All animal experiments were conducted in accordance with the guidelines and approval of the Animal Research and Ethics Committees of Sun Yat-Sen University. All efforts were made to minimize suffering.

Healthy E. coioides weighing approximately 500 g were purchased from the Guangdong Daya Bay Fishery Development Center (Huizhou, Guangdong, P. R. China). The fish were maintained in a recirculating seawater system, with a 12 h light/12 h dark cycle, at 25–30°C for 7 days before use. The fish were fed with commercial pellets twice daily. Food was withheld from the fish for 24 h prior to sample collection. All of the fish used in this study appeared to be healthy before the experiments were conducted. Before sample collection, the fish were anesthetized with MS-222 in dechlorinated water for 2 min.

The head kidney was dissected from healthy E. coioides, and total RNA was extracted by TRIzol reagent (Invitrogen, USA). The first strand of cDNA was synthesized with ReverTra Ace qPCR RT Kit (TOYOBO, Japan) according to the manufacturer’s instructions.

The open reading frame (ORF) regions of IFNγ1 and IFNγ2 were amplified with the primers listed in Table 1. PCR amplification were performed at 94°C for 5 min, followed by 40 cycles at 94°C for 20 s, 55°C for 20 s, and 72°C for 45 s, with 72°C for a final 10 min at the end of the last cycle. All PCR products were ligated into the pTZ57R/T Vector (Fermentas, USA) after analyzing on 1.5% agarose gels, finally sequenced by Invitrogen Bioengineering Corporation, Guangdong, China.

The upstream sequence of pre-miR-146a was cloned from E. coioides genomic DNA using the primers of miR-146a F/R (Table 1). The pre-miR-146a upstream fragment was ligated into pGL4.10[luc2] vector (Promega, USA). Mutated versions of these constructs were obtained by site-directed mutagenesis using Mut Express II Fast Mutagenesis Kit (Vazyme, China).

TNF receptor-associated factor 6 3ʹUTR was cloned from head kidney cDNA by PCR with the primers of TRAF6 F/R (Table 1). To create 3ʹUTR luciferase reporter constructs, fragments of 3ʹUTR of TRAF6 gene were sub-cloned downstream of CMV-driven firefly luciferase cassette in pMIR-REPORT vector (Ambion, USA).

BLAST was used for identifying cDNA and deducing amino acid sequences in the NCBI.¹ Prediction of ORF were performed in the DNAssist 2.0 software. Multiple-sequence alignment of the EcIFNγ with other vertebrate IFNγs were performed with the Clustal Omega.² A vertebrata IFNγ phylogenetic tree was constructed with MEGA6 software using the maximum likelihood (ML) method, with a bootstrap of 100 times to verify its credibility. The SignalP program³ was applied to search for the signal peptide of vertebrata IFNγ. N-glycosylation sites were predicted by NetNGlyc⁴ and nuclear localization sequences (NLS) were predicted by Brameier et al. (35). Target prediction between miR-146a and the 3ʹUTR region of TRAF6 was performed using FINDTAR3. Sequences of mature miR-146a were obtained from our lab by whole genome sequencing. The pre-miR-14a was confirmed by structure prediction using RNAfold WebServer.⁵

Twelve tissues including the thymus, head kidney, trunk kidney, spleen, heart, gill, eye, skin, intestine, stomach, skin, and liver were aseptically dissected from three independent individuals. The tissue expression profiles were detected by real-time PCR performed with SYBR Green PCR Master Mix (Life Technologies, USA) and the primers of IFNγ1 F/R or IFNγ2 F/R, respectively. E. coioides 18S rRNA (18S-F/R) was amplified as an internal control (Table 1).

