Tumor necrosis factor α1 decreases mucosal immune and antioxidant response in the midgut of hybrid fish (white crucian carp ♀ × red crucian carp ♂).

Tumor necrosis factor α1 (TNFα) is a pleiotropic cytokine involved in immune regulation and cellular homeostasis, but the crucial role of TNFα in fish gut remained unclear. The current study aimed to evaluate the immunoregulatory function of TNFα1 on gut barrier in a novel hybrid fish (WR), which was produced by crossing white crucian carp (Carassius cuvieri, ♀) with red crucian carp (Carassius auratus red var, ♂). In this study, WR-tnfα1 sequence was identified, and a high-level expression was detected in the intestine. Elevated levels of WR-tnfα1 expressions were detected in immune-related tissues and cultured fish cells on stimulation. The appearance of vacuolization and submucosal rupture was observed in TNFα1-treated midgut of WR, along with elevated levels of goblet cell atrophy, whereas no significant changes were detected in most expressions of tight-junction genes and mucin genes. In contrast, WR receiving gut perfusion with WR-TNFα1 showed a remarkable decrease in antioxidant status in midgut, whereas the expression levels of apoptotic genes and redox responsive genes increased sharply. These results suggested that TNFα1 could exhibit a detrimental effect on antioxidant defense and immune regulation in the midgut of WR.

Alteration of synergistic immune response in gut-liver axis of white crucian carp (Carassius cuvieri) after gut infection with Aeromonas hydrophila.

Aeromonas hydrophila can pose a great threat to the survival of farmed fish. In current study, we investigated the pathological characteristics and immune response in gut-liver axis of white crucian carp (WCC) upon gut infection. WCC anally intubated with A. hydrophila exerted a tissue deformation in damaged midgut with elevated levels of goblet cells along with a significant decrease in tight junction proteins and villi length-to-width ratios. In addition, immune-related gene expressions and antioxidant properties increased dramatically in gut-liver axis of WCC following gut infection with A. hydrophila. These results highlighted the immune modulation and redox alteration in gut-liver axis of WCC in response to gut infection.

Integrated Transcriptomic and Metabolomic Analysis Reveals the Mechanism of Gibberellic acid Regulates the Growth and Flavonoid Synthesis in Phellodendron chinense Schneid Seedlings.

The phytohormone gibberellic acids (GAs) play a crucial role in the processes of growth, organ development, and secondary metabolism. However, the mechanism of exogenous GA3 regulating the growth and flavonoid synthesis in Phellodendron chinense Schneid (P. chinense Schneid) seedlings remains unclear. In this study, the physicochemical properties, gene expression level, and secondary metabolite of P. chinense Schneid seedlings under GA3 treatment were investigated. The results showed that GA3 significantly improved the plant height, ground diameter, fresh weight, chlorophyll content, soluble substance content, superoxide dismutase, and peroxidase activities. This was accompanied by elevated relative expression levels of Pc(S)-GA2ox, Pc(S)-DELLA, Pc(S)-SAUR50, Pc(S)-PsaD, Pc(S)-Psb 27, Pc(S)-PGK, Pc(S)-CER3, and Pc(S)-FBA unigenes. Conversely, a notable reduction was observed in the carotenoid content, catalase activity and the relative expression abundances of Pc(S)-KAO, Pc(S)-GID1/2, and Pc(S)-GH 3.6 unigenes in leaves of P. chinense Schneid seedlings (p < 0.05). Furthermore, GA3 evidently decreased the contents of pinocembrin, pinobanksin, isosakuranetin, naringin, naringenin, (-)-epicatechin, tricetin, luteolin, and vitexin belonged to flavonoid in stem bark of P. chinense Schneid seedlings (p < 0.05). These results indicated that exogenous GA3 promoted growth through improving chlorophyll content and gene expression in photosynthesis and phytohormone signal pathway and inhibited flavonoid synthesis in P. chinense Schneid seedlings.

Comparative analysis on the immunoregulatory roles of ferritin M in hybrid fish (Carassius cuvieri ♀ × Carassius auratus red var ♂) and its parental species after bacterial infection.

