Xinyan Wang

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 two heat shock proteins (Hsp70/Hsc70) from grass carp (Ctenopharyngodon idella): evidence for their differential gene expression, protein synthesis and secretion in LPS-challenged peripheral blood lymphocytes.

Two cDNAs, encoding the stress-inducible 70-kDa heat shock protein (Hsp70) and the constitutively expressed 70-kDa heat shock cognate protein (Hsc70), were isolated from grass carp. The Hsp70 and Hsc70 cDNAs were 2250 bp and 2449 bp in length and contained 1932 bp and 1953 bp open reading frames, respectively. Tissue distribution results showed that Hsp70/Hsc70 was highly expressed in gill, kidney, head kidney and peripheral blood lymphocytes (PBLs). Using grass carp PBLs as a cell model, effects of lipopolysaccharide (LPS) on the mRNA and protein levels of Hsp70/Hsc70 were examined. In this case, LPS increased the mRNA expression of Hsp70 in a time- and dose-dependent manner, but had no effect on Hsc70 mRNA expression. In agreement with this, LPS elevated the intracellular Hsp70 markedly, but not the Hsc70 protein levels in parallel experiments. Furthermore, Hsp70 protein was also detected in culture medium. Moreover, inhibition of LPS on Hsp70 release in a time-dependent manner was observed, indicating that there may be a dynamic balance between Hsp70 stores and Hsp70 release in grass carp PBLs following exposure to LPS. Taken together, these results not only shed new insights into the different regulations of LPS on Hsp70/Hsc70 gene expression, protein synthesis and release, but also provide a basis for further study on the functional role of Hsp70 in fish immune response.

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.

Grass carp somatolactin: II. Pharmacological study on postreceptor signaling mechanisms for PACAP-induced somatolactin-α and -β gene expression

Somatolactin (SL), the latest member of the growth hormone/prolactin family, is a novel pituitary hormone with diverse functions. However, the signal transduction mechanisms responsible for SL expression are still largely unknown. Using grass carp as an animal model, we examined the direct effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on SL gene expression at the pituitary level. In primary cultures of grass carp pituitary cells, SLalpha and SLbeta mRNA levels could be elevated by PACAP via activation of PAC-I receptors. With the use of a pharmacological approach, the AC/cAMP/PKA and PLC/inositol 1,4,5-trisphosphate (IP(3))/PKC pathways and subsequent activation of the Ca(2+)/calmodulin (CaM)/CaMK-II cascades were shown to be involved in PACAP-induced SLalpha mRNA expression. Apparently, the downstream Ca(2+)/CaM-dependent cascades were triggered by extracellular Ca(2+) ([Ca(2+)](e)) entry via L-type voltage-sensitive Ca(2+) channels (VSCC) and Ca(2+) release from IP(3)-sensitive intracellular Ca(2+) stores. In addition, the VSCC component could be activated by cAMP/PKA- and PLC/PKC-dependent mechanisms. Similar postreceptor signaling cascades were also observed for PACAP-induced SLbeta mRNA expression, except that [Ca(2+)](e) entry through VSCC, PKC coupling to PLC, and subsequent activation of CaMK-II were not involved. These findings, taken together, provide evidence for the first time that PACAP can induce SLalpha and SLbeta gene expression in fish model via PAC-I receptors through differential coupling to overlapping and yet distinct signaling pathways.

Functional expression and characterization of grass carp IL-10: An essential mediator of TGF-β1 immune regulation in peripheral blood lymphocytes

