Grass Carp Head Kidney Leukocytes Research Articles

Identification, Expression, Characteristic Analysis, and Immune Function of Two Akirin Genes in Grass Carp (Ctenopharyngodon idella).

Intensive aquaculture of grass carp often leads to decreased immunity and increased disease prevalence, resulting in economic losses. Improving grass carp immunity is therefore a critical strategy for addressing these challenges. Akirin reportedly participates in myogenesis, growth, and immune responses. However, its role in grass carp remains unclear. Herein, we isolated akirins from the spleen of grass carp and analyzed their tissue-specific expression. Akirin expression was detected following treatment with poly (I:C), LPS, and Aeromonas hydrophila (A. hydrophila). The immunological function of the akirin protein was evaluated in head kidney leukocytes (HKLs). The results revealed that the coding sequence (CDS) of akirin1 is 570 bp, encoding 189 amino acids. There was one predicted nuclear localization signal (NLS) and two predicted α- helix domains. The CDS of akirin2 is 558 bp, encoding 185 amino acids. There were two predicted NLSs and two predicted α-helix domains. Tissue-specific expression analysis showed that akirins are widely detected in grass carp tissues. akirin1 was highly detected in the brain, kidneys, heart, spleen, and gonads, while akirin2 was highly detected in the brain, liver, gonads, kidneys, spleen, and heart. The mRNA levels of akirins were promoted after treatment with poly (I:C), LPS, and A. hydrophila. Recombinant akirin proteins were produced in Escherichia coli (E. coli). il-1β, ifnγ, il-6, tnfα, il-4, iκbα, and nfκb were markedly increased in grass carp HKLs by treatment with the akirin protein. These results suggest that akirins play a role in the immunological regulation of grass carp.

The Teleost CXCL13-CXCR5 Axis Induces Inflammatory Cytokine Expression through the Akt-NF-κB, p38-AP-1, and p38-NF-κB Pathways.

The ancestors of chemokines originate in the most primitive of vertebrates, which has recently attracted great interest in the immune functions and the underlying mechanisms of fish chemokines. In the current study, we identified an evolutionarily conserved chemokine, CiCXCL13, from a teleost fish, grass carp. CiCXCL13 was characterized by a typical SCY (small cytokine CXC) domain and four cysteine residues (C34, C36, C61, C77), with the first two cysteines separated by a random amino acid residue, although it shared 24.2-54.8% identity with the counterparts from other vertebrates. CiCXCL13 was an inducible chemokine, whose expression was significantly upregulated in the immune tissues of grass carps after grass carp reovirus infection. CiCXCL13 could bind to the membrane of grass carp head kidney leukocytes and promote cell migration, NO release, and the expression of >15 inflammatory cytokines, including IL-1β, TNF-α, IL-10 and TGF-β1, thus regulating the inflammatory response. Mechanistically, CiCXCL13 interacted with its evolutionarily conserved receptor CiCXCR5 and activated the Akt-NF-κB and p38-AP-1 pathways, as well as a previously unrevealed p38-NF-κB pathway, to efficiently induce inflammatory cytokine expression, which was distinct from that reported in mammals. Zebrafish CXCL13 induced inflammatory cytokine expression through Akt, p38, NF-κB, and AP-1 as CiCXCL13. Meanwhile, the CiCXCL13-CiCXCR5 axis-mediated inflammatory activity was negatively shaped by grass carp atypical chemokine receptor 2 (CiACKR2). The present study is, to our knowledge, the first to comprehensively define the immune function of CXCL13 in inflammatory regulation and the underlying mechanism in teleosts, and it provides a valuable perspective on the evolution and biology of fish chemokines.