The putative mature peptides of E. coioides IFNγ1 and IFNγ2 were predicted by the SignalP program. Then, the cDNA fragments encoding the putative mature peptide with deletion of the the signal peptide from the N terminus were amplified by PCR using the primers of IFNγ1 NdeI-F/IFNγ1 XhoI-R and IFNγ2 EcoRI-F/IFNγ2 XhoI-R (Table 1), respectively. The fragments were separated on a 1.5% agarose gel and purified by Qiagen gel extraction kit (Qiagen, Germany). After subcloning into pTZ57R/T vector for sequencing, the fragments were digested with restriction enzymes and then inserted into the pET22b expression vector (Novagen, USA). The E. coli BL21 (DE3) cells transformed with IFNγ1 recombinant construct and E. coli Rosetta (DE3) cells transformed with IFNγ2 recombinant construct were induced by different concentrations of IPTG. The cells were collected by centrifugation and the resultant recombinant proteins named rEcIFNγ1 and rEcIFNγ2 were purified using His·Bind Column (Novagen) according to the manufacturer’s protocol. The purity of rEcIFNγs was checked on SDS-PAGE gel stained with coomassie brilliant blue R-250 (Sigma, USA), and the size of target proteins was measured by comparing the protein band location with a standard protein (Fermentas). Both proteins were detected by western blotting using a primary anti-His-tag monoclonal antibody (Novagen) and a secondary goat anti-mouse IgG (Amersham Biosciences, UK).

The whole blood of fish was directly obtained from three fish (n = 3) using 5 mL syringe. Blood lymphocytes were isolated from whole blood using lymphocyte separation medium. The primary head kidney cells were obtained as previously described (36). Head kidney monocytes were isolated from primary head kidney cells using monocyte isolating kit (TBDscience, China). The isolated cells were washed and enumerated on a hemocytometer with trypan blue and resuspended at a concentration of 10⁶ cells/mL in tissue culture medium (TCM). The TCM was prepared from RPMI-1640 medium by adding 10% fetal bovine serum (Life Technologies), 2 mM l-glutamine (Sigma), and penicillin/streptomycin (Sigma).

Primary blood lymphocytes were distributed into 96-well plates at a density of 10⁶ cells/mL. rEcIFNγ1 or rEcIFNγ2 were respectively added to the culture medium to reach a working concentration of 1, 10, and 100 ng/mL, and the control group was treated with TCM. These cells were incubated at 28°C in 5% CO₂ for 72 h. Nitrite production was determined based on the Griess reaction with a NO determination kit (Beyotime Institute of Biotechnology, China).

Primary blood lymphocytes cultivation and in vitro stimulation were performed as described in Nitric oxide assay. The respiratory burst assay was performed as previously described (8). These cells were incubated at 28°C in 5% CO₂ for 18 h. Then, NBT (2 mg/mL, Sigma) and PMA (final concentration, 100 ng/mL, Sigma) were added to the cell cultures at room temperature. Absolute methanol was applied to fix the pelleted cells, and 70% methanol was applied to remove the non-reduced NBT. After air drying, the reduced NBT was dissolved using 2 M KOH and the blue crystals in the cytoplasm were dissolved by DMSO. Finally, the OD values were detected at 630 nm.

Primary head kidney cells were distributed into 6-well plates at a density of 10⁶ cells/mL. rEcIFNγ1 or rEcIFNγ2 were, respectively, added to the culture medium to reach a working concentration of 1, 10, and 100 ng/mL, and the control group was treated with TCM. After incubation for 3 h at 28°C in 5% CO₂, these cells were washed with PBS and lysed in a lysis buffer (Beyotime), which contained protease inhibitors (Sigma) and phosphatase inhibitors (Sigma). The protein lysates were separated by SDS-PAGE, then transferred onto nitrocellulose membranes. The membranes were blocked in 5% BSA in TBST for 1 h at room temperature followed by incubations with primary antibodies and the relevant HRP-conjugated secondary antibodies. The membranes were processed for ECL Western Blotting Detection Reagents (Pierce). Meanwhile, the TRAF6 expression levels in primary head monocytes after rEcIFNγ1 or rEcIFNγ2 treatment were also detected by western blot. Antibodies used in the study were TRAF6 (Santa Cruz, CA, USA) and β-actin (Proteintech, USA). The bands were analyzed semiquantitively by densitometry (grayscale analysis) with ImageJ software and normalized to their controls.