In mammals, interleukin-10 (IL-10) is known as a potent regulatory cytokine with pleiotropic effects on various leukocytes. Although teleost IL-10 has been identified in several fish species, its immunoregulatory role is still poorly understood. In the present study, grass carp IL-10 (gcIL-10) has been identified and recombinant gcIL-10 (rgcIL-10) was prepared by using a prokaryotic expression system. Using grass carp peripheral blood lymphocytes (PBLs) as a cell model, rgcIL-10 was shown to up-regulate the cell activity, which was consistent with the regulatory tone of transforming growth factor β1 (TGF-β1). An interesting question that follows would be whether IL-10 and TGF-β1 redundantly regulate PBL activity. To address this question, we investigated the effect of IL-10 on the cell activity of grass carp PBLs challenged by TGF-β1 using immunoneutralization, showing that removal of endogenous IL-10 could abolish TGF-β1-induced cell activity, indicating an essential role of IL-10 in TGF-β1 immune regulation. Furthermore, to examine the connection between TGF-β1 and IL-10 during immunoregulatory process, effect of TGF-β1 on IL-10 expression was investigated. Results showed that TGF-β1 displayed stimulatory effects on gcIL-10 mRNA expression and protein secretion in the same cell model, suggesting that TGF-β1 may function through its effect on IL-10 production in fish immunity. These results provide clues to the potential immunoregulatory role and production of IL-10 in teleost.

Dual-parallel inhibition of IL-10 and TGF-β1 controls LPS-induced inflammatory response via NF-κB signaling in grass carp monocytes/macrophages.

In fish, the knowledge on the regulation of inflammatory responses is limited. In the present study, LPS rapidly increased the mRNA levels of grass carp pro-inflammatory factors, including tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxides synthase (iNOS) and IL-8 in monocytes/macrophages, indicating the occurrence of innate inflammatory responses in fish as seen in mammals. Intriguingly, the gene expression and protein secretion of grass carp IL-10 (gcIL-10) and TGF-β1 (gcTGF-β1) were induced by LPS in the same cell model, promoting us to clarify their roles in regulating inflammatory response. Results revealed that grass carp IL-10 polyclonal antibody (anti-gcIL-10 pAb) and grass carp TGF-β1 monoclonal antibody (anti-gcTGF-β1 mAb) could amplify the stimulation of LPS on the mRNA levels of tnfα, il1β, inos and il8, suggesting the inhibitory tone of endogenous IL-10 and TGF-β1 in LPS-challenged immune responses. This notion was further supported by the fact that recombinant grass carp IL-10 (rgcIL-10) and recombinant grass carp TGF-β1 (rgcTGF-β1) attenuated LPS-stimulated tnfα, il1β, inos and il8 gene expression in monocytes/macrophages. Further study revealed that rgcIL-10 and rgcTGF-β1 impaired NF-κB activation by blocking LPS-induced grass carp IκBα (gcIκBα) protein degradation in the cells. In addition, the correlation between gcIL-10 and gcTGF-β1 in this regulation was examined by immunoneutralization, unveiling that anti-gcTGF-β1 mAb and anti-gcIL-10 pAb were unable to alter the inhibitory effects of rgcIL-10 and rgcTGF-β1 on pro-inflammatory factors expression in grass carp monocytes/macrophages, respectively. This dual and parallel effect of gcIL-10 and gcTGF-β1 strengthened their importance in controlling inflammatory responses. Taken together, our findings shed a light on the functional role, regulatory mechanism and relationship of fish IL-10 and TGF-β1 in regulating inflammatory response.

TGF-β1 Exerts Opposing Effects on Grass Carp Leukocytes: Implication in Teleost Immunity, Receptor Signaling and Potential Self-Regulatory Mechanisms