Interleukin-1β enhances the expression of two antimicrobial peptides in grass carp (Ctenopharyngodon idella) against Vibrio mimicus via activating NF-κB pathway

Vibrio mimicus (V. mimicus) is a pathogen causing serious vibriosis in aquatic animals. Hepcidin and β-Defensin1 are two important antibacterial peptides (AMPs) with broad-spectrum antibacterial activity in fish. In mammals, some evidences demonstrated that interleukin-1β (IL-1β) primarily promote AMPs expression via activating classical NF-κB pathway, but it still remains unclear in fish. Here, the temporal and spatial expression patterns of grass carp IL-1β (gcIL-1β) gene and two AMPs genes (gchepcidin and gcβ-defensin1) in tissues post-V. mimicus infection and anti-V. mimicus activity of these two AMPs in vitro were detected, showing that V. mimicus infection significantly elevated the mRNA levels of these three genes in the immune-related tissues although their expression patterns were not entirely consistent, and both gcHepcidin and gcβ-Defensin1 possessed anti-V. mimicus activity in vitro. Subsequently, the recombinant gcIL-1β (rgcIL-1β) was expressed prokaryotically in an inclusion body, which could promote proliferation of grass carp head kidney leukocytes (gcHKLs) and enhance respiratory burst activity and phagocytic activity of head kidney macrophages. Stimulation with rgcIL-1β was able to significantly regulate the mRNA expression of key regulatory genes (il-1RI, traf6, tak1, ikkβ, iκBα and p65) involved in the activation of classical NF-κB pathway, and then induce gcTAK1 phosphorylation, promote gcp65 nuclear translocation and enhance endogenous gcIL-1β expression at both mRNA and protein levels, implying NF-κB pathway was activated. More importantly, exogenous rgcIL-1β stimulation also significantly up-regulated both gcHepcidin and gcβ-Defensin1 mRNA levels against V. mimicus, and the regulatory effect was blocked or inhibited by NF-κB inhibitor PDTC. Taken together, our results demonstrated for the first time that grass carp IL-1β stimulation could significantly enhance the expression of these two anti-V.mimicus AMPs via activating classical NF-κB pathway.

Insights into the functional role of grass carp IL-8 in head kidney leukocytes: pro-inflammatory effects and signalling mechanisms.

Interleukin-8 (IL-8) is a critical chemokine regulating immune cells' chemotaxis as well as their physiological or pathological activations. In fish cells, recombinant IL-8 proteins induced transcriptions of pro-inflammatory cytokines. Nonetheless, the exact mechanisms underlying the function of fish IL-8 as a pro-inflammatory cytokine are still unclear. In this paper, the authors first prepared recombinant grass carp IL-8 (rgcIL-8) using an Escherichia coli expression system, and later confirmed rgcIL-8 increased gene expression of il8, il1β and tumour necrosis factor alpha (tnfα) in grass carp head kidney leukocytes (HKLs). Using signalling pathway inhibitors, the authors showed that rgcIL-8 regulated transcriptions of pro-inflammatory cytokines via MAPK and/or NF-κB signalling pathways. They cloned gcIL-8-specific receptor CXCR1 and subsequently discovered that gcIL-8 could increase the activity of NF-κB and the transcription of IL-1β via CXCR1. Simultaneously, antibody neutralization assay showed that endogenous IL-8 is partially relevant to the self-regulation of IL-1β. Moreover, rgcIL-8 led to the expression of inducible nitric oxide synthase gene, causing an accumulation of nitric oxide in the culture medium of HKLs, suggesting the potential of gcIL-8 to mediate inflammatory response. This study not only enriched the function of IL-8 in teleost but also revealed it as a potential target for the inflammatory control in grass carp.

Characterization and bioactivity of grass carp (Ctenopharyngodon idella) interleukin-21: Inducible production and involvement in inflammatory regulation

In mammals, interleukin 21 (IL-21) is a broad pleiotropic cytokine that plays critical roles in the development of several inflammatory and autoimmune diseases. In fish, functional information of Il-21 is limited, and its role in immune response is largely unknown. In the present study, we cloned a coding sequence of grass carp (Ctenopharyngodon idella) il21 gene (gcil21). To characterize the release patterns and biological activity of gcIl-21, we prepared recombinant gcIl-21 (rgcIl-21) and obtained the polyclonal antibody with gcIl-21 specificity. Western blotting analysis showed that in grass carp head kidney leukocytes (HKLs), gcIl-21 was undetected in culture supernatant of untreated cells but drastically induced by heat-killed Aeromonas hydrophila (A. hydrophila), uncovering the release features of gcIl-21 and its possible involvement in immune response. Subsequent functional experiments revealed that rgcIl-21 did not affect the mRNA expression of grass carp il1b and tgfb, but induced a strong expression of grass carp il10, and to a lesser extent of grass carp tnfa in HKLs, suggesting a dominant effect of gcIl-21 in modulating Il-10 signaling as seen in rainbow trout and mammals. Furthermore, in vivo studies showed that intraperitoneal injection of rgcIl-21 was able to increase the survival rate of grass carp infected with live A. hydrophila, and reduce the pathological responses caused by the same pathogenic bacteria in head kidney and intestine. Taken together, these results for the first time revealed the close relationship of fish Il-21 production and function with inflammatory responses, and highlighted its anti-bacterial and anti-inflammatory ability, thereby providing a new insight into host defense mechanisms 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.

