Fish arginase constrains excessive production of nitric oxide and limits mitochondrial damage during Aeromonas hydrophila infection.

Bacteria-enhanced inducible nitric oxide synthase (iNOS) overproduces nitric oxide (NO) leading to mitochondrial and cellular damage. In mammals, arginase (ARG), the enzyme consuming the same substrate l-arginine with iNOS, was believed to inhibit iNOS activity by competing the substrate. But in fish, this conception has been widely challenged. In this study, the gene expression using real-time quantitative PCR (RT-qPCR) technology showed that when stimulated by Aeromonas hydrophila (A. hydrophila), grass carp (gc) iNOS was up-regulated in head kidney monocytes/macrophages (M0/MФ), and its changes were not detected in the whole tissue of liver or spleen, showing a high degree of cell-specific expression pattern. At the same time, gcARG2 had a high basal expression in tissues and was up-regulated by A. hydrophila stimulation. Next, phthalaldehyde-primaquine reaction was first used in the determination of intracellular urea in fish cells. It was found that the induced gcARG2 led to an increase in the intracellular urea content. Moreover, urea and NO production in M0/MФ were increased in a substrate dose-dependent manner from 30 to 100 µM of l-arginine and reached the highest yield at 300 and 3000 µM of l-arginine, respectively. Furthermore, head kidney M0/MФ was cultured in RPMI1640 medium containing physiological concentration (500 µM) of l-arginine to evaluate the effect of ARG. Under A. hydrophila stimulation, treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine (BEC) showed that inhibition of arginase could further enhance the NO production stimulated by A. hydrophila. This in turn led to a cumulation in peroxynitrite (ONOO-) content and an injury of the mitochondrial membrane potential. Our study showed for the first time that fish ARG in head kidney M0/MФ can limit excessive production of NO and harmful products by iNOS to maintain mitochondrial and cellular homeostasis.

Dietary intakes of vitamin D promote growth performance and disease resistance in juvenile grass carp (Ctenopharyngodon idella).

Vitamin D3 (VD3) is an essential nutrient for fish and participates in a variety of physiological activities. Notably, both insufficient and excessive supplementation of VD3 severely impede fish growth, and the requirements of VD3 for fish vary considerably in different species and growth periods. The present study aimed to evaluate the appropriate requirements of VD3 for juvenile grass carp (Ctenopharyngodon idella) according to growth performance and disease prevention capacity. In this study, diets containing six supplemental levels of VD3 (0, 300, 600, 1200, 2400, and 4800 IU/kg diet) were formulated to investigate the effect(s) of VD3 on the growth performance, antioxidant enzyme activities, and antimicrobial ability in juvenile grass carp. Compared with the VD3 deficiency group (0 IU/kg), the supplementation of 300-2400 IU/kg VD3 significantly enhanced growth performance and increased antioxidant enzyme activities in the fish liver. Moreover, dietary supplementation of VD3 significantly improved the intestinal health by manipulating the composition of intestinal microbiota in juvenile grass carp. In agreement with this notion, the mortality of juvenile grass carp fed with dietary VD3 was much lower than that in VD3 deficient group upon infection with Aeromonas hydrophila. Meanwhile, dietary supplementation of 300-2400 IU/kg VD3 reduced bacterial load in the spleen and head kidney of the infected fish, and 1200 IU/kg VD3 supplementation could decrease enteritis morbidity and increase lysozyme activities in the intestine. These findings strengthened the essential role of dietary VD3 in managing fish growth and antimicrobial capacity. Additionally, based on weight gain ratio and lysozyme activities, the appropriate VD3 requirements for juvenile grass carp were estimated to be 1994.80 and 2321.80 IU/kg diet, respectively.

Grass carp Il-2 promotes neutrophil extracellular traps formation via inducing ROS production and autophagy in vitro.

