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Characterization and bioactivity of grass carp (Ctenopharyngodon idella) interleukin-21: Inducible production and involvement in inflammatory regulation
Fish and Shellfish Immunology 99 (2020) 19–26
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Characterization and bioactivity of grass carp (Ctenopharyngodon idella) interleukin-21: Inducible production and involvement in inflammatory regulation
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Anying Zhang1, Xiaoyu Jian1, Dan Wang, Jingqi Ren, Xinyan Wang, Hong Zhou∗ School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, People's Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Grass carp Il-21 Protein release Anti-bacterial activity Anti-inflammatory property
In mammals, interleukin 21 (IL-21) is a broad pleiotropic cytokine that plays critical roles in the development of several inflammatory and autoimmune diseases. In fish, functional information of Il-21 is limited, and its role in immune response is largely unknown. In the present study, we cloned a coding sequence of grass carp (Ctenopharyngodon idella) il21 gene (gcil21). To characterize the release patterns and biological activity of gcIl21, we prepared recombinant gcIl-21 (rgcIl-21) and obtained the polyclonal antibody with gcIl-21 specificity. Western blotting analysis showed that in grass carp head kidney leukocytes (HKLs), gcIl-21 was undetected in culture supernatant of untreated cells but drastically induced by heat-killed Aeromonas hydrophila (A. hydrophila), uncovering the release features of gcIl-21 and its possible involvement in immune response. Subsequent functional experiments revealed that rgcIl-21 did not affect the mRNA expression of grass carp il1b and tgfb, but induced a strong expression of grass carp il10, and to a lesser extent of grass carp tnfa in HKLs, suggesting a dominant effect of gcIl-21 in modulating Il-10 signaling as seen in rainbow trout and mammals. Furthermore, in vivo studies showed that intraperitoneal injection of rgcIl-21 was able to increase the survival rate of grass carp infected with live A. hydrophila, and reduce the pathological responses caused by the same pathogenic bacteria in head kidney and intestine. Taken together, these results for the first time revealed the close relationship of fish Il21 production and function with inflammatory responses, and highlighted its anti-bacterial and anti-inflammatory ability, thereby providing a new insight into host defense mechanisms in fish.
1. Introduction In mammals, interleukin 21 (IL-21) is a type I cytokine with four-αhelix bundle produced by activated CD4+ T cells and natural killer T (NKT) cells [1]. IL-21 mediates its biological effects through a specific receptor chain (IL-21R) and a common receptor γ chain (γc) which shares with IL-2 family cytokines such as IL-4, IL-7, IL-9, and IL-15 [2]. The wide distribution of IL-21R and γc transcripts in a variety of lymphoid tissues such as thymus, spleen and lymph nodes, as well as peripheral blood leukocytes indicates the pleiotropic functions of IL-21 [3–5]. Actually, upon binding to its receptors, IL-21 activates the Janus family tyrosine kinases members JAK1 and JAK3, with subsequent phosphorylation of STAT3 and STAT1 [6,7]. Additionally, the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK) pathways contribute to IL-21 signal transmission [8,9]. For
functionality, IL-21 is suggested to mediate the differentiation of T cells, the maturation of B cells, as well as the proliferation and cytotoxicity of NK cells [10,11]. Besides, IL-21 plays critical roles in the development of several inflammatory and autoimmune diseases such as inflammatory bowel diseases (IBD), rheumatoid arthritis and cancers [12,13]. For example, IL-21 shows critical immunosuppressive effects in IBD because of its ability to induce IL-10 [14], a crucial regulator of immune homeostasis. However, in dentritic cells (DC), IL-21 induces IL1β expression in NF-ΚB independent way [15]. These studies suggest the pleiotropic properties of IL-21 in immune response [13]. Considering the important role of IL-21 in mammalian immunity, the fish Il-21 has also attracted increasing attention. At present, il21 gene has been identified in a few fish species including fugu [16], spotted green pufferfish [17], zebrafish [18], and rainbow trout [19], and most efforts have been focused on the gene expression of il21 in
∗
Corresponding author. School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, People's Republic of China. E-mail address: [email protected] (H. Zhou). 1 Anying Zhang and Xiaoyu Jian contributed equally to this article. https://doi.org/10.1016/j.fsi.2020.01.059 Received 17 September 2019; Received in revised form 27 January 2020; Accepted 28 January 2020 Available online 31 January 2020 1050-4648/ © 2020 Elsevier Ltd. All rights reserved.
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Phylogenetic analysis was conducted using the neighbor-joining method in MEGA X (http://www.megasoftware.net/) using 1000 repetitions for bootstrapping.
response to various immune stimuli such as phytohaemagglutinin (PHA), lipopolysaccharide (LPS), T cell stimulant phorbol myristate acetate (PMA) and calcium ionophore [16,17,19]. However, the protein expression pattern of fish Il-21, which is the important property of Il-21 as a cytokine, has still not been investigated. For functionality, Il-21 is only characterized in rainbow trout, showing that the recombinant Il-21 proteins are capable to induce the expression of some cytokines and T/B cell marker genes in head kidney cells [19]. Accordingly, the functional information of fish Il-21 is limited and its role in fish immunity so far remains to be elucidated. The coding sequence of grass carp il21 (gcil21) gene was isolated and identified in the present study. Subsequently, recombinant grass carp Il-21 (rgcIl-21) was prepared, and the polyclonal antibody specific for gcIl-21 was generated. By using this antibody, Western blotting (WB) analysis revealed the inducible release of gcIl-21 by the heatkilled (HK) Aeromonas hydrophila (A. hydrophila)in grass carp head kidney leukocytes (HKLs), suggesting possible involvement of gcIl-21 in the immune responses. In agreement with this notion, rgcIl-21 showed the bioactivity to regulate the mRNA expression of inflammation-related cytokines in a time- and dose-dependent manner. To reveal the significance of rgcIl-21 in mediating these effects, in vivo studies were performed and results proved that intraperitoneal injection of rgcIl-21 was effective in increasing the survival rate, and reducing the pathological changes of tissues in grass carp infected with live A. hydrophila. These results highlight the role and potential of fish Il-21 as an inflammation regulator during bacterial infection.
2.4. Recombinant expression of gcIl-21 Recombinant gcIl-21 (rgcIl-21) was expressed by BL21 (DE3)/pET30a (+) (Merck Millipore, Billerica, USA) system. Briefly, the DNA fragment encoding mature gcIl-21 was subcloned into the Nde Ⅰ and BamH Ⅰ sites of pET-30a (+). After the plasmid was amplified in E. Coli and extracted by using a TIANprep Rapid Mini Plasmid Kit (Tiangen), it was transformed into E. Coli BL21 (DE3) strain. The positive clone was grown in Luria Broth (LB) medium containing kanamycin (30 ng/mL) for 3 h with shaking at 37 °C. When the OD at 600 nm reached 0.6, Isopropyl-β-D-Thiogalactopyranoside (IPTG, Sigma-Aldrich, St. Louis, USA) was added to a final concentration of 1 mM to induce rgcIl-21 expression. After 4 h of induction, the bacterial cells were collected by centrifugation (4000×g, 10 min) at 4 °C and washed by 10 mM ice-cold phosphate buffer (pH 7.4) twice. The cells were resuspended in 20 mL of 10 mM phosphate buffer supplemented with 1 mM PMSF and 1 mg/ mL Lysozyme and finally sonicated on ice. The sediment was collected by centrifugation at 10,000×g for 20 min at 4 °C and resuspended in washing buffer (20 mM Tris-HCl, 10 mM EDTA and 1% Triton X-100, pH7.5). The inclusion body was washed by washing buffer for three times and 3 M urea once, via centrifugation (10,000×g, 10 min) at 4 °C and then dissolved in ice-cold denaturation buffer (30 mg inclusion body/ml, 8 M urea, 10% glycerol, 50 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA and 10 mM DTT, pH8.0). The supernatant was collected after centrifugation at 10,000×g for 10 min at 4 °C. About 1 mg protein of the supernatant was added into 50 mL of renaturation buffer (100 mM Tris-HCl, 400 mM L-Arg-HCl, 2 mM EDTA, 5 mM oxidized and reduced glutathione each and 0.5 mM PMSF, pH8.0) slowly (5–6 drops/ min) with magnetic stirring at 100 rpm and the solution was incubated at 4 °C overnight. After that, the renaturation buffer containing rgcIl-21 was ultrafiltered using an Amicon Ultra-15 Centrifugal Filter Unit (Amicon®Ultra 10 K device, Merck Millipore). LPS (endotoxin) was removed by Endotoxin Removal Agarose Resin (Yeasen, Shanghai, China). The molecular weight and purity of rgcIl-21 were evaluated by SDS-PAGE and identified by Western blotting using an anti-His antibody (1:1000, Boster, Wuhan, China). The endotoxin levels in rgcIl-21 were determined using an endotoxin assay kit (Chinese Horseshoe Crab Reagent Manufactory Co. Ltd., Xiamen, China). The purified rgcIl-21 protein was lyophilized and then stored at −80 °C for further use.