After treatment with rEcIFNγ1 or rEcIFNγ2, head kidney monocytes were collected. Total RNA was extracted by TRIzol reagent (Invitrogen), then reverse-transcribed and amplified with the Hairpin-it miRNAs RT-PCR Quantitation kit (GenePharma, China). E. coioides U6 snRNA served as control. The relative level of miR-146a expression was calculated using the comparative threshold (2^−ΔΔCt) method (37).

In order to confirm whether miR-146a could directly target the mRNA of TRAF6, HEK-293T cells were transfected with 40 nM miR-146a mimics (GenePharma) or negative control (nc) (GenePharma) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. 24 h after transfection, luciferase activity in 293T cells was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. The luciferase data were normalized by dividing the firefly luciferase activity by the activity of Renilla luciferase.

Primary head kidney monocytes were also transfected with miR-146a mimics or nc. The cells were lysed and the TRAF6 protein levels in the lysates were detected with western-blot.

Mutant or wild-type pre-miR-146a upstream-reporter vectors were transfected into 293T cell using Lipofectamine 2000. After 6 h, the cells were stimulated with rEcIFNγ1 or rEcIFNγ2 and luciferase activities were measured.

All data were expressed as mean values ± SEM. Statistical analysis was carried out by one-way analysis of variance (ANOVA) or t-test. Differences were considered significant with a p-value less than 0.05 and were marked with asterisks.

The ORF of the EcIFNγ1 and EcIFNγ2 genes transcript was obtained (Figure 1). EcIFNγ1 ORF was 567 bp in length and translated into a 188-aa precursor molecule with a 19-aa signal peptide, while EcIFNγ2 ORF was 603 bp in length and encoded a 200-aa putative protein with a 19-aa signal peptide. The predicted mature EcIFNγ1 peptides contain two potential N-glycosylation sites (NTS and NVT), while EcIFNγ2 have one N-glycosylation site (NRT). Furthermore, EcIFNγ1 and EcIFNγ2 both possess an IFNγ signature sequence ([I/V]-Q-X-[K/Q]-A-X2-E-[L/F]-X2-[I/V]) at the C-terminus, which was conserved among known IFNγ molecules. In particular, a conserved motif RRRRRR similar to the nuclear localization signal of the known IFNγ molecules is found in EcIFNγ2 at its C-terminal tail, which was absent in EcIFNγ1.

Both EcIFNγ1 and EcIFNγ2 sequences and the IFNγs from other species were subjected to multiple alignment (Figure 2). The alignment confirms that EcIFNγ1 and EcIFNγ2 have relatively low sequence similarity compared with their vertebrate counterparts, respectively. EcIFNγ1 has 18.42–44.89% amino acid identity with other known fish IFNγ1, which shared the highest degree with tetraodon (Tetraodon nigroviridis) (44.89%). EcIFNγ2 shared a higher similarity with teleosts IFNγ2 sequences varied from 25.15 to 61.86% than with other vertebrates (12.41–22.02%), being most similar to IFNγ from Japanese flounder (Paralichthys olivaceus) (61.86%).

A phylogenetic tree was also constructed based on the alignments of EcIFNγ1 and EcIFNγ2 sequences with that of other species (Figure 3). The EcIFNγ1 was branched with T. nigroviridis. EcIFNγ2 was most closely related to those of P. olivaceus and T. nigroviridis, all of which belonged to marine teleost IFNγ2 clade. The previously reported rainbow trout (Oncorhynchus mykiss) IFNγ1 might also belong to the IFNγ2 family based on the phylogenetic tree and protein alignments.

Quantitative expression analysis of EcIFNγ1 or EcIFNγ2 in tissues of healthy fish revealed that EcIFNγ1 and EcIFNγ2 mRNA were expressed in different patterns. The highest mRNA levels of EcIFNγ1 were in the thymus and intestine, while the expression was relatively low in stomach, heart, eye, and spleen (Figure 4A). EcIFNγ2 was globally expressed in almost all tissues, and its expression levels were higher in gills and spleen, but lower in intestine, head kidney and eye (Figure 4B).