Interleukin (IL)-2 has been reported to regulate neutrophil functions in humans, mice, pigs and chicken although it is a key regulator of T cells. Consistently, we found that grass carp (Ctenopharyngodon idellus) interleukin-2 (gcIl-2) is capable of modulating the antimicrobial activities of neutrophils via regulating granzyme B- and perforin-like gene expression in our previous study. In the present study, stimulation of gcIl-2 on neutrophil extracellular traps (NETs) formation in grass carp neutrophils was demonstrated by detecting free DNA release, histone H3 citrullination and morphological changes of the cells. Further investigation revealed that reactive oxygen species (ROS) production from NADPH oxidase but not mitochondria was involved in NETosis induced by gcIl-2. Aside from ROS, autophagy was disclosed to be indispensable for NETosis induced by gcIl-2. These converging lines of evidence suggested that fish Il-2 could induce NETs formation via NADPH oxidase-derived ROS- and autophagy-dependent pathways in fish species which is evolutionarily conserved with that in mammals. It is noteworthy that these two pathways did not interplay with each other in Il-2-stimulated NETosis. The mechanisms governing autophagy induced by Il-2 were also explored in the present study, showing that Il-2 modulated the action of high mobility group box 1 (HMGB1) protein to stimulate autophagy, leading to NETs formation in fish neutrophils. These results provided a new insight to the function of Il-2 in fish neutrophils, and a clue about the regulation of NETosis in the lower vertebrates.

Characterization and in vitro antibacterial activity of grass carp (Ctenopharyngodon idella) serum amyloid A.

Serum amyloid A (SAA) predominantly synthesized by hepatocytes is a classical acute phase protein and has been extensively studied in mammals. However, the studies on the structure and properties of fish SAA are limited although SAA genes have been cloned and identified from various fishes. In the present study, a cDNA of grass carp (Ctenopharyngodon idella) SAA (gcSAA) was cloned and characterized, displaying a high homology with its counterparts in vertebrates. gcSAA mRNA was expressed with highest abundance in the liver and its levels were increased by a 24-hour infection of Aeromonas hydrophila (A. hydrophila) for more than 5 folds in the intestine, 15 folds in the spleen, 75 folds in the head kidney and 100 folds in the liver, implying that it is an acute phase protein in grass carp. Subsequently, recombinant gcSAA protein (rgcSAA) was prepared from a prokaryotic expression system after codon optimization of its coding sequence. The direct antibacterial activity assay and the plate count assay disclosed that gcSAA inhibited the growth and survival of A. hydrophila but not Edwardsiella piscicida (E. piscicida) which both are common bacterial pathogens in aquaculture. The propidium iodide (PI) uptake assay confirmed the bactericidal property of gcSAA, showing that it is able to enhance the uptake of PI in A. hydrophila but not E. piscicida. These findings revealed the molecular features of gcSAA and its roles in host defense against bacterial infection.

Vitamin D ameliorates Aeromonas hydrophila-induced iron-dependent oxidative damage of grass carp splenic macrophages by manipulating Nrf2-mediated antioxidant pathway.

Aeromonas hydrophila (A. hydrophila) is one of major pathogenic bacteria in aquaculture and potentially virulent to grass carp (Ctenopharyngodon idella). As an essential nutrient for fish, vitamin D3 (VD3) has been reported to play a role against oxidative stress, but the exact mechanism remains to be elusive. In this study, we found that A. hydrophila induced ferrugination and macrophage aggregation in the spleen of grass carp. Along this line, using the splenic macrophages as the model, the effects of VD3 on A. hydrophila-caused iron deposition and subsequent injuries were determined. In the context, 1,25D3 (the active form of VD3) significantly reduced cellular free Fe2+, lipid peroxidation and lactic dehydrogenase (LDH) release induced by A. hydrophila in the splenic macrophages, indicating the protective effects of VD3 on A. hydrophila-led to ferroptosis-related injuries. In support of this notion, 1,25D3 was effective in hindering ferroptosis inducers-stimulated LDH release in the same cells. Mechanically, 1,25D3 enhanced iron export protein (ferroportin1) and glutathione peroxidase 4 (GPX4) protein levels, and glutathione (GSH) contents via vitamin D receptor (VDR). Moreover, NF-E2-related factor 2 (Nrf2) pathway mediated the regulation of 1,25D3 on GPX4 protein expression and GSH synthesis. Meanwhile, 1,25D3 maintained the stability of Nrf2 proteins possibly by attenuating its ubiquitination degradation. Furthermore, in vivo experiments showed that 1,25D3 injection could not only improve the survival of fish infected by A. hydrophila, but also enhance GSH amounts and decrease malonaldehyde (MDA) contents and iron deposition in the spleen. In summary, our data for the first time suggest that VD3 is a potential antioxidant in fish to fight against A. hydrophila induced-ferroptotic damages.