Ferritin M is involved in the regulation of fish immunity. In this study, open reading frame (ORF) sequences of ferritin M from hybrid fish and its parental species were 534 bp. Tissue-specific analysis indicated that the highest level of ferritin M from red crucian carp was observed in kidney, while peaked expressions of ferritin M from white crucian carp and hybrid carp were observed in gill. Elevated levels of ferritin M from hybrid carp and its parental species were detected in immune-related tissues following Aeromonas hydrophila infection or in cultured fish cell lines after lipopolysaccharide (LPS) challenge. Ferritin M overexpression could attenuate NF-κB and TNFα promoter activity in their respective fish cells. Purified ferritin M fusion proteins elicited in vitro binding activity to A. hydrophila and Edwardsiella tarda, lowered bacterial dissemination to tissues and alleviated inflammatory response. Furthermore, treatment with ferritin M fusion proteins could mitigate bacteria-induced liver damage and rescue antioxidant activity. These results suggested that ferritin M in hybrid fish showed a similar immune defense against bacteria infection in comparison with those of its parental species.

A novel ferritin L (FerL) in hybrid crucian carp could participate in host defense against Aeromonas hydrophila infection and diminish inflammatory signals.

FerL, a multifunctional iron-storage polypeptide, not only exhibited a regulatory role in iron metabolism, but also participated in the regulation of fish immunity. In this study, ORF sequence of WR-FerL was 522 bp, encoding 173 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-FerL was detected in spleen. A. hydrophila challenge and LPS stimulation could sharply enhance WR-FerL mRNA expression in tissues and fish cells, respectively. Purified WR-FerL fusion peptide exhibited in vitro binding activity to A. hydrophila and endotoxin, limited bacterial dissemination to tissues as well as attenuated A. hydrophila-induced production of pro-inflammatory cytokines. Moreover, WR-FerL overexpression could abrogate NF-κB and TNFα promoter activity in fish cells. These results indicated that WR-FerL could play an important role in host defense against A. hydrophila infection.

Blood cell characterization and transcriptome analysis reveal distinct immune response and host resistance of different ploidy cyprinid fish following Aeromonas hydrophila infection.

Aeromonas hydrophila can pose a great threat to survival of freshwater fish. In this study, A. hydrophila infection could decrease blood cell numbers, promote blood cell damage as well as alter the levels of alkaline phosphatase (ALP), lysozyme (LZM), aspartate aminotransferase (AST), total antioxidant capacity (T-AOC), total superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) in immune-related tissues of red crucian carp (RCC, 2 N = 100) and triploid cyprinid fish (3 N fish, 3 N = 150). In addition, the significant alternation of antioxidant status was observed in PBMCs isolated from RCC and 3 N following LPS stimulation. The core differential expression genes (DEGs) involved in apoptosis, immunity, inflammation and cellular signals were co-expressed differentially in RCC and 3 N following A. hydrophila challenge. NOD-like receptor (NLR) signals appeared to play a critical role in A. hydrophila-infected fish. DEGs of NLR signals in RCCah vs RCCctl were enriched in caspase-1-dependent Interleukin-1ß (IL-1ß) secretion, interferon (IFN) signals as well as cytokine activation, while DEGs of NLR signals in 3Nah vs 3Nctl were enriched in caspase-1-dependent IL-1ß secretion and antibacterial autophagy. These results highlighted the differential signal regulation of different ploidy cyprinid fish to cope with bacterial infection.

Transcriptome analysis and co-expression network reveal the mechanism linking mitochondrial function to immune regulation in red crucian carp (Carassius auratus red var) after Aeromonas hydrophila challenge.

Aeromonas hydrophila is a common pathogen of freshwater fish. In this study, A. hydrophila infection was shown to cause tissue damage, trigger physiological changes as well as alter the expression profiles of immune- and metabolic-related genes in immune tissues of red crucian carp (RCC). Transcriptome analysis revealed that acute A. hydrophila infection exerted a profound effect on mitochondrial oxidative phosphorylation linking metabolic regulation to immune response. In addition, we further identified cellular senescence, apoptosis, necrosis and mitogen-activated protein kinase signal pathways as crucial signal pathways in the kidney of RCC subjected to A. hydrophila infection. These findings may have important implications for understanding modulation of immunometabolic response to bacterial infection.

