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Fish & Shellfish Immunology 30 (2011) 324e329 Contents lists available at ScienceDirect Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Molecular cloning, characterization and expression analysis of macrophage migration inhibitory protein (MIF) in Chinese mitten crab, Eriocheir sinensis Wei-Wei Li, Xing-Kun Jin, Lin He, Ying Wang, Li-Li Chen, Hui Jiang, Qun Wang* School of Life Science, East China Normal University, North Zhong-Shan Road, Shanghai, China a r t i c l e i n f o a b s t r a c t Article history: Received 13 September 2010 Received in revised form 21 October 2010 Accepted 4 November 2010 Available online 16 November 2010 Macrophage migration inhibitory factor (MIF) as a multi-functional cytokine mediating both innate and adaptive immune responses, however, their function within the innate immune system of invertebrates remains largely unknown. Therefore, we investigated the immune functionality of MIF in Chinese mitten crab (Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The fulllength MIF cDNA (704 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a E. sinensis cDNA library. The MIF cDNA contained a 363 bp open reading frame (ORF) that encoded a putative 120 amino acid (aa) protein. Comparisons with other reported invertebrate and vertebrate MIF sequences revealed conserved enzyme active sites. MIF mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in hepatotpancreas, and (b) responsive in hemocytes, hepatopancreas and gill to a Vibrio anguillarum challenge, with peak exposure observed 8 h, 12 h and 12 h post-injection, respectively. Collectively, data demonstrate the successful isolation of MIF from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate. Ó 2010 Elsevier Ltd. All rights reserved. Keywords: Eriocheir sinensis Macrophage migration inhibitory factor mRNA expression profile Real-time PCR 1. Introduction Since found first in 1966 [1,2], the mammalian macrophage migration inhibitory factor (MIF), has been shown to correlate with the regulation of macrophage functions [3,4], lymphocyte immunity [5,6], endocrine function [7e11] and a number of immune and inflammatory diseases [7,8,12e22]. MIF initially identified as an inhibitor of random migration of macrophages, has now emerged to be a multi-functional factor involved in immune response, glucose and lipid metabolism. In addition, MIF can modulate glycolysis and insulin resistance in insulin target cells including adipocytes and myocytes [6]. MIF was constitutively expressed by a variety of immune and some non-immune cells and released into the circulation from preformed intracellular stores by a non-conventional leaderless pathway [7,23,24]. As one of the first lymphocyte-derived cytokines, MIF was identified as a soluble factor produced by antigenactivated T lymphocytes that inhibited the random migration of macrophages [1,2,25]. Furthermore, recent research shows a more prominent role of MIF as a multi-functional cytokine mediating both innate and adaptive immune responses. To data, mammalian MIF is an immunomodulator that controls macrophage functions, * Corresponding author. Fax: þ86 21 62233754. E-mail address: [email protected] (Q. Wang). 1050-4648/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2010.11.008 resulting in the promotion of proinflammatory cytokine expression (TNF-, IL-1, IL-2, IL-6, IL-8, and IFN-) [3,5,20], nitric oxide (NO) release [26] and COX-2 activity [27]. In activated macrophages, MIFinduced TNF- leads to further MIF release, resulting in optimal expression of TNF- by macrophages [3]. MIF also up-regulates the expression of Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharide (LPS) and induces the activation of monocytes/ macrophages, suggesting potential involvement of MIF in innate immune responses [28]. Homologues of mammalian MIF have been detected in chicken, fish, ticks, nematode and even plant [29]. All evidences available up to now imply that MIFs might exert