Highly efficient metal-organic framework based intumescent poly(L-lactic acid) towards fire safety, ignition delay and UV resistance.

Developing multifunctional biodegradable PLA with ignition delay, high efficient fire retardancy, and UV resistance properties is a challenging task owing to its high flammability, and mutually exclusive phenomenon between the latter two properties. In this work, we report a superior efficient synergistic action combining piperazine pyrophosphate (PAPP) and a Co based metal-organic framework (ZIF-67). Results illustrated that with merely 0.06 wt% ZIF-67, intumescent PLA containing 4.96 wt% PAPP reached UL-94 V0 rating. The PLA/4.9PAPP/0.1MOF sample possessed a limiting oxygen index (LOI) value at 33 %, exhibited a 28 % reduction in peak heat release rate (pHRR) and a 67 % increase in fire propagation index (FPI). Moreover, the presence MOF delayed the ignition time of PLA by 12 s due to the highly porous structure of MOF and its chemical heat-sink performance. Insightful reaction to fire mechanism in the condensed phase via TG-FTIR and Raman revealed that a crack free protective intumescent char layer with higher graphitization degree was formed, which effectively enhanced the barrier effect and minimize the heat and fuel transfer. In addition, the UV resistance of PLA composites is enhanced, remaining at and below 5 % transmittance in the UVA and UVB areas. This work provides a green production way of multifunctional degradable materials and broadens their application fields.

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.

Solvothermal preparation of nitrogen-doped carbon dots with PET waste as precursor and their application in LEDs and water detection.

Developing novel, alternative ways to recycle PET waste, which has an important influence on reduction of landfilling and CO2 emissions, has always been a research hot spot for industry and academy. In this work, PET waste was adopted as precursor for the preparation of nitrogen-doped Carbon Dots (NCDs). Firstly, PET oligomers were obtained by alcoholysis of PET waste with ethylene glycol. Then, the mixture without isolation and purification as well as pyromellitic acid dianhydride and urea were adopted as precursors for the preparation of NCDs by solvothermal method with tetrahydrofuran (THF) as solvent. The as-prepared NCDs has a spherical structure with an average particle size of 2.3 nm. What is more, NCDs exhibit excitation-independent emission properties, the largest excitation peak and emission peak of NCDs located in 360 nm and 470 nm, and the fluorescence quantum yield is 48.16 %. In term of application, NCDs are dispersed in PMMA and loaded on 365 nm and 430 nm LED chips to obtain LED devices emitting yellow light ((0.55, 0.44), 2018 K) and warm white light ((0.37, 0.31), 3783 K), respectively. In addition, NCDs could be adopted as fluorescent probe for the construction of sensor for water in organic solvents based on dynamic quenching of NCDs, and the limit of detection (LOD) is 0.00001 %.

Mechanical, Thermal Stability, and Flame Retarding Properties of Phosphorus-Modified PET Blended with DOPO-POSS.

