Linyong Du

Functional characterization of TNF-α in grass carp head kidney leukocytes: induction and involvement in the regulation of NF-κB signaling.

Tumor necrosis factor-alpha (TNF-α) is a potent regulatory cytokine, which serves as a key mediator of inflammation, immunity and apoptosis in mammals. Identification, expression and regulatory effects of TNF-α have been reported in various fish species, showing the structural and functional similarity or discrepancy between each other. In this study, TNF-α was identified from grass carp (Ctenopharyngodon idella) and the deduced grass carp TNF-α (gcTNF-α) protein possessed the TNF family signature motifs, a protease cleavage site, a transmembrane domain and two conserved cysteine residues. Further studies showed that gcTNF-α expression was induced with a rapid kinetics by immune challenge in vitro and in vivo. To characterize the function of gcTNF-α, recombinant gcTNF-α (rgcTNF-α) was prepared by using the Escherichia coli expression system. It was shown to enhance the mRNA expression of gcTNF-α and gcIL-1β in head kidney leukocytes (HKLs), confirming the biological activity of rgcTNF-α. In the same model, NF-κB inhibitor (PDTC) was able to attenuate rgcTNF-α-induced gcTNF-α mRNA expression, implying the involvement of NF-κB pathway in fish TNF-α action. This notion was reinforced by the finding that rgcTNF-α could induce the phosphorylation of IκBα in a time-dependent oscillation in HKLs, indicating a dynamical variation of NF-κB activity as seen in mammals. In addition, rgcTNF-α could up-regulate the expression of two TNF receptor-associated factors (TRAF), TRAF1 and TRAF2, in a time- and dose-dependent manner, suggesting that gcTNF-α may function as a regulator of fish NF-κB pathway. These results for the first time reveal the link of gcTNF-α to the NF-κB pathway and provide a better understanding of TNF-α signaling in teleost immunity.

Characterization of grass carp (Ctenopharyngodon idella) IL-17D: Molecular cloning, functional implication and signal transduction

Although the roles of IL-17 family members during inflammation have been extensively studied in mammals, their knowledge in lower vertebrates is limited. In particular, the biological activities of fish IL-17 and their functional roles are largely unknown. In this study, we cloned grass carp IL-17D (gcIL-17D) and found that its putative protein possessed the conserved features of IL-17 family members. Tissue distribution analysis showed that gcIL-17D was preferentially expressed in the mucosal tissues, including skin, gill and intestine. Subsequently, the involvement of gcIL-17D in inflammatory response was demonstrated by examining the expression profiles of gcIL-17D in head kidney and head kidney leukocytes following in vivo bacterial infection and in vitro LPS treatment, respectively. Furthermore, recombinant gcIL-17D (rgcIL-17D) was prepared in grass carp kidney cells and was able to promote the gene expression of some pro-inflammatory cytokines (IL-1β, TNF-α and CXCL-8) in grass carp primary head kidney cells, revealing gcIL-17D can function as a pro-inflammatory cytokine. Moreover, rgcIL-17D appeared to activate NF-κB signaling by modulating the phosphorylation of IκBα and up-regulated CXCL-8 mRNA expression possibly through NF-κB pathway. Our data shed new light on the functional role of teleost IL-17D in inflammatory response.

Molecular evidence for the involvement of RORα and RORγ in immune response in teleost.

In mammals, retinoid-related orphan receptors (ROR) consist of three members as RORα, RORβ and RORγ. It is well known that RORα plays a critical role in cerebellum development while RORγt directs T helper 17 (Th17) cell differentiation. So far, the knowledge on fish ROR family is limited as only zebrafish ROR family members have been characterized, showing that they play roles in embryonic and cerebellar development. In this study, we have cloned two paralogues for RORα (RORα1 and RORα2) and RORγ (RORγ1 and RORγ2) from grass carp (Ctenopharyngodon idellus). Phylogenetic analysis showed that grass carp RORα and RORγ were grouped in the clade of zebrafish RORα and RORγ, respectively. Real-time RT-PCR assay revealed that these four ROR transcripts exhibited similar expression patterns, in particular the high levels in pituitary, brain and some immune-related tissues, suggesting that all of them may play a role in endocrine and immune system of teleost. To explore the immune roles of grass carp RORα and RORγ, their expression was detected in periphery blood lymphocytes (PBLs) challenged by immune stimuli. Results showed that both RORα and RORγ mRNA levels were up-regulated by PHA but not LPS in PBLs, suggesting that their expression may be subject to different immune processes. In the same cell model, poly I:C stimulation induced RORγ1/2 but not RORα1/2 expression, pointing to the different roles of grass carp RORα and RORγ in immune response. Consistently, bacterial challenge significantly up-regulated the expression of these four ROR genes in spleen, headkidney and thymus. These results not only contribute to elucidate the roles of ROR in fish immunity but also facilitate to further clarify the existence of Th17-like cells in fish.