Functional characterization of three fish-specific interleukin-23 isoforms as regulators of Th17 signature cytokine expression in grass carp head kidney leukocytes

Mammalian Interleukin (IL)-23 is a heterodimeric cytokine with an IL-23-specific P19 subunit and a P40 subunit shared with IL-12, and plays a key role in the regulation of cell differentiation as well as inflammation. We previously demonstrated the existence of three soluble fish Interleukin (Il)-23 isoforms consist of a single P19 and one of three P40 isoforms (P40a/b/c) in grass carp. In the present study, three recombinant grass carp Il-23 (rgcIl-23) isoforms were prepared by linking gcP19 and gcP40a/b/c in a prokaryotic expression system, and then their functional properties were verified in grass carp head kidney leukocytes (HKLs). All three rgcIl-23 isoforms showed the bioactivities to divergently upregulate the mRNA expression of Th17 signature cytokines (il17a/f1, il21, il22 and il26) as well as Il-23 receptor (il23r) in HKLs. Moreover, they also promoted gcIl-17a/f1 secretion in a dose-dependent manner, strengthening their roles in Th17-like response. Furthermore, induction of il17a/f1 and il23r transcription by rgcIl-23 was blocked by a STAT3 inhibitor in grass carp HKLs, suggesting the involvement of STAT3 signaling in these inductions. Taken together, we for the first time identified the bioactivities of fish Il-23 isoforms and particularly revealed the existence of Il-23/Il-17a/f1 axis in fish, thereby advancing our understanding of Th17-like responses in fish 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-1β 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.

Identification and functional characterization of grass carp (Ctenopharyngodon idella) tumor necrosis factor receptor 2 and its soluble form with potentiality for targeting inflammation

Tumor necrosis factor-alpha (TNF-α) signals through two distinct cell surface receptors, TNFR1 and TNFR2 in mammals. In the present study, grass carp Tnfr2 (gcTnfr2) was isolated and characterized. Sequence alignment and phylogenetic analysis suggested that gcTnfr2 was a homolog of goldfish and zebrafish Tnfr2. Tissue distribution assay showed gctnfr2 transcripts were expressed in all examined tissues similar to gctnfr1. To functionally characterize the newly cloned molecule, gcTnfr2 was overexpressed in COS7 cell lines and it showed the ability to mediate the recombinant grass carp Tnf (rgcTnf)-α-triggered NF-κΒ activity and gcil1b promoter activity, clarifying its role in mediating Tnf-α signaling. The recombinant soluble form of gcTnfr2 (rgcsTnfr2) was prepared and it was able to interact with rgcTnf-α with higher affinity than that of rgcsTnfr1. Moreover, grass carp soluble Tnfr2 (gcsTnfr2) were detected in the culture medium of grass carp head kidney leukocytes (HKLs) and heat-inactivated A. hydrophila challenge significantly induced its production, indicating involvement of gcsTnfr2 in inflammation response. In agreement with this notion, rgcsTnfr2 effectively antagonized the effect of rgcTnf-α on il1b mRNA expression in HKLs, suggesting anti-Tnf-α property of gcsTnfr2. To strengthen the anti-inflammatory role of soluble Tnfr2, bacteria were injected intraperitoneally in grass carp followed by rgcsTnfr2. Hematoxylin-eosin (HE) staining of head kidney, spleen and intestine showed that rgcsTnfr2 could significantly improve infection-induced histopathological changes. These results functionally identified gcTnfr2 and its soluble form, particularly highlighting the role of gcsTnfr2 against Tnf-α-triggered inflammatory signaling. In this line, rgcsTnfr2 displayed anti-inflammatory potentiality during infection, thereby providing a powerful mediator of inflammation control in fish.

