Stimulus-Specific Expression, Selective Generation and Novel Function of Grass Carp (Ctenopharyngodon idella) IL-12 Isoforms: New Insights Into the Heterodimeric Cytokines in Teleosts

Abstract

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of a p35 subunit specific to IL-12 and a p40 subunit shared with IL-23. In this study, we unveiled the existence of two p35 paralogues in grass carp (named gcp35a and gcp35b). Notably, gcp35a and gcp35b displayed distinct inducible expression patterns, as poly I:C merely induced the gene expression of gcp35a but not gcp35b, while recombinant grass carp interferon-gamma (rgcIfn-γ) only enhanced the transcription of gcp35b but not gcp35a. Moreover, the signaling mechanisms responsible for the inducible expression of gcp35a and gcp35b mRNA were elucidated. Because of the existence of three grass carp p40 genes (gcp40a, gcp40b and gcp40c) and two p35 paralogues, six gcIl-12 isoforms were predicted by 3D modeling. Results showed that gcp40a and gcp40b but not gcp40c had the potential for forming heterodimers with both gcp35 paralogues via the disulfide bonds. Non-reducing electrophoresis experiments further disclosed that only gcp40b but not gcp40a or gcp40c could form heterodimers with gcp35 to produce secretory heterodimeric gcp35a/gcp40b (gcIl-12AB) and gcp35b/gcp40b (gcIl-12BB), which prompted us to prepare their recombinant proteins. These two recombinant proteins exhibited their extensive regulation on Ifn-γ production in various immune cells. Intriguingly, both gcIl-12 isoforms significantly enhanced the transcription of il-17a/f1 and il-22 in lymphocytes, and their regulation on il-17a/f1 expression was mediated by Stat3/Rorγt signaling, supporting the potential of gcIl-12 isoforms for inducing Th17-like responses. Additionally, stimulatory effects of gcIl-12 isoforms on il-17a/f1 and ifn-γ expression were attenuated by gcTgf-β1 via suppressing the activation of Stat3 signaling, implying that their signaling could be manipulated. In brief, our works provide new insights into the inducible expression pattern, heterodimeric generation and functional novelty of Il-12 isoforms in teleosts.

Keywords: Il-12; Th17-like response; grass carp; heterodimeric form; p35 paralogues.

Figure 1

Gene synteny and phylogenetic tree analysis of p35 homologues. (A) Gene synteny analysis of grass carp p35a, grass carp p35b, fugu p35b, zebrafish p35b, and Atlantic salmon p35b. The direction of the box arrow indicated gene transcription orientation. (B) An unrooted phylogenetic analysis of p35 amino acid sequences in various vertebrates. The tree was constructed by the neighbor joining method by using MEGA 7 software. The numbers indicate the bootstrap confidence values obtained for each node after 1000 replications. The GenBank accession numbers of p35 are as follows: human (NM_000882), mouse (NM_008351), chicken (NM_213588), frog (XM_012963291), fugu p35a (H2SI185), fugu p35b (NM_001078598), Atlantic salmon p35a1 (HG917954.1), Atlantic salmon p35a2 (HG917955.1), Atlantic salmon p35b (HG917956.1), zebrafish p35a (NM_001007107), zebrafish p35b (XM_017352586), grass carp p35a (KF944667) and grass carp p35b (MZ393470).

Figure 2

Inductive expression of gcp35a and gcp35b transcripts after different immune challenges in grass carp monocytes/macrophages. (A) The monocytes/macrophages were treated with poly I:C (50 µg/mL), LPS (30 µg/mL) and rgcIfn-γ (500 ng/mL) for 6 h. ***p < 0.001 (vs. p35a ctrl), ^###p < 0.001 (vs. p35b ctrl). Data are shown as mean ± SEM (N = 4). (B, C) The inhibitors for p38 (SB202190, 30 µM), JNK (SP600125, 6 µM), ERK (PD98059, 30 µM) and NF-κB (PDTC, 0.25 µM) were added with poly I:C (50 g/mL), or rgcIfn-γ (500 ng/mL) for 12 h, separately. The gcp35a and gcp35b mRNA was normalized relative to β-actin and expressed as fold changes compared with the control group. Different letters indicate significant differences at p < 0.05. Data are shown as mean ± SEM (N = 4). Activation of Jnk, Erk1/2 and IκBα by poly I:C (50 µg/mL) (D) and IκBα and p38 by rgcIfn-γ (500 ng/mL) (E) for 0, 10, 30, 60, 90 and 120 min in grass carp monocytes/macrophages.

Figure 3

3D structural models of grass carp Il-12 isoforms. The structural models of grass carp Il-12 isoforms were constructed based on the human IL-12 crystal structure (PDB: 1F45). The exposed cysteines at the contact surface of gcp35 paralogues are marked with black arrows and the red arrows indicated the cysteines of gcp40 paralogues.