2. Materials and methods 2.1. Animals Healthy grass carp, weighing 0.5–1.0 kg or 50–60 g, were purchased from Chengdu Tongwei Aquatic Science and Technology Company (Chengdu, China). The fish were reared in aquaria with circulating, filtered and well-aerated tap water at 25 ± 2 °C in our laboratory at least 2 weeks before experimental procedures. The tissues were obtained from the freshly killed fish and all animal experiments were performed in accordance with the Regulation for Animal Experimentation of Sichuan province, China, and were allowed by the ethics committee of the University of Electronic Science and Technology of China. 2.2. Cloning and sequencing of gcil21 coding sequence (CDS)
2.5. Isolation and culture of grass carp HKLs
Total RNA was extracted from grass carp HKLs by using Tripure Isolation Reagent (Roche, Basel, Switzerland) and about 2 μg of total RNA was converted into cDNA using M-MLV Reverse Transcriptase (Promega, Madison, USA) with oligo (dT)18 as the primer. According to the uploaded gcil21 gene sequence (GenBank accession no.: KP226585), the primers (Supplementary Table 1) were designed and used to clone gcil21 CDS. Finally, the CDS of gcil21 was validated by PCR amplification using Phusion DNA polymerase (NEB, Beverly, USA) and subcloned into pGM-T vector (Tiangen, Beijing, China). The ligation products were transformed into JM109 competent cells and the positive clones were selected for sequencing.
Healthy grass carp with body weight of 0.5–1 kg were kept in laboratory for two weeks before experiments and sacrificed for HKLs collection. HKLs were obtained by discontinuous density gradient [20]. In brief, grass carp head kidney was obtained from freshly killed fish, washed twice and gently pressed in RPMI-1640 medium (Gibco, NY, USA). The cell suspension was filtrated by a 200-gauge stainless steel mesh before centrifuging at 2800 rpm for 30 min at 20 °C in a density gradient centrifugation (Ficoll-Hypaque 1.083 kg/L, TBD, Tianjin, China). After that, the cells were obtained from the interface and washed twice, then resuspended in the RPMI-1640 medium supplemented 24 mM NaHCO3, 25 mM HEPES, 1% antibiotic-antimycotic and 10% fetal bovine serum (Gibco). In the 24-well plate (BD Biosciences, San Jose, USA), approximately 6 × 105 cells per well were seeded and incubated at 26 °C overnight under 5% CO2 for drug treatment. Alternatively, 1 × 107 cells per well were planted in the 6-well plate (BD Biosciences) with the same culture condition.
2.3. Sequence analysis The deduced amino acid sequence was analyzed using the DNAMAN 8.0 software (Lynnon Biosoft, Pointe-Claire, Canada) and signal peptide was predicted by SignalP-5.0 Server (http://www.cbs.dtu.dk/services/ SignalP/). Isoelectric point (pI) and molecular weight (MW) of protein were calculated by Compute pI/MW in ExPASy (https://web.expasy. org/compute_pi/). Protein hydrophobicity was calculated by ProtParam tool in ExPASy (https://web.expasy.org/protparam/). The multiple sequence alignments were generated by DNAMAN 8.0 software.
2.6. Western blot assay of gcIl-21 in HKLs Heat-killed A. hydrophila (HK-A. hydrophila) were prepared in 20
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Fig. 1. Recombinant expression of gcIl-21. (A) SDSPAGE analysis of rgcIl-21. M, molecular weight marker; lane 1, crude lysate of inclusion body from the cells without IPTG induction; lane 2, crude lysate of inclusion body from the cells with IPTG induction; line 3, the purified rgcIL-21. (B) Western blotting analysis of rgcIl-21 using the antibody for His tag. M, molecular weight marker; lane 1, the purified rgcIl21.
Fig. 2. Validation of anti-gcIl-21 pAb and the protein release patterns of gcIl-21 in grass carp HKLs. (A) Western blotting analysis of anti-gcIl-21 pAb. Lane 1, culture medium of grass carp HKLs; lane 2, culture medium of grass carp HKLs challenged by HK-A. hydrophila; lane 3, rgcIl-21. (B) Total proteins from culture medium of grass carp HKLs were used to verify the specificity of anti-gcIl-21 pAb pre-absorbed with an excess of rgcIl-21 with the molar mass ratio of 1:30. Lane 1, culture medium of grass carp HKLs without treatment; lane 2, culture medium of grass carp HKLs challenged with HK-A. hydrophila; lane 3, rgcIl-21. (C) Effect of A. hydrophila treatment on gcIl-21 secretion in grass carp HKLs. HKLs were seeded in 6-well plate with density of 1 × 107 cells per well and incubated at 28 °C under 5% CO2 and saturated humidity. After the cells were challenged with or without HK-A. hydrophila at concentration of 1 × 107 CFU/well for 6 h (three replications), the medium of each well with the same volume was collected and analyzed by Western blotting assay using anti-gcIl-21 pAb (1:3000).
boiling water for 30 min at concentration of 1 × 107 CFU/well. The HKLs were treated with or without 1.0 multiplicity of infection (MOI) HK-A. hydrophila. Subsequently, the supernatant of culture medium was harvested for WB assay. In this assay, the gcIl-21 polyclonal antibodies (anti-gcIl-21 pAb) were customized from Biogot (Nanjing, China). To validate its specificity, recombinant gcIl-21 and the concentrated culture medium of HKLs were analyzed by WB in which the membrane was incubated with anti-gcIl-21 pAb or the antibody pre-absorbed with rgcIl-21 with the molar mass ratio of 1:30. The proteins in culture medium were separated by SDS-PAGE gel and WB assay was used to detect gcIl-21. The membrane was incubated with anti-gcIl-21 pAb (1:3000) at 4 °C overnight, and then exposed to horseradish peroxidaseconjugated goat anti-rabbit secondary antibody (1:5000, ZSGB-BIO, Beijing, China) for 2 h at room temperature. Finally, the signals were detected using an ECL kit (Roche) according to the manufacturer's instruction.