important and similar functions among different species. Chinese mitten crab, Eriocheir sinensis, is an economically important freshwater species for aquaculture in China. However, bacterial- and viral-born disease have blossomed within booming E. sinensis cultures, incurring catastrophic losses to crab aquaculture [30]. Crab, as well as other invertebrates, do not possess an adaptive immune system and must rely on efficient innate immune defenses [31]; therefore, obtaining a better understanding of the innate immune ability of crab and their defense mechanisms has become a research priority. In order to determine the participation of MIF in innate immune responsiveness in crustaceans, and identify key mechanistic steps for improving immunity and associated mortality in aquaculture E. sinensis, we (1) cloned the fulllength Es-MIF cDNA using EST sequences identified from a E. sinensis hepatopanreatic cDNA library previously constructed by W.-W. Li et al. / Fish & Shellfish Immunology 30 (2011) 324e329 our laboratory [32], (2) examined mRNA tissue-dependent expression patterns, and (3) determined the temporal response of Es-MIF expression in response to an immune challenge via Vibrio anguillarum exposure. 2. Method and materials 325 Table 1 Primer sequences. Primers Primers for 50 RACE PCR MIF 50 first GSP primer MIF 50 nested GSP primer Sequences Code 50 -GCTCTGATTTGC CAAGCATCTCACTA30 50 -GGAACATTTGTG AACACCTCCAGCA30 GSP-50 -f 50 -GATGGTGGACAA TCGGCTGTGGe30 50 -TTTCCATGCAATA CTTGGCCGCTGAA30 GSP-30 -f GSP-50 -n 2.1. Sample preparation Healthy E. sinensis (mean weight ¼ 100 g) were collected from the Tongchuan aquatic product market in Shanghai, China. Crabs were placed in an ice bath for 1e2 min until lightly anesthetized prior to sacrifice. The following tissues were harvested, snap frozen in liquid nitrogen, and stored at 80 C until nucleic acid analysis, hepatopancreas, brain, gills, intestine, hemocytes, muscle, stomach, heart, testis and ovarian. For cloning and expression analysis, tissues from 10 crabs were pooled and ground with a mortar and pestle prior to extraction. Primers for 30 RACE PCR MIF 30 first GSP primer MIF 30 nested GSP primer ClontechÔ Kit primesr Universal primer A mix Nested universal primer A 2.2. Nucleic acid extraction Total RNA was extracted from E. sinensis using TrizolÒ reagent (Invitrogen) according to the manufacturer’s protocol. The concentration and quality of total RNA were estimated by spectrophotometry (absorbance at 260 nm) and agarose-gel electrophoresis, respectively. 2.3. First-strand cDNA synthesis Total RNA (5 mg) isolated from hepatopancreas was reverse transcribed using the SMARTÔ cDNA kit (Clonetech) for cDNA cloning. For expression analysis, total RNA (4 mg) was reverse transcribed using the PrimeScriptÔ RT-PCR Kit (TaKaRa) for semiquantitative RT-PCR analysis or the PrimeScriptÔ Real-time PCR Kit (TaKaRa) for real-time quantitative RT-PCR (qRT-PCR) analysis. 2.4. The cDNA full-length clone by RACE The Es-MIF cDNA sequence was extended using 50 and 30 RACE and a total of four gene-specific primers (Table 1) based upon the original EST sequence (as described above; GenBank accession no. GE340285). The initial 30 RACE PCR reaction was carried out in a total volume of 50 ml that contained 2.5 ml of the first-strand cDNA reaction as template, 5 ml of 10 Advantage 2 PCR buffer, 1 ml of 10 mM dNTPs, 5 ml of 10 mM gene-specific primer (GSP-30 -f and GSP-30 -n; Table 1), 1 ml of Universal Primer A Mix (UPM; Clonetech, USA), 34.5 ml of sterile deionized water, and 1 U 50 Advantage 2 polymerase mix (Clonetech). Diluted products of the first PCR 30 RACE reaction (1:50 with TricineeEDTA buffer) served as template for the 30 nested PCR reaction. With the exception of the template and nested primer (NUP), contents of the nested PCR reaction were identical to the initial reaction. For 50 RACE, SMARTÔ cDNA kit UPM and NUP were used as forward primers in initial and nested PCR reactions in conjunction with the reverse