In this study, an antidroplet flame retardant system based on FRPET (phosphorus-containing copolyester) is constructed with DOPO-POSS (polyhedral oligomeric silsesquioxane containing DOPO) as an additive flame retardant. It is demonstrated that DOPO-POSS has good dispersibility at a lower amount. When the amount of DOPO-POSS is 9 wt %, the residual char of DOPO-POSS/FRPET at 700 °C increases to 23.56 from 18.16% of FRPET, and the maximum thermal weight loss rate also reduces. What is more is that the limiting oxygen index increases to 33 from 26% of FRPET. The flame burning time is shortened to 4.95 from 20.8 s, the phenomenon of self-extinguishing of the fire occurs, and the vertical combustion level is increased from V-2 to V-0. Compared with FRPET, the peak of the heat release rate decreases by 66.0%, the total heat release decreases by 32.4%, the flame retardancy index (FRI) reaches an excellent value, and the condensed-phase products significantly improve. The Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy-energy dispersive X-ray spectrometry (SEM-EDX), thermogravimetric-FTIR (TG-FTIR), and pyrolysis-gas chromatograph/mass spectrometry (Py-GC/MS) results indicate that DOPO-POSS contributes to the formation of char layers and decomposes to generate free radicals with a quenching effect. In a word, DOPO-POSS is an effective radical trapper and charring agent for PET and exerts a flame retardancy effect in gaseous and condensed phases simultaneously.

Preparation of Carbon Dots with Ultrahigh Fluorescence Quantum Yield Based on PET Waste.

Environmentally friendly polyethylene terephthalate-based carbon dots (PET-CDs) with ultrahigh fluorescence quantum yield were prepared with waste PET textiles as raw materials. First, oligomers were prepared from the reaction of waste PET textile and ethylene glycol by the microwave method. Then, the mixture without isolation and purification as well as pyromellitic acid and urea were adopted as precursors for the preparation of PET-CDs by the hydrothermal method. It was found that the as-prepared PET-CDs had a spherical structure with an average particle size of 2.8 nm. The carbon core of PET-CDs was a graphene-like structure doped with nitrogen atoms in the form of pyrrole nitrogen and the surface contained -NH2, which is convenient for modification and functionalization with various materials in the form of chemical bonds. The as-prepared PET-CDs exhibit excitation-independent emission properties in the range from 340 to 440 nm, and the best excitation and emission wavelengths of PET-CDs are 406 and 485 nm, respectively, while the fluorescence quantum yield is 97.3%. In terms of the application, the as-prepared PET-CDs could be adopted as a fluorescence probe for the detection of Fe3+, and the limit of detection is as low as 0.2 µmol/L. The mechanism of PET-CDs by Fe3+ was found to be the static quenching mechanism. In addition, PET-CDs can be used in LEDs and fluorescent anticounterfeiting.

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.

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, il1ß 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-1ß via CXCR1. Simultaneously, antibody neutralization assay showed that endogenous IL-8 is partially relevant to the self-regulation of IL-1ß. 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.

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.

One-step preparation of red-emitting carbon dots for visual and quantitative detection of copper ions.

A one-step solvothermal method for the preparation of carbon dots with red fluorescence (R-CDs) was put forward, in which sodium citrate and formamide were chosen as precursors, while formamide was adopted as the solvent. The fluorescence emission peak of the as-prepared R-CDs remained the same (600 nm) when the excitation wavelength increased from 490 nm to 560 nm, and the fluorescence quantum yield is 35.3%. Furthermore, the fluorescence intensity of the as-prepared R-CDs could be selectively quenched by copper ions, and the mechanism of Cu2+ quenching R-CDs is the combination of static and dynamic quenching. As a result, the R-CDs were applied for the construction of a fluorescent sensor without any modification for the quantitative and visual detection of copper ions, which is a typical contaminant in water. The limit of detection for the fluorescent sensor was as low as 5 nmol/L, and it can be used to fast and directly confirm whether the content of copper ions in drinking water meets the criteria of the United States Environmental Protection Agency and the World Health Organization.

Molecular Dynamics Simulation on Thin-Film Lubrication of a Mixture of Three Alkanes.

We used the COMPASS forcefield to perform molecular dynamics (MD) simulation of a mixture composed of three alkanes as the lubricant for the thin-film lubrication. The viscosity of the lubrication film in the non-working state, the final film thickness, and density distribution were investigated. The results reveal that the viscosity error among different initial film thicknesses in the non-working state is within 5%, which confirms the applicability of the model and the forcefield. The viscosity decreases oscillating as temperature increases. Whatever the initial film thickness is, the film thickness change rate with respect to pressure load is almost the same. When pressure increases, the density peaks increase. As the initial film thickness increases, the normalized thicknesses of adsorption and ordered layers decrease. In nanoscale, the density predicted by the MD simulation is higher than the prediction of the Tait equation, even if the adsorption layers is excluded.