2.7. Dose response and time course experiments To determine the optimal concentration, HKLs were treated with serial dilutions of rgcIl-21 from 0.3 to 10 ng/mL. The cells were collected at 3 h or 12 h later and dissolved in TriPure Isolation Reagent. Total RNA was extracted to obtain the cDNA by reverse transcription as described above, and then the expression of gcil10, gctgfb, gctnfa and gcil1b were measured by the real-time quantitative PCR (RT-qPCR). To investigate the kinetics of rgcIl-21-modulated gene expression, an optimal dose of rgcIl-21 (10 ng/mL) was used to stimulate HKLs, the expression of gcil10, gctgfb, gctnfa and gcil1b were assessed at 1, 3, 6 and 12 h by RT-qPCR. RT-qPCR was run on Bio-Rad CFX96 Real-time detection system (Bio-Rad, Hercules, USA) by Real Master Mix (SYBR Green) Kit with gene specific primers (Table 1). Parallel measurement of bactin was conducted to serve as an internal control to exclude the deviations during RNA extraction and cDNA synthesis. The pGEM-T easy vectors containing the target sequence were used as a standard to make a standard curve by using the 10-fold serial dilutions (from 10−1 21
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Fig. 3. Effect of rgcIl-21 on the mRNA expression of various cytokines in HKLs. The HKLs were seeded at the density of 6 × 105 cells/well in 24-well plates. The cells were treated with 0.3–10 ng/mL of rgcIl-21 or heat inactivated (HI) proteins (10 ng/mL) for 3 h for expression detection of il10 (A), tgfb (B) and il1b (C) or 12 h for tnfa (D). The transcript levels of bactin, il10 (A), tgfb (B), il1b (C) and tnfa (D) were determined by RT-qPCR. The mRNA levels of each gene were normalized by bactin and expressed as fold changes of the mean value in the control group. Data are shown as mean ± SEM (N = 4). The “*” denotes significant differences (p < 0.05) relative to the control group. “**” means p < 0.01.
to 10−6 fmol/μL) of plasmid to eliminate the influence of the efficiency of different gene primers.
embedded in paraffin and sectioned with the rotary microtome (Leica Microsystems, Wetzlar, German). The slices were stained with hematoxylin-eosin as reported previously [21]. The stained sections were visualized under the light microscope (BX51, OLYMPUS, Tokyo, Japan).
2.8. Effect of rgcIl-21 on the survival of grass carp infected with A. hydrophila Healthy grass carp with body weight of 50 g were kept in laboratory for two weeks before experiments and then transferred into four tanks (15 fish/tank) containing 1000 L water. The live A. hydrophila used for infection were prepared as described before [21]. All fish were anesthetized with 0.01% MS-222 (Sigma-Aldrich) before injection. In the four tanks, two groups of fish were injected intraperitoneally (i.p.) with rgcIl-21 (0.1 mL/fish, 20 μg/fish) or sterilized PBS (0.1 mL/fish), respectively. In parallel, the other two groups were received i.p. injections with the bacteria (0.1 mL/fish; 3 × 108 CFU/mL in PBS) 1 h prior to treatment with or without rgcIl-21 (20 μg/fish). After that, the time and number of fish deaths were counted for the survival rate of grass carp.
2.10. Data analysis
2.9. Hematoxylin-eosin (HE) staining of grass carp tissues
3.1. Isolation and sequence analysis of gcil21 CDS
Similar to in vivo studies except for the dose of A. hydrophila (0.1 mL/fish; 1 × 108 CFU/mL in PBS) were performed as described. After 24 h of treatment (5 fish/group), tissue samples (head kidney and intestine) were collected and fixed in 4% neutral buffered paraformaldehyde (Sigma-Aldrich) for 24 h and dehydrated in ascending concentrations of alcohol and cleared in xylene. These tissues were
The CDS of gcil21 was 450 bp, encoding 149 amino acids (aa). Apart from the signal peptide (19 aa), mature peptide was 130 aa and the molecular weight of it was 14.78 kDa with a pI of 8.92. The aliphatic index was 79.38 and the proportion of hydrophobic amino acid was 39.9%. Alignment of amino acid sequences indicated that the identity of gcIl-21 with other species was 15.43–41.14%, and lowest identity
Statistical analysis was conducted by one-way analysis of variance ANOVA followed by Fisher's least significance difference (LSD) using the SPSS Statistics 10.0 software. For comparison between two groups, Student's t-test was performed. Data were expressed as mean ± SEM with four independent replicates (N = 4), and significant difference and highly significant difference were considered at p < 0.05 and p < 0.01, respectively. All experiments were repeated at least three times. 3. Results
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Fig. 4. Time-course study of rgcIl-21 on the mRNA expression of several cytokines in HKLs. Freshly prepared HKLs were seeded at the density of 6 × 105 cells/well in 24-well plates and were incubated in the presence or absence of 10 ng/mL of rgcIl-21 or HI proteins (10 ng/mL) for 1–12 h. The transcript levels of bactin, il10 (A), tgfb (B), il1b (C), tnfa (D) were determined by RT-qPCR. The mRNA levels of each gene were normalized by bactin and expressed as fold changes of the mean value in the control group. Data are shown as mean ± SEM (N = 4). The “*” denotes significant differences (p < 0.05) relative to the control group. “**” means p < 0.01.
was chicken and highest was zebrafish. The results illustrated that all the Il-21 had four relative conservative cysteine residues as being pointed in the figure (Supplemental Fig. 1A). As shown in rootless phylogenetic tree (Supplemental Fig. 1B), Il-21 in different fishes were clustered into one branch, in which grass carp, rainbow trout and zebrafish clustered into a small branch, showing that the kinship of grass carp was closer with zebrafish and rainbow trout.
grass carp HKLs challenged by HK-A. hydrophila (Fig. 2A). Moreover, in the antibody pre-absorption experiment, the binds for both rgcIl-21 and natural gcIl-21 were not observed (Fig. 2B). Additionally, HK-A. hydrophila markedly stimulated the release of gcIl-21 from HKLs at 6 h but gcIl-21 was undetected in the control group (Fig. 2C).
3.2. Recombinant expression and purification of rgcIl-21
To explore the biological activity of rgcIl-21 grass carp HKLs, influences of rgcIl-21 on the expression of gcil10, gctgfb, gctnfa and gcil1b were detected by RT-qPCR. Results showed that a 3 h-incubation of HKLs with increasing doses (0.3–10 ng/mL) of rgcIl-21 resulted in a dose-dependent increase in gcil10 mRNA levels (Fig. 3A), but did not alter the mRNA expression of gctgfb (Fig. 3B) and gcil1b (Fig. 3C). Additionally, a 12 h-treatment of rgcIl-21 (3–10 ng/mL) was effective in enhancing the gctnfa transcript levels (Fig. 3D). A time course study was conducted by incubation of grass carp HKLs with 10 ng/mL of rgcIl-21 for 1, 3, 6 and 12 h (Fig. 4). When compared the time-matched controls, rgcIl-21 stimulated gcil10 mRNA expression in a time-dependent manner from 1 to 12 h (Fig. 4A) and increased the mRNA levels of gctnfa only at 12 h (Fig. 4D). However, the rgcIl-21 had no effect on the expression of gctgfb and gcil1b from 1 to 12 h (Fig. 4B and C).
3.4. Effects of rgcIl-21 on the cytokine expression in HKLs
The rgcIl-21 was produced in the BL21 (DE3)/pET-30a (+) system. After IPTG induction, 6 × His-tagged rgcIl-21 protein was successfully produced in the inclusion bodies of the crude protein extraction of E. coli cells (Fig. 1A, line 2). The proteins of inclusion body was denatured and refolded, and subsequently purified by washing buffer and desalted by centrifugal ultrafiltration. The purified rgcIl-21 was visualized as a single band around 17 kDa on the gel of SDS-PAGE (Fig. 1A, line 3). Besides, the rgcIl-21 was further verified by Western blotting using Histag antibody and only one band with the same size as the SDS-PAGE was observed (Fig. 1B). The purified rgcIl-21 contained 4.81 EU/mg of endotoxin. 3.3. Determination of gcIl-21 release in the culture supernatant of grass carp HKLs
3.5. In vivo protection of rgcIl-21 against A. hydrophila in grass carp
The specificity of anti-gcIl-21 pAb was verified by WB assay, showing that it could specifically recognize rgcIl-21 with a size of about 17 kDa and a single band with similar size from the culture medium of
To assess in vivo anti-bacterial activity of rgcIl-21, it was injected i.p. into live A. hydrophila (0.1 mL/fish; 3 × 108 CFU/mL in PBS) pre-infected grass carp and the survival rate within 36 h was counted. The 23
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3.6. Effect of rgcIl-21 on A. hydrophila infection-induced pathological changes in vivo Four groups of grass carp were treated by PBS (0.1 mL/fish), A. hydrophila (0.1 mL/fish; 1 × 108 CFU/mL in PBS), rgcIl-21 (0.1 mL/ fish, 10 μg/fish) and rgcIl-21 following live A. hydrophila infection, separately, and then the pathological changes of head kidney and intestine were observed by HE staining. As shown in Figs. 6 and 7, the head kidney and intestine in both PBS (A) and rgcIl-21 (C) groups presented as normal status. However, A. hydrophila treatment resulted in severe hyperaemia and loose organizational structure of head kidney (Fig. 6B). Obviously, rgcIl-21 was effective in mitigating the hyperaemia symptom of head kidney (Fig. 6D). In parallel, mucosal disruption, shedding of villus and epithelial cells, goblet cell reduction and tissue hyperaemia were observed in the intestine of grass carp infected by the bacteria (Fig. 7B), and rgcIl-21 could improve these symptoms caused by A. hydrophila infection (Fig. 7D).