Identification and functional characterization of grass carp IL-17A/F1: An evaluation of the immunoregulatory role of teleost IL-17A/F1

In mammals, IL-17A and IL-17F are hallmark cytokines of Th17 cells which act significant roles in eradicating extracellular pathogens. IL-17A and IL-17F homologs nominated as IL-17A/F1-3 have been revealed in fish and their functions remain largely undefined. Here we identified and characterized grass carp IL-17A/F1 (gcIL-17A/F1) in fish immune system. In this regard, both tissue distribution and inductive expression of gcIL-17A/F1 indicated its possible involvement in immune response. Moreover, recombinant gcIL-17A/F1 (rgcIL-17A/F1) was prepared and displayed an ability to enhance pro-inflammatory cytokines (IL-1β, TNF-α and IL-6) mRNA expression in head kidney leukocytes. It is suggestive of that gcIL-17A/F1 may act as a proinflammatory cytokine in fish immunity. Besides, rgcIL-17A/F1 induced gene expression and protein release of grass carp chemokine CXCL-8 (gcCXCL-8) in head kidney cells (HKCs), probably via NF-κB, p38 and Erk1/2 pathways. In particular, culture medium from the HKCs treated by rgcIL-17A/F1 could stimulate peripheral blood leukocytes migration and immunoneutralization of endogenous gcCXCL-8 could partially attenuate this stimulation, suggesting that rgcIL-17A/F1 could recruit immune cells through producing gcCXCL-8 as mammalian IL-17 A and F. Taken together, we not only identified the pro-inflammatory role of gcIL-17A/F1 in host defense, but also provided the basis for clarifying Th17 cells in teleost.

Identification of grass carp IL-10 receptor subunits: Functional evidence for IL-10 signaling in teleost immunity

Although the functions of teleost IL-10 have been preliminarily determined, functional evidence for its receptor signaling is lacking. Particularly, the identity of fish IL-10 receptor 2 (IL-10R2) is ambiguous. Cytokine receptor family member b4 (CRFB4) and CRFB5 are likely the ortholog of mammalian IL-10R2. In this study, grass carp CRFB4 (gcCRFB4) and gcCRFB5 cDNAs were isolated and characterized. The relatively high expression levels of grass carp IL10 receptor 1 (gcIL-10R1), gcCRFB4 and gcCRFB5 in immune tissues and cells implied their importance in fish immunity. Accordingly, gcIL-10R1, gcCRFB4 and gcCRFB5 were overexpressed in a grass carp kidney cell line to identify the IL-10 receptor subunits upon grass carp IL-10 (gcIL-10) treatment. Results showed that gcIL-10R1 was essential for gcIL-10 stimulation on STAT3 activation and grass carp suppressor of cytokine signaling 3 (gcSOCS3) promoter activity, and also indicated that gcCRFB4 but not gcCRFB5 might be the ortholog of mammalian IL-10R2. Furthermore, mutation of a putative STAT3-binding element in gcSOCS3 promoter attenuated the stimulation of gcIL-10 on gcSOCS3 promoter activity, indicating that gcIL-10 may modulate gcSOCS3 transcription at least partly via STAT3 activation. This notion was further supported by our observation that gcIL-10 was able to induce STAT3 phosphorylation and STAT3 inhibitor could abolish the upregulation of gcSOCS3 mRNA expression by gcIL-10 in grass carp head kidney leukocytes. Taken together, this study for the first time functionally characterized the teleost IL-10 receptor subunits and clarified the conservation of fish IL-10 signaling during evolution, thus laying the ground for further understanding the critical immune events led by IL-10 in teleost.

Molecular and functional characterization of IL-1 receptor type 2 in grass carp: a potent inhibitor of IL-1β signaling in head kidney leukocytes.

