Developmental and Comparative Immunology 49 (2015) 207–216
Contents lists available at ScienceDirect
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
Molecular and functional characterization of an IL-1β receptor antagonist in grass carp (Ctenopharyngodon idella) Fuli Yao, Xiao Yang, Xinyan Wang, He Wei, Anying Zhang, Hong Zhou * School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China
A R T I C L E
I N F O
Article history: Received 25 July 2014 Revised 24 November 2014 Accepted 25 November 2014 Available online 2 December 2014 Keywords: IL-1β receptor antagonist Receptor binding assay Functional identification Head kidney leukocytes Grass carp
A B S T R A C T
In the present study, we discovered a novel IL-1 family member (nIL-1F) from grass carp that possessed the ability to bind with grass carp IL-1β receptor type 1 (gcIL-1R1) and attenuate grass carp IL-1β activity in head kidney leukocytes (HKLs), suggesting that it may function as an IL-1β receptor antagonist. Grass carp nIL-1F transcript was constitutively expressed with the highest levels in some lymphoid organs, including head kidney, spleen and intestine, implying its potential in grass carp immunity. In agreement with this notion, in vitro and in vivo studies showed that nIL-1F mRNA was inductively expressed in grass carp with a rapid kinetics, indicating that it may be an early response gene during immune challenges. In addition, recombinant grass carp IL-1β (rgcIL-1β) induced nIL-1F mRNA expression via NF-κB and MAPK (JNK, p38 and p42/44) signaling pathways in HKLs. Particularly, the orthologs of nIL-1F found in other fish species, including zebrafish, pufferfish and rainbow trout are not homologous to mammalian IL-1β receptor antagonist (IL-1Ra), indicating that fish nIL-1F and mammalian IL-1Ra may not share a common evolutionary ancestor. Taken together, our data suggest the existence of a naturally occurring fish nIL-1F, which may function like mammalian IL-1Ra, being beneficial to understand the autoregulatory mechanism of IL-1β activity in fish immunity. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction As a major proinflammatory cytokine, interleukin-1β (IL-1β) plays pleiotropic roles in host defense by inducing a cascade of reactions leading to inflammation (Sims and Smith, 2010). In addition to its induction of other proinflammatory cytokines (e.g. IL-6, IL-8 and TNF-α), IL-1β rapidly stimulates its own production, which amplifies IL-1β response via a positive-feedback loop (Sims and Smith, 2010). In parallel, the potent and extensive proinflammatory activities of IL-1β are tightly controlled by several naturally occurring IL-1β signaling inhibitors, including IL-1 receptor antagonist (IL1Ra), IL-1 receptor type 2 (IL-1R2) and other soluble receptors (Weber et al., 2010). Among these endogenous inhibitors, IL-1Ra binds to IL-1β receptor type 1 (IL-1R1) but does not transduce the signal as it lacks the IL-1 receptor accessory protein interacting domain, while IL-1R2 acts as a decoy receptor for IL-1 activity, thereby constituting two fundamental regulatory mechanisms for the blockade of IL-1β signaling (Nambu and Nakae, 2010). Alternatively, the role of IL-1Ra in autoimmune diseases has been well documented (Jesus and Goldbach-Mansky, 2014; Molto and Olive, 2010). It has become
* Corresponding author. School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China. Tel: +86 28 83206437; fax: +86 28 83208238. E-mail address: [email protected] (H. Zhou). http://dx.doi.org/10.1016/j.dci.2014.11.016 0145-305X/© 2014 Elsevier Ltd. All rights reserved.
evident that hereditary autoinflammatory diseases are caused by excessive IL-1 signaling due to the mutations or deletions of the IL1Ra gene (Aksentijevich et al., 2009; Jesus and Goldbach-Mansky, 2014; Reddy et al., 2009). In animal models, it has been further implicated in IL-1Ra’s contributions to autoinflammatory syndromes. As predicted, IL-1Ra-deficient mice spontaneously develop chronic inflammatory polyarthropathy, such as arthritis, skin inflammation and arteritis (Gabay et al., 2010; Nicklin and Shepherd, 2003). In contrast, administration of IL-1Ra has resulted in marked improvements in experimental models of inflammatory arthritis and osteoarthritis (Jacques et al., 2006). Importantly, the recombinant IL-1Ra named anakinra has been approved for the treatment of patients with rheumatoid arthritis (Jesus and Goldbach-Mansky, 2014; Molto and Olive, 2010). Taken together, these findings highlight the key regulatory role and clinical implication of IL-1Ra in IL-1related diseases. Considering its clinical application, IL-1Ra has been extensively studied in mammals, showing that it has four distinct isoforms derived from the same gene by alternative mRNA splicing and alternative translation initiation (Gabay et al., 2010). One isoform is secreted, whereas the others lack a signal peptide and are localized within the cells (Arend et al., 2008). These four isoforms are produced by different types of cells and all of them can inhibit IL-1 effects (Arend et al., 2008). Like IL-1β production, IL-1Ra expression is induced by IL-1β and other inflammatory stimuli including IgG complexes, bacterial or viral components (Sims and Smith, 2010).
