Healthy grass carp weighing about 0.75-1.00 kg was obtained from Chengdu Tongwei Aquatic Science and Technology Company (Chengdu, China). After an adaptation period of 7 days, the fish was anaesthetized in 0.05% MS222 (Sigma-Aldrich, MO, USA) and sacrificed. The head kidney was taken from the fish for head-kidney leukocytes (HKLs), lymphocytes and monocytes/macrophages isolation. All animal experiments were reviewed and conducted according to the Regulation of Animal Use in Sichuan province, China, and were approved by the ethics committee of the University of Electronic Science and Technology of China.

The recombinant gcIfn-γ (rgcIfn-γ, 500 ng/mL) (17), rgcTgf-β1 (100 ng/mL) and anti-gcTgf-β1 mAb (1:2000 diluted) were prepared referring to our previous studies (18, 19). LPS from Escherichia coli O55:B5 and poly I:C were purchased from Sigma Aldrich, and their doses used in this study were determined according to our previous studies (17, 18, 20, 21). Inhibitors for NF-κB (PDTC, 0.25 µM, Sigma Aldrich, St. Louis, USA), ERK1/2 (PD98059, 30 μM, Merck, Bad Soden, Germany), JNK (SP600125, 6 μM, Merck), P38 (SB202190, 30 µM, Merck), activin receptor-like kinase 5 (ALK5, TGF-β1 RI Kinase inhibitor VIII, 2 µM, Calbiochem, EMD Chemicals, San Diego, USA), RORγt (SR1001, 15 μM, Sigma-Aldrich) and STAT3 (STAT3 VI, 30 μM, Sigma-Aldrich) were used and an equal amount of solvent was used in the control groups in the experiments. The use of these inhibitors referred to previous studies (18, 22–25). Among them, anti-gcTgf-β1 mAb and ALK5 inhibitor were used to confirm the role of Tgf-β1 signaling in limiting rgcIl-12BB actions.

Total RNA was extracted from the grass carp head kidney with TriPure Isolation Reagent (Roche, Basel, Switzerland) and then reverse transcribed to cDNA by using M-MLV reverse transcriptase (Promega, Madison, USA) with oligo d(T)₁₈ as the primer. The potential sequence encoding gcp35b was obtained by searching the grass carp genome sequence (http://www.ncgr.ac.cn/grasscarp/) with the offline BLAST tool based on the sequence of zebrafish p35b (GenBank ID: XM_017352586). The full-length gcp35b cDNA sequence was amplified from the grass carp head kidney cDNA by Phusion High-Fidelity DNA Polymerase (Vazyme, Nanjing, China) and sequenced. The cDNA and deduced amino acid sequence of gcp35b were analyzed by using the ExPASy Molecular Biology server (http://www.expasy.org). The molecular weight and isoelectric point of the putative gcp35b were predicted by Compute PI/Mw tool (http://web.expasy.org/compute pi/) and the deduced signal peptide was predicted using SignalP (http://www.cbs.dtu.dk/services/SignalP/). The potential N-glycosylation sites of grass carp Il-12 subunits were predicted by NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/). Gene synteny of the p35 loci was analyzed using the data from the NCBI database (https://www.ncbi.nlm.nih.gov/). The multiple amino acid alignments and the phylogenetic tree were constructed by MEGA7.1 software (https://www.megasoftware.net/).

The structural models of grass carp Il-12 heterodimer were constructed by SWISS-MODEL (http://swissmodel.expasy.org) based on the human Il-12 crystal structure (PDB:1F45). The molecular graphics visualization tool RasMol 2.7.2.1 (http://www.openrasmol.org/) was used to display the structural models. These predicted models were evaluated by the Qualitative Mean Energy Analysis Distance Constraint Global (QMEANDis Co Global) of SWISS-MODEL (https://swissmodel.expasy.org/qmean/).