IL-1 receptor type 2 (IL-1R2) is known as one of natural IL-1β singling inhibitors in mammals. However, the functional role of IL-1R2 in fish remains largely unknown. In this study, grass carp (Ctenopharyngodon idellus) IL-1R2 (gcIL-1R2) was identified and functionally characterized. Similar to its fish homologs, the deduced protein of gcIL-1R2 possessed two Ig-like domains in its extracellular region but lacked an intracellular signaling domain. The involvement of gcIL-1R2 in immune response was demonstrated by investigating its expression profiles in head kidney and head kidney leukocytes (HKLs) following in vivo bacterial infection and in vitro LPS treatment, respectively. Moreover, recombinant grass carp IL-1β (rgcIL-1β) was able to stimulate gcIL-1R2 mRNA expression with a rapid kinetics. This stimulation was possibly dependent on p38, JNK, p42/44 and NF-κB pathways in grass carp HKLs, revealing a new regulatory point of IL-1β signaling at receptor level in fish. Furthermore, recombinant protein of the gcIL-1R2 extracellular region (rgcIL-1R2) was demonstrated to interact with rgcIL-1β by using ELISA, elucidating the binding specificity of gcIL-1R2. Importantly, the stimulatory effect of rgcIL-1β on its own mRNA expression was blocked by rgcIL-1R2 in a dose-dependent manner in grass carp HKLs, providing the evidence for a functional role of IL-1R2 in IL-1β signaling in teleost. These findings suggested that teleost IL-1R2 may serve as a local naturally occurring inhibitor involving in IL-1β signaling as seen in mammals.

Cellular activation, expression analysis and functional characterization of grass carp IκBα: evidence for its involvement in fish NF-κB signaling pathway.

IκBα is a well-known member of the inhibitors of kappa B (IκB) family that controls NF-κB signaling by blocking NF-κB translocation from cytoplasm to nucleus. In the present study, an IκBα homologue was identified from grass carp (gcIκBα), showing the structural characteristics of IκB family. Moreover, mRNA expression of this molecule in grass carp periphery blood lymphocytes (PBLs) was enhanced significantly by both LPS and PHA in a time- and dose-dependent manner, indicating the involvement of gcIκBα in fish immune response. Further analysis demonstrated that LPS but not PHA induced gcIκBα phosphorylation and protein degradation in PBLs, implying different signaling pathways mediated by LPS and PHA in gcIκBα expression regulation in grass carp PBLs. In particular, the time-dependent oscillation of gcIκBα phosphorylation and total protein levels induced by LPS is in accordance with the characteristics of mammalian IκBα phosphorylation followed by protein degradation during NF-κB activation. In support of this notion, overexpression of gcIκBα was able to block both basal and LPS-stimulated NF-κB activity in grass carp kidney cell line, indicating the negatively regulatory role of gcIκBα in NF-κB activity as seen in mammals. Therefore, our results not only reveal a dynamic variation of NF-κB activity based on the activation and expression of IκBα for the first time, but also provide the direct evidence for the involvement of IκBα in NF-κB signaling in fish immune cells.

Reciprocal interaction between fish TGF-β1 and IL-1β is responsible for restraining IL-1β signaling activity in grass carp head kidney leukocytes

In the present study, we found that recombinant grass carp IL-1β (rgcIL-1β) simultaneously up-regulated grass carp IL-1β (gcIL-1β) and TGF-β1 (gcTGF-β1) expression via NF-κB and MAPK signaling in grass carp head kidney leukocytes (HKLs), promoting us to clarify whether TGF-β1 is an effective antagonist in IL-1β expression and activity. Our results showed that a stimulation of gcIL-1β on its own expression was noted within 6 h, but gcTGF-β1 neutralizing antibody prolonged gcIL-1β autostimulation up to 12 h, indicating a possible inhibitory role of gcTGF-β1 in regulating gcIL-1β effect. This notion was reinforced by the fact that recombinant grass carp TGF-β1 (rgcTGF-β1) could impede rgcIL-1β-induced gcIL-1β gene expression and secretion in a reciprocal manner. Further studies revealed that rgcTGF-β1 was able to attenuate rgcIL-1β-induced mRNA expression of its own receptor signaling molecules and the activation of NF-κB. By contrast, rgcIL-1β significantly amplified rgcTGF-β1-mediated gcTGF-β1 type I receptor (ALK5) expression and Smad2 phosphorylation in the same cell model. Taken together, these data shed light on an intrinsic mechanism for controlling inflammatory response by the reciprocal interaction between TGF-β1 and IL-1β in teleost.