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F. Yao et al./Developmental and Comparative Immunology 49 (2015) 207–216
Actually, IL-1Ra is produced and released by almost all cells expressing IL-1β, being considered as the third member of the IL-1 family (Molto and Olive, 2010). However, the existing knowledge on fish IL-1Ra is limited as there is only a single report about nIL1F in rainbow trout, showing its ability to inhibit IL-1β effect in a trout macrophage cell line and interact with IL-1R1 in protein modeling although it has low homology to the known mammalian IL-1 family members (Wang et al., 2009). The cDNAs homologous to trout nIL-1F (GenBank ID: NM _001124396.2) can be found from NCBI database in other fish species, including zebrafish (GenBank ID: JF436963.1) and pufferfish (GenBank ID: JF714481.1), all of which are termed IL-Ra probably based on their homology to nIL-1F. However, direct evidence for IL1Ra and IL-1R1 binding in fish is still lacking. In this study, we discovered a new molecule from grass carp that is homologous to the so-called fish IL-Ra described above. Its structural characterization and expression patterns were revealed as well. Notably, in accordance with the natural characteristics of mammalian IL-1Ra, its recombinant protein displayed the ability to bind with grass carp IL-1R1 and attenuate grass carp IL-1β activity, providing the functional evidence for this molecule serving as IL-1Ra. Given that it is the ortholog of trout nIL-1F (Wang et al., 2009), the newly identified molecule was nominated as grass carp nIL-1F. Our data, together with the findings in other fish species, suggest the existence of nIL1F serving as an IL-1β receptor antagonist in fish, although their gene features such as genomic location and sequence homology differ from mammalian IL-1Ra.
www.ncbi.nlm.nih.gov). The similarity and identity of the protein sequences were calculated by MatGAT 2.0 software (Campanella et al., 2003). A phylogenetic tree was constructed by using MEGA 4 software based on Neighbor-Joining method with the bootstrapping of 1000 repetitions (Kumar et al., 2004). The molecular weight and isoelectric point of the protein were predicted by the Compute pI/Mw tool (http://www.expasy.ch/) and the signal peptide prediction was performed with SignalP 3.0 (Bendtsen et al., 2004). The ICE cut site and thrombin cut site were determined by referring to the cleavage specificity of ICE (Howard et al., 1991) and thrombin (Chang, 1986). The potential N-glycosylation sites were predicted using NetNGlyc 1.0 Server. 2.4. Tissue distribution of nIL-1F in grass carp
Healthy grass carp (Ctenopharyngodon idella) weighing 0.75– 1.0 kg were purchased from Chengdu Tongwei Aquatic Science and Technology Company (Chengdu, China). The fish were acclimated to the laboratory environment (1000-L aquaria) for at least 2 weeks prior to use in experiments. During the procedures of cell preparation, the fish were killed by anesthesia in 0.05% MS222 (SigmaAldrich, St. Louis, MO) and the head kidneys were collected for subsequent HKL isolation. All animal experiments complied with the Regulation of Animal Experimentation of Sichuan province, China.
About 2 μg RNA samples from each selected tissue (brain, thymus, gill, heart, kidney, head kidney, liver, spleen and intestine) were digested with RNase-free DNase I (Promega, Madison, WI) and then subjected to reverse transcription as described in section 2.2. The levels of nIL-1F and β-actin transcripts were quantitated by real time quantitative PCR (qPCR) following MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and the primers were listed in Appendix: Supplementary Table S1. In brief, qPCR was performed on a Bio-Rad CFX96 Real-time detection system (Bio-Rad Laboratories, Inc., Hercules, CA) in a final volume of 20 μl containing 10 μl of Real Master Mix (Tiangen, Beijing, China), 2 μl of the cDNA, and 0.5 μl of each of forward and reverse primers (10 μM). The amplification program was 94 °C for 2 min, followed by 35 cycles of 94 °C for 20 s, 59 °C (β-actin) or 64.5 °C (nIL1F) for 20 s and 66 °C for 20 s. After the amplification phase, a melting analysis (65–95 °C with a heating rate of 0.5 °C per second and a continuous fluorescence measurement) was routinely carried out to confirm the presence of a single PCR product. For data calibration, a standard curve was generated for nIL-1F and β-actin gene from 10-fold serial dilutions (10−1–10−6 femtomole/2 μl) of plasmids containing the target gene sequences. The primer efficiency was calculated according to the equation E = 10(−1/slope) (Higuchi et al., 1993). The PCR reaction for each sample was duplicated. In these experiments, β-actin was amplified as the internal control. Data were analyzed using the CFX manager (Bio-Rad) and normalized to β-actin after correcting for differences in amplification efficiency.
2.2. Molecular cloning of grass carp nIL-1F and gcIL-1R1 cDNA
2.5. Expression analysis of nIL-1F in vivo after bacterial infection
Total RNA was extracted from grass carp head kidney with TRIzol Reagent (Invitrogen, Carlsbad, CA). About 2 μg of RNA was subjected to reverse transcription by using the SuperScript III RTPCR system (Invitrogen, Carlsbad, CA) with Oligo (dT)18 as the primer. Partial cDNA sequences of nIL-1F and gcIL-1R1 were obtained by PCR using the degenerated primers listed in Appendix: Supplementary Table S1, which were designed based on the conserved regions of the known fish IL-1Ra so-called in GenBank database and IL-1R1. Then, 5′- and 3′-RACE PCRs were performed to obtain the full-length cDNA of nIL-1F, and the 5′-RACE PCR was carried out to obtain partial cDNA of gcIL-1R1 (containing the extracellular domain) by using the primers listed in Appendix: Supplementary Table S1. Finally, all of the assembled cDNA sequences of nIL-1F and gcIL-1R1 were confirmed with Phusion HighFidelity DNA Polymerase (Finnzymes, Espoo, Finland) by using the primers for sequence validation (Appendix: Supplementary Table S1).
