The IL-12 family cytokines in fish: Molecular structure, expression profile and function.

Interleukin (IL)-12 family cytokines including IL-12, IL-23, IL-27, IL-35 and IL-39 are heterodimeric cytokines composed of two subunits, an α-chain (entitled p35, p19 and p28) and a ß-chain (namely p40 and Epstein-Barr virus-induced gene 3, EBI3). Unlike in mammals, specific whole genome duplication events in fish may generate more paralogues of these subunits as the components of IL-12 family cytokines. Although all subunit genes of IL-12 family have been isolated and identified in various fish species, some important issues on fish IL-12 family are needed to be addressed compared to the extensive study in mammals: Whether the expansion of these subunit genes results in the generation of multiple isoforms of the family cytokines; Whether the related receptor genes have similar complex repertoire corresponding to their ligands; How about the expression kinetics of these subunit paralogues particularly under the circumstance of pathogen infection and immune stimulation; How about the functional properties of IL-12 family in fish. In the past ten years, these concerns have received increasing attentions to establish the biological significance of this family cytokines in fish immunity. In this review, we summarized the current understanding of IL-12 family with a special focus on the molecular structures, inducible expression profiles and functions of IL-12 family members in fish.

Expression analysis of grass carp Foxp3 and its biologic effects on CXCL-8 transcription in non-lymphoid cells.

Teleost Forkhead box protein P3 (Foxp3) expression was discovered not only in regulatory T cells (Tregs) but also in other cells. Compared to the extensive study on its roles in lymphoid cells, the expression pattern and biological roles of Foxp3 in non-lymphoid cells have not been elucidated in both mammals and fish species. In the present study, grass carp Foxp3 (gcFoxp3) mRNA expression was detected in different cell types, showing that it has a moderate expression level in peripheral blood leukocytes (PBLs), head kidney leukocytes (HKLs) and grass carp fibroblast-like kidney cells (CIK cells). Interestingly, gcFoxp3 mRNA and protein expression could be significantly stimulated by polyinosinic-polycytidylic acid (poly I:C) in CIK cells, indicating its participation in poly I:C-induced immune response in non-lymphoid cells. To further investigate the function of gcFoxp3, its overexpression plasmid was constructed and transfected into CIK cells. After 24 h of transfection, grass carp C-X-C chemokine ligand (CXCL) 8 (gcCXCL-8) mRNA expression was elevated, implying the modulatory role of gcFoxp3 in gcCXCL-8 mRNA expression. This notion was further supported by the features of gcCXCL-8 promoter which contained a putative Foxp3 binding site at -2196 to -2190 region. Poly I:C or overexpression of gcFoxp3 obviously stimulated gcCXCL-8 promoter activity and deletion of gcFoxp3 binding region on the promoter abolished this stimulation, revealing that Foxp3 is pivotal for transcription of CXCL-8 induced by poly I:C. In conclusion, our results collectively demonstrate expression pattern of teleost Foxp3, and illuminate novel immune function of fish Foxp3 in regulating chemokine transcription in non-lymphoid cells.

Regulation of Il-2 on the expression of granzyme B- and perforin-like genes and its functional implication in grass carp peripheral blood neutrophils.

Granzyme (Gzm) B and perforin, both as cytotoxic proteins, can collaborate to induce the death of target cells as well as the microbes. They were originally discovered in cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells and confer the cytotoxic activities of these cells. In the present study, the coding sequences of a granzyme b-like (gcgzmbl) and a perforin-like (gcprfl) genes were cloned from grass carp (Ctenopharyngodon idellus) and their specific antibodies were subsequently prepared and validated. The mRNA and protein expression of these two cytotoxic proteins in grass carp peripheral blood neutrophils was demonstrated by quantitative PCR (qPCR) and immunofluorescence staining, respectively. In the same cell model, expression of gcGzmbl and gcPrfl was stimulated by grass carp interleukin (Il)-2 in a dose- and time-dependent manners and Erk, NF-κB and Stat5 pathways were found to be involved in the regulation of Il-2 on the genes' expression. Additionally, glycolysis was proved to play a role in the stimulation of Il-2 on gcGzmbl and gcPrfl expression in peripheral blood neutrophils. As combating the invading microorganisms is one of the main functions of neutrophils, the roles of gcGzmbl and gcPrfl in the anti-bacterial activities of grass carp peripheral blood neutrophils were explored. Results showed that immunoneutralization of gcGzmbl or gcPrfl significantly attenuated the antimicrobial abilities of the neutrophils enhanced by Il-2. These findings shed a light on the expression, regulation and functions of granzyme B- and perforin-like proteins in fish peripheral blood neutrophils and enrich the understanding of Il-2 function in fish innate immunity.