Comparative analysis of erythrocyte hemolysis, plasma parameters and metabolic features in red crucian carp (Carassius auratus red var) and triploid hybrid fish following Aeromonas hydrophila challenge.

Aeromonas hydrophila can pose a great threat to survival of freshwater fish. In this study, A. hydrophila challenge could promote the erythrocyte hemolysis, increase free hemoglobin (FHB) level and generate malondialdehyde (MDA) production in plasma but decrease the levels of total antioxidant capacity (T-AOC), total superoxide dismutase (SOD), catalase (CAT), alkaline phosphatase (ALP) and lysozyme (LZM) of red crucian carp (RCC, 2 N = 100) and triploid hybrid fish (3 N fish, 3 N = 150) following A. hydrophila challenge. Elevated expression levels of heat shock protein 90 alpha (HSP90α), matrix metalloproteinase 9 (MMP-9), free fatty acid receptor 3 (FFAR3), paraoxonase 2 (PON2) and cytosolic phospholipase A2 (cPLA2) were observed in A. hydrophila-infected fish. In addition, A. hydrophila challenge could significantly increase expressions of cortisol, leucine, isoleucine, glutamate and polyunsaturated fatty acids (PUFAs) in RCC and 3 N, while glycolysis and tricarboxylic acid cycle appeared to be inactive. We identified differential fatty acid derivatives and their metabolic networks as crucial biomarkers from metabolic profiles of different ploidy cyprinid fish subjected to A. hydrophila infection. These results highlighted the comparative metabolic strategy of different ploidy cyprinid fish against bacterial infection.

A novel NK-lysin in hybrid crucian carp can exhibit cytotoxic activity in fish cells and confer protection against Aeromonas hydrophila infection in comparison with Carassius cuvieri and Carassius auratus red var.

NK-lysin, an effector of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), not only exhibits cytotoxic effect in fish cells, but also participates in the immune defense against pathogenic infection. In this study, ORF sequences of RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin were 369 bp. Tissue-specific analysis revealed that the highest expressions of RCC-NK-lysin and WCC-NK-lysin were observed in gill, while the peaked level of WR-NK-lysin mRNA was observed in spleen. A. hydrophila infection sharply increased RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin mRNA expression in liver, trunk kidney and spleen. In addition, elevated levels of NK-lysin mRNA were observed in cultured fin cell lines of red crucian carp (RCC), white crucian carp (WCC) and their hybrid offspring (WR) after Lipopolysaccharide (LPS) challenge. RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin exerted regulatory roles in inducing ROS generation, modulating mitochondrial membrane potential, decreasing fish cell viability and antagonizing survival signalings, respectively. RCC/WCC/WR-NK-lysin-overexpressing fish could up-regulate expressions of inflammatory cytokines and decrease bacterial loads in spleen. These results indicated that NK-lysin in hybrid fish contained close sequence similarity to those of its parents, possessing the capacities of cytotoxicity and immune defense against bacterial infection.

FABP regulates fatty acid metabolism and oxidative response via PPARα/RXR signaling in Litopenaeus vannamei following environmental exposure of clofibric acid.

Clofibric acid (CFA), a drug and personal care product, has been identified as ubiquitous in the aquatic system and surface water, causing pollution to the environment. In this study, after environmental (4 µg/L) levels of CFA challenge, the LvFABP, LvACS gene expressions, total haemocyte count (THC), relative enzymes (SOD1 and GST) activities in Litopenaeus vannamei were observed to decrease. In the meantime LvFATP, LvRXR expression and the level of NEFA were upregulated in L. vannamei body. LvFABP expression in vivo was knocked down by dsRNA-mediated RNA interference (RNAi), which led to significantly decreased levels of PPARα (including LvFATP, LvRXR and LvACS). When exposed to environmental CFA after 4 days, LvFABP knocked down group had a sharp upregulation of LvFATP, LvRXR, LvACS expression, GST activity and NEFA amount, following decreased THC and SOD1 activity. These results suggested that environmental concentration CFA may have some toxicological effect on L. vannamei, following fatty acids metabolism and oxidative stress responses by LvFABP via the PPARα/RXR signaling pathway, including LvFATP, LvRXR and LvACS.