A pathogenic strain of Aeromonas hydrophila (A. hydr) was supplied by State Collection Center of Aquatic Pathogen, Shanghai Ocean University (Shanghai, China) and the re-isolated pathogen from the liver of the infected grass carp was identified by PCR assay. Two weeks before bacterial infection, healthy grass carps weighing about 0.75 kg were transferred into 2 clean tanks (at least 24 fish/tank) containing 1000 L running water. A single colony of A. hydr was picked and cultured in 200 ml TSB medium [0.5% soya peptone (Sigma-Aldrich), 1.5% tryptone (Sigma-Aldrich), 3% NaCl] at 28 °C with shaking at 180 rpm for approximately 18 h during exponential growth. The bacteria were collected by centrifugation and resuspended with PBS. The fish were injected intraperitoneally (i.p.) with bacteria (1 ml/kg; 1010 CFU/ml in PBS) or with PBS (1 ml/kg). After infection, head kidneys were collected at 3, 6, 12, 24, 48 and 72 h from the infection group or control group (N = 4). RNA extraction, reverse transcription and qPCR were performed as described in tissue distribution assay.
2.3. Sequence analysis of grass carp nIL-1F and gcIL-1R1
2.6. Expression analysis of nIL-1F in HKLs
The cDNA and deduced amino acid sequences of nIL-1F and gcIL1R1 were analyzed using the BLAST program from NCBI (http://
Grass carp HKLs were isolated using a discontinuous density gradient (Histopaque 1.083 kg/L, Sigma-Aldrich) according to the method
2. Materials and methods 2.1. Fish
F. Yao et al./Developmental and Comparative Immunology 49 (2015) 207–216
reported before (Wang et al., 2010). Briefly, head kidneys were removed from the fish under sterile conditions and gently filtrated through a 200 μm nylon mesh. Subsequently, the leukocytes were isolated by density gradient centrifugation at 400 × g for 30 min at 18 °C. After centrifugation, the cells at the interface were collected and washed twice. The cells were resuspended in RPMI1640 (Gibco BRL, NY) supplemented with 10% fetal bovine serum (PAA, Haidmannweg, GM) and seeded in a 24-well plate (Becton Dickinson, NJ) at a density of 6 × 105 cells/well, if not stated otherwise. Finally, the cells were incubated at 27 °C under 5% CO2 and saturated humidity. On the following day, HKLs were exposed to 10 μg/ml of LPS (Sigma-Aldrich) or 100 ng/ml of rgcIL-1β that has been prepared in our previous study (Yang et al., 2013) for 1, 3, 6, 12 and 24 h. In parallel experiments, HKLs were treated with rgcIL1β (100 ng/ml) in the presence or absence of the JNK inhibitor (100 μM, SP600125, Merck, Bad Soden, German), MEK inhibitor (100 μM, PD98059, Merck), p38 inhibitor (20 μM, SB202190, Merck) and NF-κB inhibitor (0.5 μM, PDTC, Sigma-Aldrich) for 4 h. After drug treatment, the mRNA levels of nIL-1F were quantified by qPCR. 2.7. Recombinant expression of nIL-1F and gcIL-1R1 Recombinant grass carp nIL-1F (rnIL-1F) and IL-1R1 (rgcIL1R1) were obtained by using yeast expression system pPICZα/X33 and prokaryotic expression system pGEX-4T-1/BL21 (DE3), respectively. To construct a plasmid for nIL-1F protein expression, the primers of nIL-1F-EcoRI/nIL-1F-NotI (Appendix: Supplementary Table S1) were designed to amplify the DNA fragment at aa position of 158–347 encoding the putative mature protein by PCR with the Phusion High-Fidelity DNA Polymerase. The fragment was inserted into the expression vector pPICZα (Invitrogen) after digestion with EcoR I and Not I. The linearized construct (pPICZα-nIL-1F, ~ 5– 20 μg) was transformed into Pichia pastoris (X33) using the Gene Pulser Xcell Electroporation System (Bio-Rad) according to the manufacturer’s instructions. Transformant colonies were selected on Zeocin (Invitrogen) plates and screened by PCR analysis. The selected clone was cultured in 50 ml BMGY (100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 × 10−5% biotin, 1% glycerol) at 28 °C with shaking at 250 rpm until the OD600 of the culture reached 2–6 (log-phase growth, approximately 16~18 hours). The cells were collected by centrifugation at 1500 × g for 5 minutes at room temperature and resuspended in BMMY (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 × 10−5% biotin, 0.5% methanol) with OD600 at 1.0, and then cultured at 28 °C with shaking at 250 rpm to induce protein expression for approximately 72 h. One hundred percent of methanol was added with a final concentration of 0.5% every 24-hour to maintain induction. The culture media were collected and the protein was purified by chromatography on a HisTrap affinity column (GE Healthcare, Waukesha, USA) and a Superdex 25 prep grade column (GE Healthcare). Additionally, gcIL-1R1 DNA fragment (GenBank ID: KM066967) at aa position of 21–317 encoding its extracellular domain was amplified by PCR using the primers of IL-1R1-BamHI/IL-1R1-XhoI (Appendix: Supplementary Table S1) and then subcloned into pGEX4T-1 expression vector (GE Healthcare) after digestion with BamH I and Xho I. The construct pGEX-4T-1-IL-1R1 was transformed into BL21 (DE3), and protein expression was induced with 1 mM IPTG (Merck, Darmstadt, Germany) for 4 h at 30 °C. The rgcIL-1R1 with GST-tag was purified following the same procedures for rnIL-1F as described above instead of using a GST affinity column (GE Healthcare). Following the same procedures in our previous study (Wei et al., 2013), the molecular weight and purity of rnIL-1F and rgcIL1R1 were analyzed by SDS-PAGE. Western blotting (WB) analysis was performed to confirm their production by using an anti-His tag monoclonal antibody (1:600, ZSGB-BIO, Beijing, China) and an antiGST tag monoclonal antibody (1:1000, CST, USA), respectively.