To study the existence of gcIl-12 isoforms, the coding sequences (CDS) of grass carp p35a and p35b were amplified by Phusion High-Fidelity DNA Polymerase (Vazyme) with the primers listed in Supplementary Table 1. The CDS of gcp35a and gcp35b were separately subcloned into p3×FLAG-CMV-7.1 (Promega) to obtain the N-terminal FLAG-tagged gcp35a (gcp35a-FLAG) and gcp35b (gcp35b-FLAG) plasmids. The C-terminal HIS-tagged gcp40a/b/c (gcp40a/b/c-HIS) expression plasmids have been used in our previous study in which gcp19 and gcp40a/b/c heterodimeric assembly is investigated (16). The integrity of the inserted DNA fragments was verified by sequencing. The highly purified and endotoxin-free DNA plasmids were extracted from Escherichia coli (E. coli) by using a TIAN prep Mini Plasmid Kit (Tiangen, Beijing, China) for subsequent transfection. The HEK293 cells (2 × 10⁴ cells/35 mm culture dish) were transiently transfected with different combinations of gcp35a/b-FLAG and gcp40a/b/c-HIS plasmids (1.2 µg of each plasmid/35 mm culture dish) by using Lipofectamine 2000 Reagent (Thermo Scientific, Carlsbad, USA), separately. Forty-eight hours after transfection, the cell culture supernatants were collected and examined by a non-reducing Western Blotting (WB) assay.

To acquire the recombinant gcIl-12 isoforms, a (GGGGS)₃ linker was used to link the p35 subunit and p40 subunit (p40-(GGGGS)₃-p35). Briefly, the cDNA sequences encoding mature gcp35a/b were amplified by primers of p35a/b-HindIII G4S3 linker F, p35a-C-myc+6*his TGA XhoI R and p35b-XhoI R (Supplementary Table 1) with Phusion High-Fidelity DNA Polymerase (Vazyme) and then cloned into dhfr-deficient CHO (CHO-dhfr^-/^-) cell expression vector pSV2-dhfr (Youbio, Changsha, China) after digested with HindIII and XhoI (NEB). Next, the DNA fragment encoding the gcp40b was amplified by PCR using the primers with HindIII restriction site (Supplementary Table 1) and subsequently inserted into the expression vector containing gcp35a or gcp35b after the digestion with HindIII. The integrity of the inserted DNA fragments was verified by sequencing. Then the highly purified and endotoxin-free DNA plasmids were extracted from E. coli by using a TIAN prep Mini Plasmid Kit (Tiangen).

CHO cells were seeded at a density of 2 × 10⁴ cells/35 mm culture dish in IMDM medium (Gibco, NY, USA) supplemented with 10% FBS (Gibco), 1% HT (Gibco) and 1% antibiotic-antimycotic (Thermo Scientific) for 24 h before transfection. The plasmids for two gcIl-12 isoforms were separately transfected into the CHO cells by Lipofectamine 2000 according to the manufacturer’s instructions (Thermo Scientific). Forty-eight hours after transfection, cells were selected in the IMDM medium with 10% FBS (Gibco) and 1% antibiotic-antimycotic (Thermo Scientific) which contains different concentrations of methotrexate (MTX) (Life Technologies, Gaithersburg, MD). MTX is commonly used as a selective antibiotic in the dihydrofolate reductase (dhfr) selection system. After cultured in high dose of MTX (500 nM), the transformed clones were isolated using 10 µL pipette tips and screened by WB assay. The selected clones were cultured in 5 mL SFM4CHO medium (Gibco) in 50 mL BD tubes at 37°C with shaking at 180 rpm for 48 h and then those cells were transferred to a 125 mL flask and cultured with 30 mL SFM4CHO medium at 32°C with shaking at 180 rpm for a week. The rgcIl-12 isoforms in the culture medium were purified by His Trap affinity column (GE Healthcare, Waukesha, USA) and desalted by the Superdex-G25 prep grade column (GE Healthcare). The molecular weight and purity of purified proteins were analyzed on SDS-PAGE and WB. Finally, the rgcIl-12 isoforms were lyophilized and then stored at −80°C for further use.