4. Discussion In the present study, we cloned a CDS of gcil21 gene with a total length of 450 bp that encodes 149 amino acids. Alignment of amino acid sequences and phylogenetic tree analysis revealed the closer relationship of gcIl-21 to fish (particularly zebrafish and rainbow trout) Il21 compared with the homologs in other species (Supplementary Fig. 1), suggesting that the newly cloned gcil21 is indeed a homolog of fish il21. However, the functional role of Il-21 in fish immunity including its production patterns and immune significance is not completely clear. In this regard, rgcIl-21 and anti-gcIl-21 pAb were prepared. Subsequently, WB assay showed that the amount of gcIl-21 was rare in the medium of untreated HKLs but largely induced in the medium of HKLs challenged by HK-A. hydrophila. To our knowledge, this is the first time to reveal the production characteristics of fish Il-21 at protein level. In particular, the highly inducible release of gcIl-21 under immune insults strongly suggested its role under inflammatory conditions, and provided a clue for targeting fish inflammation. In mammals, IL-21 shows the ability to induce the expression of various cytokines, including IFN-γ [22], IL-10 [23], IL-6 [24], TNF-α [25] and IL-1β [26], indicating the pleiotropic effect of IL-21 in immunity. In this study, the functional role of gcIl-21 was evaluated in
Fig. 5. Effect of rgcIl-21 on the survival rate of grass carp infected with A. hydrophila. After anesthetization with 0.01% oxazocaine, two groups of fish were injected i.p. with rgcIl-21 (0.1 mL/fish, 20 μg/fish) and sterilized PBS (0.1 mL/fish), respectively. In parallel, another two groups were received i.p. injections with the bacteria (0.1 mL/fish; 3 × 108 CFU/mL in PBS) 1 h prior to treatment in the presence or absence rgcIl-21 (0.1 mL/fish, 20 μg/fish).
results showed that there was no death in the groups injected with PBS or rgcIl-21 within 36 h. However, a 16 h-infection with A. hydrophila was sufficient to induce the death of grass carp, and the fish only achieved a survival rate of 40% within 36 h (Fig. 5). By comparison, the combination of rgcIl-21 (20 μg/fish) with A. hydrophila resulted in an obvious increase of survival rate (66.7%) after a 20-h challenge (Fig. 5).
Fig. 6. The representative photos of the HE stain in head kidney at 1-day post-infection (magnification, × 400; scale bar, 50 μm). (A) PBS treatment group. (B) A. hydrophila (0.1 mL/fish, 1 × 108 CFU/mL in PBS) infection group. (C) rgcIl-21 treatment group. (D) rgcIl-21 injection group following A. hydrophila infection. H, hemorrhage. 24
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Fig. 7. The representative photos of the histological alterations in intestine at 1-day post-infection (magnification, × 400; scale bar, 50 μm). (A) PBS treatment group. (B) A. hydrophila (0.1 mL/fish, 1 × 108 CFU/mL in PBS) infection group. (C) rgcIl-21 treatment group. (D) rgcIl-21 injection group following A. hydrophila infection. H, hemorrhage. VEF, villus and epithelial cell fall off. G, goblet cells.
isolated from healthy fish [33]. Moreover, in vitro and in vivo studies reveal that the gene expression of pufferfish il21 is induced by immune stimuli in head kidney cells and kidney, respectively [33]. Similarly, the inducible expression of il21 by LPS is reported in the kidney of spotted green pufferfish [17]. In particular, IL-21 has drawn increased attention to its involvement in the gut inflammation in mammals [for literature review, see Ref. [34, 35]. In accordance with this, the up-regulation of il21 transcription by immune stimuli is detected in the intestine of fish species [17,33]. Actually, it is well known that both head kidney and intestine are considered as the major lymphoid tissues in fish. These findings suggest the possible role of Il-21 in the head kidney and intestine of fish, prompting us to evaluate the morphological effects of rgcIl-21 on head kidney and intestine in live A. hydrophila-infected grass carp. As expected, HE staining showed that rgcIl-21 was effective in ameliorating A. hydrophila infection-triggered damage and inflammation symptoms in both head kidney and intestine, confirming the antibacterial activity of rgcIl-21. In agreement with our findings, IL-21 is able to suppress intestinal inflammation in mice [36]. Accordingly, our results highlighted the role of Il-21 in modulating fish inflammation. In conclusion, this study firstly reveals the immunomodulatory function of gcIL-21, especially its immunosuppression in the inflammatory response during bacterial infection as seen in mammals.
grass carp HKLs. Interestingly, rgcIl-21 displayed different effects on the gene expression of various inflammation-related cytokines. Compared with effects of rgcIl-21 on the expression of gctgfb, gcil1b and gctnfa, a strong induction of expression of gcil10 was observed by rgcIl-21. In this context, 0.3 ng/mL of rgcIl-21 was sufficient to induce gcil10 expression after a 3 h-treatment (Fig. 3) and gcil10 mRNA levels were elevated significantly as early as 1 h post-treatment with rgcIl-21, with increase expression lasting for at least 12 h (Fig. 4). In agreement with our findings, rainbow trout Il-21 also exhibits a prominent effect on the up-regulation of il10 expression in HKLs [17]. The potential of Il-21 markedly enhancing il10 expression may imply the anti-inflammatory property of Il-21 in fish. In support of this notion, the immunosuppressive activity of mouse IL-21 has been revealed by its induction of IL-10 [27]. In addition, IL-21 also presents the inhibitory effects on LPS-induced inflammatory responses in mouse peritoneal macrophages [28]. These findings suggested the possible involvement of gcIl-21 in inflammatory regulation, promoting us to address the activity and role of gcIl-21 in response to bacterial infection. Along this line, an in vivo study was carried out to investigate the effect of rgcIl-21 on grass carp survival following exposure to live A. hydrophila infection. Results showed that i.p. injection of rgcIL-21 was able to increase the survival rate of grass carp infected with A. hydrophila, suggesting the ability of fish Il-21 against bacterial infection in vivo. Similarly, human IL-21 has been proved to enhance host resistance to Mycobacterium tuberculosis [29,30]. Notably, the gcIl-21 was positive charge (pI of 8.92 and trout Il-21 with a pI of 9.16) and contained a substantial proportion (39.9%) of hydrophobic residues, which are similar to the molecular characteristics of antimicrobial peptides, such as defensin, plectasin, protegrin-1, cecropins and magainins [31]. These antimicrobial peptides with overall positive charge can accumulate at polyanionic microbial cell surfaces, form separate patches rich in hydrophobic amino acids on the bilayer, and disrupt the integrity of the membrane [31]. Thus, we cannot exclude the possibility that the antibacterial ability of gcIl-21 is associated with its molecular nature. This hypothesis is supported by the findings in which human IL-26 with positive charge is involved in host defense against bacteria [32]. In order to support our speculation, the effects of gcIl-21 on bacterial infection-caused immune tissue damage were examined. In the Japanese pufferfish, the constitutive expression of il21 was detected only in the head kidney, but not other tissues (e.g. spleen and liver)
Acknowledgments This work was supported by the grants from the National Natural Science Foundation of China (31572650) and the Applied Basic Research Program of Sichuan Province, China (19YYJC0036). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fsi.2020.01.059. References [1] J. Parrish-Novak, S.R. Dillon, A. Nelson, A. Hammond, C. Sprecher, J.A. Gross, et al., Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function, Nature 408 (6808) (2000) 57–63. [2] T. Habib, S. Senadheera, K. Weinberg, K. Kaushansky, The common gamma chain (gamma c) is a required signaling component of the IL-21 receptor and supports IL21-induced cell proliferation via JAK3, Biochemistry 41 (27) (2002) 8725–8731.
