SmMyD88 cDNA The SMART cDNAs were generated from 1 μg of total RNA extracted from head kidney of a turbot using a cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA). Based on the conserved sequences of known fish MyD88s, degenerate primers (Supplementary Table S1) were designed. A 539-bp partial cDNA of SmMyD88 was obtained by homology cloning, while the 5′- and 3′-end fragments, with lengths of 305 bp and 1008 bp, respectively, were obtained by a rapid amplifications of cDNA ends (RACE). The full length cDNA sequence was assembled and its continuity was confirmed by sequencing the cloned PCR product amplified with a pair of terminal primers. PCRs across this group were carried out with Ex Taq DNA polymerase (TaKaRa, Dalian, Liaoning, China) under the following condition: initial denaturation at 94 °C for 4 min, then 25–40 cycles of 94 °C for 40 s, 48.9–61 °C for 30 s and 72 °C for 40 s–2 min, and final extension at 72 °C for 7 min. The PCR products were isolated using an E.Z.N.A Gel Extraction Kit (Omega Bio-tek, Doraville, GA, USA), cloned into pMD18-T vector (TaKaRa) and sequenced with an ABI PRISM 3100 DNA sequencer (Applied Biosystem, Foster City, CA). 2.4. Sequence analysis Sequence result of SmMyD88 was compared with the GenBank/ EMBL database by using the BLASTX and BLASTP search programs (http://blast.genome.ad.Jp). The nucleotide sequence was trans- Q3 lated to protein sequence using Translate Tool DNAman. The multiple alignment of protein sequences was produced by the Clustal W (www.ddbj.nig.ac.jp/E-mail/clustalw-e.html). The phylogenetic tree was created using the neighbor-joining (NJ) method by MEGA version 5.0. Bootstrap values were calculated with 1000 replications to estimate the robustness of internal branches. 2.5. Quantitative real time PCR (qPCR) qPCR analysis was employed to study SmMyD88 mRNA tissue distribution and gene expression in response to TRBIV, CpG-ODN 2395 or LPS stimulation in specific organs. Five individuals were studied for tissue distribution and, also, five individuals for each time point of gene expression assay. One microgram of total RNA from each tissue was reverse-transcribed into cDNA by random primers using Superscript First Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). qPCR was conducted in 20 μl volume containing 1 × SYBR Green Real Time PCR Master Mix (Toyobo, Osaka, Japan), 0.2 μM each of gene-specific forward and reverse primers (Supplementary Table S1) and 1.0 μl diluted cDNA (50 ng/μl). PCR conditions were 94 °C for 4 min, followed by 40 cycles of 94 °C for 40 s, 61.5 °C for 30 s, 72 °C for 20 s, and final elongation at 72 °C for 7 min. Turbot 18S rRNA (GenBank accession number: EF126038) was used as endogenous control. All samples were amplified in triplicates. Fluorescent detection was performed after each extension step. A dissociation protocol was performed after thermo cycling to verify that a single amplicon of expected size was amplified. The expression levels of the target gene were normalized to 18S rRNA, and further expressed as fold change relative to the expression level in control according to the 2−ΔΔCT method (Livak and Schmittgen, 2001) in the gene expression assay upon immune stimulation. 2.6. Statistical analysis Statistical analysis was performed using SPSS13.0 software (SPSS Inc., Chicago, IL, USA). Differences in the data were compared by oneway analysis of variance (ANOVA) followed by Duncan’s post hoc
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test for multiple comparisons. Differences were considered significant at P < 0.05. 3. Results and discussion MyD88 in mammals is one of the key adapter proteins to IL-1R/TLR signal transduction that triggers downstream cascades involved in innate immunity (Krishnan et al., 2007). After activation by PAMPs, almost all TLRs and IL-1Rs directly or indirectly recruit MyD88 to propagate the downstream signaling that orchestrate the immuno-inflammatory responses (Yamamoto et al., 2003). In this study, a MyD88 ortholog in turbot (SmMyD88) was identified and characterized. The full-length cDNA sequence of SmMyD88 is composed of 1619 bp, containing an 858-bp open reading frame that encodes a putative protein of 285 amino acid residues, a 31-bp 5′-untranslated region (UTR) and a 733-bp 3′-UTR (Supplementary Fig. S1). In the 3′-UTR, four putative polyadenylation consensus signals (958ATTAAA963, 1087ATTAAA1092, 1572AATAAA1577 and 1578AATAAA1583) and a RNA instability motif (1214ATTTA1218) were found. The deduced protein shows a relatively high sequence homology to other known MyD88s with the highest identity (97.9%) to MyD88 from its close relative Japanese flounder (Supplementary Table S2). Protein domain prediction by Simple Modular Architecture Reach Tool (SMART) (http://smart.embl-heidelberg.de/) revealed that SmMyD88 possesses a death domain at position 12–103 of the amino terminus and a TIR domain at position 149–285 of the carboxyl terminus. The multiple alignment showed a significant conservation for the two domains in fish species and mammals, with the TIR domain being more conservative that harbors three highly conserved regions, Boxes 1, 2 and 3 (Fig. 1). The death domain is characterized by involvement in the signaling leading to apoptosis and inflammatory responses (Hofmann and Tschopp, 1995; Itoh and Nagata, 1993).