Identification and functional characterization of interleukin-12 receptor beta 1 and 2 in grass carp (Ctenopharyngodon idella).

Interleukin 12 (IL-12) binds its receptor complex of IL-12 receptor beta 1 (IL-12Rß1) and IL-12Rß2 to transduce cellular signaling in mammals. In teleosts, the function of Il-12 is drawing increasing attention, but molecular and functional features of Il-12 receptors remain obscure. Especially, the existence of multiple Il-12 isoforms in some fish species elicits the requirement to clarify their receptors. In this study, we isolated three cDNA sequences as Il-12 receptor candidates from grass carp, entitled as grass carp Il-12rß1 (gcIl-12rß1), gcIl-12rß2a and gcIl-12rß2b. In silico analysis showed that gcIl-12rß1 and gcIl-12rß2a shared the conserved gene locus and similar structure characteristics with their orthologues of zebrafish, frog, chicken, mouse and human, respectively. However, the Il-12rß2b of grass carp and zebrafish was similar to IL-27Ra in non-fish species. Further locally installed BLAST and gene synteny analysis uncovered three gcIl-12 receptors being single copied genes. Tissue distribution assay revealed that gcil12rß1 and gcil12rß2a transcripts were predominantly expressed in head kidney, differing from the even distribution of gcil12rß2b transcripts in all detected tissues. Subsequently, the binding ability and antagonistic effects of recombinant extracellular region of gcIl-12rß1 with recombinant grass carp Il-12 (rgcIl-12) isoforms were explored, providing functional evidence of the newly cloned gcIl-12rß1 being genuine orthologues of mammalian IL-12Rß1. Moreover, our data showed that gcIl-12rß1 and gcIl-12rß2a but not gcIl-12rß1 and gcIl-12rß2b mediated the effects of rgcIl-12 isoforms on ifn-γ promoter activity, thereby revealing Il-12 receptor signaling in fish. These results identified grass carp Il-12 receptors, thereby advancing our understanding of Il-12 isoform signaling in fish.

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

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-ß1 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.

IFN-γ Manipulates NOD1-Mediated Interaction of Autophagy and Edwardsiella piscicida to Augment Intracellular Clearance in Fish.

Edwardsiella piscicida is an intracellular pathogenic bacterium accounting for significant losses in farmed fish. Currently, cellular and molecular mechanisms underlying E. piscicida-host cross-talk remain obscure. In this study, we revealed that E. piscicida could increase microtubule-associated protein L chain 3 (LC3) puncta in grass carp (Ctenopharyngodon idella) monocytes/macrophages and a carp cell line, Epithelioma papulosum cyprini The autophagic response was confirmed by detecting the colocalization of E. piscicida with LC3-positive autophagosomes and LysoTracker-probed lysosomes in the cells. Moreover, we unveiled the autophagic machinery targeting E. piscicida by which the nucleotide-binding oligomerization domain receptor 1 (NOD1) functioned as an intracellular sensor to interact and recruit autophagy-related gene (ATG) 16L1 to the bacteria. Meanwhile, E. piscicida decreased the mRNA and protein levels of NOD1 and ATG16L1 in an estrogen-related receptor-α-dependent manner, suggesting a possible mechanism for this bacterium escaping autophagy. Subsequently, we examined the effects of various E. piscicida virulence factors on NOD1 expression and found that two of them, EVPC and ESCB, could reduce NOD1 protein expression via ubiquitin-dependent proteasomal degradation. Furthermore, an intrinsic regulator IFN-γ was found to enhance the colocalization of E. piscicida with NOD1 or autophagosomes, suggesting its involvement in the interaction between autophagy and E. piscicida Along this line, a short-time treatment of IFN-γ caused intracellular E. piscicida clearance through an autophagy-dependent mechanism. Collectively, our works demonstrated NOD1-mediated autophagy-E. piscicida dialogues and uncovered the molecular mechanism involving autophagy against intracellular bacteria in fish.