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Full length article
Characterization and bioactivity of grass carp (Ctenopharyngodon idella) interleukin-21: Inducible production and involvement in inflammatory regulation
T
Anying Zhang1, Xiaoyu Jian1, Dan Wang, Jingqi Ren, Xinyan Wang, Hong Zhou∗ School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, People's Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Grass carp Il-21 Protein release Anti-bacterial activity Anti-inflammatory property
In mammals, interleukin 21 (IL-21) is a broad pleiotropic cytokine that plays critical roles in the development of several inflammatory and autoimmune diseases. In fish, functional information of Il-21 is limited, and its role in immune response is largely unknown. In the present study, we cloned a coding sequence of grass carp (Ctenopharyngodon idella) il21 gene (gcil21). To characterize the release patterns and biological activity of gcIl21, we prepared recombinant gcIl-21 (rgcIl-21) and obtained the polyclonal antibody with gcIl-21 specificity. Western blotting analysis showed that in grass carp head kidney leukocytes (HKLs), gcIl-21 was undetected in culture supernatant of untreated cells but drastically induced by heat-killed Aeromonas hydrophila (A. hydrophila), uncovering the release features of gcIl-21 and its possible involvement in immune response. Subsequent functional experiments revealed that rgcIl-21 did not affect the mRNA expression of grass carp il1b and tgfb, but induced a strong expression of grass carp il10, and to a lesser extent of grass carp tnfa in HKLs, suggesting a dominant effect of gcIl-21 in modulating Il-10 signaling as seen in rainbow trout and mammals. Furthermore, in vivo studies showed that intraperitoneal injection of rgcIl-21 was able to increase the survival rate of grass carp infected with live A. hydrophila, and reduce the pathological responses caused by the same pathogenic bacteria in head kidney and intestine. Taken together, these results for the first time revealed the close relationship of fish Il21 production and function with inflammatory responses, and highlighted its anti-bacterial and anti-inflammatory ability, thereby providing a new insight into host defense mechanisms in fish.
1. Introduction In mammals, interleukin 21 (IL-21) is a type I cytokine with four-αhelix bundle produced by activated CD4+ T cells and natural killer T (NKT) cells [1]. IL-21 mediates its biological effects through a specific receptor chain (IL-21R) and a common receptor γ chain (γc) which shares with IL-2 family cytokines such as IL-4, IL-7, IL-9, and IL-15 [2]. The wide distribution of IL-21R and γc transcripts in a variety of lymphoid tissues such as thymus, spleen and lymph nodes, as well as peripheral blood leukocytes indicates the pleiotropic functions of IL-21 [3–5]. Actually, upon binding to its receptors, IL-21 activates the Janus family tyrosine kinases members JAK1 and JAK3, with subsequent phosphorylation of STAT3 and STAT1 [6,7]. Additionally, the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK) pathways contribute to IL-21 signal transmission [8,9]. For
functionality, IL-21 is suggested to mediate the differentiation of T cells, the maturation of B cells, as well as the proliferation and cytotoxicity of NK cells [10,11]. Besides, IL-21 plays critical roles in the development of several inflammatory and autoimmune diseases such as inflammatory bowel diseases (IBD), rheumatoid arthritis and cancers [12,13]. For example, IL-21 shows critical immunosuppressive effects in IBD because of its ability to induce IL-10 [14], a crucial regulator of immune homeostasis. However, in dentritic cells (DC), IL-21 induces IL1β expression in NF-ΚB independent way [15]. These studies suggest the pleiotropic properties of IL-21 in immune response [13]. Considering the important role of IL-21 in mammalian immunity, the fish Il-21 has also attracted increasing attention. At present, il21 gene has been identified in a few fish species including fugu [16], spotted green pufferfish [17], zebrafish [18], and rainbow trout [19], and most efforts have been focused on the gene expression of il21 in
∗
Corresponding author. School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, People's Republic of China. E-mail address: [email protected] (H. Zhou). 1 Anying Zhang and Xiaoyu Jian contributed equally to this article. https://doi.org/10.1016/j.fsi.2020.01.059 Received 17 September 2019; Received in revised form 27 January 2020; Accepted 28 January 2020 Available online 31 January 2020 1050-4648/ © 2020 Elsevier Ltd. All rights reserved.
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Phylogenetic analysis was conducted using the neighbor-joining method in MEGA X (http://www.megasoftware.net/) using 1000 repetitions for bootstrapping.
response to various immune stimuli such as phytohaemagglutinin (PHA), lipopolysaccharide (LPS), T cell stimulant phorbol myristate acetate (PMA) and calcium ionophore [16,17,19]. However, the protein expression pattern of fish Il-21, which is the important property of Il-21 as a cytokine, has still not been investigated. For functionality, Il-21 is only characterized in rainbow trout, showing that the recombinant Il-21 proteins are capable to induce the expression of some cytokines and T/B cell marker genes in head kidney cells [19]. Accordingly, the functional information of fish Il-21 is limited and its role in fish immunity so far remains to be elucidated. The coding sequence of grass carp il21 (gcil21) gene was isolated and identified in the present study. Subsequently, recombinant grass carp Il-21 (rgcIl-21) was prepared, and the polyclonal antibody specific for gcIl-21 was generated. By using this antibody, Western blotting (WB) analysis revealed the inducible release of gcIl-21 by the heatkilled (HK) Aeromonas hydrophila (A. hydrophila)in grass carp head kidney leukocytes (HKLs), suggesting possible involvement of gcIl-21 in the immune responses. In agreement with this notion, rgcIl-21 showed the bioactivity to regulate the mRNA expression of inflammation-related cytokines in a time- and dose-dependent manner. To reveal the significance of rgcIl-21 in mediating these effects, in vivo studies were performed and results proved that intraperitoneal injection of rgcIl-21 was effective in increasing the survival rate, and reducing the pathological changes of tissues in grass carp infected with live A. hydrophila. These results highlight the role and potential of fish Il-21 as an inflammation regulator during bacterial infection.
2.4. Recombinant expression of gcIl-21 Recombinant gcIl-21 (rgcIl-21) was expressed by BL21 (DE3)/pET30a (+) (Merck Millipore, Billerica, USA) system. Briefly, the DNA fragment encoding mature gcIl-21 was subcloned into the Nde Ⅰ and BamH Ⅰ sites of pET-30a (+). After the plasmid was amplified in E. Coli and extracted by using a TIANprep Rapid Mini Plasmid Kit (Tiangen), it was transformed into E. Coli BL21 (DE3) strain. The positive clone was grown in Luria Broth (LB) medium containing kanamycin (30 ng/mL) for 3 h with shaking at 37 °C. When the OD at 600 nm reached 0.6, Isopropyl-β-D-Thiogalactopyranoside (IPTG, Sigma-Aldrich, St. Louis, USA) was added to a final concentration of 1 mM to induce rgcIl-21 expression. After 4 h of induction, the bacterial cells were collected by centrifugation (4000×g, 10 min) at 4 °C and washed by 10 mM ice-cold phosphate buffer (pH 7.4) twice. The cells were resuspended in 20 mL of 10 mM phosphate buffer supplemented with 1 mM PMSF and 1 mg/ mL Lysozyme and finally sonicated on ice. The sediment was collected by centrifugation at 10,000×g for 20 min at 4 °C and resuspended in washing buffer (20 mM Tris-HCl, 10 mM EDTA and 1% Triton X-100, pH7.5). The inclusion body was washed by washing buffer for three times and 3 M urea once, via centrifugation (10,000×g, 10 min) at 4 °C and then dissolved in ice-cold denaturation buffer (30 mg inclusion body/ml, 8 M urea, 10% glycerol, 50 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA and 10 mM DTT, pH8.0). The supernatant was collected after centrifugation at 10,000×g for 10 min at 4 °C. About 1 mg protein of the supernatant was added into 50 mL of renaturation buffer (100 mM Tris-HCl, 400 mM L-Arg-HCl, 2 mM EDTA, 5 mM oxidized and reduced glutathione each and 0.5 mM PMSF, pH8.0) slowly (5–6 drops/ min) with magnetic stirring at 100 rpm and the solution was incubated at 4 °C overnight. After that, the renaturation buffer containing rgcIl-21 was ultrafiltered using an Amicon Ultra-15 Centrifugal Filter Unit (Amicon®Ultra 10 K device, Merck Millipore). LPS (endotoxin) was removed by Endotoxin Removal Agarose Resin (Yeasen, Shanghai, China). The molecular weight and purity of rgcIl-21 were evaluated by SDS-PAGE and identified by Western blotting using an anti-His antibody (1:1000, Boster, Wuhan, China). The endotoxin levels in rgcIl-21 were determined using an endotoxin assay kit (Chinese Horseshoe Crab Reagent Manufactory Co. Ltd., Xiamen, China). The purified rgcIl-21 protein was lyophilized and then stored at −80 °C for further use.