In fish immunity, the regulatory role of transforming growth factor-β1 (TGF-β1) has not been fully characterized. Here we examined the immunoregulatory effects of TGF-β1 in grass carp peripheral blood leukocytes (PBL) and head kidney leukocytes (HKL). It is interesting that TGF-β1 consistently stimulated the cell viability and the mRNA levels of pro-inflammatory cytokines (Tnfα and Ifnγ) and T/B cell markers [Cd4-like (Cd4l), Cd8α, Cd8β and Igμ] in PBL, which contrasted with its inhibitory tone in HKL. Further studies showed that grass carp TGF-β1 type I receptor, activin receptor-like kinase 5 (ALK5), was indispensable for the immunoregulatory effects of TGF-β1 in PBL and HKL. Notably, TGF-β1 persistently attenuated ALK5 expression, whereas immunoneutralization of endogenous grass carp TGF-β1 could increase ALK5 mRNA and protein levels. It is consistent with the observation that TGF-β1 decreased the number of ALK5+ leukocytes in PBL and HKL, revealing a negative regulation of TGF-β1 signaling at the receptor level. Moreover, transient treatment with TGF-β1 for 24 h was sufficient to induce similar cellular responses compared with the continuous treatment. This indicated a possible mechanism by which TGF-β1 triggered the down-regulation of ALK5 mRNA and protein, leading to the desensitization of grass carp leukocytes toward TGF-β1. Accordingly, our data revealed a dual role of TGF-β1 in teleost immunity in which it can serve as a positive or negative control device and provided additional mechanistic insights as to how TGF-β1 controls its signaling in vertebrate leukocytes.

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.

Characterization of grass carp (Ctenopharyngodon idellus) Foxp1a/1b/2: evidence for their involvement in the activation of peripheral blood lymphocyte subpopulations.

Foxp subfamily belongs to the Fox family of winged-helix transcription factors and plays critical roles in multiple biological processes including development and immunoregulation. However, little is known about the regulation and function of Foxp subfamily in fish immune system. In this study, we obtained the complete cDNAs of grass carp Foxp1a, Foxp1b and Foxp2. They possess the conserved leucine zipper domain, zinc finger domain and forkhead domain when compared with their mammalian counterparts, except that Foxp1a lacks the forkhead domain. Real-time RT-PCR analysis showed that their transcripts were mainly found in thymus, spleen and peripheral blood lymphocytes (PBLs). In grass carp PBLs, both LPS and PHA were effective in elevating Foxp1b mRNA levels but had no effect on Foxp1a mRNA, while only PHA affected Foxp2 mRNA expression. Using the same cell model, PHA was revealed to up-regulate mRNA expression of T-cell marker genes (CD4-like, CD8alpha and CD8beta) but not B-cell marker gene (IgM). Unlike PHA, LPS increased IgM mRNA level but did not affect T-cell marker gene expression. These findings suggest that PHA and LPS may act on distinct lymphocyte subpopulations in grass carp PBLs and provide evidence for the involvement of Foxp1b and Foxp2 in the activation of different lymphocyte subpopulations in grass carp.

A newly identified epithelial cell adhesion molecule (EpCAM) from grass carp (Ctenopharyngodon idellus): Cloning, tissue distribution and lipopolysaccharide-induced expression in head kidney leucocytes

Epithelial cell adhesion molecule (EpCAM) was initially identified as a tumor-associated antigen in human. Recent studies show that EpCAM is a transmembrane glycoprotein with diverse functions in cell adhesion and other processes such as cellular signaling, cell migration, proliferation and differentiation. In fish, the knowledge on the structure and function of EpCAM is limited as only zebrafish EpCAM has been characterized and shown to play a role in embryonic development. In the present study, we isolated a cDNA encoding a homologue of zebrafish EpCAM from grass carp. The deduced grass carp EpCAM (gcEpCAM) protein of 306 amino acids shared 59-85% of similarity with the EpCAMs in other vertebrates. Protein sequence analysis revealed a transmembrane domain and two epidermal growth factor (EGF)-like repeats in the extracellular region of gcEpCAM which were both conserved in EpCAM from fish to mammal. RT-PCR analysis showed that gcEpCAM transcript was expressed with high abundance in skin, pituitary, kidney, head kidney, gill, thymus and head kidney leucocyte (HKL). Furthermore, real time PCR assay was developed to assess gcEpCAM mRNA expression in HKLs stimulated by lipopolysaccharide (LPS). Results showed that LPS significantly increased gcEpCAM mRNA expression in a time- and dose-dependent manner and the peak response was observed at 12h after treatment with 30 μg/ml of LPS. These results indicated that gcEpCAM may be a LPS-induced gene involved in the immune response in fish head kidney.