Functional characterization of grass carp (Ctenopharyngodon idella) interleukin-2 in head kidney leukocytes.

Interleukin (IL)-2 belongs to the four-helix bundle cytokine family and plays key roles in growth, survival, activation-induced cell death and differentiation of the immune cells. In cyprinid fish, only common carp interleukin-2 (il2) has been cloned because of relatively low sequence homology between carp Il-2 and its homologs in other fish species. In the present study, the coding sequence of grass carp Il-2 (gcIl-2) was cloned and its identity was verified via bioinformatic analysis. Tissue distribution study showed that grass carp il2 (gcil2) mRNA was expressed in thymus, head kidney and gill with relatively high levels. Recombinant gcIl-2 (rgcIl-2) protein was subsequently prepared by using a prokaryotic expression system followed by a refolding method. The purified rgcIl-2 displayed an ability to stimulate the cell proliferation along with an increased mRNA expression of cd4l but not cd8a, igm or mcsfr in grass carp head kidney leukocytes (HKLs), suggesting the possible involvement of gcIl-2 in T helper (Th) cell proliferation. In the same cell model, rgcIl-2 significantly enhanced mRNA expression of some cytotoxic molecules including perforin-like protein 2, granzyme B-like and Fas ligand, indicating the modulation of cytotoxic cells by gcIl-2 in grass carp HKLs. Besides, gene expression of regulatory T (Treg) cell- and Th1/2 cell-related cytokines or transcription factors was detected in grass carp HKLs treated by rgcIl-2. Results showed that rgcIL2 treatment increased the mRNA expression of foxp3, cd25l, ifng2, il12p35, tbet, tnfa, il2, il4/13a, il4/13b and gata3l in HKLs, implying the regulatory roles of Il-2 in the expression of these immune genes and its possible involvement in differentiation of Treg and Th1/2 cells. These observations together with the related studies in other fishes suggest the existence of cytotoxic cells, Treg and Th1/2 subpopulations in fish species and the functional roles of Il-2 in these cells.

Novel functions of grass carp three p40 isoforms as modulators of Th17 signature cytokine expression in head kidney leukocytes.

Interleukin (IL)-12p40, a component of IL-12 and IL-23, can be secreted as monomer and homodimer in mammals. Our previous study has proved the existence of natural three p40 isoforms and their proinflammatory properties in grass carp. In the present study, we unexpectedly found that recombinant grass carp p40a/b/c (rgcp40a, rgcp40b and rgcp40c) were able to enhance the mRNA levels of grass carp il-17a/f1 (gcil-17a/f1) in a dose- and time-dependent manner in head kidney leukocytes (HKLs). In agreement with these findings, the enzyme-linked immunosorbent assay (ELISA) showed that rgcp40a, rgcp40b and rgcp40c markedly stimulated gcIl-17a/f1 secretion from the HKLs. Together with their stimulatory effects on grass carp gcil-22 and gcil-26 expression, our data suggested their potential to mediate Th17-like response in grass carp. To support this notion, we investigated the underlying mechanisms for the regulation of rgcp40 isoforms on gcil-17a/f1 expression, and found that three rgcp40 isoforms significantly induced the activation of Erk, Jnk and Stat3 pathways in a time-dependent oscillation in the same cell model. Moreover, three rgcp40 isoforms-induced gcil-17a/f1 mRNA expression was suppressed by the inhibition on Erk, Jnk and Stat3 pathways, suggesting the signaling pathways in the p40 isoforms-mediating il-17a/f1 transcription. These studies for the first time proved the involvement of three gcp40 isoforms in mediating Th17 signature cytokine expression in fish immune cells, therefore providing new insights into the roles of p40 in teleost immunity.

Production and functional insights into the potential regulation of three isoforms of grass carp p40 subunit in inflammation.