2. Materials and methods 2.1. Animals Healthy grass carp, weighing 0.5–1.0 kg or 50–60 g, were purchased from Chengdu Tongwei Aquatic Science and Technology Company (Chengdu, China). The fish were reared in aquaria with circulating, filtered and well-aerated tap water at 25 ± 2 °C in our laboratory at least 2 weeks before experimental procedures. The tissues were obtained from the freshly killed fish and all animal experiments were performed in accordance with the Regulation for Animal Experimentation of Sichuan province, China, and were allowed by the ethics committee of the University of Electronic Science and Technology of China. 2.2. Cloning and sequencing of gcil21 coding sequence (CDS)
2.5. Isolation and culture of grass carp HKLs
Total RNA was extracted from grass carp HKLs by using Tripure Isolation Reagent (Roche, Basel, Switzerland) and about 2 μg of total RNA was converted into cDNA using M-MLV Reverse Transcriptase (Promega, Madison, USA) with oligo (dT)18 as the primer. According to the uploaded gcil21 gene sequence (GenBank accession no.: KP226585), the primers (Supplementary Table 1) were designed and used to clone gcil21 CDS. Finally, the CDS of gcil21 was validated by PCR amplification using Phusion DNA polymerase (NEB, Beverly, USA) and subcloned into pGM-T vector (Tiangen, Beijing, China). The ligation products were transformed into JM109 competent cells and the positive clones were selected for sequencing.
Healthy grass carp with body weight of 0.5–1 kg were kept in laboratory for two weeks before experiments and sacrificed for HKLs collection. HKLs were obtained by discontinuous density gradient [20]. In brief, grass carp head kidney was obtained from freshly killed fish, washed twice and gently pressed in RPMI-1640 medium (Gibco, NY, USA). The cell suspension was filtrated by a 200-gauge stainless steel mesh before centrifuging at 2800 rpm for 30 min at 20 °C in a density gradient centrifugation (Ficoll-Hypaque 1.083 kg/L, TBD, Tianjin, China). After that, the cells were obtained from the interface and washed twice, then resuspended in the RPMI-1640 medium supplemented 24 mM NaHCO3, 25 mM HEPES, 1% antibiotic-antimycotic and 10% fetal bovine serum (Gibco). In the 24-well plate (BD Biosciences, San Jose, USA), approximately 6 × 105 cells per well were seeded and incubated at 26 °C overnight under 5% CO2 for drug treatment. Alternatively, 1 × 107 cells per well were planted in the 6-well plate (BD Biosciences) with the same culture condition.
2.3. Sequence analysis The deduced amino acid sequence was analyzed using the DNAMAN 8.0 software (Lynnon Biosoft, Pointe-Claire, Canada) and signal peptide was predicted by SignalP-5.0 Server (http://www.cbs.dtu.dk/services/ SignalP/). Isoelectric point (pI) and molecular weight (MW) of protein were calculated by Compute pI/MW in ExPASy (https://web.expasy. org/compute_pi/). Protein hydrophobicity was calculated by ProtParam tool in ExPASy (https://web.expasy.org/protparam/). The multiple sequence alignments were generated by DNAMAN 8.0 software.
2.6. Western blot assay of gcIl-21 in HKLs Heat-killed A. hydrophila (HK-A. hydrophila) were prepared in 20
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Fig. 1. Recombinant expression of gcIl-21. (A) SDSPAGE analysis of rgcIl-21. M, molecular weight marker; lane 1, crude lysate of inclusion body from the cells without IPTG induction; lane 2, crude lysate of inclusion body from the cells with IPTG induction; line 3, the purified rgcIL-21. (B) Western blotting analysis of rgcIl-21 using the antibody for His tag. M, molecular weight marker; lane 1, the purified rgcIl21.
Fig. 2. Validation of anti-gcIl-21 pAb and the protein release patterns of gcIl-21 in grass carp HKLs. (A) Western blotting analysis of anti-gcIl-21 pAb. Lane 1, culture medium of grass carp HKLs; lane 2, culture medium of grass carp HKLs challenged by HK-A. hydrophila; lane 3, rgcIl-21. (B) Total proteins from culture medium of grass carp HKLs were used to verify the specificity of anti-gcIl-21 pAb pre-absorbed with an excess of rgcIl-21 with the molar mass ratio of 1:30. Lane 1, culture medium of grass carp HKLs without treatment; lane 2, culture medium of grass carp HKLs challenged with HK-A. hydrophila; lane 3, rgcIl-21. (C) Effect of A. hydrophila treatment on gcIl-21 secretion in grass carp HKLs. HKLs were seeded in 6-well plate with density of 1 × 107 cells per well and incubated at 28 °C under 5% CO2 and saturated humidity. After the cells were challenged with or without HK-A. hydrophila at concentration of 1 × 107 CFU/well for 6 h (three replications), the medium of each well with the same volume was collected and analyzed by Western blotting assay using anti-gcIl-21 pAb (1:3000).
boiling water for 30 min at concentration of 1 × 107 CFU/well. The HKLs were treated with or without 1.0 multiplicity of infection (MOI) HK-A. hydrophila. Subsequently, the supernatant of culture medium was harvested for WB assay. In this assay, the gcIl-21 polyclonal antibodies (anti-gcIl-21 pAb) were customized from Biogot (Nanjing, China). To validate its specificity, recombinant gcIl-21 and the concentrated culture medium of HKLs were analyzed by WB in which the membrane was incubated with anti-gcIl-21 pAb or the antibody pre-absorbed with rgcIl-21 with the molar mass ratio of 1:30. The proteins in culture medium were separated by SDS-PAGE gel and WB assay was used to detect gcIl-21. The membrane was incubated with anti-gcIl-21 pAb (1:3000) at 4 °C overnight, and then exposed to horseradish peroxidaseconjugated goat anti-rabbit secondary antibody (1:5000, ZSGB-BIO, Beijing, China) for 2 h at room temperature. Finally, the signals were detected using an ECL kit (Roche) according to the manufacturer's instruction.