The non-reducing electrophoresis with SDS but without β-mercaptoethanol (β-ME) was used to determine the gcIl-12 heterodimer composition. In this scenario, the cell culture media of HEK293 cells transfected with gcp35a/b and gcp40a/b/c expression plasmids were harvested at 48 h after transfection and added a 5×loading buffer without β-ME, and then the samples were boiled at 70°C for 10 min. After that, these samples were separated on 10% SDS-PAGE, and then electrophoretically transferred to a PVDF membrane (Millipore, Billerica, MA). The membrane was blocked by TBST buffer (1% Tween) containing 10% (wt/vol) defatted dry milk for 2 h at room temperature and then incubated with anti-HIS mAb (1:600, ZSGB-BIO, Beijing, China) or anti-FLAG mAb (1:5000, Cell Signaling Technology, MA, USA) overnight at 4°C. The membrane was exposed to horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:5000, ZSGB-BIO) for 2 h at room temperature. Finally, signals were detected using an ECL kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions.

To detect the activation of signaling pathways, the cell lysates were separated on 12% SDS-PAGE and the signaling molecules were detected by WB assay by using specific primary antibodies for phosphorylated ERK (anti-pERK1/2, 1:1000, Cell Signaling Technology), JNK (anti-pJNK, 1:1000, Cell Signaling Technology), p38 (anti-p-p38, 1:1000, Cell Signaling Technology), IκBa (anti-p-IκBa, 1:1000, Cell Signaling Technology), STAT3 (anti-pSTAT3, 1:1000, Anaspec, Fremont, CA, USA), and the β-actin (anti-β-actin 1:5000, Boster, Wuhan, China) as the loading control. These antibodies detecting the activated state of signaling molecules are raised against peptides based on the phosphorylation sites of each signaling molecule (Figure S5B), and they have been applied and effectively recognized the corresponding molecules in grass carp and other fish species (23, 26–28). The predicted sizes and amino acid sequence alignment analysis of these signaling molecules in grass carp are shown in Figure S5.

The molecular weight and purity of two rgcIl-12 isoforms were evaluated by SDS-PAGE and verified by Western blotting using anti-gcp35a pAb (1:1000), anti-gcp35b pAb (1:1000) and anti-gcp40b pAb (1:1000, Biogot Technology, Nanjing, China). In the experiments, anti-gcp35a pAb and anti-gcp35b pAb were custom products from Abmart Inc. (Shanghai, China). To validate anti-gcp35a pAb specificity, the recombinant gcp35a and grass carp HKLs lysates were analyzed by WB in which the membrane was incubated with anti-gcp35a pAb (1:1000) or the antibody pre-absorbed with rgcp35a (Figures S6A, B). Following the same procedures, the lysates of grass carp HKLs treated with or without heat-killed Aeromonas hydrophila (A. hydrophila) [MOI 1:1, which has been described previously (29)] for 6 h were used to verify the specificity of the gcp35b antibody (Figures S6C, D). Additionally, the specificity of anti-gcp40b pAb has been demonstrated and used in previous research (30).

Grass carp HKLs were prepared by discontinuous density gradient centrifugation with Ficoll-Hypaque (1.083 kg/L, TBD science, Tianjin, China) referring to our previous studies (23). Briefly, head kidney was obtained from freshly sacrificed fish and then squeezed to release the cells. After the tissue debris was removed, the cells were layered on Ficoll-Hypaque and centrifuged at 1580 × g for 30 min at 20°C. After centrifugation, the leukocytes at the interface were collected and washed twice with PBS. The cells were resuspended in RPMI-1640 medium (Gibco) with 10% FBS (Gibco) and 1% antibiotic-antimycotic (Thermo Scientific). About 6 × 10⁵ cells/well were seeded in a 24-well plate (Nunc-Intermed, Roskilde, Denmark) and incubation overnight at 26°C under 5% CO₂ and saturated humidity. For lymphocytes and monocytes/macrophages isolation, the cell suspension was centrifuged at 400 × g for 25 min in a density gradient column formed by two solutions with different densities from fish lymphocyte and monocyte preparation kit (TBD, Tianjin, China) according to the previous study (17). About 6 × 10⁵ cells/well lymphocytes were seeded in a 24-well plate and incubated overnight at 26°C under 5% CO₂ and saturated humidity. The collected monocytes/macrophages were resuspended in RPMI-1640 medium (Gibco) with 1% FBS (Gibco) and 1% antibiotic-antimycotic (Thermo Scientific) and seeded in a 24-well plate with 5 × 10⁶ cells/well. After 2 hours of incubation, the unattached cells on the plate were washed away with PBS. Then the cells were cultured in RPMI-1640 medium (Gibco) with 10% FBS (Gibco) and 1% antibiotic-antimycotic (Thermo Scientific). In the following day, the cells were treated with different drugs in individual experiments.

