Developmental and Comparative Immunology 43 (2014) 54–58

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Molecular cloning and expression analysis of mannose receptor C type 1 in grass carp (Ctenopharyngodon idella) Li Wang a, Lichun Liu a, Yang Zhou a, Xiaoheng Zhao a, Mingjun Xi a, Shun Wei a, Rui Fang a, Wei Ji a, Nan Chen a, Zemao Gu a, Xueqin Liu a, Weimin Wang b,c, Muhammad Asim a, Xiaoling Liu a,⇑, Li Lin a,b,c,d,⇑ a

Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China Hubei Collaborative Innovation Center for Freshwater Aquaculture, Wuhan 430070, People’s Republic of China Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Wuhan 430070, People’s Republic of China d State Key Laboratory of Agricultural Microbiology, Wuhan 430070, People’s Republic of China b c

a r t i c l e

i n f o

Article history: Received 17 September 2013 Revised 21 October 2013 Accepted 21 October 2013 Available online 31 October 2013 Keywords: Grass carp (Ctenopharyngodon idella) Mannose receptor C type 1 (MRC1) Expression Aeromonas hydrophila

a b s t r a c t Mannose receptor C type 1 (MRC1) is a pattern-recognition receptor (PRR) which plays a significant role in immune responses. Much work on MRC1 has been done in mammals and birds while little in fish. In this study, we cloned and characterized MRC1 in grass carp (gcMR). The full-length gcMR contained 5291 bp encoding a putative protein of 1432 amino acids. The predicted amino acid sequences showed that gcMR contained a signal peptide, a cysteine-rich (CR) domain, a fibronectin type II (FN II) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short cytoplasmic domain. gcMR were constitutively expressed in different organs with the higher expression in spleen and head kidney. During embryonic development, gcMR transcript levels were highest at cleavage stage. The up-regulation expression of gcMR, IL-1b and TNF-a in liver, spleen, head kidney and intestine after Aeromonas hydrophila infection indicating it involved in innate immune regulation during bacterial infections. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction Mannose receptor C type 1 (MRC1) is one of pattern-recognition receptors (PRRs) which not only play a significant role in innate immune responses but also in acquired immune responses (Gazi and Martinez-Pomares, 2009). The structure of MRC1 contains extracellular, transmembrane and cytoplasmic regions. In the extracellular region, it consists of a signal peptide, an N-terminal cysteine-rich (CR) domain, a fibronectin type II (FN II) domain and eight tandemly arranged C-type lectin-like domains (CTLDs) (Gazi and Martinez-Pomares, 2009). MRC1 recognizes surface polysaccharides of various pathogens, such as viruses, bacteria, yeasts and parasites and plays an important role in the anti-inflammatory procedure (Chieppa et al., 2003; Taylor et al., 2005; Zhang et al., 2005). During bacterial infections, pro-inflammatory cytokines are always involved in innate immune responses. It has been reported that MRC1 is involved in synthesis of pro-inflammatory cytokines such as IL-1b and TNF-a by binding or internalization of natural or synthetic ligands (Xu et al., 2010; Yamamoto et al., ⇑ Corresponding authors at: Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China. Tel.: +86 27 87282113; fax: +86 27 87282114. E-mail addresses: [email protected] (X. Liu), [email protected] (L. Lin).

1997; Zhang et al., 2005). Furthermore, the release of IL-1b, TNFa requires the collaboration of MRC1 and TLR4 (Zhang et al., 2005). Due to the critical role in innate immune responses, there have been many reports about MRC1 in human and mice but little work has been done in fish. There are five fish MRC1 gene sequences in GenBank, however all these sequences were predicted by automated computational analysis of fish chromosome sequences and no one was verified at mRNA level. Noticeable, there are two types of mannose receptors, named MRC1 and MRC2. Despite of sharing similar domains, such as fibronectin type II domain and multiple C-type lectin-like domains, they play different roles in cells. Compared to MRC1 involving in innate immune responses, MRC2 mediates collagen degradation in lysosome (Sulek et al., 2007). The grass carp (Ctenopharyngodon idella) accounts for the third biggest value (USD 4.8 billion) at single species level of major cultured fish species in the world (FAO, 2010). However, it suffered from bacterial infections, such as Aeromonas hydrophila which can cause high mortality and considerable economic losses in China. Therefore the studies of grass carp MRC1 will fill up the knowledge gap in respects of the structure and function of fish MRC1, provide an insight into the understanding of pathogen recognition responses in fish and will be useful for developing strategies to control fish diseases.