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In most reported death domain-containing proteins, it is typically located at the extreme carboxyl terminus (Weber and Vincenz, 2001). In contrast, it is located at the N-terminus in MyD88 and mediates downstream interactions with the IRAK family (Medzhitov et al., 1998). The TIR domain interacts with its cognate domains located in the cytoplasmic tails of activated TLRs or IL-1R. This process is mediated primarily through Box 1 (Xu et al., 2000), while Box 2 is important for the formation of loop structure that probably regulates downstream elements (Takano et al., 2006) and Box 3 is involved in controlling trafficking and localization of the receptor in IL-1R (Slack et al., 2000), but whether it has such a function in MyD88 remains unclear. Taken together, the structural characteristics of SmMyD88 suggest that it may share similar functions with its mammalian orthologs. To determine the evolutionary position of SmMyD88, a phylogenetic tree containing 22 MyD88 protein sequences from various vertebrates was constructed (Supplementary Fig. S2). The MyD88 proteins were separated into three clades, mammalian MyD88, amphibian MyD88 and fish MyD88. SmMyD88 was grouped into the clade of fish MyD88. It exhibited the closest relationship to MyD88s from two other flatfishes, Japanese flounder and tongue sole, followed by other percomorpha fishes. A less close relationship was seen to basal teleosts like zebrafish and a chondrostei species Chinese sturgeon and a remote relationship to mammals. This result matches the evolutionary relationships among various species. qPCR analysis showed that SmMyD88 mRNA was constitutively expressed in all examined tissues of healthy turbots (Supplementary Fig. S3). The strong expression was detected in the heart, head kidney, kidney, spleen and liver, while a weak expression in the intestine and gonad. This result is in general consistent with the reports for MyD88s from mammals and other teleosts which also have a broad tissue expression spectrum (Burns et al., 1998; Janssens et al., 2002). MyD88 is a universal intracellular immune
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Fig. 1. Multiple alignment of SmMyD88 and other known MyD88 proteins. The death domain and TIR domain are indicated by lines above the aligned sequences. The high highly conserved regions Box 1, Box 2 and Box 3 in the TIR domain are boxed. The accession numbers of the sequences are presented in Supplementary Table S2.
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mediator, functioning as a signal transduction adapter downstream of multiple TLRs that are expressed ubiquitously. This may explain the ubiquitous expression of SmMyD88 in turbot tissues. The higher expression levels were observed in the leukocyte-rich organs, indicating an important role of SmMyD88 in immune system. Interestingly, SmMyD88 was also strongly expressed in non-immune tissues such as the brain, suggesting that its function is not limited to the immune system. Additionally, MyD88 has been reported to participate in the activation of several members of interferon regulatory factor (IRF) family transcription factors, namely, IRF3, -5 and -7 (Yamamoto et al., 2002). Since these IRFs were constitutively expressed also in most tissue types of turbots with higher levels in the leukocyte-rich organs (Hu et al., 2011a, 2011b; Xia et al., 2012), it can be inferred that SmMyD88 has a close relationship with them. To explore the potential role of SmMyD88 in immune responses of turbot, its gene expression profile was studied over a 7-day time course in the gills, head kidney, spleen and muscle following stimulation with LPS, CpG-ODN or TRBIV. The up-regulation of SmMyD88 was observed in all four tested tissues after treatment with each stimulant (Fig. 2), suggesting its general inducibility in the immune and non-immune organs by bacterial or viral infections. The gram-negative endotoxin LPS has been reported to be a powerful stimulator of innate immunity in diverse eukaryotic species (Lemaitre et al., 1996; Ulevitch and Tobias, 1995). Here, its effect on
SmMyD88 expression was investigated (Fig. 2 A1–4). Upon LPS stimulation, SmMyD88 was markedly induced in the head kidney with a peak transcript level of 5.6-fold over control arising at day 1 postinjection and less induced in the gills and spleen with a peak level of 4.2- and 3.2-fold arising at hour 6 and day 1, respectively. The induction in the muscle was weak with a peak level of 1.5-fold arising at day 3, suggesting a weak Toll/IL-1R signal pathway response taking place in this tissue where lymphomyeloid cells are scarce. This expression pattern generally agrees with the reports for MyD88s from Japanese flounder, rock bream and scallop upon LPS treatment (Qiu et al., 2007; Takano et al., 2006; Whang et al., 2011). In mammals, stimulation with LPS leads to a cellular response where TLR4 in membrane recruits MyD88 that acts as an adapter to propagate the downstream signaling after binding to LPS (Netea et al., 2004; Takano et al., 2006). However, fish TLR4 does not recognize LPS (Sepulcre et al., 2009) and some fish species lack a TLR4 ortholog. Further, the results from zebrafish demonstrated that LPS was signaled by a TLR4and MyD88-independent pathway (Sepulcre et al., 2009). Thus, the up-regulation of SmMyD88 is probably mediated independently of SmMyD88 activation, i.e., via a TLR4- and MyD88-independent pathway. Additionally, some studies suggested that fugu TLR23 may participate in LPS recognition and alternative signaling receptors like beta-2 integrins may play a pivotal role in the activation of piscine leukocytes by LPS (Iliev et al., 2005). These findings suggest that
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Fig. 2. Time-course expression profiles of SmMyD88 gene in gills, head kidney, spleen and muscle of turbots injected with 280 ng LPS, 14.25 ng CpG-ODN 2395 or 2.4 × 105 TCID50 TRBIV per fish. A1–4 show fold changes of SmMyD88 expression with LPS stimulation, B1–4 with CpG-ODN 2395 stimulation and C1–4 with TRBIV infection. Values are means ± standard error (S.E.), n = 5. The level of significance of the comparison to the control is indicated by *P < 0.05 and **P < 0.01.