Figure 4

Identification of the grass carp Il-12 heterodimer composition. HEK293 cells were transfected with 1.2 µg of plasmids encoding the individual subunits alone or with a mixture of 1.2 µg each of the two plasmids as indicated in picture. The cell culture media samples were separated under non-reducing conditions (without β-ME). The molecular mass standards (kDa) are indicated. The lane of ctrl means that HEK293 cells were transfected with a mixture of 1.2 µg each of the two vectors [p3×FLAG-CMV-7.1 and pcDNA 3.1/myc-His (–)]. (A) Non-reducing WB assay by using anti-FLAG antibody. (B) Non-reducing WB assay by using anti-HIS antibody.

Figure 5

Effects of rgcIl-12 isoforms on Ifn-γ mRNA expression and protein release in grass carp HKLs. HKLs were treated with rgcIl-12AB or rgcIl-12BB (30-1000 ng/mL) for 12 h (A, B). The mRNA levels of ifn-γ mRNA were detected by qPCR. The mRNA levels were normalized by β-actin and expressed as fold changes compared with the control group. The protein levels of gcIfn-γ in HKLs culture medium were detected by competitive-inhibition ELISA after the HKLs were treated with rgcIl-12AB or rgcIl-12BB (30-1000 ng/mL) for 12 h (C, D). Data are shown as mean ± SEM (N = 4). *p < 0.05 and ***p < 0.001.

Figure 6

Effects of rgcIl-12 isoforms on ifn-γ mRNA expression in grass carp lymphocytes and monocytes/macrophages. Lymphocytes and monocytes/macrophages were treated with 30-1000 ng/mL rgcIl-12AB (A, B) or rgcIl-12BB (C, D) for 12 h, respectively. The mRNA expression of ifn-γ was detected by qPCR and they were normalized by β-actin and expressed as fold changes compared with the control group. Data are shown as mean ± SEM (N = 4). *p < 0.05, **p < 0.01, and ***p < 0.001. The “n.s.” indicates no significant.

Figure 7

Effects of rgcIl-12 isoforms on Th17-related cytokines mRNA expression in grass carp lymphocytes. Lymphocytes were treated with 30-1000 ng/mL rgcIl-12AB (A, B) or rgcIl-12BB (C, D) for 12 h. The mRNA expression of il-17a/f1 and il-22 mRNA were detected by qPCR and normalized by β-actin and expressed as fold changes compared with the control group. Data are shown as mean ± SEM (N = 4). *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 8

Signaling pathways of rgcIl-12 isoforms in regulating il-17a/f1 in lymphocytes. (A) Lymphocytes were incubated with rgcIl-12AB (1000 ng/mL) in the presence or absence of STAT3 VI (STAT3 inhibitor, 30 µM). (B) Lymphocytes were incubated with RgcIl-12AB (1000 ng/mL) in the presence or absence of SR1001 (RORγt inhibitor, 15 µM). The il-17a/f1 mRNA was normalized by β-actin and expressed as fold changes compared with the control group. (C) Phosphorylation of Stat3 was detected in the lymphocytes treated with rgcIl-12AB (1000 ng/mL) for 0-60 min. (D) Lymphocytes were incubated with rgcIl-12BB (1000 ng/mL) in the presence or absence of STAT3 VI (STAT3 inhibitor, 30 µM). (E) Lymphocytes were incubated with rgcIl-12BB (1000 ng/mL) in the presence or absence of SR1001 (RORγt inhibitor, 15 µM). The il-17a/f1 mRNA was normalized by β-actin and expressed as fold changes compared with the control group. Data are shown as mean ± SEM (N = 4). Different letters indicate significant differences at p < 0.05. (F) Phosphorylation of Stat3 was detected in lymphocytes treated with rgcIl-12BB (1000 ng/mL) for 0-60 min.

Figure 9

The effects of gcTgf-β1 on rgcIl-12AB- and rgcIl-12BB-induced il-17a/f1 and ifn-γ mRNA expression. Lymphocytes were treated with 1000 ng/mL of rgcIl-12AB (A, B) and rgcIl-12BB (C, D) in the presence or absence of rgcTgf-β1 (100 ng/mL) for 12 h. (E, F) Lymphocytes were exposed to rgcIl-12BB (1000 ng/mL) in the presence or absence of gcTgf-β1 mAb (1:1000 diluted) for 12 h. (G, H) Lymphocytes were incubated with rgcIl-12BB (1000 ng/mL) in the presence or absence of ALK5 inhibitor (TGF-β1 RI Kinase inhibitor VIII, 2 µM) for 12 h. Then the levels of il-17a/f1 and ifn-γ mRNA were detected by qPCR. Relative mRNA levels were analyzed using β-actin as an internal reference and expressed as the fold changes of the control group (blank column). Data are shown as mean ± SEM (N = 4). Different letters indicate significant differences at p < 0.05. (I) Phosphorylation of Stat3 was detected in lymphocytes incubated with rgcIl-12BB (1000 ng/mL) in the presence or absence of rgcTgf-β1 (100 ng/mL) for 20 min.