gene-specific primers GSP-50 -f and GSP-50 -n, respectively (see Table 1 for sequence). PCR amplification conditions for initial 30 and 50 RACE were as follows: 5 cycles at 94 C for 30 s, 72 C for 3 min; 5 cycles at 94 C for 30 s, 70 C for 30 s, and 72 C for 3 min; 20 cycles at 94 C for 30 s, 68 C for 30 s, and 72 C for 3 min. Nested PCR amplification conditions were as follows: 20 cycles at 94 C for 30 s, 68 C for 30 s, and 72 C for 3 min. PCR amplicons were size separated and visualized on an ethidium bromide stained 1.2 % agarose gel. Amplicons of expected sizes were purified with E.Z.N.AÒ Gel Extraction Kit (Omiga BioTek), and inserted into a pTZ57RÒ Vector (Promega). Positive clones GSP-30 -n 50 -CTAATACGACTCACT ATAGGGCAAGCAGTGG TATCAACGCAGAGT-30 50 -CTAATACGACTCA CTATAGGGC-30 50 -AAGCAGTGGTAT CAACGCAGAGT-30 Primers for RT-PCR and Real-time PCR analysis MIF 50 primer 50 -TGACTTGTTTTC CTCCACTCCC-30 MIF 30 primer 50 -GGTGTTCACAA ATGTTCCCAAGG-30 b-actin 50 primer 50 -CTCCTGCTTGCT GATCCACATC-30 b-actin 30 primer 50 -GCATCCACGAG ACCACTTACA-30 M-RT-R M-RT-F b-RT b-FT containing inserts of the expected size were sequenced using T7 and SP6 primers. 2.5. Sequence analysis Es-MIF full-length cDNA and deduced amino acid sequences were compared with other sequences reported in NCBI’s GenBank using the BLAST program. MIF cDNA and deduced amino acid sequences from E. sinensis and representative vertebrates and invertebrates were compared by multiple sequence alignment using ClustalX. Anphylogenetic treewas constructed with MEGA4.0. E. sinensis MIF cDNA and deduced amino acid sequence were deposited under GenBank accession numbers HM775084. 2.6. Immune challenge and hemocyte isolation Chinese mitten crabs (n ¼ 155; 70 5 g wet weight) were acclimated for 1 week at 20e25 C in filtered, aerated freshwater prior to injection with approximately 70 ml live V. anguillarum resuspended in 0.1 mol L1 PBS (pH ¼ 7.0, 109 CFU mL1) or a vehicle control of PBS (pH ¼ 7.0). Five crabs were randomly selected 2, 4, 6, 8, 12, 16, 32 and 48 h post-injection from experimental and control groups for hemolymph collection using a syringe (approximately 2.0 ml per crab). An equal volume of anticoagulant solution was then added to each hemolymph sample, and centrifuged at 800 g at 4 C to isolate hemocytes. Hemocytes were stored at 80 C after addition of 1 mL Trizol reagent (Invitrogen) for subsequent RNA extraction. 2.7. Real-time qRT-PCR analysis The mRNA expression of MIF was measured by real-time RTPCR. Briefly, total RNA was isolated from hepatopancreas, gill, ovary, testis, muscle, heart, brain, stomach, intestine and hemocytes of 326 W.-W. Li et al. / Fish & Shellfish Immunology 30 (2011) 324e329 2 SYBR Premix Ex Taq (TaKaRa), 0.5 ml diluted cDNA template, 11.0 ml PCR-Grade water, and 0.5 ml of each primer. PCR conditions were as follows, 95 C for 30 s; followed by 40 cycles of 95 C, and a 0.5 C/5 s incremental increase from 60 C to 95 C that lasted 30 s per cycle. Resultant data was analyzed using the CFX ManagerÔ software (Version 1.0). 2.8. Statistical analysis Statistical analysis was performed using SPSS software (Ver11.0). Data represent the mean standard error (S.E.). Statistical significance was determined by one-way ANOVA [33] and post hoc Duncan multiple range tests. Significance was set at P < 0.05. 3. Results 3.1. Cloning and characterization of Es-MIF cDNA The full-length Es-MIF cDNA sequence cloned from E. sinensis hepatopancreas was 704 bp long and contained a 363 bp ORF that encoded a 120 amino acid protein, a 103 bp 50 UTR, and a 235 bp 30 UTR (Fig. 1). 3.2. Amino acid sequence alignment Alignment of the Es-MIF amino acid sequence with those reported for other organisms demonstrated conservation of sites with demonstrated catalytic activity (Fig. 2). Overall homology was high among other species in reference to Es-MIF, with Maconellicoccus hirsutus and Acyrthosiphon pisum exhibiting the greatest percent identity (56%), followed by Bombyx mori (52%), Ascaris suum (46%), Amblyomma americanum (46%) and Xenopus tropicalis (45%). 