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2.8. Direct and competitive binding assay of nIL-1F with gcIL-1R1 ELISA was utilized to analyze the direct binding of rnIL-1F and rgcIL-1R1. Briefly, a 96-well plate was coated with 25 pmol/well of rgcIL-1R1 or GST (negative control, Sigma-Aldrich) at 4 °C overnight. After blocking with 5% non-fat milk plus 0.3% BSA in PBS at room temperature for 3 h, increasing doses (5, 15 and 25 pmol/ well) of rnIL-1F were added to each well and incubated at room temperature for 3 h. The wells were washed with PBST (0.05% Tween20 in PBS) three times and then incubated with anti-His Tag monoclonal antibody (1:600, ZSGB-BIO) or an isotype-matched antibody (IgG, 1:600, ZSGB-BIO) at room temperature for 3 h. The samples were washed with PBST and incubated with HRP conjugated secondary antibody (1:1000, goat anti mouse IgG, ZSGBBIO) at 4 °C overnight. After washing, the signals were developed by adding substrate buffer (1 mg/ml of 3,3′,5,5′-Tetramethylbenzidine, TMB, Tiangen) at 37 °C for 1 h. The reactions were terminated by adding 2 M H2SO4 and the results were analyzed by using a BioRad iMark Microplate Reader (Bio-Rad) at 450 nm. All samples were run in triplicate. In a competitive binding assay, a 96-well plate was coated with 5 pmol/well of rgcIL-1R1 or GST at 4 °C overnight. After blocking, increasing doses (5, 15 and 25 pmol/well) of rnIL-1F were added to each well and incubated at room temperature for 3 h. The wells were washed with PBST three times and then incubated with 5 pmol/well of rgcIL-1β at room temperature for a further 3 h. After washing, HRP conjugated anti-gcIL-1β antibody (1:1000) that has been used in our previous paper (Yang et al., 2013) was added and the plates were incubated at 4 °C overnight. After washing, the signals were developed and analyzed following the same procedures as described above. 2.9. Antagonistic effect of rnIL-1F on rgcIL-1β actions in grass carp HKLs Grass carp HKLs were plated into a 24-well plate with a density of 1.5 × 105 cells/well. After overnight incubation, the cells were incubated with rgcIL-1β (40 ng/ml) in the presence or absence of increasing doses (1000–4000 ng/ml) of rnIL-1F for 4 h and the mRNA levels of grass carp TNF-α and IL-1β were detected by qPCR using gene-specific primers (Appendix: Supplementary Table S1). 2.10. Data transformation and statistics In a tissue distribution study, gcIL-1Ra mRNA levels were expressed as a ratio to β-actin mRNA levels in the same sample. All data were normalized against the expression level in the thymus. Data presented (mean ± SEM, N = 3) are pooled results from three fishes. In the in vitro assay, the relative mRNA levels were expressed as mean ± SEM of four individual samples (N = 4 wells) and the value of each sample was the mean of the duplicates. Each experiment was repeated at least twice. Statistical analysis was conducted by using one-way ANOVA followed by Student’s test for the comparison between two groups or Duncan’s shortest significant range test for multiple comparisons by using the Statistical Product and Service Solutions 13.0 (SPSS, Chicago). Differences were considered statistically significant at P < 0.05. 3. Results 3.1. Molecular cloning of grass carp nIL-1F The nIL-1F cDNA (GenBank ID: KM066966) consisted of 1260 bp containing a 1044 bp open reading frame (ORF) with a 42 bp 5′ untranslated region (UTR) and a 174 bp 3′ UTR (Fig. 1). The eukaryotic polyadenylation signal (aataaa) was present at 26 bp upstream of a poly(A) tail within the 3′ UTR. The putative peptide contained
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Fig. 1. Nucleotide and deduced amino acid sequences of grass carp nIL-1F. The ORF is shown in upper case and the 5′-UTR and 3′-UTR sequences are presented in lower case. The nucleotides in bold indicate the start codon (ATG), the stop codon (TAA) and the polyadenylation signal (aataaa). The putative amino acid sequence is shown under the triplet codon. The potential glycosylation sites (NXT) are in bold and italicized. The thrombin cut site (VVARG) is underlined.