Total RNA was extracted from the cells and then reverse transcribed to cDNA by using M-MLV reverse transcriptase (Promega). The gcp35a, gcp35b, ifn-γ, il-17a/f1, il-22 and β-actin mRNA levels were assessed by using RT-qPCR. In brief, RT-qPCR was performed on the Bio-Rad CFX96™. Real-time detection system (Bio-Rad, Hercules, CA) in a total volume of 10 µL with 4 µL of 2.5 × RealMasterMix (Tiangen, Beijing, China), 0.5 µL of 20 × SYBR Green, 1 µL of cDNA, 0.2 µL of each of forward primer and reverse primer and 4.1 µL of deionized water. The amplification program was 94°C for 2 min, followed by 35 cycles of 94°C for 20 s, 54-60°C (54°C for p35a, 60°C for p35b, 60°C for ifn-γ, 60°C for il-17a/f1, 60°C for il-22 and 59°C for β-actin) for 20 s and 65°C for 20 s. All PCR products were visualized on a 2% agarose gel to check the PCR amplification. To estimate the amplification efficiency, the standard curve for each target molecule was generated by 10-fold serial dilutions (from 10^-1 to 10^-6 fmol/µL) of a plasmid containing the individual target gene sequences as the PCR template. The melting analysis was routinely performed to check the authenticity of the PCR products (31). The relative expression levels of target genes were analyzed using the 2^-ΔΔCt method (32) by normalization with β-actin gene expression and presented as fold changes compared with the matched controls. The primers for the RT-qPCR are listed in Supplementary Table 1.

The grass carp HKLs culture medium was collected to measure the concentration of grass carp Ifn-γ by competitive-inhibition ELISA. In the assay, anti-gcIfn-γ pAb was a custom product from Abmart (Shanghai, China). To validate anti-gcIfn-γ pAb specificity, the recombinant gcIfn-γ (rgcIfn-γ) was used and analyzed by SDS-PAGE (Figure S10A) and WB in which the membrane was incubated with anti-gcIfn-γ pAb (1:1000) (Figure S10B) or the antibody pre-absorbed with rgcIfn-γ (Figure S10C). To establish a competitive-inhibition ELISA, an orthogonal experiment was designed by setting different coating concentrations of gcIfn-γ, and the dilution ratios of primary antibody and secondary antibody. The optimized amount of coating antigen was 100 ng/well of rgcIfn-γ and concentrations of the antibodies were 1:1000 (v/v) of anti-gcIfn-γ pAb (1:1000) and HRP-conjugated goat anti-rabbit secondary antibody. Subsequently, a standard curve was built according to the inhibition ratio and the corresponding protein concentrations (Figure S11).

In this experiment, a 96-well polystyrene plate (Costar, Cambridge, MA, USA) was coated with 100 ng/well of rgcIfn-γ at 4°C for 16 h and then blocked with 5% defatted milk plus 0.3% BSA in PBS for 3 h at room temperature. At the same time, 50 µL of culture medium or the titrated rgcIfn-γ and 50 µL of anti-gcIfn-γ pAb (1:1000) (Abmart) were mixed and incubated at room temperature for 2 h. After that, the plate was washed with PBST (0.05% Tween-20 in PBS) for three times and 100 µL of the medium-antibody mixture was added into each well and further incubated at room temperature for 2 h. The plate was washed with PBST for five times, and then 100 µL of HRP-conjugated goat anti-rabbit secondary antibody (1:1000, ZSGB-BIO) was added into each well. After 2 h incubation at room temperature, the plate was washed with PBST for five times and 100 µL of substrate buffer (TMD, Tiangen) was added into the wells and incubated for 20 min at 37°C. The reaction was stopped by 2 M H₂SO₄ and the absorbance values were measured at 450 nm with the iMark Microplate Reader (Bio-Rad). Control groups were pre-coated with BSA followed by the same procedures as described above. The concentrations of samples were extrapolated from a standard curve for gcIfn-γ inhibition.