For further study of the biological activities of EcIFNγ1 and EcIFNγ2, the two putative mature peptides were expressed as C-terminal 6 His-tagged fusion protein in BL21 (DE3) and Rosetta (DE3) cells, respectively, followed by purification with affinity chromatography. SDS-PAGE analysis (Figures 5A,B) indicated that the purified sample exhibited a single protein band after induction by IPTG, and the molecular weights were close to the predicted size of the 6-His fused rEcIFNγ1 and rEcIFNγ2 peptides, respectively. Besides, both rEcIFNγ1 and rEcIFNγ2 were confirmed by Western blot assay with monoclonal antibody against the His-tag, and a single band was detected for the each of fused proteins (Figure 5C).

Blood lymphocytes were exposed to different concentrations of rEcIFNγ1 or rEcIFNγ2, followed by detection of the respiratory burst or nitric oxide levels. Both rEcIFNγ1 and rEcIFNγ2 at concentrations of 1 and 10 ng/mL induced nitric oxide responses in blood lymphocytes that were significantly higher than that of none recombinant protein treatment (Figure 6A). In addition, rEcIFNγ1 or rEcIFNγ2 at 1 ng/mL induced an obvious respiratory burst response in blood lymphocytes (Figure 6B).

The miR-146a expression level was detected by real-time PCR. Compared to the control group, the expression level of miR-146a in group with rEcIFNγ1 or rEcIFNγ2 treatment was significantly increased at 0.5 and 2 h (Figure 7A). miR-146a expression level reached a peak (increased approximately 11-fold) at about 2 h after rEcIFNγ2 treatment. The rapid induction of miR-146a in response to rEcIFNγ1 or rEcIFNγ2 suggested that miR-146a might be involved in response to IFNγ.

We first identified the stem loop of E. coioides pre-miR-146a (Figure 7B). Then, approximately 2.5 kb genomic region upstream of mature miR-146a was analyzed. Two gamma-activated sequence (GAS, TTNCTTTAA) were found, namely 300 GAS (−198 to −190 bp) and 1300 GAS (−1,136 to −1,128 bp) (Figure 7C). To explore the relationship between GAS and E. coioides IFNγs, the miR-146a upstream approximately 2.5 kb fragment was inserted into pGL4.10 vector. Dual-luciferase report assay was conducted after transfection with wild-type or mutant pGL4-miR-146a vectors followed by rEcIFNγ1 or rEcIFNγ2 stimulation. The miR-146a upstream region luciferase activity significant increased after rEcIFNγ2 treatment (Figure 7D). Upon mutantion of either GAS, rEcIFNγ2 had no effect on luciferase activity of miR-146a upstream region.

To further investigate the effect of miR-146a induction, we predicted its mRNA target sites. 3ʹUTR of TRAF6 mRNA was found to contain miR-146a target sequences (Figure 8A). In mammals, TRAF6 are key adapter molecules in TLR receptor signaling cascades, mediating activation of NF-κB pathway. To test the possibility that the TRAF6 expression may be regulated posttranscriptionally by miR-146a, we constructed a reporter vector that contained the firefly luciferase gene fused to 900 bp of the 3ʹUTR region of TRAF6 containing putative miR-146 target site. The reporter construct was transiently transfected into 293T cells together with miR-146a mimics. Compared to nc, a significant downregulative effect in the relative luciferase activity was observed when cells were transfected with miR-146a mimics (Figure 8B). We further examined the effect of miR-146a on TRAF6 protein expression in primary head kidney monocytes of E. coioides. After miR-146a mimic treatment, miR-146a mRNA expression was found to increase, which suggested that miR-146a mimics was successfully transfected into the primary head kidney monocytes (Figure 8C). The western blot results showed that TRAF6 protein level decreased after miR-146a mimics transfection (Figure 8D). These data suggested that the TRAF6 gene might be the target for posttranscriptional repression by miR-146a.

Compared to the control group, both rEcIFNγ1 and rEcIFNγ2 treatments obviously induced TRAF6 protein expression from 0.5 to 2 h. After 4 h, rEcIFNγ1 treatment slightly increased the TRAF6 protein expression, while rEcIFNγ2 treatment resulted in slight decrease (Figure 9).