Cloning and identification of grass carp transcription factor HSF1 and its characterization involving the production of fish HSP70.

In mammals, heat shock transcription factor 1 (HSF1) is well documented as the critical transcript factor to regulate heat shock protein 70 (HSP70) expression under different stresses, such as heat shock or bacterial infection. In fish, Hsf1 responses to physiological and environmental stresses and regulates Hsp70 expression under thermal exposure. However, the functional role of Hsf1 in Hsp70 production is still elusive under bacterial infection. In the present study, a coding sequence of grass carp hsf1 (gchsf1) gene was cloned and identified. Using Ctenopharyngodon idellus kidney (CIK) cells as the model, we found that lipopolysaccharide (LPS) exerted stimulatory effects on the expression of grass carp hsp70 (gchsp70) and hsf1, implying possible relationship of Hsp70 and Hsf1 under immune stimulation in fish. To validate the hypothesis, overexpression of gcHsf1 was performed in CIK cells, and the effects of overexpressing gcHsf1 on the expression of gcHsp70 in the absence or presence of LPS were examined. Results showed that LPS significantly upregulated the transcription and protein synthesis of gcHsp70, and these stimulatory effects were further amplified when overexpression of gcHsf1 was performed. Furthermore, luciferase reporter assays in CIK cells revealed that both overexpression of Hsf1 and LPS upregulated gchsp70 transcription, and their combined treatment further enhanced the gchsp70 promoter activity. Moreover, the regions responsive to these treatments were mapped to the promoter of gchsp70. Besides transcriptional level and cellular protein contents, gcHsp70 secretion was measured by competitive ELISA, uncovering that gcHsf1 enhanced the release of gcHsp70 induced by LPS in the same cells. These data not only demonstrated the enhancement of Hsf1 in Hsp70 production but also initially revealed the involvement of Hsf1-Hsp70 axis in mediating inflammatory response in fish.

Regulation of Il-10 gene expression by Il-6 via Stat3 in grass carp head kidney leucocytes.

Interleukin (IL)-10 is a critical anti-inflammatory and late cytokine being produced after the proinflammatory mediators while IL-6 is a promptly synthesized cytokine in response to inflammation in mammals. This chronological expression of interleukin (Il)-6 and Il-10 was also found in grass carp head kidney leucocytes (HKLs) treated by heat-killed Aeromonas hydrophila, supporting the possible interplay between grass carp (gc)Il-6 and gcIl-10 in HKLs. Our further findings were in agreement with this hypothesis that recombinant gcIl-6 (rgcIl-6) promptly and transiently increased gcil10 mRNA levels in grass carp HKLs. Moreover, rgcIl-6 enhanced its own mRNA level and this self-enhancement of gcil6 mRNA level could be partially blocked by rgcIl-10. These results collectively suggest that gcIl-10 production stimulated by gcIl-6 may provide a negative feedback to gcIl-6 production. Interestingly, rgcIl-6 significantly decreased gcil10 mRNA levels in grass carp HKLs after the treatment for 12 and 24 h in contrast to its enhancement of gcil10 levels after the treatment for 3 h. Involvement of Stat3 but not MEK, p38 MAPK or JNK pathway in the increase of gcil10 mRNA levels by rgcIl-6 was revealed by using the signaling pathway inhibitors. This was supported by the fact that rgcIl-6 stimulated Stat3 phosphorylation in grass carp HKLs. Furthermore, rgcIl-6 had no effect on the stability of gcil10 mRNA after the treatment for 3 to 36 h while it increased gcil10 promoter activity after the treatment for 24 h. Taken these data together, gcIl-6 can stimulate Il-10 production at early stage but subsequently inhibit il10 mRNA expression in grass carp HKLs, shedding light on the dynamic regulation of il10 mRNA expression by Il-6 in fish immune cells.

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.

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.