0145-305X/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dci.2013.10.006

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2. Materials and methods 2.1. Sample collection Grass carp ranging from 25 to 30 g in weight, about 9–10 months old, were obtained from a fish farm located at Xiantao city in Hubei Province, China. The fish were maintained at 25–26 °C in a recirculating freshwater system and acclimatized in laboratory for 2 weeks before carrying out experiments. Gill, heart, head kidney, blood, brain, intestine, liver, muscle, spleen and skin from three healthy fish were frozen in liquid nitrogen immediately after collection, and stored at 80 °C until used. Embryos and fries were reared in a hatching trough with constant pool water flow at 24 ± 1 °C. On day 5 post-hatching, the fries were fed with freshwater rotifers captured from pool. Embryos and early larvae were collected and stored in liquid nitrogen until used. Embryonic stages included: cleavage, blastula, gastrula, neurula, somite, eye sac appearance, caudal fin appearance, muscular effect, heart-beating and hatching stages. Fries were also collected everyday for 15 days post-hatching. 2.2. Full-length gcMR cDNA sequences obtained by RACE Core gcMR sequences were obtained by RT-PCR using degenerate primers shown in Supplementary Table 1. cDNAs were synthesized using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Dalian, China) following the manufacturer’s protocol. PCR reactions were carried out in a volume of 20 ll containing 10 ll of Premix Ex Taq (Takara, Dalian, China), 1 ll of 10 lM of each forward and reverse primers, 7 ll of nuclease-free water, and 1 ll of cDNA. Cycling parameters were 94 °C for 3 min followed by 30 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 2 min; and final extension at 72 °C for 10 min followed by cooling down to 16 °C. The PCR products were purified and ligated into pMD-18T vectors (Takara, Dalian, China) and transformed in Escherichia coli DH5a cells. Positive clones were sequenced. Based on the obtained gcMR fragment sequences, primers for 50 -RACE and 30 -RACE (Supplementary Table 1) to obtain the entire gcMR cDNA sequences were designed. The RACE reactions were performed by using a 50 -Full RACE Core Set and a 30 -Full RACE Core Set (Takara, Dalian, China) according to the manufacturer’s protocols. 2.3. Sequence and phylogenetic analysis The MR amino acid sequences from various species were obtained from NCBI (http://www.ncbi.nlm.nih.gov/index.html). The cDNA and deduced amino acid sequences of gcMR were analyzed using BLAST algorithm (http://www.ncbi.nlm.nih.gov/blast) and Expert Protein Analysis System (http://www.expasy.org/). The presence and location of the signal peptide were predicted using SignalP 4.0 (http://www.cbs.dtu.dk/services/SignalP/) and the protein domains were predicted with the simple modular architecture research tool (SMART) version 4.0 (http://smart.embl-heidelberg.de/). Multiple sequence alignment was performed using the Vector NTI Suite 8 program. A phylogenetic tree was constructed using neighbour-joining method in the Molecular Evolutionary Genetics Analysis (MEGA 4.0) package (Tamura et al., 2007). Data were analyzed using Poisson correction, and gaps were removed by complete deletion. The topological stability of the trees was evaluated by 1000 bootstrap replications. 2.4. Tissue distribution and expression profiles of gcMR during embryonic development The expression profiles of gcMR-RNA expression in embryos and tissues were assessed using real-time quantitative reverse