347 amino acids with a molecular mass of 39.5 kDa and a theoretical isoelectric point (pI) of 5.3. No signal peptide or transmembrane domain was found in this predicted protein, but there was a potential interleukin-converting enzyme (ICE) cut site (CAD) at the position of 115–117, a thrombin cut site (VVARG) at the position of 153–157, and three potential N-glycosylation sites (NXT) at the positions of 49–51, 226–228 and 251–253 (Fig. 1). Moreover, the thrombin cut site divided the protein into an N-terminal acidic domain (pI of 4.8, 17.5 kDa) and a C-terminal basic domain (pI of 9.1, 22 kDa). Comparing the position-specific iterated (PSI)-BLAST search (20 iterations) with nonredundant protein sequence database, nIL-1F was similar to IL-1β, IL-18, and other vertebrate IL-1
family (IL-1F) cytokines (the expect value
Contents lists available at ScienceDirect
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
Molecular and functional characterization of an IL-1β receptor antagonist in grass carp (Ctenopharyngodon idella) Fuli Yao, Xiao Yang, Xinyan Wang, He Wei, Anying Zhang, Hong Zhou * School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China
A R T I C L E
I N F O
Article history: Received 25 July 2014 Revised 24 November 2014 Accepted 25 November 2014 Available online 2 December 2014 Keywords: IL-1β receptor antagonist Receptor binding assay Functional identification Head kidney leukocytes Grass carp
A B S T R A C T
In the present study, we discovered a novel IL-1 family member (nIL-1F) from grass carp that possessed the ability to bind with grass carp IL-1β receptor type 1 (gcIL-1R1) and attenuate grass carp IL-1β activity in head kidney leukocytes (HKLs), suggesting that it may function as an IL-1β receptor antagonist. Grass carp nIL-1F transcript was constitutively expressed with the highest levels in some lymphoid organs, including head kidney, spleen and intestine, implying its potential in grass carp immunity. In agreement with this notion, in vitro and in vivo studies showed that nIL-1F mRNA was inductively expressed in grass carp with a rapid kinetics, indicating that it may be an early response gene during immune challenges. In addition, recombinant grass carp IL-1β (rgcIL-1β) induced nIL-1F mRNA expression via NF-κB and MAPK (JNK, p38 and p42/44) signaling pathways in HKLs. Particularly, the orthologs of nIL-1F found in other fish species, including zebrafish, pufferfish and rainbow trout are not homologous to mammalian IL-1β receptor antagonist (IL-1Ra), indicating that fish nIL-1F and mammalian IL-1Ra may not share a common evolutionary ancestor. Taken together, our data suggest the existence of a naturally occurring fish nIL-1F, which may function like mammalian IL-1Ra, being beneficial to understand the autoregulatory mechanism of IL-1β activity in fish immunity. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction As a major proinflammatory cytokine, interleukin-1β (IL-1β) plays pleiotropic roles in host defense by inducing a cascade of reactions leading to inflammation (Sims and Smith, 2010). In addition to its induction of other proinflammatory cytokines (e.g. IL-6, IL-8 and TNF-α), IL-1β rapidly stimulates its own production, which amplifies IL-1β response via a positive-feedback loop (Sims and Smith, 2010). In parallel, the potent and extensive proinflammatory activities of IL-1β are tightly controlled by several naturally occurring IL-1β signaling inhibitors, including IL-1 receptor antagonist (IL1Ra), IL-1 receptor type 2 (IL-1R2) and other soluble receptors (Weber et al., 2010). Among these endogenous inhibitors, IL-1Ra binds to IL-1β receptor type 1 (IL-1R1) but does not transduce the signal as it lacks the IL-1 receptor accessory protein interacting domain, while IL-1R2 acts as a decoy receptor for IL-1 activity, thereby constituting two fundamental regulatory mechanisms for the blockade of IL-1β signaling (Nambu and Nakae, 2010). Alternatively, the role of IL-1Ra in autoimmune diseases has been well documented (Jesus and Goldbach-Mansky, 2014; Molto and Olive, 2010). It has become
* Corresponding author. School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China. Tel: +86 28 83206437; fax: +86 28 83208238. E-mail address: [email protected] (H. Zhou). http://dx.doi.org/10.1016/j.dci.2014.11.016 0145-305X/© 2014 Elsevier Ltd. All rights reserved.
evident that hereditary autoinflammatory diseases are caused by excessive IL-1 signaling due to the mutations or deletions of the IL1Ra gene (Aksentijevich et al., 2009; Jesus and Goldbach-Mansky, 2014; Reddy et al., 2009). In animal models, it has been further implicated in IL-1Ra’s contributions to autoinflammatory syndromes. As predicted, IL-1Ra-deficient mice spontaneously develop chronic inflammatory polyarthropathy, such as arthritis, skin inflammation and arteritis (Gabay et al., 2010; Nicklin and Shepherd, 2003). In contrast, administration of IL-1Ra has resulted in marked improvements in experimental models of inflammatory arthritis and osteoarthritis (Jacques et al., 2006). Importantly, the recombinant IL-1Ra named anakinra has been approved for the treatment of patients with rheumatoid arthritis (Jesus and Goldbach-Mansky, 2014; Molto and Olive, 2010). Taken together, these findings highlight the key regulatory role and clinical implication of IL-1Ra in IL-1related diseases. Considering its clinical application, IL-1Ra has been extensively studied in mammals, showing that it has four distinct isoforms derived from the same gene by alternative mRNA splicing and alternative translation initiation (Gabay et al., 2010). One isoform is secreted, whereas the others lack a signal peptide and are localized within the cells (Arend et al., 2008). These four isoforms are produced by different types of cells and all of them can inhibit IL-1 effects (Arend et al., 2008). Like IL-1β production, IL-1Ra expression is induced by IL-1β and other inflammatory stimuli including IgG complexes, bacterial or viral components (Sims and Smith, 2010).