Antibacterial and immunoregulatory activity of a novel hepcidin homologue in diploid hybrid fish (Carassius auratus cuvieri ♀ × Carassius auratus red var ♂).

Hepcidin, a multifunctional hormone oligopeptide, not only exhibits a regulatory role in iron metabolism, but also participates in the regulation of teleostean immunity. In this study, ORF sequence of WR-hepcidin was 258 bp and encoded 85 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-hepcidin was observed in liver. Aeromonas hydrophila challenge can sharply increased WR-hepcidin mRNA expression in liver, trunk kidney and spleen. The purified WR-hepcidin fusion peptide can directly bind to A. hydrophila and Streptococcus agalactiae, reduce the relative bacterial activity, limit bacterial growth and attenuate their dissemination to tissues in vivo. In addition, the treatment of WR-hepcidin fusion protein can diminish the production of pro-inflammatory cytokines. These results indicated that WR-hepcidin can play a negative regulatory role in bacteria-stimulated pro-inflammatory cytokines production and MyD88-IRAK4 activation.

Molecular characterization of complement 9 in Epinephelus coioides and differential expression analysis of classical complement genes following Vibrio alginolyticus challenge.

Vibrio alginolyticus is posting an increasing threat to survival of grouper. Classical complement cascade can trigger initiation of immunity, while complement 9 (C9) is a major complement molecule involved in final step of membrane attack complex (MAC) formation. In this study, full-length EcC9 contained an ORF sequence of 1779 bp, encoding a polypeptide of 592 amino acids. A high-level expression of EcC9 mRNA was observed in liver. Following vibrio challenge, increased expression levels of EcC1q, EcBf/C2, EcC4, EcC6, EcC7 and EcC9 mRNA were detected in liver and kidney. These results implied that elevated expression level of classical complement pathway (CCP) and terminal complement components (TCCs) may assess toxicological effect of V. alginolyticus.

N-terminal domain of EcC1INH in Epinephelus coioides can antagonize the LPS-stimulated inflammatory response.

Complement 1 inhibitor (C1INH) serving as a multifunctional factor can participate in the regulation of complement cascades and attenuate the activation of various proteases. In this study, we obtained EcC1INH cDNA and the tissue-specific analysis indicate that the highest expression level of EcC1INH mRNA was detected in liver. Moreover, Vibrio alginolyticus challenge can significantly increase EcC1INH mRNA expression in liver and kidney. N-terminal domain of EcC1INH could decrease LPS binding activity to cell surface, while loss of positively charged residues (PCRs) Arg21, His22, Lys50, Arg61 in N-terminal domain of EcC1INH can significantly reduce its interaction with LPS. Furthermore, LPS injection experiment indicated that the binding of EcC1INH N-terminal domain to LPS can antagonize LPS-induced inflammatory signaling pathway and attenuate the production of proinflammatory cytokines in vivo, indicating that EcC1INH was involved in negative regulation of inflammatory response.

Characterization of a CD59 in orange-spotted grouper (Epinephelus coioides).

CD59, a multifunctional glycoprotein, not only plays a regulatory role in complement cascades, but also participates in modulation of teleostean immunity. In this study, full length sequence of EcCD59 was obtained, comprising a 5'UTR of 163 bp, an ORF of 354 bp and a 3'UTR of 559 bp. EcCD59 gene encoded a polypeptide of 117 amino acids. Tissue-specific analysis revealed that the highest expression of EcCD59 mRNA was observed in muscle. Vibrio alginolyticus challenge can significantly increase EcCD59 mRNA expression in liver, kidney and spleen. EcCD59 distribution was detected by a combined approach using GFP-overexpression, immunofluorescence and ELISA assay, indicating that EcCD59 may be predominantly aggregated in cellular membrane. Both EcCD59 and EcCD59delGPI can directly bind to V. alginolyticus and decrease the in vitro growth of V. alginolyticus. Additionally, vibrio injection experiment indicated that the binding of EcCD59 or EcCD59delGPI to V. alginolyticus can restrict its growth rate in vivo. In this study, we found that EcCD59 may be involved in immune defense against vibrio infection in a complement-independent manner.