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transcription PCR (qRT-PCR). The cDNA was synthesized as above. b-actin was used as an internal control. The assay was conducted on Rotor-Gene Q Series Software 1.7 Real-Time PCR System (Roche Molecular Systems, USA) in a final volume of 20 ll containing 1 ll cDNA sample, 10 ll SYBRÒ Premix Ex Taq™ (Takara, Dalian, China), 0.8 ll of each forward and reverse primers (10 lM) and 7.4 ll nuclease-free water. Cycling parameters were 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s, 55 °C for 20 s, 72 °C for 20 s, finally at 4 °C for 5 min. All reactions were done in triplicate. Dissociation curve analysis was performed after each assay to determine target specificity. The relative expression ratio of the gcMR gene versus the b-actin gene was calculated using 2DDCT method, and all data were given in terms of relative gcMR-RNA expressed as mean ± SE (N = 3). The data were submitted to one-way analysis of variance (one-way ANOVA) followed by Fisher’s LSD test using SPSS 17.0. Differences were considered significant at P < 0.05 and extremely significant at P < 0.01. 2.5. Analysis of gcMR, IL-1b and TNF-a expression after A. hydrophila infection Challenge experiments were conducted on the fish aquariums following established protocols for A. hydrophila challenges with slight modification (Zhang et al., 2011). In brief, grass carp were randomly divided into 2 groups and were injected intraperitoneally with 100 ll bacterial suspension (4.8  107 CFU/ml) whereas control fish were injected equal volume of sterile 0.65% NaCl. Head kidney, intestine, liver and spleen were collected from each group at 3, 6, 12, 24, 48 and 72 h post-injection. At each time point, tissues from three fishes were frozen in liquid nitrogen immediately after collection, and stored at 80 °C until used. RNA extraction, cDNA synthesis, qRT-PCR assay and data analysis were carried out as described above. 3. Results 3.1. Identification and characterization of gcMR cDNA The full-length gcMR cDNA sequences were shown in Supplementary Fig. 1. The complete sequence of gcMR cDNA consisted of a 50 -terminal untranslated region (UTR) of 88 bp, a 30 -UTR of 904 bp with a poly (A) tail, and an open reading frame (ORF) of 4299 bp. The typical polyadenylation signal AATAAA was found 18 bp before the poly A signal. The ORF encoded a polypeptide of 1432 amino acids, with calculated molecular weight of 163.7 kDa and the predicted isoelectric point of 5.84. As shown in Supplementary Fig. 2, the deduced amino acid sequences of gcMR shared the same domain structures with other MRC1s: an extracellular region containing a signal peptides, an N-terminal cysteine-rich (CR) domain, a fibronectin type II (FN II) domain and eight tandemly arranged C-type lectin-like domains (CTLDs), a transmembrane domain and a short cytoplasmic tail. 3.2. Multiple sequence alignments and phylogenetic analysis The deduced amino acid sequences of the gcMR showed the highest amino acid identity of 93% to that of Megalobrama amblycephala MRC1 followed by Danio rerio MRC1 (78%), while had lowest identity of 29% to that of D. rerio MRC2 (Supplementary Table 2). As shown in Fig. 1, the phylogenetic tree showed that MRC1 and MRC2 were grouped into two clusters. In the case of MRC1, C. idella, M. amblycephala and D. rerio were clustered together in one sub-clade and they all belonged to Cypriniformes. Furthermore, Dicentrarchus labrax and Oreochromis niloticus were grouped together in one sub-clade (Perciformes). Finally, the clade