To examine if the rgcIl-12AB and rgcIl-12BB expressed by CHO cells were glycosylated, PNGase F (NEB), a glycosidase, was used to digest rgcIl-12AB and rgcIl-12BB following the manufacturer’s instructions. According to the method in a previous study (33), 10 µg of rgcIl-12AB or rgcIl-12BB was denatured in 1 × Glycoprotein Denaturing buffer (0.5% SDS, 40 mM DTT) for 10 min in boiling water, and added to 10 × G2 Reaction buffer (500 mM PBS, pH 7.5) containing 2 µL of 10% NP-40 and 2 µL of PNGase F. The mixture was incubated at 37°C for 2 h and the samples were assayed by SDS-PAGE and WB.

Data were collected from at least three independent experiments and all results were expressed as mean ± SEM with four independent replicates (N = 4). GraphPad Prism 7 software (GraphPad Inc., San Diego, CA, USA) was used to test the normality and homogeneity of variance of all data according to the instructions of GraphPad Prism (https://www.graphpad-prism.cn/guides/prism/8/statistics/index.htm), and then perform statistical analyses. For comparison between two groups, Student’s t-test was performed. Multiple group comparison was conducted by one-way ANOVA followed by a Tukey’s multiple comparisons test. Significant differences and highly significant differences were considered at p < 0.05 and p < 0.01, respectively.

Although two or three p35 paralogues were found in a variety of teleosts, only one p35 gene has been reported so far in grass carp (nominated as gcp35a in the present study). In this study, another grass carp p35 cDNA (named gcp35b) was isolated, which contains 582 bp nucleotides encoding a 193-aa polypeptide (Figure S1). Moreover, gcp35b loci exhibited a conserved synteny with its homologues in other teleost species like fugu, Atlantic salmon and zebrafish, and both gcp35 paralogues were in proximity with the conserved gene schip1 (Figure 1A). Subsequent phylogenetic analysis of p35 showed three sub-clades: teleost p35a, p35b branches, and the branch consisted of frog, chicken, mouse and human p35 genes (Figure 1B). The multiple amino acid sequences alignment of gcp35b with its homologues showed that it had the highest identity with zebrafish p35b (50.93%) and shared low identities with other homologues in teleosts (fugu p35a 17.47%, fugu p35b 19.70%, zebrafish p35a 18.22% and grass carp p35a 20.07%). In addition, gcp35b also shared lower identities (14.87%, 12.64%, 14.50% and 13.01%) with the human, mouse, chicken and frog p35 homologues (Figure S2A). Furthermore, the cysteine residues (C²⁸, C⁶⁰, C⁷⁷, C⁸⁷, C¹⁰⁰, C¹³⁸ and C¹⁶⁸ of gcp35a, C⁴¹, C⁶⁷, C⁸⁴, C⁹¹, C¹⁰⁴, C¹⁴² and C¹⁷⁰ of gcp35b) were conserved with p35 homologues in other vertebrate species (Figure S2A). The cysteine residues (C⁸⁷ of gcp35a and C⁹¹ of gcp35b) that form the inter-chain disulfide bond were marked with a star (Figure S2A). The glycosylation sites of gcp35a/b were highlighted in Figure S3.