208
F. Yao et al./Developmental and Comparative Immunology 49 (2015) 207–216
Actually, IL-1Ra is produced and released by almost all cells expressing IL-1β, being considered as the third member of the IL-1 family (Molto and Olive, 2010). However, the existing knowledge on fish IL-1Ra is limited as there is only a single report about nIL1F in rainbow trout, showing its ability to inhibit IL-1β effect in a trout macrophage cell line and interact with IL-1R1 in protein modeling although it has low homology to the known mammalian IL-1 family members (Wang et al., 2009). The cDNAs homologous to trout nIL-1F (GenBank ID: NM _001124396.2) can be found from NCBI database in other fish species, including zebrafish (GenBank ID: JF436963.1) and pufferfish (GenBank ID: JF714481.1), all of which are termed IL-Ra probably based on their homology to nIL-1F. However, direct evidence for IL1Ra and IL-1R1 binding in fish is still lacking. In this study, we discovered a new molecule from grass carp that is homologous to the so-called fish IL-Ra described above. Its structural characterization and expression patterns were revealed as well. Notably, in accordance with the natural characteristics of mammalian IL-1Ra, its recombinant protein displayed the ability to bind with grass carp IL-1R1 and attenuate grass carp IL-1β activity, providing the functional evidence for this molecule serving as IL-1Ra. Given that it is the ortholog of trout nIL-1F (Wang et al., 2009), the newly identified molecule was nominated as grass carp nIL-1F. Our data, together with the findings in other fish species, suggest the existence of nIL1F serving as an IL-1β receptor antagonist in fish, although their gene features such as genomic location and sequence homology differ from mammalian IL-1Ra.
www.ncbi.nlm.nih.gov). The similarity and identity of the protein sequences were calculated by MatGAT 2.0 software (Campanella et al., 2003). A phylogenetic tree was constructed by using MEGA 4 software based on Neighbor-Joining method with the bootstrapping of 1000 repetitions (Kumar et al., 2004). The molecular weight and isoelectric point of the protein were predicted by the Compute pI/Mw tool (http://www.expasy.ch/) and the signal peptide prediction was performed with SignalP 3.0 (Bendtsen et al., 2004). The ICE cut site and thrombin cut site were determined by referring to the cleavage specificity of ICE (Howard et al., 1991) and thrombin (Chang, 1986). The potential N-glycosylation sites were predicted using NetNGlyc 1.0 Server. 2.4. Tissue distribution of nIL-1F in grass carp
Healthy grass carp (Ctenopharyngodon idella) weighing 0.75– 1.0 kg were purchased from Chengdu Tongwei Aquatic Science and Technology Company (Chengdu, China). The fish were acclimated to the laboratory environment (1000-L aquaria) for at least 2 weeks prior to use in experiments. During the procedures of cell preparation, the fish were killed by anesthesia in 0.05% MS222 (SigmaAldrich, St. Louis, MO) and the head kidneys were collected for subsequent HKL isolation. All animal experiments complied with the Regulation of Animal Experimentation of Sichuan province, China.
About 2 μg RNA samples from each selected tissue (brain, thymus, gill, heart, kidney, head kidney, liver, spleen and intestine) were digested with RNase-free DNase I (Promega, Madison, WI) and then subjected to reverse transcription as described in section 2.2. The levels of nIL-1F and β-actin transcripts were quantitated by real time quantitative PCR (qPCR) following MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and the primers were listed in Appendix: Supplementary Table S1. In brief, qPCR was performed on a Bio-Rad CFX96 Real-time detection system (Bio-Rad Laboratories, Inc., Hercules, CA) in a final volume of 20 μl containing 10 μl of Real Master Mix (Tiangen, Beijing, China), 2 μl of the cDNA, and 0.5 μl of each of forward and reverse primers (10 μM). The amplification program was 94 °C for 2 min, followed by 35 cycles of 94 °C for 20 s, 59 °C (β-actin) or 64.5 °C (nIL1F) for 20 s and 66 °C for 20 s. After the amplification phase, a melting analysis (65–95 °C with a heating rate of 0.5 °C per second and a continuous fluorescence measurement) was routinely carried out to confirm the presence of a single PCR product. For data calibration, a standard curve was generated for nIL-1F and β-actin gene from 10-fold serial dilutions (10−1–10−6 femtomole/2 μl) of plasmids containing the target gene sequences. The primer efficiency was calculated according to the equation E = 10(−1/slope) (Higuchi et al., 1993). The PCR reaction for each sample was duplicated. In these experiments, β-actin was amplified as the internal control. Data were analyzed using the CFX manager (Bio-Rad) and normalized to β-actin after correcting for differences in amplification efficiency.
2.2. Molecular cloning of grass carp nIL-1F and gcIL-1R1 cDNA
2.5. Expression analysis of nIL-1F in vivo after bacterial infection
Total RNA was extracted from grass carp head kidney with TRIzol Reagent (Invitrogen, Carlsbad, CA). About 2 μg of RNA was subjected to reverse transcription by using the SuperScript III RTPCR system (Invitrogen, Carlsbad, CA) with Oligo (dT)18 as the primer. Partial cDNA sequences of nIL-1F and gcIL-1R1 were obtained by PCR using the degenerated primers listed in Appendix: Supplementary Table S1, which were designed based on the conserved regions of the known fish IL-1Ra so-called in GenBank database and IL-1R1. Then, 5′- and 3′-RACE PCRs were performed to obtain the full-length cDNA of nIL-1F, and the 5′-RACE PCR was carried out to obtain partial cDNA of gcIL-1R1 (containing the extracellular domain) by using the primers listed in Appendix: Supplementary Table S1. Finally, all of the assembled cDNA sequences of nIL-1F and gcIL-1R1 were confirmed with Phusion HighFidelity DNA Polymerase (Finnzymes, Espoo, Finland) by using the primers for sequence validation (Appendix: Supplementary Table S1).