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from fish was clustered with avian, amphibian and mammalian clades successively. Similar patterns were also observed in cluster of MRC2. Since there is only one computational predicted MRC2 from zebra fish available, we can not get too much information about MRC2 in teleost and need to investigate teleost MRC2 in the future. Overall, the relationships of MR displayed in the phylogenic tree were generally consistent with the results of traditional taxonomy, indicating that MR should be a good evolutionary marker for vertebrates. 3.3. Tissue distribution and expression profiles of gcMR during embryonic development The expression patterns of gcMR during embryogenesis and in healthy fish were analyzed by qRT-PCR. As shown in Supplementary Fig. 3, gcMR transcript levels were high in early embryos at the cleavage and blastula stages, then reduced sharply at the gastrula stage and kept a relatively lower expression level until the heart-beating stage, which suggested that gcMR transcription was maternally inherited because it was already existed in embryos before the mid-blastula transition (Xu et al., 2011). It significantly increased at the hatching stage and kept a relatively high level until the early larval stage, which indicated that gcMR might play an important role in host defense against the pathogen infection at early larval stages. In healthy grass carp, the mRNA transcripts of gcMR were found to be constitutively expressed in a wide range of tissues with different levels, including gill, heart, head kidney, intestine, liver, muscle, blood, brain, skin and spleen (Supplementary Fig. 4). However, higher expression levels of gcMR were observed in spleen and head kidney which were 13.91-, 12.55-fold of that in intestine, indicating that gcMR might play a role in the innate immune response.

3.4. Temporal expression of gcMR, IL-1b and TNF-a after A. hydrophila infection In order to examine the specific role of gcMR in response to A. hydrophila challenge, the level of gcMR regulation as well as the release of proinflammatory cytokines IL-1b and TNF-a mRNAs were measured and qRT-PCR was performed in 4 major immune-related organs, including liver, spleen, head kidney and intestine. As shown in Fig. 2, there were basically one-peak and two-peak expression patterns of gcMR in the four organs during infection. In the liver and intestine, the gcMR expression fitted two-peak pattern. In the liver it was initially down-regulated at 3 h post-infection (poi), and then first peak appeared at 6 h poi (6.94-fold, P < 0.01). Subsequently, it sharply dropped to the level of 3 h poi. The second peak was observed at 24 h poi (4.07-fold, P < 0.01) and eventually decreased to the lowest level at 72 h poi (0.27-fold, P < 0.01) (Fig. 2A). In case of intestine, gcMR gene expression pattern was similar to that in liver except the first peak came up early at 3 h poi (1.98-fold), subsequently lower expressions were observed at 12 h (0.32-fold) and 48 h (0.32-fold) poi (Fig. 2D). By contrast, the gcMR expression matched one-peak pattern in spleen and head kidney. The expression levels of gcMR were initially down-regulated at 3 h, then up-regulated drastically and reached the peak at 12 h poi, with 8.23- and 6.59-fold in the spleen and head kidney, respectively (all P < 0.01), thereafter significantly decreased at 48 h poi and recovered near to the pre-injection level at 72 h poi (Fig. 2B and C). Overall, temporal expression of gcMR mRNA transcripts in the tested tissues were significantly up-regulated after A. hydrophila infection, indicating that A. hydrophila might be recognized by gcMR. The expression levels of gcMR were initially down-regulated at 3 h poi in liver, spleen and head kidney, which suggested that gcMR might also be down-regulated by A.

mammal

avian

C type 1

amphibian

teleost teleost amphibian

mammal

C type 2 avian

Fig. 1. Phylogenetic tree of gcMR and other MRs were constructed using MEGA 4.0 with neighbor-joining method. Numbers of each node indicated the percentage of bootstrapping of a 1000 replications. The protein sequences used for phylogenetic analysis were shown in Supplementary Table 2.

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Fig. 2. Expression analysis of gcMR, IL-1b and TNF-a after A. hydrophila infection. Expression patterns were determined in liver (A), spleen (B), head kidney (C) and intestine (D) by qRT-PCR method. The samples were analyzed at 3, 6, 12, 24, 48 and 72 h post-treatment. Expression of b-actin was as an internal control. Each experiment was performed at least in triplicate. Data were shown as mean ± SE (N = 3).The asterisk indicated a statistically significant difference (⁄⁄P < 0.01, ⁄P < 0.05) compared with the control (0 h).