Regulation of Th17 Differentiation by IKKα-Dependent and -Independent Phosphorylation of RORγt.

Transcription factor retinoid acid-related orphan receptor (ROR)γt transcriptionally regulates the genes required for differentiation of Th17 cells that mediate both protective and pathogenic immunity. However, little is known about the function of posttranslational modifications in the regulation of RORγt activity. Mass spectrometric analysis of immunoprecipitated RORγt from Th17 cells identified multiple phosphorylation sites. Systematic mutation analysis of the identified phosphorylation sites found that phosphorylation of S376 enhances whereas phosphorylation of S484 inhibits Th17 differentiation. IκB kinase (IKK)α binds and phosphorylates RORγt at S376 but not S484. Knockdown of IKKα, dominant-negative IKKα, and RORγt mutants incapable of interacting with IKKα all decrease Th17 differentiation. Furthermore, nonphosophorylatable RORγt mutant (S376A) impairs whereas phosphomimetic mutant (S376E) stimulates Th17 differentiation independent of IKKα. Therefore, IKKα-dependent phosphorylation of S376 stimulated whereas IKKα-independent phosphorylation of S484 inhibited RORγt function in Th17 differentiation.

Effects of high temperature on biochemical parameters, oxidative stress, DNA damage and apoptosis of pufferfish (Takifugu obscurus).

Water temperature is an important environmental factor that affects physiology and biochemical activities of fish. In this study, we investigated of high temperature on biochemical parameters, oxidative stress, DNA damage and apoptosis of pufferfish. Thermal stress could significantly increase the levels of AST, ALT, LDH, GLU and TG, whereas the levels of ALP and TP decrease significantly. In addition, thermal stress also decreased total blood cell count, inhibited cell viability, and subsequently lead to DNA damage and apoptosis. The mRNA levels of p53, caspase-9 and caspase-3 were up-regulated under thermal stress. These results suggested that caspase-dependent and p53 signaling pathways could play important roles in thermal stress-induced apoptosis in fish. Furthermore, the gene expression of SOD, CAT, HSP90 and C3 were induced by thermal stress. This study provides new insights into the mechanism whereby thermal stress affects physiological responses and apoptosis in pufferfish.

The protective effects of vitamin C on apoptosis, DNA damage and proteome of pufferfish (Takifugu obscurus) under low temperature stress.

The aim of this study was to investigate the protective effects of vitamin C on apoptosis, DNA damage and proteome of pufferfish under low temperature stress. Six diets were formulated to contain 2.60, 48.90, 95.50, 189.83, 382.40, 779.53mg/kg vitamin C. After 8-week feeding trial, fish were exposed to low temperature challenge. The results showed that pufferfish receiving vitamin C diet exhibited a significant decrease in ROS production (48.9-189.83mg/kg vitamin C diet groups), cytoplasmic free-Ca2+ concentration (48.9-779.53mg/kg vitamin C diet groups), apoptotic cell ratio (95.5-779.53mg/kg vitamin C diet groups) and DNA damage (189.83-779.53mg/kg vitamin C diet groups) under low temperature stress in comparison with those of control. We also investigated the alteration in protein expression under low temperature stress by a comparative proteomic analysis. The results demonstrated that 24 protein spots showed significantly differential expression in the cold-stress-treated group compared with those of the control group, and 5 protein spots were successfully identified. Furthermore, comparative proteomic analysis revealed that vitamin C could increase expressed proteins related to energy metabolism, immune responses and cytoskeleton. These findings would be helpful to understand the protective effects of vitamin C against cold stress.

Molecular and immune response characterizations of a novel Bax inhibitor-1 gene in pufferfish, Takifugu obscurus.