In grass carp monocytes/macrophages, poly I:C (50 µg/mL) significantly up-regulated the mRNA expression of gcp35a but not gcp35b, while rgcIfn-γ (500 ng/mL) induced the transcription of gcp35b but not gcp35a (Figure 2A). Meanwhile, LPS (30 µg/mL) did not affect the transcript levels of two gcp35 paralogues (Figure 2A). The time course experiments showed that a 12-h treatment with poly I:C and rgcIfn-γ was sufficient to induce a marked expression of gcp35a and gcp35b (Figures S4A, B), and this time point was chosen for later studies. Subsequently, the signaling mechanisms responsible for the regulation of poly I:C and rgcIfn-γ on gcp35a and gcp35b transcription were elucidated, respectively. The poly I:C-induced mRNA expression of gcp35a was partially attenuated by NF-κB, JNK and ERK inhibitors (Figure 2B). Meanwhile, the stimulation of rgcIfn-γ on the gcp35b mRNA expression was totally blocked by NF-κB inhibitor and partially suppressed by p38 inhibitor (Figure 2C). Moreover, poly I:C was effective in stimulating the phosphorylation of Jnk, Erk and IκBα from 30 to 120 min (Figure 2D). Meanwhile, rgcIfn-γ significantly induced the phosphorylation of p38 at 10 min and showed a slight stimulation of IκBα phosphorylation at 30 min (Figure 2E). Referring to the information available at Cell Signaling Technology’s website (https://www.cellsignal.com/), the molecular sizes of p-JNK, p-ERK1/2, p-IκBα and p-p38 in grass carp were similar to that in mammals (Figure S5A).

To learn the structural information of theoretical gcIl-12 heterodimers, their 3D models were constructed based on the human Il-12 crystal structure (PDB: 1F45), thereby revealing the location of those conserved cysteine residues on the contact surface of the predicted heterodimers consisting of gcp35 and gcp40 paralogues (Figure 3). The QMEANDisCo Global scores of all gcIl-12 heterodimer 3D models were higher than 0.5 (Supplementary Table 2). As shown in Figure 3, the conserved cysteine residues on the contact surfaces of the predicted heterodimers included gcp35a (C⁸⁷) and gcp35b (C⁹¹) marked by black arrows, as well as C¹⁸⁴ and C³⁰⁰ of gcp40a and the C¹⁷⁶ and C²³⁷ of gcp40b indicated by red arrows. However, no cysteine residue was found on the contact surface of gcp40c.

To clarify the composition of the gcIl-12 heterodimer, different combinations of gcp35a/b-FLAG and gcp40a/b/c-HIS were overexpressed in HEK293 cells, and then the cell culture media were collected and analyzed under non-reducing conditions. As shown in Figure 4, anti-FLAG antibody and anti-HIS antibody could detect the monomeric gcp35a/b (35 kDa and 25 kDa in Figure 4A) and gcp40a/b/c (55 kDa, 50 kDa and 43 kDa in Figure 4B) showing weak and clear bands, respectively. Notably, anti-FLAG antibody recognized an obvious band with the MW of 75 kDa in overexpressing gcp35a/gcp40b and gcp35b/gcp40b lanes (indicated by “heterodimer”, Figure 4A). Consistently, this band was also detected by anti-HIS antibody in the same lanes (indicated by “heterodimer”, Figure 4B). In addition, homodimeric gcp35b (50 kDa) was detected by anti-FLAG antibody (right panel, Figure 4A). Similarly, homodimeric gcp40a (more than 100 kDa) was detected by anti-HIS antibody (Figure 4B).

To explore the biological activity of two main gcIl-12 isoforms as described above, the recombinant gcIl-12AB (rgcIl-12AB) and gcIl-12BB (rgcIl-12BB) were prepared by the CHO cell expression system and purified by His Trap affinity column, and their purity was evaluated by SDS-PAGE and WB. SDS-PAGE analysis showed that the purified rgcIl-12AB (Figure S7A) and rgcIl-12BB (Figure S7D) were visualized as a single band about 75 kDa and 70 kDa, respectively, and they were larger than their predicted MW (rgcIl-12AB with 58 kDa and rgcIl-12BB with 57 kDa). Furthermore, the single band of purified rgcIl-12AB was verified by WB analysis with anti-gcp35a and anti-gcp40b pAb (Figures S7B, C), while a single band for rgcIl-12BB was recognized by both anti-gcp35b and anti-gcp40b pAb (Figures S7E, F). The glycosylation of rgcIl-12AB and rgcIl-12BB was assessed by glycosidase digestion. SDS-PAGE analysis showed the MWs of rgcIl-12AB and rgcIl-12BB digested with glycosidase were corresponding to their predicted sizes, respectively (Figures S8A, B) and these results were further confirmed by WB assay (Figures S8C, D).