3.3. Phylogenetic analysis of Es-MIF Fig. 1. E. sinensis MIF cDNA and deduced amino acid sequences. The triad of conserved catalytic active sites (P2, K33 and Cys57) are shaded. unchallenged crabs (used as a pool) and hemocytes, hepatopancreas and gill of bacteria-challenged crabs (used as individually). Real-time qRT-PCR was conducted using the CFX96Ô Real-Time System (Bio-Rad). Gene-specific primers (Table 1) were designed based upon the cloned MIF cDNA to produce a 168 bp amplicon. Samples were run in triplicate and normalized to the control gene b-actin, Es-MIF expression levels were calculated by the 2DDCt comparative CT method. Real-time qPCR amplification reactions were carried out in a final volume of 25 ml, which contained 12.5 ml Phylogenetic analysis of Es-MIF from representative crustaceans, mammals, and pisces (Fig. 3) produced an NJ-phylogenetic tree, that contained two distinct branches, suggesting a phylogenetic relationship and shared common ancestor among MIF genes in different species. The first branch was comprised by vertebrate including mammals and fish, the second branch was composed by invertebrate include E. sinensis, which supporting traditional taxonomic relationships. 3.4. Tissue distribution of Es-MIF expression As determined by Real-time qRT-PCR, detectable Es-MIF expression was widely observed in the hepatopancreas, hemocytes, gill, brain, muscle, heart, intestine, stomach testis and ovarian of E. Fig. 2. Multiple alignment of MIF amino acid sequences. Identical (*) and similar (. or :) amino acid residues are indicated. Gaps () were introduced to maximize the alignment. Protein abbreviations and corresponding GenBank accession numbers are as follows: Eriocheir sinensis (HM775084); Homo sapiens (CAG30406.1); Danio rerio (NP_001036786.1); Mus musculus (AAA91638.1); Ornithorhynchus anatinus (XP_001507338.1); Salmo salar (NP_001117081.1); Cyprinus carpio (ABY71027.1); Oncorhynchus mykiss (ACO08446.1); Tetraodon nigroviridis (AAW50793.1); Branchiostoma belcheri tsingtauense (AAT77698.1); Sus scrofa (NP_001070681.1); Bos taurus (NP_001028780.1); Ovis aries (NP_001072123.1); Rattus norvegicus (AAB04024.1); Acyrthosiphon pisum (NP_001156107.1); Bombyx mori (NP_001040199.1); Ascaris suum (49257069); Trichuris trichiura (5327286); Strongyloides ratti (198448303). W.-W. Li et al. / Fish & Shellfish Immunology 30 (2011) 324e329 Fig. 3. Neighbor-joining phylogenetic tree of MIF amino acid sequences reported in representative taxa. See Fig. 2 for GenBank accession numbers. sinensis (Fig. 4). Expression was highest in hepatopancreas, and comparable among heart, gill, intestine and ovarian. 3.5. Temporal expression of Es-MIF in immune challenged hemocytes Es-MIF expression level, as measured by real-time qRT-PCR, was induced in hemocytes, hepatopancreas and gill following exposure to V. anguillarum (Fig. 5 and Fig. 6). Es-MIF expression in hemocyte was significantly greater than the vehicle control after 4 h, 6 h, 8 h and 12 h post V. anguillarum stimulation (P < 0.05). Es-MIF expression in hemocyte was up-regulated at 4 h post-injection, and peaked to 16.5-fold that of the vehicle control after 8 h, and then decreased to levels with no significant different with the vehicle 16 h post-injection (Fig. 5). The expression of Es-MIF after immune challenge was determined with real-time PCR in both hepatopancreas and gill tissues in E. sinensis (Fig. 6). Es-MIF mRNA levels in hepatopancreas and gill increased sharply 12 h after exposure to V. anguillarum, and decreased down after 16 h post-exposure. Control reactions, in which were induced with PBS, yielded no significant increase in expression levels. 