Lipopolysaccharide-induced autophagy participates in the control of pro-inflammatory cytokine release in grass carp head kidney leukocytes

Microtubule-associated protein 1 light chain 3B (LC3B) is a known marker of autophagy in mammals. In the present study, we isolated and identified grass carp LC3B (gcLC3B) cDNA and found its inductive expression in response to bacterial infection in vivo. To assess the occurrence of autophagy in immune response, the role of gcLC3B as an autophagy marker in grass carp was characterized. Accordingly, grass carp kidney cells (CIKs) with stable expression of GFP-gcLC3B were established and GFP-gcLC3B puncta were counted under a confocal fluorescence microscope. Results showed that starvation, a conventional inducer of autophagy, significantly enhanced GFP-gcLC3B puncta number, indicating the existence of gcLC3B-linked autophagy in fish cells. Moreover, a commercial antibody recognizing gcLC3B and 3-methyladenine (3-MA) were validated in grass carp CIKs, and used to evaluate autophagy in grass carp head kidney leukocytes (HKLs) in response to LPS. Western blotting assay showed that LPS significantly induced the conversion of gcLC3B protein, providing the evidence for autophagy induced by LPS in fish immune cells. Importantly, autophagy inhibition by 3-MA enhanced grass carp IL-1β and TNF-α secretion, indicating the involvement of autophagy in pro-inflammatory cytokine production. Besides, 3-MA could amplify LPS-induced IL-1β and TNF-α release, implying that autophagic induction may drive a mechanism for controlling inflammatory response in fish. Thus, our data highlight the role of autophagy in fish immunity and provide new insight into the mechanism for the regulation of inflammation 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-1β (gcIL-1β) promoter activity was determined in COS7 cells. Results showed that gcTNFR1 was involved in the regulation of rgcTNF-α on gcIL-1β transcription. Consistently, rgcTNFR1 was effective in attenuating the effect of rgcTNF-α on IL-1β 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.

Grass carp TGF-β1 impairs IL-1β signaling in the inflammatory responses: Evidence for the potential of TGF-β1 to antagonize inflammation in fish

In the present study, effects of TGF-β1 on IL-1β signaling during inflammatory response were examined in grass carp. In grass carp head kidney leukocytes (HKLs), LPS significantly induced the mRNA expression of grass carp TGF-β1 (gcTGF-β1) and IL-1β, indicating the involvement of TGF-β1 and IL-1β in inflammatory process. Using anti-IL-1β antibody to neutralize the endogenous IL-1β, we found that stimulation of IL-1β mRNA expression by LPS was independent on IL-1β itself. Interestingly, recombinant gcTGF-β1 (rgcTGF-β1) suppressed basal and LPS-stimulated IL-1β mRNA expression in spite of immunoneutralizing endogenous IL-1β or not. Given that IL-1β receptor signaling molecule and natural IL-1β inhibitors are the important regulators in IL-1β signaling and activity, the effect of LPS on these molecules' expression was determined in HKLs. Results showed that LPS significantly enhanced the mRNA levels of IL-1 receptor type I (IL-1RI) and II (IL-1RII), IL-1R accessory protein (IL-1Racp) and novel IL-1 family member (nIL-1F). Moreover, the induction of IL-1RII, IL-1Racp and nIL-1F by LPS was IL-1β-dependent since IL-1β immunoneutralization abolished these inductions, implying the involvement of IL-1β auto-induction in these effects. Consistently, TGF-β1 could block basal IL-1RI and nIL-1F mRNA expression, and LPS-induced IL-1RI, IL-1Racp and nIL-1F mRNA expression, suggesting these molecules as the regulatory sites for TGF-β1 to modulate IL-1β signaling. Subsequent in vivo studies showed that bacterial challenge significantly up-regulated IL-1β mRNA expression with a rapid and transient pattern and TGF-β1 mRNA expression with a relatively time-delayed kinetics in head kidney. These expression patterns coincide with their pro-inflammatory and anti-inflammatory roles, respectively. As expected, rgcTGF-β1 could suppress bacterial-induced IL-1β mRNA expression, strengthening the anti-inflammatory role of TGF-β1 in vivo. Taken together, these results to our knowledge provide the first evidence for inducible TGF-β1 expression in inflammatory process, as well as the induction of inflammatory stimuli on IL-1β expression and signaling. In turn, TGF-β1 suppressed the proinflammatory process in vitro and in vivo presumably via interfering IL-1β expression and signaling in inflammatory response, highlighting the potential of TGF-β1 in the control of inflammation in fish.