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different receptors might be involved in the activation of signaling pathway by LPS in turbot that needs to be further studied. The innate immune system of vertebrates has evolved an ability to recognize unmethylated CG dinucleotides within certain sequence contexts in bacterial and viral DNA (Krieg, 2002). The synthetic oligodeoxynucleotides (ODNs) are a mimic of these microorganism DNA because of containing CpG motifs to trigger innate immune responses of hosts (Mutwiri et al., 2003). In this study, CpGODN 2395, a class C CpG-ODN with a powerful capacity of inducing IFN-α and B cell proliferation (Krug et al., 2001; Verthelyi et al., 2001; Vollmer et al., 2004), was used to stimulate turbots. Upon CpG-ODN 2395 stimulation, SmMyD88 was markedly induced in the head kidney with a peak level of 7.2-fold at hour 6 post-injection and less induced in the gills, spleen and muscle where the maximum induction were 4.3-, 6.2- and 3.2-fold arising at hours 6 and 3 and day 1, respectively (Fig. 2 B1–4). The up-regulated expression of MyD88 by CpG-ODN 2395 was also found in Atlantic salmon (Skjæveland et al., 2009; Strandskog et al., 2008). TLR9 is the key sensor for CpG signaling. Following the binding of TLR9 to CpG DNA, the MyD88 combines with its cytoplasmic part through TIR–TIR interaction to activate downstream immunological reactions. Previous studies have shown the functional IFN α/β activity following CpG stimulation (Bonnert et al., 1997; Vollmer et al., 2004), suggesting that the stimulation of IFN by CpG might be dependent on TLR9-MyD88 pathway. Therefore, the different inducibilities in the four tissues may be caused by different levels of TLR9 expression there. TRBIV, a double-stranded DNA virus, is regarded as a cause of serious systemic diseases among cultured turbots. In order to explore whether SmMyD88 is involved in antiviral immunity, fish were infected with TRBIV and SmMyD88 expression levels were assessed in different organs post infection. Upon TRBIV challenge, SmMyD88 expression level reached the peak at day 1, 1 and 3 in the gills, head kidney and muscle with 2.4-, 4.3- and 1.4-fold, respectively (Fig. 2 C1, C2 and C4). The induction in the spleen was stronger with a peak level of 6.6-fold arising at day 4 (Fig. 2 C3). These results are in agreement with the findings for MyD88s from grass carp and orangespotted grouper with virus challenge (Su et al., 2011; Yan et al., 2012). In contrast, the two synthetic immunostimulants induced higher levels of SmMyD88 transcripts in the head kidney. TRBIV is expected to possess both DNA (CpG DNA) and RNA (intermediate singleand double-stranded RNA) PAMPs (Hu et al., 2011b). Previous studies have demonstrated the involvement of TLR7/8/9-MyD88 pathways in controlling viral infection and replication through recruitment of IRAKs by MyD88 to activate NF-κB and induce proinflammatory cytokines (Pichlmair and Reise, 2007). Thus, these pathways were likely activated in the response of turbots to TRBIV infection. In summary, we identified and characterized the structure and expression pattern of a MyD88 molecule in turbot in the present study. SmMyD88 had a structural feature same with its mammalian counterpart. It was up-regulated by LPS, CpG-ODN and TRBIV with the induction by CpG-ODN being earliest and strongest and inducibility in the muscle faint. These findings may help a further understanding of the functions and evolution of vertebrate MyD88.
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Acknowledgments This work was supported by grants from the National Basic Research Program of China (2012CB114404), National Natural Science Foundation of China (81100697, 30671604), Fundamental Research Funds for the Central Universities (201362024), Program for New Century Excellent Talents in University of Ministry of Education of China (NCET-11-0467), Promotive Research Fund for Excellent Young and Middle-aged Scientists of Shandong Province
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(2011BSB01433, BS2011YY001) and Shandong Provincial Natural Science Foundation (ZR2013CM045).
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Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.dci.2015.05.013.
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