4. Discussion 327 Fig. 5. Temporal MIF mRNA expression in response to V. anguillarum challenge (black bars). Hemocytes collected from crabs injected with V. anguillarum (black bars) or vehicle (white bars) were compared with respect to MIF mRNA expression (relative to b-actin) using Students t-tests. Bars represent mean S.E. (n ¼ 6). Statistical significance is indicated with an asterisk (P < 0.05). protozoa, which may result from its functional significance. Correlation between mechanism of action and role in diseases reveals that MIF is in a cytokine network not only as an effector molecular but also as a proinflammatory mediator and serves as a potential therapeutic target [34]. The presence of Es-MIF in this study further demonstrated that MIF is evolutionarily conserved, The amino-terminal proline residue (Fig. 1, position P) which is crucial for the catalytic activity of isomerase [35e39] was found in Es-MIF (shown in Fig. 1). In addition, the invariant lysine residue (Fig. 1, position K) observed in many species include mammalian, which contributes to the isomerase activity of the protein [38], is also present in Es-MIF. Because crucial amino acid residues, thus we expected Es-MIF to have the same activity as mammalian MIF. Mammalian MIF is distinguished by the presence of three conserved cysteines (Fig. 1, Cys57, Cys60, and Cys81), the first two of which define a CXXC motif that mediates thiol protein oxidoreductase activity [35e39]. Except that Cys is conserved in Es-MIF, the latter two cysteines as well as the CXXC motif, are absent in EsMIF, and that is the same with the report in Haliotis diversicolor supertexta MIF [40]. Therefore, whether the absence of two cysteines as well as the CXXC motif has any effect on the function of ESMIF needs to be further studied. MIF was found to be conserved throughout species, from both invertebrates and vertebrates, even in single-celled parasitic Fig. 4. Tissue-dependent MIF mRNA expression in E. sinensis. MIF mRNA expression in hemocytes, heart, hepatopancreas, gill, stomach, muscle, intestine, brain, testis and ovarian. Fig. 6. Hepatopancreas and gill tissues collected from crabs injected with V. anguillarum were compared with respect to MIF mRNA expression (relative to b-actin) using Students t-tests. Bars represent mean S.E. (n ¼ 6). Statistical significance is indicated with an asterisk (P < 0.05). 328 W.-W. Li et al. / Fish & Shellfish Immunology 30 (2011) 324e329 Analysis of secondary structure suggested that Es-MIF protein possesses four a-helices and five b-sheets, in the following order: b1a1b2b3b4a2b5a3a4, which is almost similar to human MIF, whose three-dimensional structure is comprised of two a-helices and six b-sheets in the following order: b1a1b2b3b4a2b5b6 [25]. The little difference is the structure of carbon terminal, which in human MIF has a b-sheet (b6) and Es-MIF has two small a-helices (a3a4), which was found in H. diversicolor supertexta too [40]. So the putative molecular modeling of Es-MIF also has similar structure to that of human MIF. On account of the analysis above, we can assume that Es-MIF protein may have similar biological functions to human MIF. Information extracted from Es-MIF tissue-dependent mRNA expression may offer useful cues when speculating function. EsMIF transcripts were detected in all tissues examined, including hepatopancreas, hemocytes, gills, brain, muscle, heart, intestine, stomach, testis and ovarian. In mammals, MIF is ubiquitously expressed in various cells and tissues such as the lung, the skin, gastrointestinal and several tissues of the endocrine system [7, 41-43], and our study was consistent with the ubiquitous expression reported before. Notably, Es-MIF mRNA expression was highest in hepatopancreas, and the possibility exists that Es-MIF’s wide distribution among tissue types may be the result of hemocyte infiltration. Further, the high level of Es-MIF mRNA observed in hepatopancreas may imply the hepatopancreas’ participation in immune response, as does its expression in gill, which undergoes constant bacterial challenges due to direct water exposure. Current knowledge on acute response of MIF is mostly limited to cultured cells (in vitro systems). Macrophage MIF can be released after stimulation with microbial