To investigate the biological activity of two rgcIl-12 isoforms, grass carp HKLs were incubated with different concentrations of rgcIL-12AB or rgcIl-12BB for 12 h. Results showed that rgcIl-12AB (Figure 5A) and rgcIl-12BB (Figure 5B) could induce ifn-γ mRNA expression from 30 to 1000 ng/mL. In parallel, a time course experiment showed that a 12-h treatment of rgcIl-12BB (1000 ng/mL) was sufficient to induce the maximal effect on ifn-γ expression in HKLs (Figure S9). Furthermore, the competitive-inhibition ELISA results showed that rgcIl-12AB (100-1000 ng/mL) and rgcIl-12BB (100-1000 ng/mL) markedly stimulated the release of Ifn-γ (Figures 5C, D). Similarly, in lymphocytes and monocytes/macrophages, a 12-h treatment with rgcIl-12AB or rgcIl-12BB (30-1000 ng/mL) resulted in the increase of ifn-γ transcription (Figures 6A–D).

In lymphocytes, both rgcIl-12AB and rgcIl-12BB were effective in enhancing the Th17 related cytokines il-17a/f1 and il-22 mRNA expression from 30 to 1000 ng/mL (Figures 7A–D). At the same time, in monocytes/macrophages, the different concentrations (30-1000 ng/mL) of rgcIl-12AB and rgcIl-12BB had no effect on the expression of il-17a/f1 and il-22 (Figures S12A–D).

To elucidate the signaling mechanisms in the modulation of rgcIl-12 isoforms on il-17a/f1 transcription, the inhibitors for Stat3 and Rorγt signaling were used in the present study. Results showed that the stimulatory effects of rgcIl-12AB and rgcIl-12BB (1000 ng/mL) on the il-17a/f1 mRNA expression were impeded by STAT3 VI (30 μM, STAT3 inhibitor) (Figures 8A, D) and SR1001 (15 μM, RORγt inhibitor) (Figures 8B, E). In addition, rgcIl-12AB (1000 ng/mL) was able to induce the phosphorylation of Stat3 from 5 to 20 min (Figure 8C) and rgcIl-12BB (1000 ng/mL) was able to induce the phosphorylation of Stat3 from 5 to 60 min (Figure 8F).

To assess whether the regulatory effects of gcIl-12 isoforms were modified in lymphocytes, the involvement of gcTgf-β1 in mediating these effects was examined. RgcTgf-β1 (100 ng/mL) significantly inhibited rgcIl-12AB-stimulated il-17a/f1 and ifn-γ gene expression (Figures 9A, B) and rgcTgf-β1 also significantly inhibited rgcIl-12BB-elevated il-17a/f1 and ifn-γ gene expression (Figures 9C, D). Immunoneutralization of gcTgf-β1 secreted from the cells by using an anti-gcTgf-β1 mAb (1:2000 diluted) could enhance il-17a/f1 mRNA expression alone or in combination with rgcIl-12BB (Figure 9E), but immunoneutralization of gcTgf-β1 had no effect on ifn-γ mRNA expression (Figure 9F). Moreover, ALK5 inhibitor (2 µM) was able to up-regulate il-17a/f1 but not ifn-γ mRNA expression alone, and further stimulated rgcIl-12BB-induced il-17a/f1 and ifn-γ transcription (Figures 9G, H). Additionally, rgcTgf-β1 (100 ng/mL) was effective in reducing rgcIl-12BB-triggered phosphorylation of Stat3 in the cells (Figure 9I).