A pathogenic strain of Aeromonas hydrophila (A. hydr) was supplied by State Collection Center of Aquatic Pathogen, Shanghai Ocean University (Shanghai, China) and the re-isolated pathogen from the liver of the infected grass carp was identified by PCR assay. Two weeks before bacterial infection, healthy grass carps weighing about 0.75 kg were transferred into 2 clean tanks (at least 24 fish/tank) containing 1000 L running water. A single colony of A. hydr was picked and cultured in 200 ml TSB medium [0.5% soya peptone (Sigma-Aldrich), 1.5% tryptone (Sigma-Aldrich), 3% NaCl] at 28 °C with shaking at 180 rpm for approximately 18 h during exponential growth. The bacteria were collected by centrifugation and resuspended with PBS. The fish were injected intraperitoneally (i.p.) with bacteria (1 ml/kg; 1010 CFU/ml in PBS) or with PBS (1 ml/kg). After infection, head kidneys were collected at 3, 6, 12, 24, 48 and 72 h from the infection group or control group (N = 4). RNA extraction, reverse transcription and qPCR were performed as described in tissue distribution assay.
2.3. Sequence analysis of grass carp nIL-1F and gcIL-1R1
2.6. Expression analysis of nIL-1F in HKLs
The cDNA and deduced amino acid sequences of nIL-1F and gcIL1R1 were analyzed using the BLAST program from NCBI (http://
Grass carp HKLs were isolated using a discontinuous density gradient (Histopaque 1.083 kg/L, Sigma-Aldrich) according to the method
2. Materials and methods 2.1. Fish
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reported before (Wang et al., 2010). Briefly, head kidneys were removed from the fish under sterile conditions and gently filtrated through a 200 μm nylon mesh. Subsequently, the leukocytes were isolated by density gradient centrifugation at 400 × g for 30 min at 18 °C. After centrifugation, the cells at the interface were collected and washed twice. The cells were resuspended in RPMI1640 (Gibco BRL, NY) supplemented with 10% fetal bovine serum (PAA, Haidmannweg, GM) and seeded in a 24-well plate (Becton Dickinson, NJ) at a density of 6 × 105 cells/well, if not stated otherwise. Finally, the cells were incubated at 27 °C under 5% CO2 and saturated humidity. On the following day, HKLs were exposed to 10 μg/ml of LPS (Sigma-Aldrich) or 100 ng/ml of rgcIL-1β that has been prepared in our previous study (Yang et al., 2013) for 1, 3, 6, 12 and 24 h. In parallel experiments, HKLs were treated with rgcIL1β (100 ng/ml) in the presence or absence of the JNK inhibitor (100 μM, SP600125, Merck, Bad Soden, German), MEK inhibitor (100 μM, PD98059, Merck), p38 inhibitor (20 μM, SB202190, Merck) and NF-κB inhibitor (0.5 μM, PDTC, Sigma-Aldrich) for 4 h. After drug treatment, the mRNA levels of nIL-1F were quantified by qPCR. 2.7. Recombinant expression of nIL-1F and gcIL-1R1 Recombinant grass carp nIL-1F (rnIL-1F) and IL-1R1 (rgcIL1R1) were obtained by using yeast expression system pPICZα/X33 and prokaryotic expression system pGEX-4T-1/BL21 (DE3), respectively. To construct a plasmid for nIL-1F protein expression, the primers of nIL-1F-EcoRI/nIL-1F-NotI (Appendix: Supplementary Table S1) were designed to amplify the DNA fragment at aa position of 158–347 encoding the putative mature protein by PCR with the Phusion High-Fidelity DNA Polymerase. The fragment was inserted into the expression vector pPICZα (Invitrogen) after digestion with EcoR I and Not I. The linearized construct (pPICZα-nIL-1F, ~ 5– 20 μg) was transformed into Pichia pastoris (X33) using the Gene Pulser Xcell Electroporation System (Bio-Rad) according to the manufacturer’s instructions. Transformant colonies were selected on Zeocin (Invitrogen) plates and screened by PCR analysis. The selected clone was cultured in 50 ml BMGY (100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 × 10−5% biotin, 1% glycerol) at 28 °C with shaking at 250 rpm until the OD600 of the culture reached 2–6 (log-phase growth, approximately 16~18 hours). The cells were collected by centrifugation at 1500 × g for 5 minutes at room temperature and resuspended in BMMY (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 × 10−5% biotin, 0.5% methanol) with OD600 at 1.0, and then cultured at 28 °C with shaking at 250 rpm to induce protein expression for approximately 72 h. One hundred percent of methanol was added with a final concentration of 0.5% every 24-hour to maintain induction. The culture media were collected and the protein was purified by chromatography on a HisTrap affinity column (GE Healthcare, Waukesha, USA) and a Superdex 25 prep grade column (GE Healthcare). Additionally, gcIL-1R1 DNA fragment (GenBank ID: KM066967) at aa position of 21–317 encoding its extracellular domain was amplified by PCR using the primers of IL-1R1-BamHI/IL-1R1-XhoI (Appendix: Supplementary Table S1) and then subcloned into pGEX4T-1 expression vector (GE Healthcare) after digestion with BamH I and Xho I. The construct pGEX-4T-1-IL-1R1 was transformed into BL21 (DE3), and protein expression was induced with 1 mM IPTG (Merck, Darmstadt, Germany) for 4 h at 30 °C. The rgcIL-1R1 with GST-tag was purified following the same procedures for rnIL-1F as described above instead of using a GST affinity column (GE Healthcare). Following the same procedures in our previous study (Wei et al., 2013), the molecular weight and purity of rnIL-1F and rgcIL1R1 were analyzed by SDS-PAGE. Western blotting (WB) analysis was performed to confirm their production by using an anti-His tag monoclonal antibody (1:600, ZSGB-BIO, Beijing, China) and an antiGST tag monoclonal antibody (1:1000, CST, USA), respectively.
