Common carp (an average weight of 50 ± 5 g) were purchased from a commercial fish farm in Xinxiang (Henan province, China). Before the experiment, the fish were kept in freshwater tanks at 25 ± 2 °C for 2 weeks, and fed with commercial pellets twice a day.

All animal experiments in this study were performed following the protocols of the “Guidelines for Experimental Animals” of the Ministry of Science and Technology (Beijing, China), and all experiments involving animals were approved by the Animal Care and Use Ethics Committee of the Henan Normal University.

The predicted IL-15L sequence of common carp was obtained based on the transcriptional database of common carp and the sequence of zebrafish IL-15 (NM_001039565.1). Subsequently, specific primers were designed based on the predicted gene sequence of IL-15L ( Table 1 ). Total RNA of the liver from healthy common carp was extracted using TRIzol (TaKaRa, Japan), and then the first strand of cDNA was synthesized. The cDNA sequence of CcIL-15L was amplified by polymerase chain reaction. The PCR products were then examined with 1.5% agarose gel, and purified with an agarose gel DNA purification kit (OMEGA, Bio-Tek). The target sequences were ligated into the pMD19-T vector and transformed into DH-5α for sequencing.

CcIL-15L open reading frames (ORFs) were identified using NCBI ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/) and the deduced amino acid sequence was predicted with translate tool (https://web.expasy.org/translate/). SignalP (http://www.cbs.dtu.dk/services/SignalP) was used to predict signal peptides, while molecular weight and isoelectric point were calculated using online software (https://web.expasy.org/compute_pi/). ClustalW X2 was utilized for multiple sequence comparisons, and SMART (SMART, http://smart.emblheidelberg.de/) was employed to predict protein motif features. Phylogenetic analysis was performed using MEGA7.0 software with the neighbor-joining method and 10000 replicates were used to test branch reliability.

To investigate the expression pattern of CcIL-15L in different tissues (liver, head kidney, spleen, muscle, intestine, brain, gills, and skin), five healthy common carp were immobilized by MS-222 anesthesia and sacrificed. Then the tissues were sampled and placed in enzyme-free EP tubes, respectively. Then, TRizol reagent (TaKaRa, Japan) was used to extract total RNA according to the instructions of the manufacturer. The quality of extracted RNA was detected by 1% agarose gel electrophoresis and the purity was determined by the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Subsequently, the first strand of cDNA was synthesized according to Hifair^® II 1st Strand cDNA Synthesis SuperMix (Yeason, Shanghai, China) instructions. The primers are listed in Table 1 . The RT-qPCR reaction system is 10 μL, including 0.2 μL of each primer, 2.6 μL of sterile ultrapure water, 2 μL of diluted cDNA template, and 5 μL of Hieff UNICON^® Universal Blue qPCR SYBR Green Master Mix (Yeason, Shanghai, China). The reaction system was carried out according to the following steps: 95°C for 2 min, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 20 s. EF-1α was used as the internal reference gene. The relative mRNA expression levels of CcIL-15L were calculated using the 2^-ΔΔCt method (21).

The pathogen A. hydrophila was isolated from common carp and preserved in our laboratory, and they were prepared according to the previous studies (22). In brief, the bacteria were cultured in LB medium at 28°C for 12 h with constant shaking (200 rpm), and then they were centrifuged at 6000 rpm for 10 min and washed twice with PBS. The fish were randomly selected for the challenge experiment. Each common carp was intraperitoneal injected (i.p.) with 100 μL inoculum (approximately 1.5×10⁶ CFU/ml). Three common carp were randomly collected each time at 0, 6, 12, 24, and 48 h post-injection (hpi). After that, the gills, intestine, spleen, and head kidney of common carp were sampled. The RT-qPCR assay was then performed as described above. The experiments were carried out in triplicate.