Apoptosis plays a crucial role in many biological processes, including development, cellular homeostasis, and immune responses. Bax inhibitor-1 (BI-1) is an anti-apoptotic protein that protects cells from endoplasmic reticulum stress-induced apoptosis. In this study, a BI-1 gene from the pufferfish Takifugu obscurus (Pf-BI-1) was identified and characterized. The full length of Pf-BI-1 cDNA was 1387 bp, including a 5'-UTR of 82 bp, a 3'-UTR of 591 bp containing a poly-(A) tail, and an open reading frame (ORF) of 714 bp that encodes a polypeptide of 237 amino acids. Pf-BI-1 was ubiquitously expressed in various tissues, with the highest expression levels in the blood, brain, and gill. The expression of Pf-BI-1 was up-regulated in a time-dependent manner after heat shock stress, ammonia stress, and bacterial challenge. Intracellular localization revealed that Pf-BI-1 was primarily localized in the cell cytoplasm. Furthermore, over-expression of Pf-BI-1 could active NF-кB reporter genes in HeLa cells. These results indicated that Pf-BI-1 may be involved in the apoptosis and immunity process against ambient stressors in pufferfish.

Identification, characterization and expression analysis of tumor suppressor protein p53 from pufferfish (Takifugu obscurus) after the Vibrio alginolyticus challenge.

The tumor suppressor protein p53 plays a critical role in cell cycle, apoptosis and DNA repair. In this study, the full-length pufferfish p53 (Pf-p53) was obtained, containing an open reading frame of 1095 bp, a 5'UTR of 157 bp and a 3'UTR of 285 bp with a poly (A) tail. The Pf-p53 encoded a polypeptide of 364 amino acids with a theoretical isoelectric point of 8.03 and predicted molecular weight of 40.6 kDa. Pf-p53 was ubiquitously expressed in various tissues with a high-level expression in kidney, liver and gill. Vibrio alginolyticus challenge could induce ROS production and disrupt Ca2+ homeostasis, subsequently leading to the induction of DNA damage and apoptosis, while the Vibrio alginolyticus-induced oxidative stress can also increase the non-specific immunity. The pufferfish challenged with Vibrio alginolyticus showed a sharp increase of Pf-p53 transcript in liver. Subcellular localization analysis revealed that Pf-p53 was primarily localized in nucleus. Furthermore, overexpression of Pf-p53 in Hela cells could inhibit cell proliferation and the transcriptional activities of the NF-ĸB promoter. Taken together, our results indicated that Pf-p53 may play an important role in the immune response to Vibrio alginolyticus challenge.

Molecular cloning and characterization of PTEN in the orange-spotted grouper (Epinephelus coioides).

PTEN is a key tumor suppressor gene that can play a regulatory role in the cellular proliferation, survival and apoptosis. In this study, the full-length PTEN (EcPTEN) was obtained, containing a 5'UTR of 745 bp, an ORF of 1269 bp and a 3'UTR of 106 bp. The EcPTEN gene encoded a polypeptide of 422 amino acids with an estimated molecular mass of 49.14 KDa and a predicted isoelectric point (pI) of 6.34. The deduced amino acid sequence analysis showed that EcPTEN comprised the conserved residues and the characteristic domains known to the critical functionality of PTEN. qRT-PCR analysis revealed that EcPTEN mRNA was broadly expressed in all the examined tissues, while the highest expression level was observed in liver, followed by the expression in blood, kidney, spleen, heart, gill, muscle and intestine. The groupers challenged with Vibrio alginolyticus showed a sharp increase of EcPTEN mRNA expression in immune tissues. In addition, western blotting analysis confirmed that the up-regulation of EcPTEN protein expression was steadily induced in liver. Subcellular localization analysis indicated that EcPTEN was localized in both nucleus and cytoplasm. Overexpression of EcPTEN can activate the apoptotic cascade and abrogate NF-kB, AP-1, Stat3 and Myc promoter activity in Hela cells. These results indicated that EcPTEN harboring highly-conserved domains with a close sequence similarity to those of PTP superfamily may disrupt the mammalian signalings and play a regulatory role in the apoptotic process.