2.7. Dose response and time course experiments To determine the optimal concentration, HKLs were treated with serial dilutions of rgcIl-21 from 0.3 to 10 ng/mL. The cells were collected at 3 h or 12 h later and dissolved in TriPure Isolation Reagent. Total RNA was extracted to obtain the cDNA by reverse transcription as described above, and then the expression of gcil10, gctgfb, gctnfa and gcil1b were measured by the real-time quantitative PCR (RT-qPCR). To investigate the kinetics of rgcIl-21-modulated gene expression, an optimal dose of rgcIl-21 (10 ng/mL) was used to stimulate HKLs, the expression of gcil10, gctgfb, gctnfa and gcil1b were assessed at 1, 3, 6 and 12 h by RT-qPCR. RT-qPCR was run on Bio-Rad CFX96 Real-time detection system (Bio-Rad, Hercules, USA) by Real Master Mix (SYBR Green) Kit with gene specific primers (Table 1). Parallel measurement of bactin was conducted to serve as an internal control to exclude the deviations during RNA extraction and cDNA synthesis. The pGEM-T easy vectors containing the target sequence were used as a standard to make a standard curve by using the 10-fold serial dilutions (from 10−1 21
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Fig. 3. Effect of rgcIl-21 on the mRNA expression of various cytokines in HKLs. The HKLs were seeded at the density of 6 × 105 cells/well in 24-well plates. The cells were treated with 0.3–10 ng/mL of rgcIl-21 or heat inactivated (HI) proteins (10 ng/mL) for 3 h for expression detection of il10 (A), tgfb (B) and il1b (C) or 12 h for tnfa (D). The transcript levels of bactin, il10 (A), tgfb (B), il1b (C) and tnfa (D) were determined by RT-qPCR. The mRNA levels of each gene were normalized by bactin and expressed as fold changes of the mean value in the control group. Data are shown as mean ± SEM (N = 4). The “*” denotes significant differences (p < 0.05) relative to the control group. “**” means p < 0.01.
to 10−6 fmol/μL) of plasmid to eliminate the influence of the efficiency of different gene primers.
embedded in paraffin and sectioned with the rotary microtome (Leica Microsystems, Wetzlar, German). The slices were stained with hematoxylin-eosin as reported previously [21]. The stained sections were visualized under the light microscope (BX51, OLYMPUS, Tokyo, Japan).
2.8. Effect of rgcIl-21 on the survival of grass carp infected with A. hydrophila Healthy grass carp with body weight of 50 g were kept in laboratory for two weeks before experiments and then transferred into four tanks (15 fish/tank) containing 1000 L water. The live A. hydrophila used for infection were prepared as described before [21]. All fish were anesthetized with 0.01% MS-222 (Sigma-Aldrich) before injection. In the four tanks, two groups of fish were injected intraperitoneally (i.p.) with rgcIl-21 (0.1 mL/fish, 20 μg/fish) or sterilized PBS (0.1 mL/fish), respectively. In parallel, the other two groups were received i.p. injections with the bacteria (0.1 mL/fish; 3 × 108 CFU/mL in PBS) 1 h prior to treatment with or without rgcIl-21 (20 μg/fish). After that, the time and number of fish deaths were counted for the survival rate of grass carp.
2.10. Data analysis
2.9. Hematoxylin-eosin (HE) staining of grass carp tissues
3.1. Isolation and sequence analysis of gcil21 CDS
Similar to in vivo studies except for the dose of A. hydrophila (0.1 mL/fish; 1 × 108 CFU/mL in PBS) were performed as described. After 24 h of treatment (5 fish/group), tissue samples (head kidney and intestine) were collected and fixed in 4% neutral buffered paraformaldehyde (Sigma-Aldrich) for 24 h and dehydrated in ascending concentrations of alcohol and cleared in xylene. These tissues were
The CDS of gcil21 was 450 bp, encoding 149 amino acids (aa). Apart from the signal peptide (19 aa), mature peptide was 130 aa and the molecular weight of it was 14.78 kDa with a pI of 8.92. The aliphatic index was 79.38 and the proportion of hydrophobic amino acid was 39.9%. Alignment of amino acid sequences indicated that the identity of gcIl-21 with other species was 15.43–41.14%, and lowest identity
Statistical analysis was conducted by one-way analysis of variance ANOVA followed by Fisher's least significance difference (LSD) using the SPSS Statistics 10.0 software. For comparison between two groups, Student's t-test was performed. Data were expressed as mean ± SEM with four independent replicates (N = 4), and significant difference and highly significant difference were considered at p < 0.05 and p < 0.01, respectively. All experiments were repeated at least three times. 3. Results
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Fig. 4. Time-course study of rgcIl-21 on the mRNA expression of several cytokines in HKLs. Freshly prepared HKLs were seeded at the density of 6 × 105 cells/well in 24-well plates and were incubated in the presence or absence of 10 ng/mL of rgcIl-21 or HI proteins (10 ng/mL) for 1–12 h. The transcript levels of bactin, il10 (A), tgfb (B), il1b (C), tnfa (D) were determined by RT-qPCR. The mRNA levels of each gene were normalized by bactin and expressed as fold changes of the mean value in the control group. Data are shown as mean ± SEM (N = 4). The “*” denotes significant differences (p < 0.05) relative to the control group. “**” means p < 0.01.
was chicken and highest was zebrafish. The results illustrated that all the Il-21 had four relative conservative cysteine residues as being pointed in the figure (Supplemental Fig. 1A). As shown in rootless phylogenetic tree (Supplemental Fig. 1B), Il-21 in different fishes were clustered into one branch, in which grass carp, rainbow trout and zebrafish clustered into a small branch, showing that the kinship of grass carp was closer with zebrafish and rainbow trout.
grass carp HKLs challenged by HK-A. hydrophila (Fig. 2A). Moreover, in the antibody pre-absorption experiment, the binds for both rgcIl-21 and natural gcIl-21 were not observed (Fig. 2B). Additionally, HK-A. hydrophila markedly stimulated the release of gcIl-21 from HKLs at 6 h but gcIl-21 was undetected in the control group (Fig. 2C).
3.2. Recombinant expression and purification of rgcIl-21
To explore the biological activity of rgcIl-21 grass carp HKLs, influences of rgcIl-21 on the expression of gcil10, gctgfb, gctnfa and gcil1b were detected by RT-qPCR. Results showed that a 3 h-incubation of HKLs with increasing doses (0.3–10 ng/mL) of rgcIl-21 resulted in a dose-dependent increase in gcil10 mRNA levels (Fig. 3A), but did not alter the mRNA expression of gctgfb (Fig. 3B) and gcil1b (Fig. 3C). Additionally, a 12 h-treatment of rgcIl-21 (3–10 ng/mL) was effective in enhancing the gctnfa transcript levels (Fig. 3D). A time course study was conducted by incubation of grass carp HKLs with 10 ng/mL of rgcIl-21 for 1, 3, 6 and 12 h (Fig. 4). When compared the time-matched controls, rgcIl-21 stimulated gcil10 mRNA expression in a time-dependent manner from 1 to 12 h (Fig. 4A) and increased the mRNA levels of gctnfa only at 12 h (Fig. 4D). However, the rgcIl-21 had no effect on the expression of gctgfb and gcil1b from 1 to 12 h (Fig. 4B and C).
3.4. Effects of rgcIl-21 on the cytokine expression in HKLs
The rgcIl-21 was produced in the BL21 (DE3)/pET-30a (+) system. After IPTG induction, 6 × His-tagged rgcIl-21 protein was successfully produced in the inclusion bodies of the crude protein extraction of E. coli cells (Fig. 1A, line 2). The proteins of inclusion body was denatured and refolded, and subsequently purified by washing buffer and desalted by centrifugal ultrafiltration. The purified rgcIl-21 was visualized as a single band around 17 kDa on the gel of SDS-PAGE (Fig. 1A, line 3). Besides, the rgcIl-21 was further verified by Western blotting using Histag antibody and only one band with the same size as the SDS-PAGE was observed (Fig. 1B). The purified rgcIl-21 contained 4.81 EU/mg of endotoxin. 3.3. Determination of gcIl-21 release in the culture supernatant of grass carp HKLs
3.5. In vivo protection of rgcIl-21 against A. hydrophila in grass carp
The specificity of anti-gcIl-21 pAb was verified by WB assay, showing that it could specifically recognize rgcIl-21 with a size of about 17 kDa and a single band with similar size from the culture medium of
To assess in vivo anti-bacterial activity of rgcIl-21, it was injected i.p. into live A. hydrophila (0.1 mL/fish; 3 × 108 CFU/mL in PBS) pre-infected grass carp and the survival rate within 36 h was counted. The 23
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3.6. Effect of rgcIl-21 on A. hydrophila infection-induced pathological changes in vivo Four groups of grass carp were treated by PBS (0.1 mL/fish), A. hydrophila (0.1 mL/fish; 1 × 108 CFU/mL in PBS), rgcIl-21 (0.1 mL/ fish, 10 μg/fish) and rgcIl-21 following live A. hydrophila infection, separately, and then the pathological changes of head kidney and intestine were observed by HE staining. As shown in Figs. 6 and 7, the head kidney and intestine in both PBS (A) and rgcIl-21 (C) groups presented as normal status. However, A. hydrophila treatment resulted in severe hyperaemia and loose organizational structure of head kidney (Fig. 6B). Obviously, rgcIl-21 was effective in mitigating the hyperaemia symptom of head kidney (Fig. 6D). In parallel, mucosal disruption, shedding of villus and epithelial cells, goblet cell reduction and tissue hyperaemia were observed in the intestine of grass carp infected by the bacteria (Fig. 7B), and rgcIl-21 could improve these symptoms caused by A. hydrophila infection (Fig. 7D).