To construct a prokaryotic expression vector, the CcIL-15L was ligated into pET-32a (+) plasmids. The successfully constructed pET-32a-IL-15L was transferred into component cells of Escherichia coli BL21 (Biomed, China). The cells were cultured at 37°C until OD₆₀₀ reached 0.6, and then the recombinant proteins were induced with 1.0 mM IPTG at 37°C overnight and detected with 12.5% SDS-PAGE (Polyacrylamide gel electrophoresis). After that, the rIL-15L was lysed using 6 M urea and purified by NI-NTA nitrilotriacetic acid (NI-NTA). The dialysis tubing was first boiled in water for 15 min and then rinsed with sterile water. One end of the tubing was sealed with a clip, and the protein solution was added from the other end. The tubing was then placed in a dialysis solution at 4°C. The dialysis solution was changed every 6 h for a total of five times, with urea concentrations of 6 M, 4 M, 2 M, 1 M, and PBS, respectively. After dialysis, the protein concentration and purity were assessed. Purity was evaluated by SDS-PAGE, and protein concentration was determined using the BCA method. The protein was concentrated to 1 mg/mL using an ultrafiltration tube and stored at -80°C for future use. The thioredoxin was used as the a control protein apart from PBS. The proteins were then analyzed by 12.5% SDS-PAGE and the protein concentration was determined by the BCA method.

According to a previous study (22), primary head kidney leukocytes (HKLs) were separated from the head kidney of healthy common carp. In brief, head kidneys were collected from the fish, and they were placed on a 70 μm nylon mesh, and gently pushed through the mesh with constant dripping of cold L15 medium containing 0.1% FBS, 0.1% heparin, 100 U/mL penicillin, and 100 U/ml streptomycins. The cell suspension was centrifuged at 400 × g for 30 min on a 45% percoll gradient. The cells on the medium-percoll interface were collected by centrifugation at 400 × g at 4°C for 10 min and washed twice with the medium. The cells were seeded into 6-well cell culture plates at 1.5 × 10⁶ cells/well, and cultured at 28°C in a 5% CO₂ incubator for 2 h (23). After 2 h of cultivation, different concentrations of rCcIL-15L (2, 20, and 200 ng/mL) were added to the wells and incubated for 12 h, and the wells were added with PBS were set as the control group. The mRNA expression levels of IL-1β, TNF-α, IFN-γ, IL-6, perforin, and Eomesa were then examined by RT-qPCR. The primers were listed in Table 1 .

The specific primers of eukaryotic expression plasmids ( Table 1 ) were designed based on genes of CcIL-15L, and added Kozak sequences. The purified PCR products of CcIL-15L and expression vector pcDNA™3.1/3×FLAG were digested with the restriction enzymes EcoR I and Hind III and ligated with the T4 DNA Ligase (TaKaRa, Japan). Then, the linked products were transformed into E. coli DH-5α, which were then plated on LB-ampicillin media and were grown overnight at 37°C. The transformants were verified by sequencing and double enzymatic digestion. The successfully constructed plasmid is named pcIL-15L. Plasmid DNA was extracted using Endo-Free plasmid Kit (Tiangen, China), and removed the endotoxin.

Common carp were assigned randomly into three groups and subjected to intramuscular injection of 100 μl of pcIL-15L (200 μg/ml), 100 μl of the empty pcDNA 3.1 vector (pcN3) (200 μg/ml), or 100 μl of PBS. On fifth day after plasmid administration, the head kidney and spleen samples were collected from three fish in each group. The expression levels of immune-related genes (CD4-1, CD8β2, TNF-α, and IgM) were assessed using RT-qPCR as described above. The primer sequences used in this study are provided in Table 1 . The plasmid was introduced into common carp via intramuscular injection, and the overexpression of CcIL-15L was confirmed through RT-qPCR, western blotting, and immunofluorescence assay (IIFA) at 5 d post plasmid administration.

Five days after introducing the recombinant plasmid into the fish using the aforementioned method, each fish was intraperitoneally injected with 0.2 mL of A. hydrophila at a concentration of 5×10⁶ CFU/mL. After 12 and 24 h, the liver, spleen, and kidney were aseptically collected. The tissues were homogenized and diluted with sterile PBS, then spread in triplicate on LB agar plates. The plates were incubated at 28°C for 24 h, and the number of colonies was counted. Each colony was verified using PCR with A. hydrophila-specific primers ( Table 1 ).

1× 10⁶ HKLs in RPMI 1640 medium supplemented with 10% FBS were seeded into 24-well plates and incubated overnight at 28°C with 5% CO₂. The rCcIL-15L was added to the cultures with the final dose 200 ng/mL for 24 h. Fluorescent latex beads were then introduced at a 1:25 cell-to-bead ratio, as previously described (24). After removing non-ingested beads using BSA and D-glucose buffer, phagocytic activity was analyzed via flow cytometry (24).