The p40 subunit is known as a component of Interleukin (IL)-12 and IL-23. In mammals, p40 can be secreted as a monomer or homodimer and acts independently to mediate cellular responses. Recently, three p40 paralogues were isolated and identified from grass carp and other fish species, but whether they exist independently as well as their functional consequences and significance remain unclear. In the present study, using grass carp as the model, we for the first time demonstrated the existence of natural fish p40a, p40b and p40c (gcp40a, gcp40b and gcp40c) mainly as a monomer in culture supernatant of head kidney leukocytes (HKLs). Particularly, their excessive secretion induced by various immune stimuli suggests possible involvement of free p40s in fish immune responses. To define their functions, recombinant grass carp p40a/b/c (rgcp40a, rgcp40b and rgcp40c) were prepared by Pichia pastoris expression system, and they possessed the activities to enhance the secretion of pro-inflammatory cytokines including Il-1ß and tumor necrosis factor-α (Tnf-α) in grass carp HKLs. These pro-inflammatory properties of p40 isoforms prompted us to investigate their roles during the inflammatory process. In line with this, in vivo study revealed the pathogenic effect of rgcp40a on intestinal inflammation, whereas gcp40a polyclonal antibodies remarkably ameliorated Aeromonas hydrophila-induced intestinal histopathological changes. Taken together, our results uncover the biological significance of free p40s in teleost, and provide new clue for targeting fish intestinal inflammation.

In vitro characterization of grass carp (Ctenopharyngodon idella) IL-26 in regulating inflammatory factors.

Interleukin 26 (IL-26) gene has been identified in human, amphibian and teleost but not in rodents. It is well accepted that IL-26 was a crucial member of IL-10 family which acts as a pro-inflammatory cytokine in human. However, the role of IL-26 in regulating inflammation in lower vertebrates including teleost has not been defined yet. In the present study, grass carp IL-26 (gcIL-26) coding sequence was isolated and identified. Its chromosomal synteny was also analyzed, showing that gcIL-26 gene is flanked by IL-22 and IFN-γ genes with the same transcriptional orientation as seen in human, amphibian and zebrafish. Given that zebrafish and grass carp IL-26 shared relatively low amino acid identities with human IL-26, the functional roles of fish IL-26 are indispensable to be elucidated. Accordingly, recombinant gcIL-26 (rgcIL-26) was prepared by using Pichia pastoris expression system, and it was found to be partially glycosylated. Using grass carp head kidney leucocytes as cell model, rgcIL-26 displayed the bioactivity to stimulate the mRNA expression of some pro-inflammatory cytokines including IL-8, IL-1ß and IL-6, while inhibit mRNA expression of an anti-inflammatory cytokine, IL-10. Moreover, rgcIL-26 also up-regulated inos expression and NO production in grass carp monocytes/macrophages, strengthening its pro-inflammatory properties in fish. Those results collectively demonstrated the functional role of IL-26 in regulating inflammatory response in fish.

Identification and functional characterization of tumor necrosis factor receptor 1 (TNFR1) of grass carp (Ctenopharyngodon idella).

Tumor necrosis factor-alpha (TNF-α) exerts its regulatory effects by binding one of two TNF receptors, TNF-α receptor 1 (TNFR1) or TNFR2. In this study, we isolated and identified the cDNA sequence of grass carp TNFR1 (gcTNFR1). Similar to its homologs in other fish species, the putative protein of gcTNFR1 possessed an extracellular region containing three TNF homology domains, a transmembrane region and a cytoplasmic region with a conserved death domain. Consistent with the widespread expression of mammalian TNFR1, gcTNFR1 transcripts ubiquitously expressed in spleen, thymus, liver, heart, gill, intestine, brain and head kidney with the highest expression levels in head kidney. To reveal its inductive expression patterns in inflammatory response, effect of in vivo bacterial infection on gcTNFR1 gene expression was examined, showing a rapid increase of gcTNFR1 expression in head kidney, gill, liver and intestine, which is consistent with the role of TNF-α as an early response gene during immune challenges. To define the functional role of gcTNFR1, recombinant extracellular region of gcTNFR1 (rgcTNFR1) was prepared and used to perform in vitro binding assay, demonstrating its ability to interact with recombinant grass carp TNF-α (rgcTNF-α). Furthermore, to characterize the function of gcTNFR1 in affecting rgcTNF-α actions, the effect of overexpressing gcTNFR1 on rgcTNF-α-induced grass carp IL-1ß (gcIL-1ß) promoter activity was determined in COS7 cells. Results showed that gcTNFR1 was involved in the regulation of rgcTNF-α on gcIL-1ß transcription. Consistently, rgcTNFR1 was effective in attenuating the effect of rgcTNF-α on IL-1ß mRNA expression in grass carp head kidney leukocytes, providing evidence for the involvement of TNFR1 in TNF-α signaling in grass carp. These data facilitate a better understanding of TNF-α receptor signaling in grass carp.