Cytokine effects and cellular signaling pathways of grass carp HSP70 in head kidney leukocytes.

In mammals, the cytokine effects of heat shock protein 70 (HSP70) have been well documented. However, the precise role and molecular mechanism of fish HSP70 in cytokine production are poorly understood. In the present study, recombinant grass carp HSP70 (rgcHSP70) was prepared by using the E. coli expression system and its effect on interleukin (IL)-1β (gcil1β) and tumor necrosis factor (TNF)-α (gctnfα) mRNA expression was examined in grass carp head kidney leukocytes (HKLs). Compared with its heated-inactivated protein, rgcHSP70 enhanced the mRNA levels of these two cytokines in a dose- and time-dependent manner. More importantly, the results of enzyme-linked immunoabsorbent assay (ELISA) showed that rgcHSP70 markedly stimulated gcIL-1β and gcTNF-α secretion from the HKLs, for the first time indicating the ability of HSP70 to regulate cytokine release in fish. Moreover, rgcHSP70 displayed more potential to increase the cytokines' release instead of their transcription in the same cell model. Subsequently, the signaling mechanisms governing the cytokine effect of rgcHSP70 were investigated, showing that rgcHSP70-induced gcil1β mRNA expression was suppressed by inhibiting p38 MAPK and NF-κB pathways, while the stimulation of rgcHSP70 on gctnfα mRNA expression were blocked by inhibiting JNK MAPK and NF-κB pathways. In parallel, rgcHSP70 was also shown to activate NF-κB pathway through regulating grass carp IκBα protein levels in a time-dependent oscillation. These findings provide further understanding of cytokine effect and regulatory mechanism of HSP70 in fish and new insights into the roles of HSP70 in teleost immunity.

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 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.

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.

Carp interleukin-1β in the role of an immuno-adjuvant

Vibrio mimicus (V. mimicus) is a pathogen causing serious vibriosis in aquatic animals. Hepcidin and β-Defensin1 are two important antibacterial peptides (AMPs) with broad-spectrum antibacterial activity in fish. In mammals, some evidences demonstrated that interleukin-1β (IL-1β) primarily promote AMPs expression via activating classical NF-κB pathway, but it still remains unclear in fish. Here, the temporal and spatial expression patterns of grass carp IL-1β (gcIL-1β) gene and two AMPs genes (gchepcidin and gcβ-defensin1) in tissues post-V. mimicus infection and anti-V. mimicus activity of these two AMPs in vitro were detected, showing that V. mimicus infection significantly elevated the mRNA levels of these three genes in the immune-related tissues although their expression patterns were not entirely consistent, and both gcHepcidin and gcβ-Defensin1 possessed anti-V. mimicus activity in vitro. Subsequently, the recombinant gcIL-1β (rgcIL-1β) was expressed prokaryotically in an inclusion body, which could promote proliferation of grass carp head kidney leukocytes (gcHKLs) and enhance respiratory burst activity and phagocytic activity of head kidney macrophages. Stimulation with rgcIL-1β was able to significantly regulate the mRNA expression of key regulatory genes (il-1RI, traf6, tak1, ikkβ, iκBα and p65) involved in the activation of classical NF-κB pathway, and then induce gcTAK1 phosphorylation, promote gcp65 nuclear translocation and enhance endogenous gcIL-1β expression at both mRNA and protein levels, implying NF-κB pathway was activated. More importantly, exogenous rgcIL-1β stimulation also significantly up-regulated both gcHepcidin and gcβ-Defensin1 mRNA levels against V. mimicus, and the regulatory effect was blocked or inhibited by NF-κB inhibitor PDTC. Taken together, our results demonstrated for the first time that grass carp IL-1β stimulation could significantly enhance the expression of these two anti-V.mimicus AMPs via activating classical NF-κB pathway.