Identification of a single p19 gene and three p40 paralogues in grass carp (Ctenopharyngodon idellus): Their potential for the formation of interleukin 23 and inducible expression in vitro and in vivo

Abstract Interleukin (IL‐) 23, a member of IL‐12 family, is a composite cytokine with the subunits of p19 and p40. Although IL‐12 and IL‐23 share the p40 subunit, they play vastly different roles in immune regulation. In teleost, much emphasis has been placed on the identification of IL‐12, but evidence for the existence of IL‐23 is still lacking. In the present study, a p19 gene and three p40 paralogues were isolated and identified from grass carp, suggesting multiple assembly of IL‐23 molecules in fish species. To address this issue, the existence of different p19/p40 heterodimers were examined by Co‐Immunoprecipitation (Co‐IP) assay, showing that only co‐expression of p19 and each p40 subunit could produce the soluble proteins corresponding to three IL‐23 isoforms. Additionally, bacterial infection could up‐regulate the mRNA expression of p19, p40a and p40b but not p40c in head kidney, indicating distinct expression patterns of three p40 paralogues. Moreover, in vitro experiments demonstrated that both B‐cell stimulator, LPS and T‐cell mitogen, PHA markedly increased the mRNA levels of p19 and three p40 paralogues in grass carp periphery blood lymphocytes (PBLs). The simultaneous up‐regulation of mRNA expression of p19 and p40 paralogues in response to immune stimuli supports the idea that p19 may form heterodimeric molecules with three p40 subunits in grass carp under immune activation. These findings for the first time highlight the potential of p19 and p40 for dimerization in fish, particularly the existence of three IL‐23 isoforms as soluble heterodimeric cytokines in grass carp, thereby providing the basis for further investigating the function of IL‐23 in fish immunity. HighlightsA single p19 gene and three p40 paralogues were identified in grass carp.The p19 could separately form soluble heterodimers with three p40 paralogues.In vivo bacterial infection rapidly increased mRNA expression of p19 and p40a/b.LPS and PHA markedly enhanced mRNA levels of p19 and p40a/b/c in leukocytes.

Identification and functional evaluation of two STAT3 variants in grass carp: Implication for the existence of specific alternative splicing of STAT3 gene in teleost

Abstract A STAT family member, STAT3, becomes activated as a DNA binding protein in response to cytokines and growth factors. In teleost, STAT3 cDNA has been cloned and identified in a few species, but only a single STAT3 transcript is revealed in these studies. In the present study, two variants of STAT3 gene generated by alternative splicing were isolated from grass carp and nominated as STAT3&agr;1 and STAT3&agr;2 based on the homology with their mammalian orthologs. In particular, the homologs of STAT3&agr;1/2 were also found in various fish species, including zebrafish, takifugu, tilapia, medaka and goldfish. Intriguingly, sequence alignment and genomic structure analysis revealed that fish STAT3&agr;1/2 are generated through similar alternative splicing events, implying the potential physiological significance of generating STAT3 variants in fish. Grass carp STAT3&agr;1/2 (gcSTAT3&agr;1/2) were ubiquitously expressed although the transcript levels of STAT3&agr;2 were markedly higher than STAT3&agr;1 in all examined tissues. In vivo and in vitro studies showed that the expression patterns of these two variants were similar under the stimulation of immune stimuli. To reveal the role of gcSTAT3&agr;1/2 in fish immunity, their phosphorylation and involvement in IL‐17A/F1 mRNA expression were demonstrated in grass carp peripheral blood lymphocytes upon LPS or PHA challenge, providing evidence for the functional conservation of STAT3 signaling in fish. These findings also raise a question of whether both gcSTAT3&agr;1/2 participate in transcriptional regulation in fish. Actually, our results showed that both of them had the ability to translocate into the nucleus upon activation, and to amplify IL‐10 signaling, indicating the existence of STAT3 isoforms with functional redundancy in teleost. HighlightsTwo novel spliced isoforms of STAT3 gene are found in several fishes.They are generated via similar alternative splicing events.Their expression is induced in grass carp by immune stimuli in vitro and in vivo.Both of them can be phosphorylated and translocated into nucleus in grass carp cells.They play a role in regulating grass carp IL‐17A/F1 expression and IL‐10 signaling.