The chemotaxis assays were conducted using transwell chambers (Corning) with 13 mm filters and a pore size of 8 µm. The lower chambers were filled with rCcIL-15L (500 ng/mL) or supernatants from HKLs treated with rCcIL-15L for 24 h. Common carp HKLs (1 × 10⁶ cells/well) were added to the upper chambers. After a 4-hours incubation, migrated HKLs in the bottom wells were quantified under an Axio Observer Z1 microscope (Zeiss, Germany) in 2 min. Chemotactic activity was assessed by calculating the chemotactic index, defined as the ratio of cells migrating in response to rCcIL-15L or supernatants from HKLs treated with rCcIL-15L compared to the number of cells that migrated by PBS, which served as the negative control. Total RNA of the migrated cells were extracted as described above, and the expression levels of different cell type marker genes were detected using RT-qPCR. The marker gene primers were listed in Table 1 .

Common carp were administered with pcIL-15L, pcN3, or PBS as above. After 5 d post-plasmid administration, common carp were injected intramuscularly with 100 μl A. hydrophila (approximately 1.5×10⁶ CFU/ml). At 24 hpi, the head kidney and spleen were removed and placed with 1 ml of 4% paraformaldehyde, and then submerged in 4% paraformaldehyde overnight. The tissues were embedded in paraffin, sectioned, and stained with HE and the images were captured and processed with ZEN/ZEN lite imaging software from Zeiss.

Data was shown as mean + standard error of the mean (SEM). Statistical significance was analyzed with one-way analysis of variance (ANOVA), implemented in software GraphPad Prism 8. Statistical significance was set as p < 0.05 (*) and p < 0.01 (**).

The ORF of CcIL-15L was 552 nucleotides (NCBI accession number: OQ981449), encoding a protein 183 amino acids (aa) without a predicted signal peptide ( Figure 1A ) and with a theoretical molecular weight (MW) of 21.7 kDa and a theoretical PI of 9.17. The deduced amino acid sequence indicated an IL-15 domain at residue 49–157, which was similar to zebrafish ( Figure 1B ). In addition, the IL-15L protein tertiary structures of common carp and zebrafish were predicted by SWISS-Model, and the results showed that CcIL-15L was highly similar to the tertiary structures of IL-15L from zebrafish ( Figure 1B ). Syntenic analysis showed that common carp and other teleost IL-15L are located in the same locus adjacent plekhg2 and supt5h ( Figure 1C ). Homology comparison showed that the CcIL-15L shared sequence identity with those of other fish species ( Table 2 ). CcIL-15L indicated the highest sequence identity with Fathead minnow (Pimephales promelas) IL-15L (64.3%) and Tiger barb (Puntigrus tetrazona) IL-15L (60.1%), and the lowest sequence identity with grass carp (Ctenopharyngodon idellus) IL-15 (13.9%). Multiple alignment of IL-15L and IL-15 aa sequences showed that four cysteine residues were conserved between fish and mammals ( Figure 2 ). Four cysteine (C) residues are important for disulfide bond formation in human IL-15 (in human C⁸³-C¹³³ and C⁹⁰-C¹³⁶), and also conserved in fish IL-15 homologues. A phylogenetic tree was constructed using the known fish IL-15L protein sequences and the sequences of selected IL-2 family members from higher vertebrates ( Figure 3 ). Phylogenetic tree shows that CcIL-15L and IL-15L of other fish were clustered into a clade.

The relative expression levels of the CcIL-15L gene in different tissues and organs were detected by RT-qPCR. As shown in Figure 4 , CcIL-15L is constitutively expressed at different levels in all tested tissues and organs, including the spleen, head kidney, skin, gills, brain, liver, intestine, and muscle. The intestine showed the highest level of expression, followed by the liver, gills, brain, and skin, with lower levels of expression in the spleen, head kidney, and muscle.