4. Discussion In the present study, we cloned a CDS of gcil21 gene with a total length of 450 bp that encodes 149 amino acids. Alignment of amino acid sequences and phylogenetic tree analysis revealed the closer relationship of gcIl-21 to fish (particularly zebrafish and rainbow trout) Il21 compared with the homologs in other species (Supplementary Fig. 1), suggesting that the newly cloned gcil21 is indeed a homolog of fish il21. However, the functional role of Il-21 in fish immunity including its production patterns and immune significance is not completely clear. In this regard, rgcIl-21 and anti-gcIl-21 pAb were prepared. Subsequently, WB assay showed that the amount of gcIl-21 was rare in the medium of untreated HKLs but largely induced in the medium of HKLs challenged by HK-A. hydrophila. To our knowledge, this is the first time to reveal the production characteristics of fish Il-21 at protein level. In particular, the highly inducible release of gcIl-21 under immune insults strongly suggested its role under inflammatory conditions, and provided a clue for targeting fish inflammation. In mammals, IL-21 shows the ability to induce the expression of various cytokines, including IFN-γ [22], IL-10 [23], IL-6 [24], TNF-α [25] and IL-1β [26], indicating the pleiotropic effect of IL-21 in immunity. In this study, the functional role of gcIl-21 was evaluated in
Fig. 5. Effect of rgcIl-21 on the survival rate of grass carp infected with A. hydrophila. After anesthetization with 0.01% oxazocaine, two groups of fish were injected i.p. with rgcIl-21 (0.1 mL/fish, 20 μg/fish) and sterilized PBS (0.1 mL/fish), respectively. In parallel, another two groups were received i.p. injections with the bacteria (0.1 mL/fish; 3 × 108 CFU/mL in PBS) 1 h prior to treatment in the presence or absence rgcIl-21 (0.1 mL/fish, 20 μg/fish).
results showed that there was no death in the groups injected with PBS or rgcIl-21 within 36 h. However, a 16 h-infection with A. hydrophila was sufficient to induce the death of grass carp, and the fish only achieved a survival rate of 40% within 36 h (Fig. 5). By comparison, the combination of rgcIl-21 (20 μg/fish) with A. hydrophila resulted in an obvious increase of survival rate (66.7%) after a 20-h challenge (Fig. 5).
Fig. 6. The representative photos of the HE stain in head kidney at 1-day post-infection (magnification, × 400; scale bar, 50 μm). (A) PBS treatment group. (B) A. hydrophila (0.1 mL/fish, 1 × 108 CFU/mL in PBS) infection group. (C) rgcIl-21 treatment group. (D) rgcIl-21 injection group following A. hydrophila infection. H, hemorrhage. 24
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Fig. 7. The representative photos of the histological alterations in intestine at 1-day post-infection (magnification, × 400; scale bar, 50 μm). (A) PBS treatment group. (B) A. hydrophila (0.1 mL/fish, 1 × 108 CFU/mL in PBS) infection group. (C) rgcIl-21 treatment group. (D) rgcIl-21 injection group following A. hydrophila infection. H, hemorrhage. VEF, villus and epithelial cell fall off. G, goblet cells.
isolated from healthy fish [33]. Moreover, in vitro and in vivo studies reveal that the gene expression of pufferfish il21 is induced by immune stimuli in head kidney cells and kidney, respectively [33]. Similarly, the inducible expression of il21 by LPS is reported in the kidney of spotted green pufferfish [17]. In particular, IL-21 has drawn increased attention to its involvement in the gut inflammation in mammals [for literature review, see Ref. [34, 35]. In accordance with this, the up-regulation of il21 transcription by immune stimuli is detected in the intestine of fish species [17,33]. Actually, it is well known that both head kidney and intestine are considered as the major lymphoid tissues in fish. These findings suggest the possible role of Il-21 in the head kidney and intestine of fish, prompting us to evaluate the morphological effects of rgcIl-21 on head kidney and intestine in live A. hydrophila-infected grass carp. As expected, HE staining showed that rgcIl-21 was effective in ameliorating A. hydrophila infection-triggered damage and inflammation symptoms in both head kidney and intestine, confirming the antibacterial activity of rgcIl-21. In agreement with our findings, IL-21 is able to suppress intestinal inflammation in mice [36]. Accordingly, our results highlighted the role of Il-21 in modulating fish inflammation. In conclusion, this study firstly reveals the immunomodulatory function of gcIL-21, especially its immunosuppression in the inflammatory response during bacterial infection as seen in mammals.
grass carp HKLs. Interestingly, rgcIl-21 displayed different effects on the gene expression of various inflammation-related cytokines. Compared with effects of rgcIl-21 on the expression of gctgfb, gcil1b and gctnfa, a strong induction of expression of gcil10 was observed by rgcIl-21. In this context, 0.3 ng/mL of rgcIl-21 was sufficient to induce gcil10 expression after a 3 h-treatment (Fig. 3) and gcil10 mRNA levels were elevated significantly as early as 1 h post-treatment with rgcIl-21, with increase expression lasting for at least 12 h (Fig. 4). In agreement with our findings, rainbow trout Il-21 also exhibits a prominent effect on the up-regulation of il10 expression in HKLs [17]. The potential of Il-21 markedly enhancing il10 expression may imply the anti-inflammatory property of Il-21 in fish. In support of this notion, the immunosuppressive activity of mouse IL-21 has been revealed by its induction of IL-10 [27]. In addition, IL-21 also presents the inhibitory effects on LPS-induced inflammatory responses in mouse peritoneal macrophages [28]. These findings suggested the possible involvement of gcIl-21 in inflammatory regulation, promoting us to address the activity and role of gcIl-21 in response to bacterial infection. Along this line, an in vivo study was carried out to investigate the effect of rgcIl-21 on grass carp survival following exposure to live A. hydrophila infection. Results showed that i.p. injection of rgcIL-21 was able to increase the survival rate of grass carp infected with A. hydrophila, suggesting the ability of fish Il-21 against bacterial infection in vivo. Similarly, human IL-21 has been proved to enhance host resistance to Mycobacterium tuberculosis [29,30]. Notably, the gcIl-21 was positive charge (pI of 8.92 and trout Il-21 with a pI of 9.16) and contained a substantial proportion (39.9%) of hydrophobic residues, which are similar to the molecular characteristics of antimicrobial peptides, such as defensin, plectasin, protegrin-1, cecropins and magainins [31]. These antimicrobial peptides with overall positive charge can accumulate at polyanionic microbial cell surfaces, form separate patches rich in hydrophobic amino acids on the bilayer, and disrupt the integrity of the membrane [31]. Thus, we cannot exclude the possibility that the antibacterial ability of gcIl-21 is associated with its molecular nature. This hypothesis is supported by the findings in which human IL-26 with positive charge is involved in host defense against bacteria [32]. In order to support our speculation, the effects of gcIl-21 on bacterial infection-caused immune tissue damage were examined. In the Japanese pufferfish, the constitutive expression of il21 was detected only in the head kidney, but not other tissues (e.g. spleen and liver)
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