hydrophila in vivo and had the same effect as LPS in vitro (Gordon, 2003; Xu et al., 2010), while the molecular mechanisms that downregulated gcMR expression in vivo were still unknown and needed to be addressed in the future. During bacterial infections, pro-inflammatory cytokines are always involved in the innate immune responses. In order to obtain more information about cytokines involved, two cytokines, IL-1b and TNF-a in grass carp were chosen for the studies. As shown in Fig. 2, there were basically only one-peak expression pattern of both cytokines in the four organs during infection. Overall, IL-1b relative expression showed similar profiles in all four organs except the time of peak were different. The expression levels of IL1b were up-regulated drastically and reached the peak value with 5.77-fold in liver at 12 h poi, 13.96-fold in spleen at 6 h poi, 11.14fold in head kidney at 6 h poi and 28.90-fold in intestine at 12 poi (all P < 0.01), then rapidly decreased to the pre-injection level at 72 h poi. Similar to IL-1b, overall TNF-a relative expression showed similar profiles in all four organs except the time of peak and value were different. The expression levels of TNF-a were up-regulated and reached the peak value with 2.24-fold in liver at 48 h postinfection (poi), 6.64-fold in spleen at 3 h poi, 17.16-fold in head kidney at 3 h poi and 1.6-fold in intestine at 12 h poi, thereafter decreased to the pre-injection level at 72 h poi. Currently, we do not know how to explain the expression pattern differences among

these three genes, since complicate mechanisms of gcMR, IL-1b and TNF-a regulations have been reported. For example, by binding or internalization of natural or synthetic ligands, MRC1 involved in synthesis of pro-inflammatory cytokines such as IL-1b, IL-6 and TNF-a (Xu et al., 2010; Yamamoto et al., 1997; Zhang et al., 2005). Since MRC1 lacks of signal motifs in its cytoplasmic domain, it is very likely that it requires the assistance from other receptors in order to trigger signal cascades (Gazi and Martinez-Pomares, 2009). It has been reported that the release of IL-1b, TNF-a and IL-6 requires the collaboration of MRC1 and TLR4 (Zhang et al., 2005) and the IL-8 release in macrophages requires expression of MRC1 and TLR2 (Tachado et al., 2007). It is well established that NF-jB plays a key role in immune and inflammatory responses and is a major mechanism of LPS-induced expression of cytokines, including IL-1, TNF-a and IL-8 (Tian and Brasier, 2003). The relationship between IL-1b–TNF-a release and the involvement of gcMR–TLR4–NF-jB pathway in grass carp needed to be further characterized. In conclusion, we reported the successful cloning of gcMR in grass carp using RACE techniques. It shared important structural elements with MRC1s of other species. During embryonic development, gcMR transcript levels were highest at cleavage stage. The gcMR transcripts were predominantly expressed in spleen and head kidney. The up-regulation expression of gcMR, IL-1b and

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TNF-a in liver, spleen, head kidney and intestine after A. hydrophila challenge indicated its positive role in responding to bacterial infections. Better understanding of the immune defense mechanisms of teleost MRC1 will pave a new way for the strategy development of fish diseases prevention. Acknowledgements We would like to thank Prof. Dr. Vikram N. Vakharia from University of Maryland, USA for critical reading the manuscript. This work was jointly supported by the National Basic Research Program of China (973 Program, No. 2009CB118706), the Fundamental Research Funds for the Central Universities (52204-12020, 2013PY069, 2013PY70, 2013PY071, 2011PY023, 2011PY043, 2011SC27, 2011ZC011, 2012YB08) and Natural Science Foundation of Hubei Province of China (2009CDB424). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.dci.2013.10.006. References Chieppa, M., Bianchi, G., Doni, A., Del Prete, A., Sironi, M., Laskarin, G., Monti, P., Piemonti, L., Biondi, A., Mantovani, A., Introna, M., Allavena, P., 2003. Crosslinking of the mannose receptor on monocyte-derived dendritic cells activates an anti-inflammatory immunosuppressive program. J. Immunol. 171, 4552– 4560.

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