To assess the CcIL-15L response to bacterial infection, the fish were injected with A. hydrophila. Head kidney, spleen, gills, and intestine were sampled at 0, 6, 12, 24, and 48 h post injection (hpi) for examining gene expression. As shown in Figure 5A , in head kidney, the expression levels spiked at 24 h after injection but then returned to normal values by 48 h. In the gills and spleen, the expression levels were the highest at 12 hpi ( Figures 5B, C ). In the intestine, the expression level is also the highest at 12 hpi, and it is basically the same as the expression level at 24 hpi ( Figure 5D ).

We expressed and purified the rCcIL-15L protein (35 kDa) in E. coli to determine its function ( Figure 6 ). Then, we stimulated primary head kidney leukocytes with the rCcIL-15L protein to investigate its effects on immune gene expression, including NK cell activation factors (Perforin and Eomesa) and pro-inflammatory cytokines (IL-1β, IL-6, IFN-γ, and TNF-α). As shown in Figure 7 , when the leukocytes were stimulated with 2 ng/ml rCcIL-15L protein, there was no significant increase at gene expression levels for six cytokines. However, when stimulated with 20 ng/ml protein, five cytokine expressions were significantly increased, and only gene Eomesa expression was not significantly upregulated. When stimulated with higher concentrations of rCcIL-15L protein (200 ng/ml), NK cell activation genes (Eomesa and perforin) and IFN-γ expression were significantly up-regulated, while IL-6, IL-1β, and TNF-α gene expressions did not show significant up-regulation.

The results described above showed that the recombinant CcIL-15L indicated regulatory activity in vitro. To further study the effect of CcIL-15L in vivo, we constructed the pcIL-15L expressing vector, which could overexpress in common carp. As shown in Figure 8 , based on IIFA methods, it was showed that the proteins of 3×FLAG-tagged pcIL-15L were detected in the muscle of common carp after injected with pcIL-15L plasmids for 5 d. By contrast, no FLAG-labeled signal was detected in the control. Meanwhile, RT-qPCR detection revealed that the expression of CcIL-15L significantly increased in head kidney and spleen. Except for CcIL-15L, immune-related genes including IL-1β, CD4-1, CD8β2, IFN-γ, and TNF-α were also significantly increased. In contrast, for these genes, there was no significant difference at the mRNA expression levels, in the PBS and pcN3 groups ( Figures 9A, B ).

The histopathological change was further evaluated in head kidney and spleen of common carp injected with pcIL-15L vectors after A. hydrophila challenge. As shown in Figure 10 , the results showed that, compared with the group injected with pcIL-15L plasmid, histopathological lesions in head kidney and spleen showed to be more severe in the PBS and pcN3 group at 24 h, such as tissue fibrosis, lymphocyte infiltration, and tissue damage.

Five days after intramuscular injection of the recombinant plasmids, carp were challenged with A. hydrophila to evaluate bacterial infection by quantifying bacterial loads in the liver, spleen, and kidneys at 12 and 24 h post-infection. As shown in Figure 11 , at 12 h after infection, bacterial colonization in the liver was significantly reduced in the pcIL-15L group compared to the control group. By 24 h post-challenge, bacterial loads in the liver, spleen, and kidney were markedly lower in the pcIL-15L group than in the controls.

After treatment of common carp head kidney leukocytes HKLs with rCcIL-15L, phagocytic activity was assessed through flow cytometry ( Figure 12 ). Lymphocyte populations were identified within the gated HKLs ( Figure 12A ). Stimulation with rCcIL-15L significantly enhanced the phagocytic activity of HKLs, demonstrating its role in promoting immune function in these cells. (P < 0.01) ( Figures 12B–E ).

To further investigate the chemotactic role of rCcIL-15L, we assessed its capacity to attract common carp HKLs using a chemotaxis chamber. The findings revealed that while rCcIL-15L alone did not enhance HKL migration, the culture supernatants from HKLs treated with either PBS, Trx or rCcIL-15L significantly promoted the migration of HKLs ( Figure 13A ). We analyzed the expression levels of cell type marker genes using qPCR. The results indicated that the expression levels of macrophage marker CSF1R were significantly upregulated in the chemotaxed cells of rCcIL-15L treated medium supernatants compared to both primary cells and the medium supernatants treated by PBS ( Figure 13B ). However, there were no significant differences in the expression of the lymphocyte markers CD4-1 and CD8β2 among the groups ( Figures 13C, D ).