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2.8. Direct and competitive binding assay of nIL-1F with gcIL-1R1 ELISA was utilized to analyze the direct binding of rnIL-1F and rgcIL-1R1. Briefly, a 96-well plate was coated with 25 pmol/well of rgcIL-1R1 or GST (negative control, Sigma-Aldrich) at 4 °C overnight. After blocking with 5% non-fat milk plus 0.3% BSA in PBS at room temperature for 3 h, increasing doses (5, 15 and 25 pmol/ well) of rnIL-1F were added to each well and incubated at room temperature for 3 h. The wells were washed with PBST (0.05% Tween20 in PBS) three times and then incubated with anti-His Tag monoclonal antibody (1:600, ZSGB-BIO) or an isotype-matched antibody (IgG, 1:600, ZSGB-BIO) at room temperature for 3 h. The samples were washed with PBST and incubated with HRP conjugated secondary antibody (1:1000, goat anti mouse IgG, ZSGBBIO) at 4 °C overnight. After washing, the signals were developed by adding substrate buffer (1 mg/ml of 3,3′,5,5′-Tetramethylbenzidine, TMB, Tiangen) at 37 °C for 1 h. The reactions were terminated by adding 2 M H2SO4 and the results were analyzed by using a BioRad iMark Microplate Reader (Bio-Rad) at 450 nm. All samples were run in triplicate. In a competitive binding assay, a 96-well plate was coated with 5 pmol/well of rgcIL-1R1 or GST at 4 °C overnight. After blocking, increasing doses (5, 15 and 25 pmol/well) of rnIL-1F were added to each well and incubated at room temperature for 3 h. The wells were washed with PBST three times and then incubated with 5 pmol/well of rgcIL-1β at room temperature for a further 3 h. After washing, HRP conjugated anti-gcIL-1β antibody (1:1000) that has been used in our previous paper (Yang et al., 2013) was added and the plates were incubated at 4 °C overnight. After washing, the signals were developed and analyzed following the same procedures as described above. 2.9. Antagonistic effect of rnIL-1F on rgcIL-1β actions in grass carp HKLs Grass carp HKLs were plated into a 24-well plate with a density of 1.5 × 105 cells/well. After overnight incubation, the cells were incubated with rgcIL-1β (40 ng/ml) in the presence or absence of increasing doses (1000–4000 ng/ml) of rnIL-1F for 4 h and the mRNA levels of grass carp TNF-α and IL-1β were detected by qPCR using gene-specific primers (Appendix: Supplementary Table S1). 2.10. Data transformation and statistics In a tissue distribution study, gcIL-1Ra mRNA levels were expressed as a ratio to β-actin mRNA levels in the same sample. All data were normalized against the expression level in the thymus. Data presented (mean ± SEM, N = 3) are pooled results from three fishes. In the in vitro assay, the relative mRNA levels were expressed as mean ± SEM of four individual samples (N = 4 wells) and the value of each sample was the mean of the duplicates. Each experiment was repeated at least twice. Statistical analysis was conducted by using one-way ANOVA followed by Student’s test for the comparison between two groups or Duncan’s shortest significant range test for multiple comparisons by using the Statistical Product and Service Solutions 13.0 (SPSS, Chicago). Differences were considered statistically significant at P < 0.05. 3. Results 3.1. Molecular cloning of grass carp nIL-1F The nIL-1F cDNA (GenBank ID: KM066966) consisted of 1260 bp containing a 1044 bp open reading frame (ORF) with a 42 bp 5′ untranslated region (UTR) and a 174 bp 3′ UTR (Fig. 1). The eukaryotic polyadenylation signal (aataaa) was present at 26 bp upstream of a poly(A) tail within the 3′ UTR. The putative peptide contained
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Fig. 1. Nucleotide and deduced amino acid sequences of grass carp nIL-1F. The ORF is shown in upper case and the 5′-UTR and 3′-UTR sequences are presented in lower case. The nucleotides in bold indicate the start codon (ATG), the stop codon (TAA) and the polyadenylation signal (aataaa). The putative amino acid sequence is shown under the triplet codon. The potential glycosylation sites (NXT) are in bold and italicized. The thrombin cut site (VVARG) is underlined.
347 amino acids with a molecular mass of 39.5 kDa and a theoretical isoelectric point (pI) of 5.3. No signal peptide or transmembrane domain was found in this predicted protein, but there was a potential interleukin-converting enzyme (ICE) cut site (CAD) at the position of 115–117, a thrombin cut site (VVARG) at the position of 153–157, and three potential N-glycosylation sites (NXT) at the positions of 49–51, 226–228 and 251–253 (Fig. 1). Moreover, the thrombin cut site divided the protein into an N-terminal acidic domain (pI of 4.8, 17.5 kDa) and a C-terminal basic domain (pI of 9.1, 22 kDa). Comparing the position-specific iterated (PSI)-BLAST search (20 iterations) with nonredundant protein sequence database, nIL-1F was similar to IL-1β, IL-18, and other vertebrate IL-1
family (IL-1F) cytokines (the expect value