products that include bacterial endotoxin (LPS), exotoxins, streptococcal pyrogenic exotoxin A, malaria pigment, gram-negative and gram-positive bacteria, mycobacteria, and proinflammatory cytokines [3,12,44], The doseeresponse curves after most of these stimulating were usually bell-shaped. Moreover, time course of MIF release depends on cell type, culture conditions and stimulating cytokine [45]. In this study, we investigated the responsiveness of Es-MIF expression to an immune challenge in order to elucidate potential involvement in the invertebrate innate immune system. We observed a timedependent upregulation in Es-MIF expression following V. anguillarum stimulation, with a significant increase observed at 4 h postinjection (an 5.9-fold increase relative to the vehicle control), and we observed a total of four expression peaks in response to a single injection, after 4 h, 6 h (9.0-fold), 8 h (16.5-fold) and 12 h (4.6-fold). The expression of the Es-MIF after immune challenge was determined in both hepatopancreas and gill tissues in E. sinensis. Es-MIF mRNA levels in hepatopancreas increased sharply 6 h after exposure to V. anguillarum, and decreased down after 16 h post-exposure. In contrast, Es-MIF mRNA levels in gill were only slightly increased after 8 and 12 h post-challenge. Control reactions, in which cells were not induced with V. anguillarum, yielded no significant increase in expression levels. The results showed that Es-MIF was stimuli induced, and biologically relevant to crab immune or inflammatory response to bacterial and may also play a central role in the inflammatory response to bacterial. To data, few reports pay attention to the acute response of MIF in in vivo systems of aquatic animals, only available in vivo study was performed in fish T. nigroviridis, TnMIF mRNA levels in spleen increased sharply 3 h after exposure to LPS and decreased 12 h post-exposure, and TnMIF mRNA levels in head kidney were only slightly increased 3 and 24 h post-challenge [37]. In the study of H. diversicolor supertexta [40], MIF expression level at 24, 48 h after Vibrio parahaemolyticus injection was up-regulated significantly. Furthermore, MIF is rapidly induced by some proinflammatory effector molecules and pathogen components [7,45,46]. Recently, emerging evidence demonstrated that MIF has a central role as a regulator of innate immune and inflammatory responses, and the implications it might have for the development of new therapies for human autoimmune diseases, sepsis and other inflammatory diseases [47,48]. Considering that MIF is constitutively expressed in many cells [23,49] and exhibits catalytic properties reminiscent of certain cellular enzymes [50,51], we reasoned that it was possible that MIF interacts with intracellular proteins. JAB1 (Jun activating binding protein 1) was initially discovered as a coactivator of AP-1 transcriptional activity [52], Jab1-mediated rescue of fibroblasts from growth arrest is blocked by MIF. Amino acids 50e65 and Cys 60 of MIF are important for Jab1 binding and modulation. MIF may act broadly to negatively regulate Jab1-controlled pathways and that the MIF-Jab1 interaction may provide a molecular basis for key activities of MIF [53], which need to be further studied in E. sinensis. Our findings buttress those previously reported in other aquatic animals suggest that Es-MIF expression is immuno responsive, and Es-MIF may possess immune functionality in E. sinensis. However, its mechanism of action is incompletely understood. So, the role of MIF proteins or their therapeutic manipulation in immune diseases is largely unexplored worthy territory, and the analysis of Es-MIF action in immune responses could also yield valuable insights. In conclusion, we have cloned the Es-MIF cDNA from Chinese mitten crab E. sinensis which may help us to understand the origin and evolution of the immune system as well as structure and function of the immune system in crab. 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